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Attenuation of ischemia-induced rat brain injury by 2-(-2-benzofuranyl)-2-imidazoline, a high selectivity ligand for imidazoline I-2 receptors

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Abstract

The aim of this study was to determine whether 2-(2-benzofuranyl)-2-imidazoline, an imidazoline I(2) receptor ligand, could protect against cell death from brain injury and improve the functional outcome after focal cerebral ischemia in rats. Transient focal ischemia was induced by suture occlusion of the middle cerebral artery. Rats were intraperitoneally treated with a vehicle, 2-(2-benzofuranyl)-2-imidazoline or idazoxan immediately after focal ischemia. Infarct volume was assessed by 2,3,5-triphenyltrazolium chloride staining and neurobehavioral deficits were monitored. The volume of cell death in the penumbra after ischemia was determined by immunostaining using anti-cleaved caspase-3 antibody and terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL). Both 2-(2-benzofuranyl)-2-imidazoline and idazoxan significantly improved the neurological score compared with the vehicle at 24 hours after focal ischemia. Treatment with 2-(2-benzofuranyl)-2-imidazoline or idazoxan also significantly reduced infarct volume and the number of both caspase-3- and TUNEL-positive cells in the penumbra compared with vehicle-treated rats (p<0.01 and p<0.05, respectively). The results suggest the neuroprotective role of 2-(2-benzofuranyl)-2-imidazoline and idazoxan in focal cerebral ischemia, and may therefore represent useful targets for developing new treatments for stroke.

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... Accumulating evidence suggests that imidazoline I 2 receptor (I 2 R) ligands have various biological functions, including effective analgesia for inflammatory pain and neuroprotection (Bousquet et al., 1998;Zhu et al., 2015). We have shown in vitro and in vivo that 2-(2-benzofu-ranyl)-2-imidazoline (2-BFI) is the most effective I 2 R ligand for protecting neurons from ischemic damage and that it works by directly modulating NMDAR-mediated Ca 2+ influx into neurons (Han et al., 2009(Han et al., , 2012(Han et al., , 2013Thorn et al., 2016). 2-BFI triggers reversible blockade of Ca 2+ influx with a relatively fast off-rate, similarly to memantine (Han et al., 2013). ...
... Memantine, a noncompetitive NMDAR antagonist, is widely used in the clinic (Vogels et al., 1997;Lipton, 2006). The I 2 R ligand 2-BFI reversibly blocks NMDAR in a way similar to memantine (Jiang et al., 2010;Han et al., 2013), thereby protecting the brain from ischemic insult (Gustafson et al., 1990;Maiese et al., 1992;Han et al., 2009). Previous studies have focused only on the neuroprotective effects of 2-BFI administered immediately after ischemia onset, which is rarely feasible in clinical settings (Gustafson et al., 1990;Maiese et al., 1992;Han et al., 2009). ...
... The I 2 R ligand 2-BFI reversibly blocks NMDAR in a way similar to memantine (Jiang et al., 2010;Han et al., 2013), thereby protecting the brain from ischemic insult (Gustafson et al., 1990;Maiese et al., 1992;Han et al., 2009). Previous studies have focused only on the neuroprotective effects of 2-BFI administered immediately after ischemia onset, which is rarely feasible in clinical settings (Gustafson et al., 1990;Maiese et al., 1992;Han et al., 2009). To successfully translate 2-BFI from the laboratory to the clinic, it is necessary to determine whether the drug is effective when given with a certain delay after stroke onset. ...
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We previously demonstrated that administering 2-(2-benzofuranyl)-2-imidazolin (2-BFI), an imidazoline I2 receptor agonist, immediately after ischemia onset can protect the brain from ischemic insult. However, immediate administration after stroke is difficult to realize in the clinic. Thus, the therapeutic time window of 2-BFI should be determined. Sprague-Dawley rats provided by Wenzhou Medical University in China received right middle cerebral artery occlusion for 120 minutes, and were treated with 2-BFI (3 mg/kg) through the caudal vein at 0, 1, 3, 5, 7, and 9 hours after reperfusion. Neurological function was assessed using the Longa's method. Infarct volume was measured by 2,3,5-triphenyltetrazolium chloride assay. Morphological changes in the cortical penumbra were observed by hematoxylin-eosin staining under transmission electron microscopy . The apoptosis levels in the ipsilateral cortex were examined with terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay. The protein expression of Bcl-2 and BAX was detected using immunohistochemistry. We found the following: Treatment with 2-BFI within 5 hours after reperfusion obviously improved neurological function. Administering 2-BFI within 9 hours after ischemia/reperfusion decreased infarct volume and alleviated apoptosis. 2-BFI administration at different time points after reperfusion alleviated the pathological damage of the ischemic penumbra and reduced the number of apoptotic neurons, but the protective effect was more obvious when administered within 5 hours. Administration of 2-BFI within 5 hours after reperfusion remarkably increased Bcl-2 expression and decreased BAX expression. To conclude, 2-BFI shows potent neuroprotective effects when administered within 5 hours after reperfusion, seemingly by up-regulating Bcl-2 and down-regulating BAX expression. The time window provided clinical potential for ischemic stroke by 2-BFI.
