No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Effect of I.C.V. injection of AT4 receptor ligands, NLE 1-angiotensin IV and LVV-hemorphin 7, on spatial learning in rats
Abstract
Central administration of angiotensin IV (Ang IV) or its analogues enhance performance of rats in passive avoidance and spatial memory paradigms. The purpose of this study was to examine the effect of a single bolus injection of two distinct AT4 ligands, Nle1-Ang IV or LVV-haemorphin-7, on spatial learning in the Barnes circular maze. Mean number of days for rats treated with either Nle1-Ang IV or LVV-haemorphin-7 to achieve learner criterion is significantly reduced compared with controls (P < 0.001 and P < 0.05 respectively). This is due to enhanced ability of the peptide-treated rats to adopt a spatial strategy for finding the escape hatch. In all three measures of learning performance, (1) the number of errors made, (2) the distance travelled and (3) the latency in finding the escape hatch, rats treated with either 100 pmol or 1 nmol of Nle1-Ang IV or 100 pmol LVV-haemorphin-7 performed significantly better than the control groups. As early as the first day of testing, the rats treated with the lower dose of Nle1-Ang IV or LVV-haemorphin-7 made fewer errors (P < 0.01 and P < 0.05 respectively) and travelled shorter distances (P < 0.05 for both groups) than the control animals. The enhanced spatial learning induced by Nle1-Ang IV (100 pmol) was attenuated by the co-administration of the AT4 receptor antagonist, divalinal-Ang IV (10 nmol). Thus, administration of AT4 ligands results in an immediate potentiation of learning, which may be associated with facilitation of synaptic transmission and/or enhancement of acetylcholine release.
... It has been demonstrated that Ang IV [15] and other IRAP inhibitors have the ability to improve memory performance in several types of animal models [2,9,[16][17][18][19]37,38] and that Ang IV elicits protective effects against induced ischemia [39,40]. We previously demonstrated that the potent IRAP inhibitor and macrocyclic Ang IV analogue HA08 increases dendritic spine density in primary hippocampal cell cultures, indicating that a positive cognitive effect could be expected in vivo [30]. ...
... The endogenous IRAP inhibitor Ang IV has also been shown to have protective effects against induced ischemia, in which Ang IV-treated rats showed less neurological deficit, reduced infarct and overall reduced mortality [40]. In addition, Ang IV has been studied directly in relation to its effect on memory, where it has been shown to improve or restore memory in different animal models [2,[16][17][18][19]37,38], including long-term treatment of an Alzheimer's disease mice model [37]. It has also been shown to increase the amount of brain-derived neurotrophic factor (BDNF) and decrease GABA, the main inhibitory neurotransmitter [38]. ...
... To conclude, inhibitors of IRAP, such as Ang IV, improve memory and cognition in animal models, and the aminopeptidase IRAP is recognized as a new potential target for drugs aimed at treatment of cognitive disorders [2,9,[16][17][18][19]37,38]. As a consequence, in recent years a large number of IRAP inhibitors have been reported from different laboratories [9,22,[25][26][27][28][29][30][31][32] and HA08, a macrocyclic disulfide analogue of the unstable endogenous Ang IV, has been identified as one of the most potent IRAP inhibitors known to date [12,34]. ...
Angiotensin IV (Ang IV), a metabolite of Angiotensin II, is a bioactive hexapeptide that inhibits the insulin-regulated aminopeptidase (IRAP). This transmembrane zinc metallopeptidase with many biological functions has in recent years emerged as a new pharmacological target. IRAP is expressed in a variety of tissues and can be found in high density in the hippocampus and neocortex, brain regions associated with cognition. Ang IV is known to improve memory tasks in experimental animals. One of the most potent IRAP inhibitors known today is the macrocyclic compound HA08 that is significantly more stable than the endogenous Ang IV. HA08 combines structural elements from Ang IV and the physiological substrates oxytocin and vasopressin, and binds to the catalytic site of IRAP. In the present study we evaluate whether HA08 can restore cell viability in rat primary cells submitted to hydrogen peroxide damage. After damaging the cells with hydrogen peroxide and subsequently treating them with HA08, the conceivable restoring effects of the IRAP inhibitor were assessed. The cellular viability was determined by measuring mitochondrial activity and lactate dehydrogenase (LDH) release. The mitochondrial activity was significantly higher in primary hippocampal cells, whereas the amount of LDH was unaffected. We conclude that the cell viability can be restored in this cell type by blocking IRAP with the potent macrocyclic inhibitor HA08, although the mechanism by which HA08 exerts its effects remains unclear.
... Several behavioral studies have confirmed the beneficial effects of LVV-H7 on learning and memory. These effects were observed as improvements in spatial and conditioned learning and were manifested as avoidance memory in normal rats [63]. Moreover, it has been shown that hemorphins and other AT4 receptor agonists also reversed memory impairments caused by the administration of scopolamine and mecamylamine [64,65], ischemic injury [66], alcohol abuse [67] and bilateral perforant pathway lesions [68]. ...
... Moreover, it has been shown that hemorphins and other AT4 receptor agonists also reversed memory impairments caused by the administration of scopolamine and mecamylamine [64,65], ischemic injury [66], alcohol abuse [67] and bilateral perforant pathway lesions [68]. Similar effects of LVV-H7 and Ang IV on memory and learning were also observed [63,69], such as enhancement of spatial working memory in the plus maze spontaneous alternation task [70,71] or spatial learning in the Barnes maze task [63]. The similarity of action is probably related to the fact that both Ang IV and LVV-H7 are endogenous ligands of the AT4 receptor [72]. ...
... Moreover, it has been shown that hemorphins and other AT4 receptor agonists also reversed memory impairments caused by the administration of scopolamine and mecamylamine [64,65], ischemic injury [66], alcohol abuse [67] and bilateral perforant pathway lesions [68]. Similar effects of LVV-H7 and Ang IV on memory and learning were also observed [63,69], such as enhancement of spatial working memory in the plus maze spontaneous alternation task [70,71] or spatial learning in the Barnes maze task [63]. The similarity of action is probably related to the fact that both Ang IV and LVV-H7 are endogenous ligands of the AT4 receptor [72]. ...
During the last three decades, a variety of different studies on bioactive peptides that are opioid receptor ligands, have been carried out, with regard to their isolation and identification, as well as their molecular functions in living organisms. Thus, in this review, we would like to summarize the present state-of-the art concerning hemorphins, methodological aspects of their identification, and their potential role as therapeutic agents. We have collected and discussed articles describing hemorphins, from their discovery up until now, thus presenting a very wide spectrum of their characteristic and applications. One of the major assets of the present paper is a combination of analytical and pharmacological aspects of peptides described by a team who participated in the initial research on hemorphins. This review is, in part, focused on the analysis of endogenous opioid peptides in biological samples using advanced techniques, description of the identification of synthetic/endogenous hemorphins, their involvement in pharmacology, learning, pain and other function. Finally, the part regarding hemorphin analogues and their synthesis, has been added.
... 7,8 The aminopeptidase activity of IRAP has been shown to be competitively inhibited by angiotensin IV (AngIV, 1), a product of proteolytic processing of angiotensin II. 6,[9][10][11][12][13][14][15] AngIV has also been shown to improve performance in a number of memory tasks when injected into the brains of rats. [9][10][11][12][13][14][15] This activity has provided the impetus for the development of new long-lived pharmacological IRAP inhibitors as potential therapeutics to treat Alzheimer's disease, especially given the very short in vivo half-life of AngIV. ...
... 6,[9][10][11][12][13][14][15] AngIV has also been shown to improve performance in a number of memory tasks when injected into the brains of rats. [9][10][11][12][13][14][15] This activity has provided the impetus for the development of new long-lived pharmacological IRAP inhibitors as potential therapeutics to treat Alzheimer's disease, especially given the very short in vivo half-life of AngIV. The published IRAP inhibitors described to date ( Fig. 1) include small molecules such as HFI-419 (2), 16 AngIV peptidomimetics, such as the β-amino acid modified compound, IVDE-77 (3), 17,18 and more recently transition state mimetics such as DG026 (4) based on antigen substrate peptides, as shown in Fig. 1. 19,20 From 2006-2011, details relating to macrocyclic analogues of AngIV with N-terminal disulphide or metathesis-linked peptides were published. ...
... The structure of 5 was modified in several ways that would test the basic pharmacophore. Firstly, a range of structural modifications to 5 were made to test the postulated pharmacophoric elements such as replacing the C-terminal carboxylate with an amide (9), the phenolic tyrosine with a fluorophenyl group (10), modifying the P1-P1′ amide linkage as the secondary amine (11) and removing the N-terminal amino group (12), as outlined in Fig. 2. These compounds were all synthesized by adaptation of the reported synthesis of 5. ...
Macrocyclic analogues of the linear hexapeptide, angiotensin IV (AngIV) have proved to be potent inhibitors of insulin-regulated aminopeptidase (IRAP, oxytocinase, EC 3.4.11.3). Along with higher affinity, macrocycles may also offer better metabolic stability, membrane permeability and selectivity, however predicting the outcome of particular cycle modifications is challenging. Here we describe the development of a series of macrocyclic IRAP inhibitors with either disulphide, olefin metathesis or lactam bridges and variations of ring size and other functionality. The binding mode of these compounds is proposed based on molecular dynamics analysis. Estimation of binding affinities (ΔG) and relative binding free energies (ΔΔG) with the linear interaction energy (LIE) method and free energy perturbation (FEP) method showed good general agreement with the observed inhibitory potency. Experimental and calculated data highlight the cumulative importance of an intact N-terminal peptide, the specific nature of the macrocycle, the phenolic oxygen and the C-terminal functionality.
... Hemorphins have been shown to possess a number of biological activities including effects on spatial learning, inflammation, analgesia, and transient hypotension (Hughes et al., 1975;Moisan et al., 1998;Sanderson et al., 1998;Albiston et al., 2004;Cejka et al., 2004;Lee et al., 2004;Cheng et al., 2012) The analgesic effects of hemorphins have been attributed to its pharmacological action on the opioid receptors belonging to the G protein-coupled receptor (GPCR) family (Davis et al., 1989;Liebmann et al., 1989;Glämsta et al., 1992;Yukhananov et al., 1994;Szikra et al., 2001;Chow et al., 2018). LVV-hemorphin-7 has also been reported to bind and inhibit the angiotensin IV (AngIV) receptor, or the insulinregulated aminopeptidase, in the hippocampus with significant improvement of memory Lee et al., 2004). ...
... Hemorphins have been shown to possess a number of biological activities including effects on spatial learning, inflammation, analgesia, and transient hypotension (Hughes et al., 1975;Moisan et al., 1998;Sanderson et al., 1998;Albiston et al., 2004;Cejka et al., 2004;Lee et al., 2004;Cheng et al., 2012) The analgesic effects of hemorphins have been attributed to its pharmacological action on the opioid receptors belonging to the G protein-coupled receptor (GPCR) family (Davis et al., 1989;Liebmann et al., 1989;Glämsta et al., 1992;Yukhananov et al., 1994;Szikra et al., 2001;Chow et al., 2018). LVV-hemorphin-7 has also been reported to bind and inhibit the angiotensin IV (AngIV) receptor, or the insulinregulated aminopeptidase, in the hippocampus with significant improvement of memory Lee et al., 2004). In vitro and in vivo studies have demonstrated the beneficial effects of hemorphins in the control of blood pressure. ...
... Moreover, LVVhemorphin-6 (Yukhananov et al., 1994) and-7 (Szikra et al., 2001) showed agonist-like effect as demonstrated in guinea-pig ileum and GTPγS binding assays, respectively. This clearly demonstrated the binding of these hemorphins on the opioid receptors that explain the analgesic effects of hemorphin observed in vivo (Hughes et al., 1975;Moisan et al., 1998;Sanderson et al., 1998;Albiston et al., 2004;Cejka et al., 2004;Lee et al., 2004;Cheng et al., 2012). Of course, our data do not prove a direct binding of LVV-hemorphin-7 on AT1R, but the strong positive effects observed in IP1 and BRET assays, as well as the agonistic action of LVV-hemorphin-7, observed in ERK1/2 assay and to very less extent BRET (100 µM), FIGURE 6 | Positive effect of LVV-hemorphin-7 on AT1R-mediated ERK1/2 phosphorylation. ...
Hemorphins are hemoglobin β-chain–derived peptides initially known for their analgesic effects via binding to the opioid receptors belonging to the family of G protein–coupled receptor (GPCR), as well as their physiological action on blood pressure. However, their molecular mechanisms in the regulation of blood pressure are not fully understood. Studies have reported an antihypertensive action via the inhibition of the angiotensin-converting enzyme, a key enzyme in the renin–angiotensin system. In this study, we hypothesized that hemorphins may also target angiotensin II (AngII) type 1 receptor (AT1R) as a key GPCR in the renin–angiotensin system. To investigate this, we examined the effects of LVV–hemorphin-7 on AT1R transiently expressed in human embryonic kidney (HEK293) cells using bioluminescence resonance energy transfer (BRET) technology for the assessment of AT1R/Gαq coupling and β-arrestin 2 recruitment. Interestingly, while LVV–hemorphin-7 alone had no significant effect on BRET signals between AT1R and Gαq or β-arrestin 2, it nicely potentiated AngII-induced BRET signals and significantly increased AngII potency. The BRET data were also correlated with AT1R downstream signaling with LVV–hemorphin-7 potentiating the canonical AngII-mediated Gq-dependent inositol phosphate pathway as well as the activation of the extracellular signal–regulated kinases (ERK1/2). Both AngII and LVV–hemorphin-7–mediated responses were fully abolished by AT1R antagonist demonstrating the targeting of the active conformation of AT1R. Our data report for the first time the targeting and the positive modulation of AT1R signaling by hemorphins, which may explain their role in the physiology and pathophysiology of both vascular and renal systems. This finding further consolidates the pharmacological targeting of GPCRs by hemorphins as previously shown for the opioid receptors in analgesia opening a new era for investigating the role of hemorphins in physiology and pathophysiology via the targeting of GPCR pharmacology and signaling.
... treatment with either Ang IV or LVV-H7 im- proved performance on a plus maze task relative to vehicle controls, with peptide-treated animals showing increased alternation scores. Si- milarly, the second study by Lee et al. (2004) reported that i.c.v. treatment with Nle 1 -Ang IV or LVV-H7 was associated with significantly fewer errors, shorter distance traveled, and reduced latency in finding the escape tunnel, compared to the controls, with differences observed in the first two measures as early as the first day of testing. ...
... Nevertheless, while di- hexa's procognitive properties have been demonstrated in its reversal of scopolamine-induced deficit, it remains to be seen if dihexa would be similarly beneficial in maintaining cognition in models without im- pairment. Its parent peptide, Nle 1 -Ang IV, has been associated with better memory performance in such models ( Lee et al., 2004;Wright et al., 1999), so the potential for this is promising. ...
... Ang IV effects may not be due to its activation of the AT4R alone. While co-administration of Ang IV and the AT4R-antagonist Divalinal- Ang IV ameliorates the procognitive effects of Ang IV ( Lee et al., 2004;Paris et al., 2013;Wilson et al., 2009;Wright et al., 1999), these same effects were also surprisingly attenuated by administration of sarilesin, a non-specific angiotensin receptor antagonist which blocks both AT1Rs and AT2Rs ( Tchekalarova et al., 2001a). The role of the AT2R is less known, although it is found in lower quantities than AT1R in adults (Steckelings et al., 2005) and its differential distribution patterns in fetal and adult animals suggests a role in maturation and differentiation (Tsutsumi & Saavedra, 1991). ...
... Multiple animal and human studies help explain possible underlying mechanisms for our results. A sizable volume of animal model [19][20][21][22][23][24][25][26] and human studies [27][28][29][30] support the overarching hypothesis that beyond effects on BP, antihypertensive drugs that increase activity at the angiotensin type 2 and 4 receptors provide greater brain protection compared with those that decrease activity. Numerous studies suggest a role for angiotensin-II and angiotensin-IV activity in protecting from ischemia or enhancing cerebral blood flow, especially via activity at angiotensin type 2 and, possibly, type 4 receptors [8,10,24,31,32]. ...
... Numerous studies suggest a role for angiotensin-II and angiotensin-IV activity in protecting from ischemia or enhancing cerebral blood flow, especially via activity at angiotensin type 2 and, possibly, type 4 receptors [8,10,24,31,32]. Agonism at the angiotensin type 4 receptor may improve spatial memory processing [23,33,34]. Translating this mechanistic work to clinical practice, using existing antihypertensives, is an area of active research. ...
Background
Certain classes of antihypertensive medication may have different associations with cognitive impairment.
Objective
To examine the association between prevalent use of antihypertensive medications that stimulate (thiazides, dihydropyridine calcium channel blockers, angiotensin type I receptor blockers) versus inhibit (angiotensin-converting enzyme inhibitors, beta-blockers, non-dihydropyridine calcium channel blockers) type 2 and 4 angiotensin II receptors on cognitive impairment among older adults residing in Veterans Affairs (VA) nursing homes for long-term care.
Methods
Retrospective cohort study. Long-term care residents aged 65 + years admitted to a VA nursing home from 2012 to 2019 using blood pressure medication and without cognitive impairment at admission. Main exposure was prevalent use of angiotensin II receptor type 2 and 4-‘stimulating’ (N = 589), ‘inhibiting’ (N = 3,219), or ‘mixed’ (N = 1,715) antihypertensive medication regimens at admission. Primary outcome was any cognitive impairment (Cognitive Function Scale).
Results
Over an average of 5.4 months of follow-up, prevalent use of regimens containing exclusively ‘stimulating’ antihypertensives was associated with a lower risk of any incident cognitive impairment as compared to prevalent use of regimens containing exclusively ‘inhibiting’ antihypertensives (HR 0.83, 95% CI 0.74–0.93). Results for the comparison between ‘mixed’ versus ‘inhibiting’ regimens were in the same direction but not statistically significant (HR 0.96, 95% CI 0.88–1.06).
Conclusion
For residents without cognitive impairment at baseline, prevalent users of regimens containing exclusively antihypertensives that stimulate type 2 and 4 angiotensin II receptors had lower rates of cognitive impairment as compared to prevalent users of regimens containing exclusively antihypertensives that inhibit these receptors. Residual confounding cannot be ruled out.
... These are naturally occurring peptides produced in the brain, spinal cord, plasma and cerebrospinal fluid 5,[11][12][13][14] . Hemorphins have been shown to possess numerous biological activities including effects on spatial learning, inflammation, analgesia and transient hypotension [15][16][17][18] . Among hemorphin peptides, LVV-hemorphin-7 (LVV-H7 or LVVYPWTQRF) is the longest with 10 amino acids. ...
... Several in vivo and in vitro studies have reported the therapeutic potential of hemorphin 16,17,26,32 . The ability of LVV-hemorphin-7 to bind to several proteins with high affinity emphasizes the importance of understanding the binding mode and activity of this peptide at the molecular level. ...
Hemorphins are atypical endogenous opioid peptides produced by the cleavage of hemoglobin beta chain. Several studies have reported the therapeutic potential of hemorphin in memory enhancement, blood regulation, and analgesia. However, the mode of interaction of hemorphin with its target remains largely elusive. The decapeptide LVV-hemorphin-7 is the most stable form of hemorphin. It binds with high affinity to mu-opioid receptors (MOR), angiotensin-converting enzyme (ACE) and insulin-regulated aminopeptidase (IRAP). In this study, computational methods were used extensively to elucidate the most likely binding pose of mammalian LVV-hemorphin-7 with the aforementioned proteins and to calculate the binding affinity. Additionally, alignment of mammalian hemorphin sequences showed that the hemorphin sequence of the camel harbors a variation – a Q > R substitution at position 8. This study also investigated the binding affinity and the interaction mechanism of camel LVV-hemorphin-7 with these proteins. To gain a better understanding of the dynamics of the molecular interactions between the selected targets and hemorphin peptides, 100 ns molecular dynamics simulations of the best-ranked poses were performed. Simulations highlighted major interactions between the peptides and key residues in the binding site of the proteins. Interestingly, camel hemorphin had a higher binding affinity and showed more interactions with all three proteins when compared to the canonical mammalian LVV-hemorphin-7. Thus, camel LVV-hemorphin-7 could be explored as a potent therapeutic agent for memory loss, hypertension, and analgesia.
... The effect of insulin-regulated aminopeptidase (IRAP) inhibition on cognitive enhancement is well-documented. The peptide inhibitors of IRAP, angiotensin IV (Ang IV), and LVV hemorphin 7 (LVV-H7), have been shown to improve memory across a variety of behavioral paradigms including passive avoidance tasks (Braszko et al. 1988;Braszko et al. 1991;Wright et al. 1993) and spatial navigation tasks such as the Morris water maze (Wright et al. 1999a;Wright et al. 1999b) and Barnes circular maze (Lee et al. 2004). Furthermore, peptide IRAP inhibitors are able to improve cognitive function in rodents subjected to chronic alcohol exposure (Wisniewski et al. 1993), global ischemia (Wright et al. 1996), bilateral perforant pathway lesions (Wright et al. 1999a), and disruptions to the septohippocampal cholinergic pathway (Pederson et al. 2001;Lee et al. 2004;Olson et al. 2004;Olson and Cero 2010). ...
... The peptide inhibitors of IRAP, angiotensin IV (Ang IV), and LVV hemorphin 7 (LVV-H7), have been shown to improve memory across a variety of behavioral paradigms including passive avoidance tasks (Braszko et al. 1988;Braszko et al. 1991;Wright et al. 1993) and spatial navigation tasks such as the Morris water maze (Wright et al. 1999a;Wright et al. 1999b) and Barnes circular maze (Lee et al. 2004). Furthermore, peptide IRAP inhibitors are able to improve cognitive function in rodents subjected to chronic alcohol exposure (Wisniewski et al. 1993), global ischemia (Wright et al. 1996), bilateral perforant pathway lesions (Wright et al. 1999a), and disruptions to the septohippocampal cholinergic pathway (Pederson et al. 2001;Lee et al. 2004;Olson et al. 2004;Olson and Cero 2010). While promising, peptide inhibitors of IRAP are unsuitable in a clinical setting because of off-target effects, rapid degradation, and poor blood-brain barrier permeability. ...
Ethyl2‐acetylamino‐7‐hydroxy‐4‐pyridin‐3‐yl‐4H‐chromene‐3‐carboxylate (HFI‐419), the benzopyran‐based inhibitor of insulin‐regulated aminopeptidase (IRAP), has previously been shown to improve spatial working and recognition memory in rodents. However, the mechanism of its cognitive‐enhancing effect remains unknown. There is a close correlation between dendritic spine density and learning in vivo and several studies suggest that increases in neuronal glucose uptake and/or alterations to the activity of matrix metalloproteinases (MMPs) may improve memory and increase dendritic spine density. We aimed to identify the potential mechanism by which HFI‐419 enhances memory by utilizing rat primary cultures of hippocampal cells. Alterations to dendritic spine density were assessed in the presence of varying concentrations of HFI‐419 at different stages of hippocampal cell development. In addition, glucose uptake and changes to spine density were assessed in the presence of indinavir, an inhibitor of the glucose transporter 4 (GLUT4), or the matrix metalloprotease inhibitor CAS 204140‐01‐2. We confirmed that inhibition of IRAP activity with HFI‐419 enhanced spatial working memory in rats, and determined that this enhancement may be driven by GLUT4‐mediated changes to dendritic spine density. We observed that IRAP inhibition increased dendritic spine density prior to peak dendritic growth in hippocampal neurons, and that spine formation was inhibited when GLUT4‐mediated glucose uptake was blocked. In addition, during the peak phase of dendritic spine growth, the effect of IRAP inhibition on enhancement of dendritic spine density resulted specifically in an increase in the proportion of mushroom/stubby‐like spines, a morphology associated with memory and learning. Moreover, these spines were deemed to be functional based on their expression of the pre‐synaptic markers vesicular glutamate transporter 1 and synapsin. Overall, or findings suggest that IRAP inhibitors may facilitate memory by increasing hippocampal dendritic spine density via a GLUT4‐mediated mechanism.
image
Cover Image for this issue: doi: 10.1111/jnc.14745.
... Our lab has a longstanding interest in the hexapeptide angiotensin IV (Ang IV), a degradation fragment of angiotensin II, as intracerebroventricular injections were shown to improve memory and learning in a large variety of animal models. [2][3][4][5][6][7][8] High densities of binding sites for Ang IV are observed in regions of the brain associated with sensory, motor, and cognitive functions, for example, the hippocampus. 3,[9][10][11][12] At the time of the discovery, these sites of binding were referred to as the AT4 receptor, although the nature of this receptor was largely unknown. ...
... We have contributed in this field by variations of Ang IV itself, driven by the beneficial pharmacological effects observed on central administration of this degradation product originating from angiotensin II. [2][3][4][5][6][7][8] Based on the finding in 2001 that a central target for this effect is the aminopeptidase IRAP, 13 with activity toward cyclic peptides such as vasopressin and oxytocin, we have also worked with metabolically stable variants thereof, 21-25 with a primary focus are primarily focus on the field of cognition impairment. However, despite the possibility to generate potent peptide-based inhibitors, the majority of these suffer from preclinical challenges when administered systemically, particularly with regards to the limited cell permeability and penetrance of the blood-brain barrier. ...
Intracerebroventricular injection of angiotensin IV, a ligand of insulin-regulated aminopeptidase (IRAP), has been shown to improve cognitive functions in several animal models. Consequently, IRAP is considered a potential target for treatment of cognitive disorders. To identify nonpeptidic IRAP inhibitors, we adapted an established enzymatic assay based on membrane preparations from Chinese hamster ovary cells and a synthetic peptide-like substrate for high-throughput screening purposes. The 384-well microplate-based absorbance assay was used to screen a diverse set of 10,500 compounds for their inhibitory capacity of IRAP. The assay performance was robust with Z'-values ranging from 0.81 to 0.91, and the screen resulted in 23 compounds that displayed greater than 60% inhibition at a compound concentration of 10 μM. After hit confirmation experiments, purity analysis, and promiscuity investigations, three structurally different compounds were considered particularly interesting as starting points for the development of small-molecule-based IRAP inhibitors. After resynthesis, all three compounds confirmed low μM activity and were shown to be rapidly reversible. Additional characterization included activity in a fluorescence-based orthogonal assay and in the presence of a nonionic detergent and a reducing agent, respectively. Importantly, the characterized compounds also showed inhibition of the human ortholog, prompting our further interest in these novel IRAP inhibitors.
... In addition to the aforementioned behavioral tests highlighting the interactions of Ang IV with cholinergic and dopaminergic neurotransmission, numerous studies have demonstrated that Ang IV and its analogs may exert beneficial effects in mitigating memory deficits in various animal models [57,[59][60][61][62][63][64][65]. Additionally, IRAP has been identified as a potential pharmacological target for the modulation of cognitive functions [66][67][68]. ...
Background: N-methyl-D-aspartate type glutamate receptors (NMDARs) are fundamental to neuronal physiology and pathophysiology. The prefrontal cortex (PFC), a key region for cognitive function, is heavily implicated in neuropsychiatric disorders, positioning the modulation of its glutamatergic neurotransmission as a promising therapeutic target. Our recently published findings indicate that AT1 receptor activation enhances NMDAR activity in layer V pyramidal neurons of the rat PFC. At the same time, it suggests that alternative angiotensin pathways, presumably involving AT4 receptors (AT4Rs), might exert inhibitory effects. Angiotensin IV (Ang IV) and its analogs have demonstrated cognitive benefits in animal models of learning and memory deficits. Methods: Immunohistochemistry and whole-cell patch-clamp techniques were used to map the cell-type-specific localization of AT4R, identical to insulin-regulated aminopeptidase (IRAP), and to investigate the modulatory effects of Ang IV on NMDAR function in layer V pyramidal cells of the rat PFC. Results: AT4R/IRAP expression was detected in pyramidal cells and GABAergic interneurons, but not in microglia or astrocytes, in layer V of the PFC in 9–12-day-old and 6-month-old rats. NMDA (30 μM) induced stable inward cation currents, significantly inhibited by Ang IV (1 nM–1 µM) in a subset of pyramidal neurons. This inhibition was reproduced by the IRAP inhibitor LVVYP-H7 (10–100 nM). Synaptic isolation of pyramidal neurons did not affect the Ang IV-mediated inhibition of NMDA currents. Conclusions: Ang IV/IRAP-mediated inhibition of NMDA currents in layer V pyramidal neurons of the PFC may represent a way of regulating cognitive functions and thus a potential pharmacological target for cognitive impairments and related neuropsychiatric disorders.
... In 1988, Braszko et al. reported that intracerebroventricular injection of the endogenous hexapeptide angiotensin IV (Ang IV, Val-Tyr-Ile-His-Pro-Phe), formed after degradation of the octapeptide angiotensin II (Ang II, Asp-Arg-Val-Tyr-Ile-His-Pro-Phe), improved memory and learning in rats [11]. Subsequently, Ang IV and related peptides such as LVV-hemorphin 7 (Leu-Val-Val-Tyr--Pro-Trp-Thr-Gln-Arg-Phe) were examined in a variety of models [12][13][14][15][16][17][18][19][20], and binding sites were found to be abundantly expressed in brain areas linked to cognition, such as the amygdala, hippocampus and the cerebral cortex [21][22][23]. ...
... In the beginning of the century, IRAP was identified as the receptor for the hexapeptide Angiotensin IV (Ang IV) [7,18]. Ang IV and related analogues have demonstrated enhanced memory effects in several behavior models, as first demonstrated by Brazko et al. [19][20][21][22][23][24][25][26] and bind to the active site of the protein and thereby causing inhibition of its enzymatic activity. This inhibition is suggested to increase levels of peptides such as vasopressin and oxytocin, and also modulate glucose uptake, leading to improved cognitive functions [6,7,12,14,15,27,28]. Reported data has initiated the search for new ligands with capacity to inhibit IRAP in a pursuit for cognitive enhancers that are more suitable as pharmaceuticals compared to the endogenous Ang IV and related peptides [16,[29][30][31]. ...
The insulin-regulated aminopeptidase (IRAP; oxytocinase) is part of the M1 aminopeptidase family and is highly expressed in many tissues, including the neocortex and hippocampus of the brain. IRAP is involved in various physiological functions and has been identified as a receptor for the endogenous hexapeptide Angiotensin IV (Ang IV). Binding of Ang IV inhibits the enzymatic activity of IRAP and has proven to enhance learning and memory in animal models. The macrocyclic compound 9 (C9) is a potent synthetic IRAP inhibitor developed from the previously reported inhibitor HA08. In this study, we have examined compound C9 and its effects on cognitive markers drebrin, microtubule-associated protein 2 (MAP2), and glial fibrillary acidic protein (GFAP) in primary hippocampal and cortical cultures. Cells from Sprague Dawley rats were cultured for 14 days before treatment with C9 for 4 consecutive days. The cells were analysed for protein expression of drebrin, MAP2, GFAP, glucose transporter type 4 (GLUT4), vesicular glutamate transporter 1 (vGluT1), and synapsin I using immunocytochemistry. The gene expression of related proteins was determined using qPCR, and viability assays were performed to evaluate toxicity. The results showed that protein expression of drebrin and MAP2 was increased, and the corresponding mRNA levels were decreased, after treatment with C9 in the hippocampal cultures. The ratio of MAP2 positive neurons and GFAP positive astrocytes was altered in both hippocampal and cortical cultures, and there were no toxic effects observed. In conclusion, IRAP inhibitor compound C9 enhance the expression of pro-cognitive markers drebrin and MAP2 which further confirms IRAP as a relevant pharmaceutical target and C9 as a promising candidate for further investigation.
... Subsequently, the hexapeptide was examined in a variety of experimental models [2][3][4][5][6][7]. Moreover, the effects of structural analogs of Ang IV were also explored, e.g., Nle-Ang IV (Nle-Tyr-Ile-His-Pro-Phe) and LVV-hemorphin-7 (Leu-Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg-Phe), and these two peptides were shown to act as strong promoters of memory retention and retrieval in rats [4,7,8]. The binding sites of Ang IV in brain areas associated with cognition were early identified [9], e.g., in the hippocampus [10], and today, excellent reviews on Ang IV and its impact on cognition are available [11][12][13][14][15]. ...
With the ambition to identify novel chemical starting points that can be further optimized into small drug-like inhibitors of insulin-regulated aminopeptidase (IRAP) and serve as potential future cognitive enhancers in the clinic, we conducted an ultra-high-throughput screening campaign of a chemically diverse compound library of approximately 400,000 drug-like small molecules. Three biochemical and one biophysical assays were developed to enable large-scale screening and hit triaging. The screening funnel, designed to be compatible with high-density microplates, was established with two enzyme inhibition assays employing either fluorescent or absorbance readouts. As IRAP is a zinc-dependent enzyme, the remaining active compounds were further evaluated in the primary assay, albeit with the addition of zinc ions. Rescreening with zinc confirmed the inhibitory activity for most compounds, emphasizing a zinc-independent mechanism of action. Additionally, target engagement was confirmed using a complementary biophysical thermal shift assay where compounds causing positive/negative thermal shifts were considered genuine binders. Triaging based on biochemical activity, target engagement, and drug-likeness resulted in the selection of 50 qualified hits, of which the IC50 of 32 compounds was below 3.5 µM. Despite hydroxamic acid dominance, diverse chemotypes with biochemical activity and target engagement were discovered, including non-hydroxamic acid compounds. The most potent compound (QHL1) was resynthesized with a confirmed inhibitory IC50 of 320 nM. Amongst these compounds, 20 new compound structure classes were identified, providing many new starting points for the development of unique IRAP inhibitors. Detailed characterization and optimization of lead compounds, considering both hydroxamic acids and other diverse structures, are in progress for further exploration.
... LVV-H7 and the less-studied VV-H7 (Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg-Phe) were initially found to bind to opioid receptors, and despite their low affinity, many of its biological activities (e.g., antinociception and hypotension) are thought to be mediated by opioid receptor activation [11][12][13][14][15], further studies indicate that LVV-H7 binds to insulin-regulated aminopeptidase (IRAP) because its binding affinity for IRAP is much higher than that of opioid receptors. LVV-H7-induced blockade of IRAP in CNS, similarly to angiotensin IV, improves hippocampaldependent memory [10,[16][17][18][19]. The same hemorphins have also been reported as ligands for human bombesin subtype 3 receptor (BRS-3) and the MAS-related G protein-coupled receptor X1 (MRGPRX1) [20,21]. ...
This manuscript presented the synthesis and characterization of two new N- and C-modified analogues of VV-hemorphin-7 containing RGD (Arg–Gly–Asp) residues as potential nociceptive agents and bioactive materials. It has been shown that the addition of one or two RGD sequences to natural VV-hemorphin-7 increases its effect on acute nociception, but the reduction of the inflammatory phase depends on the concentration of the peptide. The structure–property relationship of the new peptide derivatives was highlighted by electrochemical and FT-IR methods of analysis. Because of the proven bone-structural bonds of hydroxyapatite, the simultaneous deposition of peptide/hydroxyapatite on the surface of a titanium surface was investigated. The deposition was performed in a medium of gelatin solution containing dissolved amounts of peptide and hydroxyapatite using ultrasound. SEM–EDS analyzes confirmed the presence of a layer of the studied system.