... As such, memantine is approved for treatment of Alzheimer's disease and is currently undergoing numerous clinical trials including vascular dementia and stroke. Our recent studies showed that ligands to the type 2 immidazoline receptor (I 2 R), such as 2-(2-benzofu-ranyl)-2-imidazoline (2- BFI) and Idazoxan, are potently and dose-dependently neuroprotective against transient cerebral ischemia, a rodent model of stroke151617. These compounds readily cross the blood brain barrier as they were used as probes for IR studies [18]. ...
... I 2 R ligands directly bind to NMDA receptors [19] and may block NMDA receptor-gated calcium channels20212223 in a manner similar to that of memantine [23]. Amongst I 2 R ligands, 2-BFI was the most effective in neuroprotection against glutamate toxicity in vitro [23] and cerebral ischemia in vivo151617. However, the molecular mechanisms of 2-BFI-mediated neuroprotection against excitotoxicity remain unclear. ...
... These studies therefore provided a potential molecular mechanism for 2-BFI neuroprotection against glutamate toxicity in vitro (Fig 9) and cerebral ischemia in vivo as we have previously demonstrated151617. The inability of 2-BFI to affect the membrane potential of neurons (Fig. 1), fast kinetics and reversibility of NMDA current inhibition (Fig. 3), voltage independence of the inhibitory action (Fig. 4), independence on agonist concentration (Fig. 5), and receptor selectivity at least in respect to AMPAR (Fig. 7), all supported the idea that 2-BFI is a fast non-competitive reversible inhibitor of NMDARs. ...
Article
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Excessive activation of the N-methyl-D-aspartic acid (NMDA) type glutamate receptors (NMDARs) causes excitotoxicity, a process important in stroke-induced neuronal death. Drugs that inhibit NMDA receptor-mediated [Ca(2+)]i influx are potential leads for development to treat excitotoxicity-induced brain damage. Our previous studies showed that 2-(2-benzofu-ranyl)-2-imidazoline (2-BFI), an immidazoline receptor ligand, dose-dependently protects rodent brains from cerebral ischemia injury. However, the molecular mechanisms remain unclear. In this study, we found that 2-BFI transiently and reversibly inhibits NMDA, but not AMPA currents, in a dose-dependent manner in cultured rat cortical neurons. The mechanism of 2-BFI inhibition of NMDAR is through a noncompetitive fashion with a faster on (Kon = 2.19±0.33×10(-9) M(-1) sec(-1)) and off rate (Koff = 0.67±0.02 sec(-1)) than those of memantine, a gold standard for therapeutic inhibition NMDAR-induced excitotoxicity. 2-BFI also transiently and reversibly blocked NMDA receptor-mediated calcium entry to cultured neurons and provided long-term neuroprotection against NMDA toxicity in vitro. Collectively, these studies demonstrated a potential mechanism of 2-BFI-mediated neuroprotection and indicated that 2-BFI is an excellent candidate for repositioning as a drug for stroke treatment.
... Several IR agonists such as agmatine have been shown to be neuroprotective through their blocking effects on N-methyl-D-aspartate (NMDA) receptors and hypoxia [71,72]. Further, imidazoline I 2 receptor agonists reduce the number of apoptotic events in the penumbra after the ischemic attack in rodents [73]. Moreover, a recent preclinical study [74] has shown that idazoxan, an imidazoline I 2 receptor agonist, could increase the integrity of the BBB by increasing the expression of tight junction (Tj) proteins in vascular endothelial cells and by down-regulating matrix metalloproteinase-9 (MMP-9) or MMP-9/tissue inhibitors of metalloproteinases-1 (TIMP-1) ratio. ...
... Evidence shows that imidazoline I 2 receptor agonists inhibit the monoamine oxidase (MAO) action, which has a crucial role in oxidative stress [75]. Following MAO inhibition, the oxidative stress of the brain significantly decreases and results in neuroprotection [73,75,76]. Chai et al. [77] have reported that agmatine attenuates oxidative stress in lipopolysaccharide-activated macrophages through activating the PI3K/Akt signaling pathway, which I3 [35,44,45] subsequently activates the nuclear factor (erythroid 2derived)-like 2 (Nrf2) pathway and heme oxygenase-1 (HO-1) expression. ...