Graphical abstract
... Ang IV when bound to the AT1 receptor showed the vasoconstrictor effect, and with the AT4 receptor, it improved learning and memory response [26]. Cognition enhancement is mainly because of its ability to increase cholinergic neurotransmission in the hippocampal region [27]. APA inhibitors, amastatin and RB150 (orally active), inhibit the conversion of Ang II to III in the brain thereby increasing Ang II which decreases BP and reduces the pressor response [28] via the AT2 receptor. ...
The RAS (renin-angiotensin system) is the part of the endocrine system that plays a prime role in the control of essential hypertension. Since the discovery of brain RAS in the seventies, continuous efforts have been put by the scientific committee to explore it more. The brain has shown the presence of various components of brain RAS such as angiotensinogen (AGT), converting enzymes, angiotensin (Ang), and specific receptors (ATR). AGT acts as the precursor molecule for Ang peptides—I, II, III, and IV—while the enzymes such as prorenin, ACE, and aminopeptidases A and N synthesize it. AT1, AT2, AT4, and mitochondrial assembly receptor (MasR) are found to be plentiful in the brain. The brain RAS system exhibits pleiotropic properties such as neuroprotection and cognition along with regulation of blood pressure, CVS homeostasis, thirst and salt appetite, stress, depression, alcohol addiction, and pain modulation. The molecules acting through RAS predominantly ARBs and ACEI are found to be effective in various ongoing and completed clinical trials related to cognition, memory, Alzheimer’s disease (AD), and pain. The review summarizes the recent advances in the brain RAS system highlighting its significance in pathophysiology and treatment of the central nervous system-related disorders.
... The paracrine RAS consists of the same components and acts independently of peripheral function [7]. AngIV, an important component of the brain RAS, has been linked to neuroprotective effects, such as the improvement of cognitive impairment and recovery of memory in some animal models [8][9][10]. Previous studies have shown that AngIV and its analogs could enhance cognitive ability in rodents and rescue cognitive impairment induced by cerebral ischemia [11,12]. ...
The renin-angiotensin system (RAS) is a paracrine RAS within the central nervous system (CNS) and is closely related to Alzheimer’s disease (AD). The endogenous hexapeptide angiotensin IV (Ang IV), an important component of the brain RAS, was found to rescue cognitive impairment and recover memory in previous studies. In our study, we used different doses of Dihexa, which can be orally administered and cross the BBB in APP/PS1 mice. We found that the amount of AngIV in mouse tissue increased after the administration of Dihexa compared to that in the WT group. Meanwhile, Dihexa restored spatial learning and cognitive functions in the Morris water maze test. Dihexa increased the neuronal cells and the expression of SYP protein in APP/PS1 mice in Nissl staining. Furthermore, Dihexa decreased the activation of astrocytes and microglia, markedly reduced levels of the pro-inflammatory cytokines IL-1β and TNF-α and increased the levels of the anti-inflammatory cytokine IL-10. Dihexa activated the PI3K/AKT signaling pathway, while PI3K inhibitor wortmannin significantly reversed the anti-inflammatory and anti-apoptotic effects of APP/PS1 mice. These findings highlight the brain AngIV/PI3K/AKT axis as a potential target for the treatment of AD.
... Several studies showed that intracerebroventricular (i.c.v.) injection of Ang IV or its analogues enhanced performance of rats in the MWM test (De Bundel et al., 2009;Lee et al., 2004;Wright and Harding, 2009;Wright et al., 1999). In addition, Wright and Harding (2009) suggested that Ang IV increased the escape latency in the passive avoidance task. ...
In this study, we investigated the protective effects of angiotensin IV (Ang IV) on cognitive function in streptozotocin (STZ)‑induced diabetic rats. Male Wistar albino rats, were randomly divided into four groups; control (C), diabetes (Dia, 60 mg/kg, STZ, i.p.), Ang IV (5 μg/kg, s.c.) and Dia+Ang IV. The passive avoidance and Morris water maze (MWM) tests were used to evaluate learning and memory performance. Behavioral tests were carried out between 21 and 30 days after the initial Ang IV injection. Hippocampi were dissected and retained for biochemical and Western blot analysis. The Dia group exhibited the poorest behavioral results, while the Dia+Ang IV group performed highest on the MWM task. Superoxide dismutase, glutathione peroxidase, and malondialdehyde levels increased significantly in the Dia group compared to Dia+Ang IV. Brain‑derived neurotrophic factor (BDNF) and N‑methyl‑D‑aspartate levels were significantly elevated, while levels of GABAA significantly decreased, in the Dia+Ang IV group compared to the Dia group. These findings suggest that peripheral administration of Ang IV ameliorated spatial memory in diabetic rats by decreasing hippocampal oxidative stress and BDNF levels.
... Among these Hb-derived peptides, hemorphins have been extensively studied in various contexts including the nervous, vascular, and endocrine systems [1][2][3][4]. Indeed, hemorphins have been shown to have various biological activities, including effects on spatial learning [5], analgesia [6][7][8][9][10][11][12], antihypertension [3,[13][14][15][16][17][18], constriction of coronary vessels and platelet aggregation [19], and inflammation [10,11]. Hemorphins are endogenous peptides released during the sequential cleavage of hemoglobin protein and initially reported as atypical opioid peptides [2][3][4][20][21][22][23][24][25]. ...
Hemorphins are short peptides produced by the proteolysis of the beta subunit of hemoglobin. These peptides have diverse physiological effects especially in the nervous and the renin-angiotensin systems. Such effects occur through the modulation of a diverse range of proteins including enzymes and receptors. In this review, we focus on pharmacological and functional targeting of G protein-coupled receptors (GPCRs) by hemorphins and their implication in physiology and pathophysiology. Among GPCRs, the opioid receptors constitute the first set of targets of hemorphins with implication in analgesia. Subsequently, several other GPCRs have been reported to be directly or indirectly involved in hemorphins’ action. This includes the receptors for angiotensin II, oxytocin, bombesin, and bradykinin, as well as the human MAS-related G protein-coupled receptor X1. Interestingly, both orthosteric activation and allosteric modulation of GPCRs by hemorphins have been reported. This review links hemorphins with GPCR pharmacology and signaling, supporting the implication of GPCRs in hemorphins’ effects. Thus, this aids a better understanding of the molecular basis of the action of hemorphins and further demonstrates that hemorphin-GPCR axis constitutes a valid target for therapeutic intervention in different systems.
... These unexpected observations were thought to potentially be due to altered brain development of the knockout mice, given there is high IRAP expression in the highly neurogenic ventricular and subventricular zone of the embryonic mouse brain, possibly implicating IRAP as having a role in neuronal development. Regardless of this finding, there is comprehensive evidence supporting the cognitive enhancing effect of IRAP inhibition Lee et al., 2004;De Bundel et al., 2009;Fidalgo et al., 2019;Royea et al., 2019). Ang IV has also been shown to have positive effects on other central nervous system functions. ...
As a member of the M1 family of aminopeptidases, insulin regulated aminopeptidase (IRAP) is characterized by distinct binding motifs at the active site in the C-terminal domain that mediate the catalysis of peptide substrates. However, what makes IRAP unique in this family of enzymes is that it also possesses trafficking motifs at the N-terminal domain which regulate the movement of IRAP within different intracellular compartments. Research on the role of IRAP has focused predominantly on the C-terminus catalytic domain in different physiological and pathophysiological states ranging from pregnancy to memory loss. Many of these studies have utilized IRAP inhibitors, that bind competitively to the active site of IRAP, to explore the functional significance of its catalytic activity. However, it is unknown whether these inhibitors are able to access intracellular sites where IRAP is predominantly located in a basal state as the enzyme may need to be at the cell surface for the inhibitors to mediate their effects. This property of IRAP has often been overlooked. Interestingly, in some pathophysiological states, the distribution of IRAP is altered. This, together with the fact that IRAP possesses trafficking motifs, suggest the localization of IRAP may play an important role in defining its physiological or pathological functions and provide insights into the interplay between the two functional domains of the protein.
... In particular, IRAP is a potential therapeutic target for the treatment of Alzheimer's disease and other cognitive impairments. Rodents treated with the IRAP inhibitors such as Angiotensin IV (AngIV, 1) (Figure 1) via intracranial injection or subcutaneous administration, show improved performance in learning and memory (Wright et al., 1999;Albiston et al., 2001;Pederson et al., 2001;Lee et al., 2004;Gard, 2008;Golding et al., 2010). The cellular mechanism for this increased learning is unclear and may be attributable to modulation of glucose uptake via GLUT4 containing vesicles or reduced degradation of oxytocin and vasopressin, which have both been shown to improve learning and memory (Vanderheyden, 2009;Fidalgo et al., 2019). ...
The insulin regulated aminopeptidase (IRAP) has been proposed as an important therapeutic target for indications including Alzheimer’s disease and immune disorders. To date, a number of IRAP inhibitor designs have been investigated but the total number of molecules investigated remains quite small. As a member the M1 aminopeptidase family, IRAP shares numerous structural features with the other M1 aminopeptidases. The study of those enzymes and the development of inhibitors provide key learnings and new approaches and are potential sources of inspiration for future IRAP inhibitors.
... Related Ang IV analogues were studied as well, such as Nle-Ang IV 2 (Nle-Tyr-Ile-His-Pro-Phe) and the endogenous LVV-hemorphin-7 3 (Leu-Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg-Phe), both with structural similarities to Ang IV at the N-terminal part of the peptides (Figure 1). Nle-Ang IV 2 and LVV-hemorphin-7 3, the latter formed after degradation of β-globin, were demonstrated to be strong promoters of memory retention and retrieval in rats (Lee et al., 2003;Lee et al., 2004;De Bundel et al., 2009). In 1992, Wright and Harding identified binding sites for the hexapeptide Ang IV in several brain areas associated with cognition, motor and sensory physiological functions (Swanson et al., 1992), e.g., in hippocampus (Harding et al., 1992). ...
It was reported three decades ago that intracerebroventricular injection of angiotensin IV (Ang IV, Val-Tyr-Ile-His-Pro-Phe) improved memory and learning in the rat. There are several explanations for these positive effects of the hexapeptide and related analogues on cognition available in the literature. In 2001, it was proposed that the insulin-regulated aminopeptidase (IRAP) is a main target for Ang IV and that Ang IV serves as an inhibitor of the enzyme. The focus of this review is the efforts to stepwise transform the hexapeptide into more drug-like Ang IV peptidemimetics serving as IRAP inhibitors. Moreover, the discovery of IRAP inhibitors by virtual and substance library screening and direct design applying knowledge of the structure of IRAP and of related enzymes is briefly presented.
... Furthermore, IRAP is localized in areas of the brain associated with learning and memory and is the receptor for angiotensin IV (AngIV), which inhibits its enzymatic activity 5,6 . Given the role of AngIV in improving memory tasks in experimental animals, development of IRAP inhibitors has been pursued as potential therapeutics for cognitive disorders [7][8][9] . The ability of IRAP to degrade cyclic peptides has inspired the development of macrocyclic analogues of AngIV, which act as potent IRAP inhibitors with significant selectivity and have the capacity to alter dendritic spine density and morphology in neuronal cultures [10][11][12] . ...
Insulin-regulated aminopeptidase (IRAP) is a transmembrane zinc metallopeptidase with many important biological functions and an emerging pharmacological target. Although previous structural studies have given insight on how IRAP recognizes linear peptides, how it recognizes its physiological cyclic ligands remains elusive. Here, we report the first crystal structure of IRAP with the macrocyclic peptide inhibitor HA08 that combines structural elements from angiotensin IV and physiological substrates oxytocin and vasopressin. The compound is found in the catalytic site in a near canonical substrate-like configuration and inhibits by a competitive mechanism. Comparison with previously solved structures of IRAP along with small-angle X-ray scattering experiments suggest that IRAP is in an open conformation in solution but undergoes a closing conformational change upon inhibitor binding. Stabilization of the closed conformation in combination with catalytic water exclusion by the tightly juxtaposed GAMEN loop are proposed as mechanisms of inhibition.
... Peptide fragments often exhibit very different physiological properties as compared to their parent peptides [24,25]. Hence, the nociceptive substance P is degraded to a heptapeptide with anti-nociceptive effects [26,27] and degradation of Ang II via the heptapeptide angiotensin III (Ang III) (Arg 2 -Val 3 -Tyr 4 -Ile 5 -His 6 -Pro 7 -Phe 8 ) results in the cognitive enhancer angiotensin IV (Ang IV) (Val 3 -Tyr 4 -Ile 5 -His 6 -Pro 7 -Phe 8 ) that demonstrates a very low AT2R affinity [28,29]. The short-lived Ang IV inhibits insulin-regulated aminopeptidase (IRAP), resulting in cognitive enhancement in rat [28]. ...
Angiotensin II receptor type 1 and 2 (AT1R and AT2R) are two G-protein coupled receptors that mediate most biological functions of the octapeptide Angiotensin II (Ang II). AT2R is upregulated upon tissue damage and its activation by selective AT2R agonists has become a promising approach in the search for new classes of pharmaceutical agents. We herein analyzed the chemical evolution of AT2R agonists starting from octapeptides, through shorter peptides and peptidomimetics to the first drug-like AT2R-selective agonist, C21, which is in Phase II clinical trials and aimed for idiopathic pulmonary fibrosis. Based on the recent crystal structures of AT1R and AT2R in complex with sarile, we identified a common binding model for a series of 11 selected AT2R agonists, consisting of peptides and peptidomimetics of different length, affinity towards AT2R and selectivity versus AT1R. Subsequent molecular dynamics simulations and free energy perturbation (FEP) calculations of binding affinities allowed the identification of the bioactive conformation and common pharmacophoric points, responsible for the key interactions with the receptor, which are maintained by the drug-like agonists. The results of this study should be helpful and facilitate the search for improved and even more potent AT2R-selective drug-like agonists.
... Hemorphins are a class of endogenous opioid peptides derived from hemoglobin. A number of studies have highlighted their therapeutic potential [10,[17][18][19]47,48]. We had previously reported the molecular binding behavior of camel and non-camel LVV-hemorphin-7 on multiple targets [20,25]. ...
Angiotensin-I converting enzyme (ACE) is a zinc metallopeptidase that has an important role in regulating the renin-angiotensin-aldosterone system (RAAS). It is also an important drug target for the management of cardiovascular diseases. Hemorphins are endogenous peptides that are produced by proteolytic cleavage of beta hemoglobin. A number of studies have reported various therapeutic activities of hemorphins. Previous reports have shown antihypertensive action of hemorphins via the inhibition of ACE. The sequence of hemorphins is highly conserved among mammals, except in camels, which harbors a unique Q>R variation in the peptide. Here, we studied the ACE inhibitory activity of camel hemorphins (LVVYPWTRRF and YPWTRRF) and non-camel hemorphins (LVVYPWTQRF and YPWTQRF). Computational methods were used to determine the most likely binding pose and binding affinity of both camel and non-camel hemorphins within the active site of ACE. Molecular dynamics simulations showed that the peptides interacted with critical residues in the active site of ACE. Notably, camel hemorphins showed higher binding affinity and sustained interactions with all three subsites of the ACE active site. An in vitro ACE inhibition assay showed that the IC50 of camel hemorphins were significantly lower than the IC50 of non-camel hemorphins.
... Ang IV, acting via the AT 4 receptor, presents a role in learning and memory. This peptide facilitates LTP, memory consolidation, novel object recognition, and spatial memory [60][61][62][63][64]. The role of Alatensins has not yet been evaluated in neurodegenerative disorders. ...
Huntington’s disease (HD) is an autosomal dominant, progressive neurodegenerative disorder characterized by severe symptoms, including motor impairment, cognitive decline, and psychiatric alterations. Several systems, molecules, and mediators have been associated with the pathophysiology of HD. Among these, there is the Renin-Angiotensin System (RAS), a peptide hormone system that has been associated with the pathology of neuropsychiatric and neurodegenerative disorders. Important alterations in this system have been demonstrated in HD. However, the role of RAS components in HD is still unclear and needs further investigation. Nonetheless, modulation of the RAS components may represent a potential therapeutic strategy for the treatment of HD.
... LVV-hemorphin-7, a ten-residue peptide, can decrease blood pressure through inhibiting angiotensin-converting enzymes [25]. LVV-hemorphin-7 also plays important roles in learning and memory [25,26]. Clinical studies showed that serum LVV hemorphin-7 levels were lower in patients with obesity and type 2 diabetes compared to controls [27,28]. ...
Elevated soluble (s) CD163 and free hemoglobin (Hb) levels predict fatty liver progression; however, the molecular mechanisms underlying Hb metabolism and liver injury remain undefined. We investigated the effects of endoplasmic reticular (ER) stress on red blood cell (RBC) rheology and free Hb recycling pathways. ER stress was induced in Sprague-Dawley rats by an intraperitoneal injection of tunicamycin (TM) (50, 100, and 200 μg/100 g body weight (BW)) or an intravenous injection of Hb (5 mg/100 g BW). A TM injection increased sCD163 levels, attenuated free Hb uptake, and maintained RBC aggregability. An Hb injection increased serum LVV-hemorphin-7 and total bilirubin levels, but this effect was suppressed by TM. A Western blot analysis showed that ER stress suppressed Hb degradation in the liver through downregulation of globin degradation proteins cathepsin D and glyoxalase-1, as well as heme degradation protein heme oxyganase-1 and keap-1 expression. An ER stress activator also increased the translocation of nuclear factor (NF)-κB (p65) and nuclear factor-erythroid 2-related factor 2 (Nrf2) to nuclei. In conclusion, ER stress triggers ineffective Hb metabolism via altering globin and heme iron degradation pathways. Inability to recycle and metabolize free Hb may underlie the association between iron dysfunction and liver injury.
... Moreover, ligand interactions with AT 1 R impacts learning and memory processes, as Ang II and Ang IV have been shown to play opposing roles regarding memory acquisition and consolidation. Ang IV, as well as Ang IV analogs such as Nle1-Ang IV, facilitate LTP, learning, and memory consolidation [65,66]. However, those memory-enhancing effects were initially attributed to Ang II, since conversion of Ang II to Ang IV occurs producing peptides acting as AT 4 R agonists that are responsible for cognitive facilitation [67]. ...
Retreat in psychiatric drug development results in innovative medication decline that might be at least partially overcome by adjunct therapy. New evidence from clinical studies has shown a possible role for brain Renin-Angiotensin System (RAS) in both affective and psychotic disorders. Simultaneously, rapidly accumulating data from basic studies indicate effectiveness of central RAS blockade in much broader range of neuropsychiatric disease. Recent findings implicate brain RAS, especially Angiotensin II (Ang II), in neural pathophysiology of mental disorders through neuroendocrine modulation and effects on neurotransmitter release, mostly noradrenaline, acetylcholine and dopamine. The potential effects of angiotensin-converting-enzyme (ACE) inhibition and angiotensin type 1 receptor (AT1R) blockade on treatment of mental disorders are a matter of considerable interest. This review describes involvement of brain RAS in pathophysiology of neuropsychiatric disorders and an intriguing possibilities of improvement in pharmacological treatment outcome, where using angiotensin-converting-enzyme inhibitors (ACEI) and Angiotensin Receptor Blockers (ARB), goes beyond blood pressure control.
... 29,30 An excellent example is degradation of the hypertensive peptide Ang II via the heptapeptide angiotensin III (Ang III) (Arg 2 -Val 3 -Tyr 4 -Ile 5 -His 6 -Pro 7 -Phe 8 ) to the cognitive enhancer angiotensin IV (Ang IV, Ang 3-8) (Val 3 -Tyr 4 -Ile 5 -His 6 -Pro 7 -Phe 8 ). 31,32 The latter hexapeptide, formed after actions of aminopeptidase A (APA) and aminopeptidase N (APN), blocks insulin-regulated aminopeptidase (IRAP), an inhibition that has been F I G U R E 3 Bioactive substances with biaryl scaffolds: Two selective AT1R antagonists, a selective AT1R agonist, a neprilysin inhibitor, and a selective MasR agonist F I G U R E 4 The selective AT2R agonist C21/M24 and the selective AT2R antagonist C38/M132 ...
The discovery of the first selective, small-molecule ATR receptor (AT2R) agonist compound 21 (C21) (8) that is now extensively studied in a large variety of in vitro and in vivo models is described. The sulfonylcarbamate derivative 8, encompassing a phenylthiofen scaffold is the drug-like agonist with the highest affinity for the AT2R reported to date (Ki = 0.4 nM). Structure-activity relationships (SAR), regarding different biaryl scaffolds and functional groups attached to these scaffolds and with a particular focus on the impact of various para substituents displacing the methylene imidazole group of 8, are discussed. Furthermore, the consequences of migration of the methylene imidazole group and presumed structural requirements for ligands that are aimed as AT2R agonists (e.g. 8) or AT2R antagonists (e.g. 9), respectively, are briefly addressed. A summary of the pharmacological actions of C21 (8) is also presented.
... In the present study, plasma angiotensin IV levels of SMMSE > 26 group were higher than plasma angiotensin IV levels of SMMSE ≤ 26 group but this difference did not reach a statistically significant level. Stimulation of the AT4 receptor had a positive effect on cognitive effects such as memory and learning in animal studies that administered angiotensin IV Braszko, 2004;Lee et al., 2004;Gard, 2008). Although it is too early to come to a conclusion, the cognitive improvement provided by ARBs could be related with angiotensin IV. ...
Renin Angiotensin Aldosterone System (RAAS) plays an important role in the development of hypertension. On the other hand, hypertension is a well-known and independent risk factor for cognitive impairment. The aim of the present study was to evaluate the relationship of blood pressure control, plasma angiotensin peptides and aldosterone with cognitive functions. Forty-one patients who were under treatment with the same antihypertensive medications for at least three months were included in the study. Plasma angiotensin II, angiotensin 1-7, angiotensin IV, and aldosterone concentrations were analyzed using an enzyme-linked immunosorbent assay (ELISA). Standardized Mini Mental State Examination (SMMSE) was used to evaluate cognitive functions. When the participants were grouped according to their SMMSE scores (cut-off value: 26 points), we determined significant differences between systolic blood pressure (SBP) levels, diastolic blood pressure levels, plasma angiotensin II and angiotensin 1-7 concentrations of the groups. When the participants were stratified according to their SBP levels (cut-off value: 140 mm Hg), we found significant differences in SMMSE scores and plasma angiotensin IV concentrations of the groups. A negative correlation between SBP and SMMSE scores and strong linear correlations among angiotensin peptides levels were determined. The relationship found between SBP and SMMSE in the present study was compatible with the literature. Our 33 patients were using at least one angiotensin II receptor blocker (ARB). Regarding AT1 receptor blockage, the significant association between higher SMMSE scores and increased angiotensin peptides may support a finding that ARBs prevent dementia and improve cognitive function. Further larger studies are needed to confirm and prove the relation of RAAS biochemical parameters with cognitive function. © 2017, Leibniz Research Centre for Working Environment and Human Factors. All rights reserved.
... Whereas losartan did not restore the density of cortical cholinergic innervation, it significantly reduced astrogliosis and normalized the protein levels of cortical AT1R and of the AT4R (Ongali et al. 2014) (Fig. 6d). AT4Rs, also known as insulin-regulated aminopeptidase (IRAP) (Albiston et al. 2001(Albiston et al. , 2010Vanderheyden 2009), are found in brain areas involved in mnemonic processing, and have been associated with facilitation of learning and memory (Benoist et al. 2011;Lee et al. 2004;Wayner et al. 2001;Harding 2008, 2010). Accordingly, genetically modified AT4R knock-out mice exhibit spatial memory deficits (Albiston et al. 2010). ...
Alzheimer's disease (AD) is a multifactorial and multifaceted disease for which we currently have very little to offer since there is no curative therapy, with only limited disease-modifying drugs. Recent studies in AD mouse models that recapitulate the amyloid-β (Aβ) pathology converge to demonstrate that it is possible to salvage cerebrovascular function with a variety of drugs and, particularly, therapies used to treat cardiovascular diseases such as hypercholesterolemia and hypertension. These drugs can reestablish dilatory function mediated by various endothelial and smooth muscle ion channels as well as nitric oxide availability, benefits that result in normalized brain perfusion. These cerebrovascular benefits would favor brain perfusion, which may help maintain neuronal function and, possibly, delay cognitive failure. However, restoring cerebrovascular function in AD mouse models was not necessarily accompanied by rescue of cognitive deficits related to spatial learning and memory. The results with cardiovascular therapies rather suggest that drugs originally designed to treat cardiovascular diseases that concurrently restore cerebrovascular and cognitive function do so through their pleiotropic effects. Specifically, recent findings suggest that these drugs act directly on brain cells and neuronal pathways involved in memory formation, hence, working simultaneously albeit independently on neuronal and vascular targets. These findings may help select medications for patients with cardiovascular diseases at risk of developing AD with increasing age. Further, they may identify molecular targets for recovering memory pathways that bear potential for new therapeutic avenues.
... In 2001, the hexapeptide angiotensin IV (Ang IV; Val-Tyr-Ile-His-Pro-Phe), which improves memory and learning in both rats and mice (Braszko et al., 1988;Wright et al., 1993;Wright et al., 1996;Wright et al., 1999;Lee et al., 2004;Gard et al., 2007;Braszko et al., 2008;De Bundel et al., 2009;Gard et al., 2012), was demonstrated to inhibit the membrane-bound zinc-dependent insulin-regulated aminopeptidase (EC 3.4.11.3 IRAP, oxytocinase), a member of the M1 family, which is expressed in a diversity of tissues/cells (Albiston et al., 2001;Liao et al., 2006;Saveanu and van Endert, 2012;Nikolaou et al., 2014). There has been dispute in the field as to the target that mediates the memory-enhancing effects of Ang IV as this hexapeptide has the ability to bind to other proteins (Vanderheyden, 2009;Kawas et al., 2012;Benoist et al., 2014;. ...
Angiotensin IV (Ang IV) and related peptide analogues, as well as non-peptide inhibitors of insulin-regulated aminopeptidase (IRAP), have previously been shown to enhance memory and cognition in animal models. Furthermore, the endogenous IRAP substrates oxytocin and vasopressin are known to facilitate learning and memory. In this study, the two recently synthesized 13-membered macrocylic competitive IRAP inhibitors HA08 and HA09, which were designed to mimic the N-terminal of oxytocin and vasopressin, were assessed and compared based on their ability to bind to the IRAP active site, and alter dendritic spine density in rat hippocampal primary cultures. The binding modes of the IRAP inhibitors HA08, HA09 and of Ang IV in either the extended or γ-turn conformation at the C-terminal to human IRAP were predicted by docking and molecular dynamics (MD) simulations. The binding free energies calculated with the linear interaction energy (LIE) method, which are in excellent agreement with experimental data and simulations, have been used to explain the differences in activities of the IRAP inhibitors, both of which are structurally very similar, but differ only with regard to one stereogenic center. In addition, we show that HA08, which is 100-fold more potent than the epimer HA09, can enhance dendritic spine number and alter morphology, a process associated with memory facilitation. Therefore, HA08, one of the most potent IRAP inhibitors known today, may serve as a suitable starting point for medicinal chemistry programs aided by MD simulations aimed at discovering more drug-like cognitive enhancers acting via augmenting synaptic plasticity.
The insulin-regulated aminopeptidase (IRAP; oxytocinase) is part of the M1 aminopeptidase family and is highly expressed in many tissues, including the neocortex and hippocampus of the brain. IRAP is involved in various physiological functions and has been identified as a receptor for the endogenous hexapeptide Angiotensin IV (Ang IV). The binding of Ang IV inhibits the enzymatic activity of IRAP and has been proven to enhance learning and memory in animal models. The macrocyclic compound 9 (C9) is a potent synthetic IRAP inhibitor developed from the previously reported inhibitor HA08. In this study, we have examined compound C9 and its effects on cognitive markers drebrin, microtubule-associated protein 2 (MAP2), and glial fibrillary acidic protein (GFAP) in primary hippocampal and cortical cultures. Cells from Sprague Dawley rats were cultured for 14 days before treatment with C9 for 4 consecutive days. The cells were analysed for protein expression of drebrin, MAP2, GFAP, glucose transporter type 4 (GLUT4), vesicular glutamate transporter 1 (vGluT1), and synapsin I using immunocytochemistry. The gene expression of related proteins was determined using qPCR, and viability assays were performed to evaluate toxicity. The results showed that protein expression of drebrin and MAP2 was increased, and the corresponding mRNA levels were decreased after treatment with C9 in the hippocampal cultures. The ratio of MAP2-positive neurons and GFAP-positive astrocytes was altered and there were no toxic effects observed. In conclusion, the IRAP inhibitor compound C9 enhances the expression of the pro-cognitive markers drebrin and MAP2, which further confirms IRAP as a relevant pharmaceutical target and C9 as a promising candidate for further investigation.
Inhibition of insulin-regulated aminopeptidase (IRAP) has been shown to improve cognitive functions in several animal models. Recently, we performed a screening campaign of approximately 10,000 compounds, identifying novel small-molecule-based compounds acting as inhibitors of the enzymatic activity of IRAP. Here we report on the chemical synthesis, structure-activity relationships (SAR) and initial characterization of physicochemical properties of a series of 48 imidazo [1,5-α]pyridine-based inhibitors, including delineation of their mode of action as non-competitive inhibitors with a small L-leucine-based IRAP substrate. The best compound displays an IC50 value of 1.0 µM. We elucidate the importance of two chiral sites in these molecules and find they have little impact on the compound’s metabolic stability or physicochemical properties. The carbonyl group of a central urea moiety was initially believed to mimic substrate binding to a catalytically important Zn²⁺ ion in the active site, although the plausibility of this binding hypothesis is challenged by observation of excellent selectivity versus the closely related aminopeptidase N (APN). Taken together with the non-competitive inhibition pattern, we also consider an alternative model of allosteric binding.
Hemorphins are a family of endogenous atypical opioid peptides released during the sequential cleavage of hemoglobin β-chain. Hemorphins are widely distributed in the organism and are implicated in pathophysiology of several diseases, including diabetes, cancer, Alzheimer disease and others. These peptides were identified several decades ago and their diverse therapeutic effects have been revealed. However, the exact molecular mechanisms involved in synthesis, metabolism and therapeutic effect of hemorphins are not fully understood. In this review, the current knowledge of hemorphins, focusing on their biological activity and Ca2+/CaM/CN-signaling pathway, involved in mechanisms of hemophins action is briefly summarized. These peptides have been intensively investigated at the Institute of Biochemistry (Armenia), and fundamental results are thoroughly discussed in this review.
Stroke is a leading cause of mortality and morbidity with a paucity of effective pharmacological treatments. We have previously identified insulin-regulated aminopeptidase (IRAP) as a potential target for the development of a new class of drugs for the treatment of stroke, as global deletion of this gene in mice significantly protected against ischemic damage. In the current study, we demonstrate that small molecular weight IRAP inhibitors reduce infarct volume and improve neurological outcome in a hypertensive animal model of ischemic stroke. The effects of two structurally distinct IRAP inhibitors (HFI419 or SJM164) were investigated in a model of stroke where the middle cerebral artery was transiently occluded with endothelin-1 in the conscious spontaneously hypertensive rat. IRAP inhibitor was administered into the lateral ventricle at 2 or 6 h after stroke, with subsequent doses delivered at 24, 48 and 70 h post-stroke. Functional outcomes were assessed prior to drug treatment, and on day 1 and 3 post-stroke. Histological analyses and neuroinflammatory cytokine profiling were conducted at 72 and 24 h post-stroke respectively. IRAP inhibitor treatment following stroke significantly reduced infarct volume and improved neurological and motor deficits. These protective effects were maintained even when the therapeutic window was extended to 6 h. Examination of the cellular architecture at 72 h post-stroke demonstrated that IRAP expression was upregulated in CD11b positive cells and activated astrocytes. Furthermore, IRAP inhibitor treatment significantly increased gene expression for interleukin 6 and C–C motif chemokine ligand 2 in the ischemic core. This study provides proof-of-principle that selective inhibition of IRAP activity with two structurally distinct IRAP inhibitors reduces infarct volume and improves functional outcome even when the first dose is administered 6 h post-stroke. This is the first direct evidence that IRAP inhibitors are a class of drug with potential use in the treatment of ischemic stroke.
Endogenous peptides have various modulating effects on many physiological and pathological processes, including
the processing of pain information. Hemorphins are hemoglobin-derived peptides with affinity for both opioid receptors
and insulin-regulated aminopeptidase. We recently reported on the antinociceptive effects of hemophins of different
lengths and amino acid composition after their supraspinal injection.
The present study aims to elucidate the potential effects of locally (intraplantar) injected hemorphin VV-H7
(Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg-Phe-NH2) and its analogs with one or two Arg-Gly-Asp sequence on carrageenaninduced
hyperalgesia in rats. We used indomethacin as a reference anti-inflammatory analgesic. Pain thresholds and
paw volume were measured before and at 1, 3, and 4 hours after intraplantar carrageenan injection using a paw
pressure test and plethysmometry in male Wistar rats.
The precursor peptide VV-H7 showed an antihyperalgesic effect comparable to that of indomethacin, while its
peptide analogues one or two Arg-Gly-Asp sequence showed an acceleration of the antinociceptive effect, but shorter
duration of antihyperalgesia and lack of efficacy against edema.
Keywords: hemorphins, carrageenan, hyperalgesia, RGD, rats.
Importance
Prevalent use of antihypertensive medications that stimulate type 2 and 4 angiotensin II receptors, compared with those that do not stimulate these receptors, has been associated with a lower risk of dementia. However, previous studies were limited by inclusion of individuals with prevalent hypertension and a history of antihypertensive use prior to the start of the study, which can introduce bias.
Objective
To examine the association of new use of antihypertensive medication regimens that stimulate vs inhibit type 2 and 4 angiotensin II receptors with Alzheimer disease and related dementias (ADRD) among Medicare beneficiaries.
Design, Setting, and Participants
This cohort study was conducted among 57 773 Medicare fee-for-service beneficiaries (January 1, 2006, through December 31, 2018) aged 65 years or older with incident hypertension. Data analysis was conducted from January 1 through June 30, 2022.
Exposures
Initiation of antihypertensive medication regimens that stimulate or inhibit type 2 and 4 angiotensin II receptors, or mixed regimens (both stimulating and inhibiting), with the time-dependent measure being each 30-day interval.
Main Outcomes and Measures
The primary outcome was time to first occurrence of ADRD (Centers for Medicare & Medicaid Services Chronic Conditions Data Warehouse definition). Cox proportional hazards regression modeling with time-dependent variables was performed to estimate the association between time-dependent treatment groups and time to ADRD, after adjusting for sociodemographic and clinical characteristics.
Results
The sample included 57 773 Medicare beneficiaries (36 348 women [62.9%]; mean [SD] age, 73.8 [6.3] years; 2954 [5.1%] Black, 1545 [2.7%] Hispanic; 50 184 [86.9%] White, and 3090 [5.4%] Other individuals [the Other category included individuals of American Indian, Asian, other, or unknown race and ethnicity]). During a median of 6.9 years (IQR, 4.7-9.3 years) of follow-up, the unadjusted incidence density rate of ADRD was 2.2 cases per 100 person-years (95% CI, 2.1-2.4 cases per 100 person-years) for the group receiving regimens that stimulate type 2 and 4 angiotensin II receptors compared with 3.1 cases per 100 person-years (95% CI, 3.0-3.2 cases per 100 person-years) for the group receiving regimens that inhibit type 2 and 4 angiotensin II receptors and 2.7 cases per 100 person-years (95% CI, 2.6-2.9 cases per 100 person-years) for the group receiving mixed treatment regimens. In adjusted Cox proportional hazards regression modeling, stimulating treatment was associated with a statistically significant 16% reduction in the hazard of ADRD compared with inhibiting treatment (hazard ratio, 0.84; 95% CI, 0.79-0.90). Mixed regimen use was also associated with reduced hazards of ADRD compared with the inhibiting group (hazard ratio, 0.90; 95% CI, 0.84-0.96).
Conclusions and Relevance
This cohort study of Medicare beneficiaries suggests that use of antihypertensive medications that stimulate type 2 and 4 angiotensin II receptors was associated with lower risk of ADRD compared with antihypertensive medications that inhibit these receptors. Confirmation is needed in a randomized trial.
The endogenous hemorphins are bioactive peptides with activity on opioid receptors. They are extensively studied and summarized in numerous reviews. During the last decade, several research teams have synthesized, characterized, and pharmacologically evaluated synthetic hemorphin analogs containing unusual amino acids, D-amino acids, α-aminophosphonic acids, and their derivatives. The present review summarizes the current studies on short-chain synthetic hemorphin peptide derivates containing non-natural amino acids. This review focuses on the structure–activity relationship analysis, details on specific methods for their characterization, and the advantage of synthetic hemorphin analogs compared to endogenous peptides as potent biologically active compounds with a complex mechanism of action.
Importance:
Use of antihypertensive medications that stimulate type 2 and 4 angiotensin II receptors, compared with those that do not stimulate these receptors, has been associated with a lower risk of dementia. However, this association with cognitive outcomes in hypertension trials, with blood pressure levels in the range of current guidelines, has not been evaluated.
Objective:
To examine the association between use of exclusively antihypertensive medication regimens that stimulate vs inhibit type 2 and 4 angiotensin II receptors on mild cognitive impairment (MCI) or dementia.
Design, setting, and participants:
This cohort study is a secondary analysis (April 2011 to July 2018) of participants in the randomized Systolic Blood Pressure Intervention Trial (SPRINT), which recruited individuals 50 years or older with hypertension and increased cardiovascular risk but without a history of diabetes, stroke, or dementia. Data analysis was conducted from March 16 to July 6, 2021.
Exposures:
Prevalent use of angiotensin II receptor type 2 and 4-stimulating or -inhibiting antihypertensive medication regimens at the 6-month study visit.
Main outcomes and measures:
The primary outcome was a composite of adjudicated amnestic MCI or probable dementia.
Results:
Of the 8685 SPRINT participants who were prevalent users of antihypertensive medication regimens at the 6-month study visit (mean [SD] age, 67.7 [11.2] years; 5586 [64.3%] male; and 935 [10.8%] Hispanic, 2605 [30.0%] non-Hispanic Black, 4983 [57.4%] non-Hispanic White, and 162 [1.9%] who responded as other race or ethnicity), 2644 (30.4%) were users of exclusively stimulating, 1536 (17.7%) inhibiting, and 4505 (51.9%) mixed antihypertensive medication regimens. During a median of 4.8 years of follow-up (95% CI, 4.7-4.8 years), there were 45 vs 59 cases per 1000 person-years of amnestic MCI or probable dementia among prevalent users of regimens that contained exclusively stimulating vs inhibiting antihypertensive medications (hazard ratio [HR], 0.76; 95% CI, 0.66-0.87). When comparing stimulating-only vs inhibiting-only users, amnestic MCI occurred at rates of 40 vs 54 cases per 1000 person-years (HR, 0.74; 95% CI, 0.64-0.87) and probable dementia at rates of 8 vs 10 cases per 1000 person-years (HR, 0.80; 95% CI, 0.57-1.14). Negative control outcome analyses suggested the presence of residual confounding.
Conclusions and relevance:
In this secondary analysis of SPRINT, prevalent users of regimens that contain exclusively antihypertensive medications that stimulate vs inhibit type 2 and 4 angiotensin II receptors had lower rates of incident cognitive impairment. Residual confounding cannot be ruled out. If these results are replicated in randomized clinical trials, certain antihypertensive medications could be prioritized to prevent cognitive decline.
Introduction
: The evolving COVID-19 pandemic became a hallmark in human history, not only by changing lifestyles, but also by enriching scientific knowledge on viral infection and its consequences.
Objective
: Although management of cardiorespiratory changes is pivotal to a favorable prognosis during severe clinical findings, dysregulation of other systems caused by SARS-CoV-2 infection may imbalance erythrocyte dynamics, such as a bidirectional positive feedback loop pathophysiology.
Method and Results
: Recent evidence shows that SARS-CoV-2 is capable of affecting the genetics and dynamics of erythrocytes and this coexists with a non-homeostatic function of cardiovascular, respiratory and renal systems during COVID-19. In hypothesis, SARS-CoV-2-induced systematical alterations of erythrocytes dynamics would constitute a setpoint for COVID-19-related multiple organ failure syndrome and death.
Conclusion
: The present review covers the most frequent erythrocyte-related non-homeostatic findings during COVID-19 capable of providing mechanistic clues of SARS-CoV-2-induced infection and inspiring therapeutic-oriented scientific evidence.
Alcohol can increase the sensitivity to painful stimulation or convert insensibility to pain at different stages. We hypothesized that chronic alcohol consumption changes the level of LVV-hemorphin-7 (abbreviated as LVV-H7, an opioid-like peptide generated from hemoglobin β-chain), thereby affecting pain sensation. We established a chronic alcohol-exposed rat model to investigate the effects of LVV-H7. Adult male Sprague-Dawley rats were subjected to daily intraperitoneal injection of 10 % ethanol (w/v) at 0.5 g/kg for 15 days and subsequent alcohol withdrawal for 5 days. Using different pharmacological strategies to affect the LVV-H7 level, we investigated the correlation between LVV-H7 and pain-related behavior. Tail-flick and hot plate tests were employed to investigate alcohol-induced pain-related behavioral changes. The serum level of LVV-H7 was determined by ELISA. Our results showed that alcohol first induced an analgesia followed by a hyperalgesia during alcohol withdrawal, which could be driven by the quantitative change of LVV-H7. A positive correlation between the level of LVV-H7 and Δtail-flick latency (measured latency minus basal latency) confirmed this finding. Moreover, we revealed that the LVV-H7 levels were determined by the activity of cathepsin D and red blood cell/hemoglobin counts, which could be affected by alcohol. These results suggest that the deterioration of anti-nociception induced by alcohol is correlated to the decreased level of LVV-H7, and this could be due to alcohol-induced anemia. This study may help to develop LVV-H7 structure-based novel analgesics for treating alcohol-induced pain disorders and thus ameliorate the complications in alcoholics.
Hemorphins are endogenous peptides, 4-10 amino acids long, belonging to the family of atypical opioid peptides released during the sequential cleavage of hemoglobin protein. Hemorphins have been shown to exhibit diverse therapeutic effects in both human and animal models. However, the precise cellular and molecular mechanisms involved in such effects remain elusive. In this review, we summarize and propose potential mechanisms based on studies that investigated the biological activity of hemorphins of different lengths on multiple therapeutic targets. Special emphasis is given to molecular events related to renin-angiotensin system (RAS), opioid receptors and insulin-regulated aminopeptidase receptor (IRAP). This review provides a comprehensive coverage of the molecular mechanisms that underpin the therapeutic potential of hemorphins. Furthermore, it highlights the role of various hemorphin residues in pathological conditions, which could be explored further for therapeutic purposes.
Angiotensin IV and LVV‐hemorphin 7 promote robust enhancing effects on learning and memory. These peptides are also competitive inhibitors of the insulin‐regulated membrane aminopeptidase, suggesting that the biological actions of these peptides may result from inhibition of IRAP activity. However the normal function of IRAP in the brain is yet to be determined. The present study investigated the subcellular distribution of IRAP in four neuronal cell lines and in the mouse brain. Using subcellular fractionation, IRAP was found to be enriched in low density microsomes, while lower levels of IRAP were also present in high density microsomes, plasma membrane and mitochondrial fractions. Dual‐label immunohistochemistry confirmed the presence of IRAP in vesicles co‐localized with the vesicular maker VAMP2, in the trans Golgi network co‐localized with TGN 38 and in endosomes co‐localized with EEA1. Finally using electron microscopy, IRAP specific immunoreactivity was predominantly associated with large 100 ‐ 200 nm vesicles in hippocampal neurons. The location, appearance and size of these vesicles are consistent with neurosecretory vesicles. IRAP precipitate was also detected in intracellular structures including the rough endoplasmic reticulum, Golgi stack and mitochondrial membranes. The sub‐cellular localization of IRAP in neurons demonstrated in the present study bears striking parallels with distribution of IRAP in insulin responsive cells, where the enzyme plays a role in insulin regulated glucose uptake. Therefore, we propose that the function of IRAP in neurons may be similar to that in insulin responsive cells.
The review discusses pleiotropic effects of erythrocytic hemoglobin (Hb) and their significance for human health. Hemoglobin is mostly known as an oxygen carrier, but its biochemical functions are not limited to this. The following aspects of Hb functioning are examined: (i) catalytic functions of the heme component (nitrite reductase, NO dioxygenase, monooxygenase, alkylhydroperoxidase) and of the apoprotein (esterase, lipoxygenase); (ii) participation in nitric oxide metabolism; (iii) formation of membrane–bound Hb and its role in the regulation of erythrocyte metabolism; (iv) physiological functions of Hb catabolic products (iron, CO, bilirubin, peptides). Special attention is given to Hb participation in signal transduction in erythrocytes. The relationships between various erythrocyte metabolic parameters, such as oxygen status, ATP formation, pH regulation, redox balance, and state of the cytoskeleton are discussed with regard to Hb. Hb polyfunctionality can be considered as a manifestation of the principle of biochemical economy.
The central renin angiotensin system (RAS) is one of the most widely investigated cardiovascular systems in the brain. It is implicated in a myriad of cardiovascular diseases. However, studies from the last decade have identified its involvement in several neurological abnormalities. Understanding the molecular functionality of the various RAS components can thus provide considerable insight into the phenotypic differences and mechanistic drivers of not just cardiovascular, but also neurological disorders. Since activation of one of its primary receptors, the angiotensin type-1 receptor (AT1R) results in an augmentation of oxidative stress and inflammatory cytokines, it becomes essential to investigate not just neuronal RAS, but also glial RAS as well. Glial cells are key homeostatic regulators in the brain, and are critical players in the resolution of overt oxidative stress and neuroinflammation. Designing better and effective therapeutic strategies that target the brain RAS, could well hinge on understanding the molecular basis of both neuronal and glial RAS. This review provides a comprehensive overview of the major studies that have investigated the mechanisms and regulation of brain RAS, and it also provides insight into the potential role of glial AT1Rs in the pathophysiology of cardiovascular and neurological disorders.
The insulin-regulated aminopeptidase (IRAP) is a membrane-bound zinc metallopeptidase with many important regulatory functions. It has been demonstrated that inhibition of IRAP by angiotensin IV (Ang IV) and other peptides, as well as more druglike inhibitors, improves cognition in several rodent models. We recently reported a series of aryl sulfonamides as small-molecule IRAP inhibitors and a promising scaffold for pharmacological intervention. We have now expanded with a number of derivatives, report their stability in liver microsomes, and characterize the activity of the whole series in a new assay performed on recombinant human IRAP. Several compounds, such as the new fluorinated derivative 29, present submicromolar affinity and high metabolic stability. Starting from the two binding modes previously proposed for the sulfonamide scaffold, we systematically performed molecular dynamics simulations and binding affinity estimation with the linear interaction energy method for the full compound series. The significant agreement with experimental affinities suggests one of the binding modes, which was further confirmed by the excellent correlation for binding affinity differences between the selected pair of compounds obtained by rigorous free energy perturbation calculations. The new experimental data and the computationally derived structure–activity relationship of the sulfonamide series provide valuable information for further lead optimization of novel IRAP inhibitors.
This chapter provides a brief historical perspective concerning the discovery of the renin–angiotensin system (RAS), followed by a description of the biochemical pathways that permit synthesis and degradation of active angiotensin peptides, and the three receptor subtypes thus far characterized. This is followed by a review of the physiologies and behaviors mediated by these peptides. These classic physiologies include cardiovascular control, vasopressin release, thirst, and electrolyte balance. More recently angiotensins have been implicated in the mediation of stress, anxiety, depression, learning, and memory consolidation. The chapter concludes with a water shortage scenario that is envisioned to encompass and illustrate the majority of the angiotensin mediated physiologies and behaviors.
Central infusion of Insulin-Regulated Aminopeptidase (IRAP) inhibitors improves memory in both normal rodents and in models of memory deficit. However, in contrast, the global IRAP knockout mice (KO) demonstrate age-accelerated spatial memory deficits and no improvements in performance in any memory tasks. Potentially, the observed memory deficit could be due to the absence of IRAP in the developing brain. We therefore generated apostnatal forebrain neuron-specific IRAP knockout mouse line (CamKIIalphaCre; IRAPlox/lox). Unexpectedly, we demonstrated that postnatal deletion of IRAP in the brain results in significant deficits in both spatial reference and object recognition memory at three months of age, although spatial working memory remained intact. These results indicate a significant role for IRAP in postnatal brain development and/or normal function of the hippocampus in adulthood.
A review. Modern concepts of the physiological role of basic active component of renin-angiotensin system angiotensin II and its 3-8 fragment angiotensin IV are stated. The basic ways of formation and further degradation of angiotensin II and IV in a body are described. General characteristic of physiological activity and possible mechanisms of action of angiotensin II and IV are presented. Specifically, the role of angiotensin II and IV in regulation of defensive and drinking behaviors of animals is considered.
Recently, it has been reported that brain penetrating angiotensin- converting enzyme (ACE) inhibitors such as captopril can reduce the incidence of Alzheimer's disease in elderly hypertensive patients. Pneumonia is the reported cause of death in a high proportion of Alzheimer's disease patients. It has been reported that aspiration pneumonia in ambulatory Alzheimer's disease patients is significantly and independently associated with intake of neuroleptics. Substance P (SP) is associated with swallowing function and elevating serum SP in patients contributes to decrease the risk of aspiration pneumonia Substance P is a neuropeptide and is a substrate of ACE. We have found that pumpkin, one of the major local products of Aomori prefecture, contains an ACE inhibitor (nicotianamine). The aim of this study was to elucidate whether pumpkin supplementation in the diet improves memory function and serum SP concentration. We found that pumpkin improved memory function of rat. On the other hand, serum SP concentration was not increased by pumpkin supplementation in the diet.
This study examined effects on memory formation produced by [Leu]enkephalin and [Met]enkephalin administration in 2 regions of the 2-day-old chick brain involved in memory formation: the intermediate medial hyperstriatum ventrale (IMHV) and the lobus parolfactorius (LPO). Basal concentrations of endogenous [Leu]enkephalin and [Met]enkephalin were determined for 5 brain regions, and effects of 1-trial peck-avoidance training on enkephalin concentrations were examined in the IMHV and LPO. [Leu]enkephalin was amnestic when administered in the IMHV but not in the LPO. In contrast, [Met]enkephalin may be amnestic when administered in the LPO but not in the IMHV. Training decreased [Met]enkephalin concentration in the LPO but not in the IMHV. Training had no effect on [Leu]enkephalin concentration in either the IMHV or the LPO. Thus, amnestic effects of [Leu]- or [Met]enkephalin administration are brain-region specific. Regional activity of endogenous [Met]enkephalin during memory formation is consistent with localized amnestic effects produced by [Met]enkephalin administration.
The development of navigational strategies to solve spatial problems appears to be dependent on an intact hippocampal formation. The circular water maze task requires the animal to use extramaze spatial cues to locate a pedestal positioned just below the surface of the water. Presently, we investigated the role of a recently discovered brain angiotensin receptor subtype (AT4) in the acquisition of this spatial learning task. The AT4 receptor subtype is activated by angiotensin IV (AngIV) rather than angiotensins II or III, as documented for the AT1 and AT2 receptor subtypes, and is heavily distributed in the CA1-CA3 fields of the hippocampus. Chronic intracerebroventricular infusion of a newly synthesized AT4 agonist (Norleucine1-AngIV) via osmotic pump facilitated the rate of acquisition to solve this task, whereas treatment with an AT4 receptor antagonist (Divalinal) significantly interfered with the acquisition of successful search strategies. Animals prepared with bilateral knife cuts of the perforant path, a major afferent hippocampal fiber bundle originating in the entorhinal cortex, displayed deficits in solving this task. This performance deficit could be reversed with acute intracerebroventricular infusion of a second AT4 receptor agonist (Norleucinal). These results suggest that the brain AngIV-AT4 system plays a role in the formation of spatial search strategies and memories. Further, application of an AT4 receptor agonist compensated for spatial memory deficits in performance accompanying perforant path knife cuts. Possible mechanisms underlying this compensatory effect are discussed.
The insulin-regulated aminopeptidase (IRAP) is a zinc-dependent membrane aminopeptidase. It is the homologue of the human placental leucine aminopeptidase. In fat and muscle cells, IRAP colocalizes with the insulin-responsive glucose transporter GLUT4 in intracellular vesicles and redistributes to the cell surface in response to insulin, as GLUT4 does. To address the question of the physiological function of IRAP, we generated mice with a targeted disruption of the IRAP gene (IRAP-/-). Herein, we describe the characterization of these mice with regard to glucose homeostasis and regulation of GLUT4. Fed and fasted blood glucose and insulin levels in the IRAP-/- mice were normal. Whereas IRAP-/- mice responded to glucose administration like control mice, they exhibited an impaired response to insulin. Basal and insulin-stimulated glucose uptake in extensor digitorum. longus muscle, and adipocytes isolated from IRAP-/- mice were decreased by 30-60% but were normal for soleus muscle from male IRAP-/- mice. Total GLUT4 levels were diminished by 40-85% in the IRAP-/- mice in the different muscles and in adipocytes. The relative distribution of GLUT4 in subcellular fractions of basal and insulin-stimulated IRAP-/- adipocytes was the same as in control cells. We conclude that IRAP-/- mice maintain normal glucose homeostasis despite decreased glucose uptake into muscle and fat cells. The absence of IRAP does not affect the subcellular distribution of GLUT4 in adipocytes. However, it leads to substantial decreases in GLUT4 expression.
Sigma and 5-HT1A receptor stimulation can increase acetylcholine (ACh) release in the brain. Because ACh release facilitates learning and memory, we evaluated the degree to which OPC-14523 (1-{3-[4-(3-chlorophenyl)-1-piperazinyl]propyl}-5-methoxy-3,4-dihydro-2[1H]-quinolinone monomethane sulfonate), a novel sigma and 5-HT1Areceptor agonist, can augment ACh release and improve learning impairments in rats due to cholinergic- or age-related deficits. Single oral administration of OPC-14523 improved scopolamine-induced learning impairments in the passive-avoidance task and memory impairment in the Morris water maze. The chronic oral administration of OPC-14523 attenuated age-associated impairments of learning acquisition in the water maze and in the conditioned active-avoidance response test. OPC-14523 did not alter basal locomotion or inhibit acetylcholinesterase (AChE) activity at concentrations up to 100 μM and, unlike AChE inhibitors, did not cause peripheral cholinomimetic responses. ACh release in the dorsal hippocampus of freely moving rats increased after oral delivery of OPC-14523 and after local delivery of OPC-14523 into the hippocampus. The increases in hippocampal ACh release were blocked by the sigma receptor antagonist NE-100 (N,N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)-phenyl]-ethylamine). Thus, OPC-14523 improves scopolamine-induced and age-associated learning and memory impairments by enhancing ACh release, due to a stimulation of sigma and probably 5-HT1A receptors. Combined sigma/5-HT1A receptor agonism may be a novel approach to ameliorate cognitive disorders associated with age-associated cholinergic deficits.
ENGELMANN, M., C. T. WOTJAK, I. NEUMANN, M. LUDWIG AND R. LANDGRAF. Behavioral consequences of intracerebral vasopressin and oxytocin: Focus on learning and memory. NEUROSCI BIOBEHAV REV 20(3)341–358, 1996.—Since the pioneering work of David de Wied and his colleagues, the neuropeptides arginine vasopressin and oxytocin have been thought to play a pivotal role in behavioral regulation in general, and in learning and memory in particular. The present review focuses on the behavioral effects of intracerebral arginine vasopressin and oxytocin, with particular emphasis on the role of these neuropeptides as signals in interneuronal communication. We also discuss several methodological approaches that have been used to reveal the importance of these intracerebral neuropeptides as signals within signaling cascades. The literature suggests that arginine vasopressin improves, and oxytocin impairs, learning and memory. However, a critical analysis of the subject indicates the necessity for a revision of this generalized concept. We suggest that, depending on the behavioral test and the brain area under study, these endogenous neuropeptides are differentially involved in behavioral regulation; thus, generalizations derived from a single behavioral task should be avoided. In particular, recent studies on rodents indicate that socially relevant behaviors triggered by olfactory stimuli and paradigms in which the animals have to cope with an intense stressor (e.g., foot-shock motivated active or passive avoidance) are controlled by both arginine vasopressin and oxytocin released intracerebrally. Copyright © 1996 Elsevier Science Ltd.
Administration of hypothalamic peptides has been reported to induce behavioral changes and to modify neurological functions such as locomotor activity and learning. Somatostatin (SS) and growth hormone-releasing factor (GRF) exert opposite effects on anterior pituitary secretion. Similarly, at the central nervous system (CNS) level, SS and GRF display antagonistic actions on behavioral parameters. The authors were able to confirm these effects in male Wistar rats by means of a computerized electronic maze measuring locomotor activity and learning. SS concentration is reduced in specific areas of the CNS in patients with late onset of senile dementia of the Alzheimer’s type (SDAT). In early onset SDAT a GRF test elicits a growth hormone response much greater than that observed in normal controls of the same age or in patients with late onset SDAT. Thus, administration of GRF to patients with early onset SDAT has been followed by a significant improvement in locomotion, appetite, mental performance and social interaction. A possible therapeutic role of GRF in the management of patients with dementia remains to be explored.
An implicit assumption guiding many studies of neurochemical systems involved in learning and memory in animal subjects is that animal and human memory systems use the same or similar mechanisms. Because acquisition and retention performance can be influenced by many processes other than information storage, special effort is required to distinguish influences on memory processes from other factors influencing performance. This article reviews the findings of recent studies investigating the effects, on memory, of drugs affecting adrenergic, opioid peptidergic, GABAergic and cholinergic systems. The review focuses primarily on studies using posttraining treatments and tests for retention given no sooner than a day after the training. Extensive evidence suggests that such drugs interact within the amygdaloid complex and that projections from the amygdala influence memory storage in other brain regions. The assumption that comparable processes occur in animal and human subjects is supported by evidence that, in human subjects, emotionally influenced memory is blocked by a β-adrenergic blocker and by lesions of the amygdaloid complex.
This study investigated the effect of posttraining administration (i.p.) of naloxone and [Met5]enkephalin on retention in intact and adrenal-denervated mice. Naloxone (0.3 mg/kg) enhanced and [Met5]enkephalin (0.5 or 2.0 μg/kg) impaired retention in both inhibitory avoidance and Y-maze discrimination tasks. Further, [Met5]enkephalin antagonized the facilitative effect of naloxone on retention while naloxone attenuated the retention deficit produced by [Met5]enkephalin. However, neither naloxone nor [Met5]enkephalin affected retention in adrenal denervated mice. These findings suggest that endogenous hormones of the adrenal medulla, including [Met5]enkephalin, may be involved in the modulating influence of opiate peptides on memory storage.
We report here the discovery of a unique and novel angiotensin binding site and peptide system based upon the C-terminal 3-8 hexapeptide fragment of angiotensin II (NH3(+)-Val-Tyr-Ile-His-Pro-Phe-COO-) (AII(3-8) (AIV)). This fragment binds saturably, reversibly, specifically, and with high affinity to membrane-binding sites in a variety of tissues and from many species. The binding site is pharmacologically distinct from the classic angiotensin receptors (AT1 or AT2) displaying low affinity for the known agonists (AII and AIII) and antagonist (Sar1,Ile8-AII). Although a definitive function has not been assigned to this system in many of the tissues in which it resides, AIV's interaction with endothelial cells may involve a role in endothelial cell-dependent vasodilation. Consequent to this action, AIV is a potent stimulator of renal cortical blood flow.
A unique angiotensin binding site specific for the hexapeptide, AII(3-8), has been identified in guinea pig hippocampus. This binding site, which is present in the pyramidal cell layer of CA1, CA2, CA3 of the hippocampus and dentate gyrus, binds AII(3-8) with high affinity (KD = 1.29 +/- 0.18 nM) in a saturable manner (Bmax = 449 +/- 62 fmol/mg protein). The N-terminal structure of the binding ligand is paramount in determining the binding affinity. The C-terminal requirements seem less stringent as evidenced by the binding affinity of AII(3-7) (KD = 20.9 +/- 2.1 nM). Neither AII, AIII,Sar1, Ile8-AII, Dup 753 nor CGP42112A appear to bind, indicating that this binding site is neither the AT1 nor AT2 sites described for AII/AIII. Autoradiographic analysis of hippocampus binding confirms the inability of Sar1,Ile8-AII to compete for [125I]AII(3-8) binding. Conversely AII(3-8) was unable to displace [125I]Sar1,Ile8-AII binding.
Behavioral effects of somatostatin-14, and some of its fragments [somatostatin(3-8), somatostatin(9-14), somatostatin(7-10)] after intracerebroventricular (ICV) administration have been investigated in male rats. In a passive avoidance learning test, somatostatin-14 (0.6 nM) given immediately after the learning session increased the avoidance latency at 24 hr after the injection, when compared to a somatostatin(3-8) (0.6 nM)-treated group. However, compared to a saline-treated group, the peptides did not significantly influence the avoidance latency. Somatostatin-14 administered in higher dose (6.0 nM) decreased the avoidance latency compared to the saline-treated group, while its fragments did not influence it. In an open field behavioral test, immediately after the 24-hr passive avoidance test, 6 nM of somatostatin-14 decreased the rearing activity, while the fragments did not influence this behavior. Somatostatin-14 produced barrel rotation in a dose-related manner, but after the injection of a high dose of the peptide (12 nM) all of the animals died in cardiorespiratory failure (apnea, pulmonary oedema). The fragments did not produce barrel rotation.
Administration of hypothalamic peptides has been reported to induce behavioral changes and to modify neurological functions such as locomotor activity and learning. Somatostatin (SS) and growth hormone-releasing factor (GRF) exert opposite effects on anterior pituitary secretion. Similarly, at the central nervous system (CNS) level, SS and GRF display antagonistic actions on behavioral parameters. The authors were able to confirm these effects in male Wistar rats by means of a computerized electronic maze measuring locomotor activity and learning. SS concentration is reduced in specific areas of the CNS in patients with late onset of senile dementia of the Alzheimer's type (SDAT). In early onset SDAT a GRF test elicits a growth hormone response much greater than that observed in normal controls of the same age or in patients with late onset SDAT. Thus, administration of GRF to patients with early onset SDAT has been followed by a significant improvement in locomotion, appetite, mental performance and social interaction. A possible therapeutic role of GRF in the management of patients with dementia remains to be explored.
Angiotensin II-(3-8)-hexapeptide, at the dose of 1 nmol given intracerebroventricularly, only slightly less than angiotensin II (the same dose and route) stimulated exploratory locomotor behaviour in an open field and electromagnetic motimeter. Both peptides considerably enhanced stereotyped behaviour produced by apomorphine and amphetamine. Angiotensin II-(3-8)-hexapeptide improved recall in a passive avoidance situation as well as angiotensin II. The 3-8 C-terminus of angiotensin II enhanced acquisition of active avoidance nearly as effectively as the complete peptide. The results indicate that the effectiveness of equimolar doses of angiotensin II-(3-8)-hexapeptide and angiotensin II in improving processes related to learning and memory in rats is almost identical and thus must be independent of specific angiotensin receptors in brain to which the hexapeptide binds with about 1000 times lower affinity than angiotensin II. The stimulation of stereotypy, a dopamine-controlled behaviour, by the peptides points to the possibility of dopaminergic mediation of their psychotropic effects.
'Memory' for a juvenile conspecific in male rats can be measured by variation in duration of investigation times when the same juvenile is presented at different intervals. Typically, exposure of an adult male rat to a juvenile results in transient investigatory activity that rapidly declines with repeated exposures at short interexposure intervals (30 min). Longer interexposure intervals (120 min) result in re-investigation with durations similar or greater than the original investigation. Arginine vasopressin (AVP) injected into adult male rats intracerebroventricularly in doses of 0.5-2.0 ng immediately after investigation of the juvenile decreased social investigation of the same juvenile at the long (120 min) interexposure interval. This decrease in investigatory time was similar to that observed after a 30-min interexposure interval in untreated animals. These results support the hypothesis that increasing the availability of AVP in the central nervous system can improve the consolidation of olfactory information and improve conspecific recognition in rats.
A peptide that accumulated as the major product during the proteolysis of arginine vasopressin by rat brain synaptic membranes
was isolated and its structure was shown to be the hexapeptide pGlu-Asn-Cys(Cys)-Pro-Arg-Gly-NH2. When administered intracerebroventricularly
in extremely low doses, this vasopressin fragment and its desglycinamide derivative facilitated memory consolidation in a
passive avoidance situation. These vasopressin metabolites, which are devoid of pressor activity, constitute highly potent
neuropeptides with selective effects on memory and related processes; they are activated via proteolytic processing of vasopressin
by brain peptidases.
A unique angiotensin binding site specific for the hexapeptide, angiotensin II(3-8) (AngIV), has been previously reported by our laboratory in the guinea pig brain and is presently described in the rat brain. This angiotensin receptor subtype has been termed AT4 and is prominently distributed in cerebral cortex, piriform cortex, hippocampus, habenulae, colliculi, septum, periaqueductal gray, several thalamic nuclei, the arcuate nucleus of the hypothalamus and cerebellum. In the second part of the present investigation, separate groups of rats received i.c.v. injections of angiotensin II (AngII), AngIV or artificial cerebrospinal fluid (aCSF) and were euthanized 2 h later for the purpose of evaluating for brain c-Fos expression. After i.c.v.-injected AngIV, Fos-like immunoreactivity was present in the hippocampus and piriform cortex. This immunoreactivity was unaffected by i.c.v. pretreatment with the AT1 angiotensin receptor antagonist DuP 753 (losartan) or the AT2 receptor ligand PD123177 but was blocked by the AT4 angiotensin receptor antagonist, divalanal-AngIV. I.c.v. injection of AngII resulted in Fos-like immunoreactivity in the dorsal third and lateral ventricles, subfornical organ, lateral hypothalamus and amygdala. Pretreatment with losartan or PD123177 significantly interfered with this AngII-induced immunoreactivity while divalanal-AngIV did not. These results indicate that in both guinea pig and rat brains the AT4 receptor has a distribution different than that previously reported for AT1 and AT2 receptor subtypes. The c-Fos expression results suggest that different brain neuronal pathways are activated by i.c.v. injection of AngII and AngIV.
JTP-4819 ((S)-2-[[(S)-2-(hydroxyacetyl)-1-pyrrolidinyl]carbonyl]-N- phenylmethyl)-1-pyrrolidinecarboxamide) is a potent (IC50: 0.83 +/- 0.09 nM in rat brain supernatant; 5.43 +/- 0.81 nM in Flavobacterium meningosepticum) and specific inhibitor of prolyl endopeptidase (PEP). JTP-4819 (3 mg/kg p.o.) exhibited a strong and durable ex vivo inhibitory effect on PEP in various regions of the rat brain. In addition, JTP-4819 inhibited the degradation of substance P, arginine-vasopressin, thyrotropin-releasing hormone, neurotensin, oxytocin, bradykinin, and angiotensin II by purified PEP with IC50 values of 9.6, 13.9, 10.7, 14.0, 4.5, 7.6 and 10.6 nM, respectively. In the one-trial passive avoidance test in rats with scopolamine-induced amnesia, JTP-4819 significantly prolonged the retention time when administered orally at doses of 1 and 3 mg/kg 1 hr before acquisition or at 3 and 10 mg/kg 1 hr before retention. In addition, coadministration of JTP-4819 and substance P, arginine-vasopressin or thyrotropin-releasing hormone (at doses at which each drug alone did not prolong the retention time) improved the retention time of rats with scopolamine-induced amnesia. Microdialysis studies demonstrated that JTP-4819 caused a significant increase in ACh release in the frontal cortex and hippocampus of young rats at oral doses of 1 and 3 mg/kg, as well as in both brain regions of aged rats at a dose of 3 mg/kg. These results indicate that JTP-4819 potentiates neuropeptide functions inhibiting PEP, that it activates cholinergic transmission and that it enhances learning and memory.
We assessed hippocampal-dependent memory in mice with a Ca(2+)-independent form of CaMKII generated by the introduction of an aspartate at amino acid 286. The CaMKII-Asp-286 mice show normal LTP at high frequency stimulation, but in the 5-10 Hz range, they show a shift in the frequency-response curve favoring LTD. This range of frequencies is similar to the theta rhythm, which is associated with exploration in rodents. Using the Barnes maze to assess spatial memory, we found the transgenic mice could not learn to navigate to a specific location using spatial cues. In contrast, one line of transgenic mice performed normally in contextual fear conditioning, a task that is also hippocampal dependent. This dissociation between spatial and contextual memory suggests that even though both require the hippocampus, they may be mediated by different synaptic mechanisms.
The present research characterizes a newly discovered ANG II(3-8) (ANG IV) binding site localized in structures associated with memory function (hippocampus, neocortex, cerebellum), as well as other brain stem structures (thalamus, inferior olivary nucleus). This site is not the AT1 or AT2 site that binds angiotensins II (ANG II) and III (ANG III) nor does it bind the nonpeptide AT1 or AT2 receptor antagonists DuP753 and PD123177, respectively. The intracerebroventricular (ICV) infusion of ANG IV was ineffective at inducing drinking in rats as compared with equivalent doses of ANG II and III. Although not as effective as ANG II or ANG III, ICV infusion of ANG IV did provoke a pressor response at the highest dose (100 pmol/min), which appeared to be mediated by ANG II (AT1)-type receptors and not the specific AIV binding site described here. By contrast, the ICV infusion of ANG IV resulted in greater effects upon retention and retrieval of a passive avoidance task as compared with ANG II. Specifically, ANG II was not different from the ICV infusion of artificial cerebrospinal fluid, while ANG IV improved retention and retrieval of this task.
Our laboratory has reported previously that a unique binding site specific for the hexapeptide angiotensin (A)II(3-8), now referred to as AIV, is present in a number of tissues including bovine adrenal gland, rabbit and guinea pig heart and guinea pig kidney, liver, lung, uterus and brain. The present results extend previous findings in the guinea pig brain and identify binding sites for AIV in the neocortex, paleocortex, hippocampus, medial habenula, superior and inferior colliculi, caudate putamen, thalamus, dorsal tegmentum, central gray, red nucleus, inferior olivary, oculomotor and hypoglossal nuclei and cerebellum. Binding of [125I]AIV in selected regions was shown to be of high affinity (Kd = 0.60-1.47 nM), saturable (maximal number of binding sites = 181-449 fmol/mg of protein) and specific. This binding site was shown to be distinct from the AT1 and AT2 sites with Ki values > 10(-4) M for DuP 753, CGP42112A and PD123177. Changes at the N-terminal of the peptide, either by removal of the valine or by extension of the peptide, resulted in a large decrease in binding affinity. In contrast, C-terminal extensions resulted in little change in affinity for the binding site. Guanosine 5'-0-(3-thiotriphosphate) was shown to have no effect on binding, suggesting that the guinea pig brain binding site is not G-protein-linked. Potential functions associated with this newly discovered A binding site are discussed.
Long-term potentiation of synaptic transmission in the hippocampus is the primary experimental model for investigating the synaptic basis of learning and memory in vertebrates. The best understood form of long-term potentiation is induced by the activation of the N-methyl-D-aspartate receptor complex. This subtype of glutamate receptor endows long-term potentiation with Hebbian characteristics, and allows electrical events at the postsynaptic membrane to be transduced into chemical signals which, in turn, are thought to activate both pre- and postsynaptic mechanisms to generate a persistent increase in synaptic strength.
Previous results have indicated the involvement of the hippocampus in the behavioral effect of vasopressin, with a better effect when the peptide was injected in the ventral part rather than in the dorsal part of this structure. The purpose of the present study was to determine, in mice, whether the injection of vasopressin or vasopressin antisera into the ventral hippocampus has an effect on retrieval and relearning of a Go-No Go visual discrimination task and, if so, to what extent this involvement of the vasopressin system depends on the integrity of the medial amygdaloid nucleus, the main source of vasopressin innervation in the ventral hippocampus in rats. In the first experiment, we showed that pretest microinjection of Arg8-vasopressin (25 pg per animal) in the ventral hippocampus alleviated forgetting observed after a prolonged interval of 24 days between the acquisition of information and its retrieval. This enhancing effect was characterized by better retrieval and relearning in vasopressin-treated mice than those in control mice. Conversely, an immunoneutralization of endogenous vasopressin in the ventral hippocampus by the microinjection of vasopressin antisera (1/10 dilution) resulted in the drastic impairment of retrieval and relearning. Since the lack of an observable change in a locomotor activity test might explain these results, we postulated that the vasopressin system in the ventral hippocampus is involved in retrieval processes. Moreover, the effects of these treatments in a nonassociative context suggest that the effect of vasopressin could be dependent on the contextual paradigm used. In the second experiment, we localized vasopressin immunoreactive fibers in the CA1-CA2 ventral hippocampal fields and CA4-gyrus dentatus region, and vasopressin perikarya in the medial amygdaloid nucleus. Then, the projection of vasopressin cells from the medial amygdaloid nucleus to the ventral hippocampus was evaluated by studying changes in vasopressin immunoreactive fiber density in the ventral hippocampus after a lesion of the medial amygdaloid nucleus. The results showed the almost complete disappearance of vasopressin fibers in the CA1-CA2 hippocampal fields after the medial amygdaloid lesion. In contrast, vasopressin fibers in the CA4 and gyrus dentatus region remain unchanged. On the basis of our immunohistochemical results, our third experiment tested the repercussions of the change in vasopressin innervation in the ventral hippocampus, due to the medial amygdaloid lesion, on the effects of exogenously administered vasopressin on both retrieval and relearning processes. The medial amygdaloid lesion induced a deleterious effect on retrieval without really affecting the ability to relearn. No observable change in locomotor activity could explain this impairment.(ABSTRACT TRUNCATED AT 400 WORDS)
Angiotensin IV (Val Tyr Ile His Pro Phe), administered centrally, increases memory retrieval and induces c-fos expression in the hippocampus and piriform cortex. Angiotensin IV binds to a high affinity site that is quite distinct in pharmacology and distribution from the angiotensin II AT1 and AT2 receptors and is known as the AT4 receptor. These observations suggest that the AT4 receptor may have multiple central effects. The present study uses in vitro receptor autoradiography, and employs [125I]angiotensin IV to map AT4 receptors in the macaca fascicularis brain. The distribution of the AT4 receptor is remarkable in that its distribution extends throughout several neural systems. Most striking is its localization in motor nuclei and motor associated regions. These include the ventral horn spinal motor neurons, all cranial motor nuclei including the oculomotor, abducens, facial and hypoglossal nuclei, and the dorsal motor nucleus of the vagus. Receptors are also present in the vestibular, reticular and inferior olivary nuclei, the granular layer of the cerebellum, and the Betz cells of the motor cortex. Moderate AT4 receptor density is seen in all cerebellar nuclei, ventral thalamic nuclei and the substantia nigra pars compacta, with lower receptor density observed in the caudate nucleus and putamen. Abundant AT4 receptors are also found in areas associated with cholinergic nuclei and their projections, including the nucleus basalis of Meynert, ventral limb of the diagonal band and the hippocampus, somatic motor nuclei and autonomic preganglionic motor nuclei. AT4 receptors are also observed in sensory regions, with moderate levels in spinal trigeminal, gracile, cuneate and thalamic ventral posterior nuclei, and the somatosensory cortex. The abundance of the AT4 receptor in motor and cholinergic neurons, and to a lesser extent, in sensory neurons, suggests multiple roles for the AT4 receptor in the primate brain.
Since the pioneering work of David de Wied and his colleagues, the neuropeptides arginine vasopressin and oxytocin have been thought to play a pivotal role in behavioral regulation in general, and in learning and memory in particular. The present review focuses on the behavioral effects of intracerebral arginine vasopressin and oxytocin, with particular emphasis on the role of these neuropeptides as signals in interneuronal communication. We also discuss several methodological approaches that have been used to reveal the importance of these intracerebral neuropeptides as signals within signaling cascades. The literature suggests that arginine vasopressin improves, and oxytocin impairs, learning and memory. However, a critical analysis of the subject indicates the necessity for a revision of this generalized concept. We suggest that, depending on the behavioral test and the brain area under study, these endogenous neuropeptides are differentially involved in behavioral regulation; thus, generalizations derived from a single behavioral task should be avoided. In particular, recent studies on rodents indicate that socially relevant behaviors triggered by olfactory stimuli and paradigms in which the animals have to cope with an intense stressor (e.g., foot-shock motivated active or passive avoidance) are controlled by both arginine vasopressin and oxytocin released intracerebrally.
Divalinal-Ang IV [V psi (CH2-NH2)YV psi (CH2-NH2)HPF] is being employed increasingly as a specific AT4 antagonist. This use, which necessitates a comprehensive physiological and pharmacological evaluation of Divalinal-Ang IV's functional and receptor binding characteristics in order to ensure its efficacy and specificity, was the stimulus for this study using bovine adrenal membranes. [125I]Ang IV and [125I]Divalinal-Ang IV were shown to bind with high affinity to a similar number of binding sites, suggesting that both bound the same receptor. This notion was verified by competition curves using [125I]Ang IV and [125I]Divalinal-Ang IV that indicated identical rank order affinities for several angiotensin-related peptides and 100% cross-displacement by Ang IV and Divalinal-Ang IV. Furthermore, an autoradiographic comparison of [125I]Ang IV and [125I]Divalinal-Ang IV in 20 microns sections of bovine adrenals revealed near identical binding distributions characterized by heavy binding in the glomerulosa layer and the medulla. Physiological studies in which test compounds were injected into the internal carotid of the rat and cerebral blood flor (CBF) was measured by laser Doppler flowmetry indicated that pretreatment with Divalinal-Ang IV, but not DuP 753 or PD123177, blocked the increased flow observed with Ang IV infusion. Conversely, DuP 753, but not Divalinal-Ang IV or PD123177, inhibited the decrease in flow witnessed with Ang II. Metabolic stability studies utilizing rat kidney homogenates as a peptidase source, demonstrated that the structural changes present in Divalinal-Ang IV greatly increased its resistance to metabolism as compared to Ang IV. Together, these studies show that Divalinal-Ang IV is a stable, efficacious and specific inhibitor of AT4 receptors.
The pharmacological actions of JTP-4819, a new prolyl endopeptidase (PEP) inhibitor targeted for the treatment of Alzheimer's disease, are reviewed with respect to its effects on PEP activity, brain neurotransmitters, and memory-related behaviour in rats. JTP-4819 was shown to be a very potent and specific inhibitor of PEP. At nanomolar concentration, JTP-4819 inhibited the degradation of substance P, arginine-vasopressin, and thyrotropin-releasing hormone by PEP in supernatants of the rat cerebral cortex and hippocampus. Repeated administration of JTP-4819 reversed the aging-induced decrease in brain substance P-like and thyrotropin-releasing hormone-like immunoreactivity, suggesting that this drug may be able to improve the imbalance of peptidergic neuronal systems that develops with senescense by inhibiting PEP activity. JTP-4819 increased acetylcholine release from the frontal cortex and hippocampus, regions closely associated with memory, in both young and aged rats. In addition, it improved performance in several memory and learning-related tests (e.g., the Morris water maze task in aged or MCA-occluded rats and the passive avoidance test). This memory-enhancing effect of JTP-4819 may result from prevention of the metabolic degradation of brain neuropeptides by PEP as well as from the enhancement of acetylcholine release. Taken together, these unique and potent pharmacological actions of JTP-4819 suggest that it may have the potential to be used for treating Alzheimer's disease.
The effect of arginine8-vasopressin (AVP1-9) and its metabolite C-terminal fragments on the scopolamine-induced disruption of spatial cognition were investigated using an 8-arm radial maze task in rats. AVP1-9 (10 micrograms/kg s.c.) markedly improved the disruption of spatial cognition by treatment with scopolamine (0.5 mg/kg i.p.), and 60% of the rats recovered to a normal level. The main metabolite of AVP1-9, AVP4-9 (0.5 and 1 ng/kg s.c.) also significantly improved the scopolamine-induced deficit of spatial memory. The activity of AVP4-9 was determined to be about 10000 fold greater than that of AVP1-9. An intracerebroventricular (i.c.v.) injection of 10 fg of AVP5-8, however, showed a lower activity. Both AVP6-8 and AVP5-7, which are both metabolites of AVP5-8, demonstrated no activity. The scopolamine-induced disruption of spatial memory was found to improve after a microinjection of AVP4-9 (1 fg) into the ventral hippocampus (VH) region, but not into the dorsal hippocampus (DH). In an in vivo microdialysis study, the scopolamine-induced acetylcholine (ACh) release from the VH was slightly potentiated by treatment with AVP4-9 (10 fg i.c.v.). In addition, an AVP4-9 analogue, No. 302, which is a synthetic hexapeptide and has a longer half-life, also demonstrated a markedly improved effect, which had a 10-fold higher activity than that with AVP4-9. AVP4-9 is the most potent activity of all the endogenous metabolites of the AVP1-9 and the new synthetic AVP4-9 analogue, No. 302 (obtained from Nippon Chemiphar Co.), substituting Ser for Cys-Cys in hexapeptide, has higher activity than that of AVP4-9. These results indicated [Ser6] hexapeptide has an important role in behavioral activity. Based on these results, it is possible that AVP1-9 and its metabolite AVP4-9 could, thus, be useful in treating cholinergic dysfunction diseases, such as Alzheimer's disease. Hexapeptide may play an important role in improving the spatial memory by promoting the release of ACh in the VH region.
The effects of a novel prolyl endopeptidase inhibitor (PEP), (S)-2-[[(S)-2-(hydroxyacetyl)-1-pyrrolidinyl]carbonyl]-N-(phenylmethyl)- 1-pyrrolidinecar-boxamide (JTP-4819), on performance of the Morris water maze task and on central cholinergic function were investigated in aged rats. Spatial memory (escape latency, path length, and swimming speed to the platform) was impaired in aged rats performing the Morris water maze task when compared to young rats. Administration of JTP-4819 (1 mg/kg, p.o.) for 14 days improved this memory deficit in aged rats, as shown by the decrease in escape latency and path length. In addition, when JTP-4819 (at doses of 1 and 3 mg/kg, p.o.) was administered for 3 wk, it reversed the age-related increase of ChAT activity in the cerebral cortex and the decrease of 3H-choline uptake in the hippocampus. These data suggest that JTP-4819 ameliorates age-related impairment of spatial memory and partly reverses central cholinergic dysfunction, possibly due to the enhancement of neuropeptide function by inhibition of PEP mediated degradation of substance P, arginine-vasopressin, and thyrotropin-releasing hormone.
Our laboratory has previously reported the discovery of a unique angiotensin binding site (termed AT4) specific for angiotensin IV (AngIV) in cultured vascular endothelial and smooth muscle cells. The present investigation employed laser-Doppler flowmetry to examine the effect of angiotensin II (AngII) and AngIV stimulation of these receptors on cerebral microcirculation in anesthetized Sprague-Dawley rats. Internal carotid artery infusion of AngII at a low dose (0.1 pmol min-1) revealed a 23% reduction in cerebral blood flow (CBF), while the infusion of AngIV increased CBF in a dose-dependent fashion with the highest dose (100 pmol min-1) resulting in an elevation of 30%. In a second experiment separate groups of rats were pre-treated with the AT1 receptor subtype antagonist DuP 753 (Losartan), the AT2 receptor subtype antagonist PD123177, or a newly synthesized AT4 receptor subtype antagonist Divalinal-AngIV (Divalinal), followed by AngII or AngIV for the purpose of determining which angiotensin receptor subtype is responsible for mediating these AngII- and AngIV-induced responses. Pre-treatment with Losartan completely blocked subsequent AngII-induced reductions in CBF, while both PD123177 and Divalinal failed to inhibit this response. In contrast, significant increases in CBF were measured due to AngIV stimulation following pre-treatment with Losartan and PD 123177, while Divalinal abolished this AngIV-induced response. These results suggest that AngII and IV play opposite roles in cerebral microcirculation, i.e., the AT1 receptor subtype mediates AngII-induced reductions in CBF, while the AT4 receptor subtype regulates increases in CBF.
This study examined effects on memory formation produced by [Leu]enkephalin and [Met]enkephalin administration in 2 regions of the 2-day-old chick brain involved in memory formation: the intermediate medial hyperstriatum ventrale (IMHV) and the lobus parolfactorius (LPO). Basal concentrations of endogenous [Leu]enkephalin and [Met]enkephalin were determined for 5 brain regions, and effects of 1-trial peck-avoidance training on enkephalin concentrations were examined in the IMHV and LPO. [Leu]enkephalin was amnestic when administered in the IMHV but not in the LPO. In contrast, [Met]enkephalin may be amnestic when administered in the LPO but not in the IMHV. Training decreased [Met]enkephalin concentration in the LPO but not in the IMHV. Training had no effect on [Leu]enkephalin concentration in either the IMHV or the LPO. Thus, amnestic effects of [Leu]- or [Met]enkephalin administration are brain-region specific. Regional activity of endogenous [Met]enkephalin during memory formation is consistent with localized amnestic effects produced by [Met]enkephalin administration.
We previously discovered that insulin stimulates the marked translocation of a novel membrane aminopeptidase, designated vp165 for vesicle protein of 165 kDa, to the cell surface in adipocytes. To examine the hypothesis that this enzyme acts on peptide hormones, we assessed the relative affinity of the enzyme for 22 peptide hormones by measuring the inhibitory effect of each on the hydrolysis of a fluorogenic substrate, and we directly assayed the cleavage of four of these. Angiotensin III, angiotensin IV, and Lys-bradykinin bound to the enzyme with half-saturation constants between 20 and 600 nM and were cleaved by vp165. Vasopressin bound with lower affinity but at saturation was cleaved more rapidly. Subsequently, the effect of insulin on the rates of cleavage of 125I-labeled vasopressin by intact 3T3-L1 and rat adipocytes was determined. With both cell types, vasopressin cleavage was stimulated approximately threefold. These findings indicate that a physiological role for vp165 may be the processing of peptide hormones and that insulin could enhance the cleavage of extracellular substrates by eliciting the translocation of vp165 to the cell surface.
Angiotensin IV (Val-Tyr-Ile-His-Pro-Phe) has been reported to interact with specific high-affinity receptors to increase memory retrieval, enhance dopamine-induced stereotypy behavior, and induce c-fos expression in several brain nuclei. We have isolated a decapeptide (Leu-Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg-Phe) from sheep brain that binds with high affinity to the angiotensin IV receptor. The peptide was isolated using 125I-angiotensin IV binding to bovine adrenal membranes to assay receptor binding activity. This peptide is identical to the amino acid sequence 30-39 of sheep betaA- and betaB-globins and has previously been named LVV-hemorphin-7. Pharmacological studies demonstrated that LVV-hemorphin-7 and angiotensin IV were equipotent in competing for 125I-angiotensin IV binding to sheep cerebellar membranes and displayed full cross-displacement. Using in vitro receptor autoradiography, 125I-LVV-hemorphin-7 binding to sheep brain sections was identical to 125I-angiotensin IV binding in its pattern of distribution and binding specificity. This study reveals the presence of a globin fragment in the sheep brain that exhibits a high affinity for, and displays an identical receptor distribution with, the angiotensin IV receptor. This globin fragment, LVV-hemorphin-7, may therefore represent an endogenous ligand for the angiotensin IV receptor in the CNS.
We investigated the effect of successive administrations of SA4503 (1-(3,4-dimethoxyphenethyl)-4-(3-phenylpropyl)piperazine dihydrochloride), a novel cognitive enhancer with high affinity and selectivity for the sigma1 receptor subtype, on the cortical cholinergic dysfunction-induced impairment of the spatial learning performance in the Morris water maze (MWM) task in rats. The impairment of the spatial learning performance was produced by the ibotenic acid-induced lesion of the basal forebrain (BF) area in rats. Escape latencies to find the platform during the training trials of the MWM task were significantly prolonged in the BF-lesioned rats compared with the sham-operated rats. Daily treatment with SA4503 (0.1-0.5 mg/kg, P.O./day) for 13 days ameliorated this learning deficit. In the probe trial, BF-lesioned rats reduced the number of times each rat crossed the former platform location during the training trials (goal area) in comparison with sham-operated rats. Successive administrations of SA4503 (0.25 mg/kg, P.O./day) also significantly increased the BF lesion-induced reduction of the number of times each rat crossed the goal area. These results suggest that the successive administrations of SA4503 attenuate the impairment of the spatial learning performance in rats with cortical cholinergic dysfunction, and that SA4503 is useful as a therapeutic drug for Alzheimer's disease.
The effect of a novel prolyl endopeptidase (PEP) inhibitor, (S)-2-2[[(S)-2-(hydroxyacetyl)-1-pyrrolidinyl]carbonyl]-N-(phenylmethyl) -1-pyrrolidinecarboxamide (JTP-4819), on spatial learning deficits in rats with dorsal hippocampal (DH) lesions was examined using an eight-arm radial maze. The correct performance remained at chance levels even after 18 acquisition trials in rats with DH lesions. JTP-4819 (3.0 mg/kg, p.o.) significantly ameliorated this learning impairment after 34-41 days of treatment. When DH lesions were created in rats after achievement of learning, postoperative performance deteriorated prominently, but gradually recovered with the repetition of trials. JTP-4819 (3.0 mg/kg, p.o.) significantly decreased the number of trials needed to reattain learning criterion. After the behavioral experiment, the choline acetyltransferase (ChAT) activity and [3H]-pirenzepine binding (Kd, Bmax) in the residual hippocampus and cerebral cortex were analyzed. Neither parameter was significantly affected by JTP-4819. In conclusion, JTP-4819 can improve both learning and relearning deficits of spatial memory in DH-lesioned rats, postulating that enhancement of neuropeptide activity via PEP inhibition may be involved in the mechanism of action of JTP-4819.
Recently, a receptor for the angiotensin II(3-8) (Ang IV) hexapeptide, was discovered in the hippocampus, suggesting a possible role in learning. The present study utilized intracerebroventricularly (icv) infused scopolamine hydrobromide (scop) to disrupt spatial learning in the circular water maze, followed by the Ang IV analog norleucine1-Ang IV (Nle1-Ang IV), to restore normal performance. Rats were icv pretreated with either scop or artificial cerebrospinal fluid (aCSF) followed by either icv injected Nle1-Ang IV or aCSF, and then behaviourally tested. During acquisition training, each animal's latency to locate the platform, path distance, speed, and efficiency ratios were measured. A probe trial was conducted on the final day of training and the time spent in the target quadrant and the number of crossings over the former location of the platform (annulus crossings) were observed. The results indicate that those animals treated with scop followed by aCSF performed poorly during acquisition training as compared with controls. In contrast, those animals that received scop followed by Nle1-Ang IV attained equivalent latencies, distances, and efficiency ratios to find the platform as those achieved by controls. There were no observed differences in swimming speed, thus arguing against drug-induced motor impairment. During the probe trial, animals treated with scop followed by aCSF spent less time in the target quadrant and made fewer annulus crossings as compared to controls, while the scop, Nle'-Ang IV treated animals performed equivalently to controls. These results suggest that Nle1-Ang IV acts to counteract the disruption of spatial learning induced by scopolamine.
We have previously reported that the insulin-responsive glucose transporter GLUT4 is strongly expressed by discrete areas of the rat brain (Leloup et al. [1996] Molec. Brain Res. 38:45-53). In the present study, a sensitive immunocytochemical technique has been used to analyze extensively the anatomical and ultrastructural localizations of GLUT4 in the rat central nervous system in order to gain insight into the physiological role of this transporter. We confirm that GLUT4 is expressed by numerous neurons of the brain and spinal cord, whereas glial cells are more scarcely labeled. In both light and electron microscopy, we observe that the immunoreactivity for GLUT4 is localized mainly in the somatodendritic portion of neurons, where some cisterns of rough endoplasmic reticulum, ribosomal rosettes, certain Golgi saccules, and some intracytoplasmic vesicles are labeled. In contrast, axons and nerve terminals are only occasionally immunostained in certain brain regions such as the neocortex and the ventricular surfaces for example. The GLUT4-immunoreactive structures appear concentrated and most prominently immunostained in motor areas, such as the sensorimotor cortex, most basal ganglia and related nuclei, the cerebellum and deep cerebellar nuclei, a number of reticular fields, motor nuclei of cranial nerves, and motor neurons of the ventral horn of the spinal cord. The labeled regions, which also include some sensory nuclei, are often those in which Vissing et al. ([1996] J. Cerebral Blood Flow Metab. 16:729-736) have shown that exercise stimulates local cerebral glucose utilization, so that GLUT4 might be involved in this effect. On the other hand, the fact that the anatomical localizations of GLUT4 reported here generally agree with the distribution of insulin- or insulin-receptor- related receptors is important since it indicates that the translocation of GLUT4 might also be regulated by insulin in the central nervous system.
We conducted behavioral and neurochemical studies of a novel prolyl endopeptidase inhibitor, (S)2-[[(S)-2-(hydroxyacetyl)-1pyrrolidinyl]carbonyl]-N-(phenylmeth yl)-1-pyrrolidine-carboxamide (JTP-4819), in rats with lesions of the nucleus basalis magnocellularis (NBM-lesioned rats) induced by ibotenate. Administration of JTP-4819 (1 and 3 mg/kg, p.o.), on and after the 8th day, significantly shortened the escape latency in the Morris water maze as compared to the vehicle-treated group. JTP-4819 also significantly increased the path length in the quadrant with the platform removed in the spatial probe trial. Neurochemical studies of brains removed after the Morris water maze task showed that choline acetyltransferase activity in the cerebral cortex, but not the hippocampus, was significantly reduced by NBM lesioning, while there were no changes of muscarinic M1 receptor binding activity detected using [3H]pirenzepine. JTP-4819 had almost no effect on these cholinergic parameters in NBM-lesioned rats. Substance P-like immunoreactivity (LI), thyrotropin-releasing hormone (TRH)-LI, and arginine-vasopressin-LI were not significantly changed in the cerebral cortex and hippocampus of NBM-lesioned rats as compared to sham-operated rats. However, these neuropeptide levels were significantly increased in both brain regions by repeated administration of JTP-4819 (1, 3 and/or 10 mg/kg, p.o.). These results suggest that JTP-4819 ameliorated memory impairment due to NBM lesioning by potentiating SP, TRH and AVPergic neurons secondary to PEP inhibition.
125I-Angiotensin (Ang) IV and (125)I-divalinal Ang IV [AT receptor subtype 4 (AT(4))] receptor agonist and putative antagonist, respectively] were used to characterize the AT(4) receptor in Mardin-Darby bovine kidney epithelial cells (MDBK cell line). Both (125)I-Ang IV and (125)I-divalinal Ang IV bound to a single high-affinity site (K(D) = 1.37 and 1.01 nM, respectively) and to a comparable density of binding sites (B(max) = 1335 and 1407 fmol/mg protein, respectively). Competition of either radiolabeled ligand with several Ang related peptides demonstrated similar displacement affinities in the following affinity order: Ang IV = divalinal Ang IV > Ang III > Ang II > losartan = PD 123177. Guanosine-5'-O-(3-thio)triphosphate or sulfhydryl reducing agents did not affect the binding of either radiolabeled ligand. Brief exposure of MDBK cells to Ang IV or divalinal Ang IV (0.1 nM to 1 microM) caused a concentration-dependent rise in intracellular calcium concentration levels with a reduced calcium response observed with Ang IV at micromolar concentrations. These results indicate that Ang IV and divalinal Ang IV bind with high affinity to the same receptor and that the MDBK AT(4) receptor is not coupled to a classic G protein, nor are sulfhydryl bonds important in regulation of receptor affinity. The MDBK AT(4) receptor appears to be pharmacologically similar to that described in nonrenal tissues. Functional studies suggest that AT(4) receptor activation can increase intracellular calcium concentration levels in MDBK cells and that divalinal Ang IV possesses agonist activity with respect to this particular intracellular signaling system.
We investigated age-related changes in the number and size of neurons positive for the p75 neurotrophin receptor in the cholinergic basal forebrain of female Dark Agouti rats. Since the integrity of these neurons is known to be closely associated with performance in tests of spatial learning ability, we also investigated the incidence of age-related spatial learning impairments, using the Barnes maze. Spatial learning impairments occurred with increasing frequency with age. No rats showed impairment at six months, but 50% were impaired at 14 months and 71% at 26 months. There was no correlation between age and decreased number of p75-positive neurons in the rostral basal forebrain, which consists of the medial septum and vertical limb of the diagonal band of Broca. In the caudal basal forebrain, which consists of the horizontal limb and the nucleus of Meynert, there was a 13% reduction in the number of p75-positive neurons at 17 months compared to six months, and a 30% reduction at 26 months. There was a strong correlation between the presence of spatial learning impairment and a reduction in the number of p75-positive neurons. This correlation was most evident in the rostral basal forebrain, but was also present in the caudal basal forebrain. In the rostral basal forebrain, all learning impaired rats had fewer p75-positive neurons than the average number in unimpaired rats. A close correspondence between the presence of p75 and choline acetyltransferase was evident in basal forebrain neurons of learning-impaired and unimpaired rats. Gross pathological changes to the morphology of p75-positive neurons were relatively frequent in learning-impaired rats. These changes consisted of hypertrophy, appearance of vacuoles, and marginalisation of the cytoplasm.
The identification of 'place cells' in the hippocampus has suggested a special role for this brain structure in spatial mapping, but other studies have indicated a more general role in memory. New findings on place cells point the way towards a reconciliation of the mapping and memory views.
(125)I-divalinal-angiotensin IV (metabolically resistant analog of angiotensin IV) was used as a receptor ligand to identify the expression and properties of the angiotensin AT(4) receptor in epithelial HK-2 cells (an immortalized cell line derived from adult human proximal tubules). Saturation binding isotherms revealed that HK-2 cells contain a saturable (125)I-divalinal-angiotensin IV binding site with an affinity of 3 nmol/L and a density of 508 fmol/mg protein. An analysis of ligand specificity showed that only angiotensin AT(4) receptor ligands (angiotensin IV and divalinal-angiotensin IV) competed with both a high- and low-affinity binding site. GTPgammaS and dithiothreitol did not affect (125)I-Ang IV or (125)I-divalinal-Ang IV binding, suggesting that the AT(4) receptor was not G-protein coupled and did not require sulfhydryl bonds for receptor affinity. Activation of the AT(4) receptor caused a complex concentration-dependent rise in [Ca(2+)](i), an elevation in [Na(+)](i), and increased mitogen-activated protein kinase activity. These results suggest that human proximal tubule epithelial cells contain functional AT(4) receptors that are pharmacologically similar to the AT(4) receptor described in more distal segments of the nephron. Furthermore, the AT(4) receptor uses several intracellular signaling pathways to convey information.
Angiotensin IV and other AT4 receptor agonists, improve memory retention and retrieval in the passive avoidance and swim maze learning paradigms. Angiotensin IV binding sites (also known as the AT4 receptors) are widely distributed in guinea pig and monkey (Macaca fascicularis) brains where high densities of the binding sites have been detected in the hippocampus, neocortex and motor nuclei. However, the distribution of the binding sites in the human brain is not known. We have recently localised the angiotensin IV binding sites (AT4 receptors) in post-mortem human brain using iodinated Nle-angiotensin IV, a higher affinity and more stable analogue of angiotensin IV. This radioligand bound with relatively high affinity and specificity to angiotensin IV binding sites. In competition studies on consecutive sections through the prefrontal cortex and claustrum, angiotensin IV, Nle-angiotensin IV and LVV-hemorphin 7 competed for the binding of 125I[Nle]-angiotensin IV with nanomolar affinities. Angiotensin II and the AT1 and AT2 receptor antagonists were ineffective in competing for the binding at concentrations of up to 10 microM. We found high densities of 125I[Nle]-angiotensin IV binding sites throughout the cerebral cortex including the insular, entorhinal, prefrontal and cingulate cortices. Very high densities of the binding sites were observed in the claustrum, choroid plexus, hippocampus and pontine nucleus. Some thalamic nuclei displayed high densities of binding including the anteroprincipal, ventroanterior, anteromedial, medial dorsal and ventrolateral nuclei. The caudate nucleus, putamen, many amygdaloid nuclei and the red nucleus all displayed moderate densities of binding with a higher level detected in the substantia nigra pars compacta. In the hypothalamus, high densities binding sites were found in the ventromedial nucleus with lower levels in the dorsomedial and paraventricular nuclei. The distribution of 125I[Nle]-angiotensin IV binding sites in the human brain is similar to that found in other species and supports multiple roles for the binding sites in the central nervous system, including facilitation of memory retention and retrieval.
Recent evidence demonstrates that the fragment of angiotensin II, angiotensin II (3-8) termed angiotensin IV, binds with high affinity to a specific binding site, the AT(4) receptor. Intracerebroventricular injection of AT(4) receptor agonists improves the performance of rats in passive avoidance and spatial learning paradigms. AT(4) receptors and cholinergic neurons are closely associated in regions involved in cognitive processing, such as the hippocampus and neocortex. We therefore postulated that AT(4) receptors affect cognitive processing by modulating cholinergic neurotransmission. To test this, we examined the effect of AT(4) receptor ligands, angiotensin IV and LVV-hemorphin-7, on potassium-evoked [(3)H]acetylcholine ([(3)H]ACh) release from rat hippocampal slices. Hippocampal slices from male Sprague--Dawley rats were incubated with [(3)H]choline chloride, perfused with Krebs--Henseleit solution and [(3)H]ACh release was determined. Angiotensin IV and LVV-hemorphin-7 both potentiated depolarisation-induced [(3)H]ACh release from the rat hippocampus in a concentration-dependent manner with the maximal dose (10(-7)M) of each inducing an increase of 45+/-7.5% (P<0.01) and 95.8+/-19% (P<0.01) above control, respectively. Potentiation of release by both agonists was attenuated by the AT(4) receptor antagonist, divalinal-Ang IV. Angiotensin IV-induced potentiation was not affected by AT(1) and AT(2) receptor antagonists. These results indicate that stimulation of AT(4) receptors can potentiate depolarisation-induced release of ACh from hippocampal slices and suggest that potentiation of cholinergic transmission may be a mechanism by which AT(4) receptor ligands enhance cognition.
Angiotensin IV (Ang IV), the 3-8 fragment of angiotensin II (Ang II), binds to a distinct receptor designated the AT(4) receptor. The peptide elicits a range of vascular and central actions including facilitation of memory retention and retrieval in several learning paradigms. The aim of this study was to characterize the AT(4) receptor in a human cell line of neural origin. Receptor binding studies indicate that the human neuroblastoma cell line SK-N-MC cells express a high-affinity Ang IV binding site with a pharmacological profile similar to the AT(4) receptor: (125)I]-Ang IV and (125)I]-Nle(1)-Ang IV bind specifically to the SK-N-MC cell membranes (K(d) = 0.6 and 0.1 nM) in a saturable manner (B(max) = 1.2 pmol/mg of protein). AT(4) receptor ligands, Nle(1)-Ang IV, Ang IV and LVV-haemorphin 7 (LVV-H7), compete for the binding of [(125)I]-Ang IV or [(125)I]-Nle(1)-Ang IV to the SK-N-MC cell membranes with rank order potencies of Nle(1)-Ang IV > Ang IV > LVV-H7 with IC(50) values of 1.4, 8.7 and 59 nM ([(125)I]-Ang IV) and 1.8, 20 and 168 nM ([(125)I]-Nle(1)-Ang IV), respectively. The binding of [(125)I]-Ang IV or [(125)I]-Nle(1)-Ang IV to SK-N-MC cell membranes was not affected by the presence of GTP gamma S. Both Ang IV and LVV-H7 stimulated DNA synthesis in this cell line up to 72 and 81% above control levels, respectively. The AT(4) receptor in the SK-N-MC cells is a 180-kDa glycoprotein; under non-reducing conditions a 250-kDa band was also observed. In summary, the human neuroblastoma cell line, SK-N-MC, expresses functional AT(4) receptors that are responsive to Ang IV and LVV-H7, as indicated by an increase in DNA synthesis. This is the first human cell line of neural origin shown to express the AT(4) receptor.