Article
Intracerebral hemorrhage (ICH), which accounts for 10-15% of all strokes, leads to higher morbidity and mortality compared with other stroke subtypes. Hypertension has been recognized as a major risk factor for ICH. Current antihypertensive options have not been fully effective for either prevention of ICH or ameliorating its complications. Therefore, attempts should be made to use novel antihypertensive medications for more effective management of blood pressure (BP) in the acute phase of ICH. Imidazoline receptors (IR) agonists can potentially be effective agents for BP control with the adjunctive ability to attenuate post-ICH brain injury. IR agonists render neuroprotective effects including inhibition of inflammatory reactions, apoptotic cell death, excitotoxicity, and brain edema. Given these properties, the present review aims to focus on the application of IR agonists for managing BP in ICH patients.
... In the early phase of EAE, it was noticed that activated astrocytes lose the perivascular end-feet, resulting in increased BBB permeability [34]. The appropriate dose of 2-BFI exhibits protective properties against astrocyte damage in an ischemic stroke [35,36]. It was reported that 2-BFI can enhance the intercellular tight junction via the downregulation of MMP-9 expression levels, and suppress vascular endothelial permeability to preserve cerebral microvascular endothelial integrity [37,38]. ...
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Background: Imidazoline compounds are well accepted to exhibit various pharmacological effects including antidepressant, anti-inflammatory, analgesic, anti-morphine tolerance and inhibit the activity of monoamine oxidase. 2-(-2-benzofuranyl)-2-imidazoline (2-BFI), a selective imidazoline 2 receptor (I2R) ligand, has been proven to exhibit therapeutic effects for various neuroimmunological diseases. However, the mechanism behind its neuroprotective properties remains elusive. Methods: In this study, we used 2-BFI for the treatment of mice with experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein (MOG33-55). The clinical signs of neurological deficits were evaluated daily. The demyelination and inflammatory infiltration in the CNS of mice with EAE were examined by Luxol Fast Blue (LFB) staining and hematoxylin-eosin (H&E) staining. Flow cytometry was utilized to examine the ratios of lymphocyte subsets in the periphery and CNS of mice with EAE. We also used Reverse Transcription–Polymerase Chain Reaction (RT-PCR) to observe the changes of expression of inflammatory cytokines by 2-BFI intervention. Results: We found that 2-BFI significantly reduced the incidence of EAE and attenuated the severity of neurological disability. Pathological staining showed that the infiltration of inflammatory cells and demyelination in the central nervous system (CNS) of the mice were markedly alleviated via 2-BFI intervention. To explore the mechanism of action of 2-BFI, we used flow cytometry to determine immunophenotypes in the spleen and CNS of the mice. We discovered that 2-BFI significantly decreased the ratio of CD28+ lymphocytes and B cells in the spleen of EAE mice. In the CNS, the expression of CD4+ T cells was downregulated by 2-BFI, while B cells and CD39+ lymphocytes were dramatically increased. RT-PCR also demonstrated that the level of IFN-γ mRNA secreted by CD4+T cells was lower than that in the CNS of EAE mice, while the levels of TGF- β and IL-10mRNA secreted by Treg and B cells were increased with 2-BFI intervention. Conclusion: 2-BFI could ameliorate EAE-induced neurobehavioral deficits and reduce the infiltration of inflammatory cells via regulating the activation and migration of lymphocyte subsets. This study provides a new explanation for the protective mechanism of 2-BFI in neuroimmune diseases.
... NMDARs are the most concerned neuroprotectant in recent years. Our current study extends our previous work showing that 2-BFI protects against brain injury by rapidly and reversibly blocking NMDAR-mediated calcium influx into neurons (Han et al., 2009;Han et al., 2010). 2-BFI acts similarly to memantine, another NMDAR antagonist which is approved for clinical use (Han et al., 2013). ...
Article
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Recombinant tissue plasminogen activator (rt-PA) is used to treat acute ischemic stroke but is only effective if administered within 4.5 h after stroke onset. Delayed rt-PA treatment causes blood-brain barrier (BBB) disruption and hemorrhagic transformation. The compound 2-(-2-benzofuranyl)-2-imidazoline (2-BFI), a newly discovered antagonist of high-affinity postsynaptic N-methyl-D-aspartate (NMDA) receptors, has been shown to have neuroprotective effects in ischemia. Here, we investigated whether combining 2-BFI and rt-PA can ameliorate BBB disruption and prolong the therapeutic window in a rat model of embolic middle cerebral artery occlusion (eMCAO). Ischemia was induced in male Sprague Dawley rats by eMCAO, after which they were treated with 2-BFI (3 mg/kg) at 0.5 h in combination with rt-PA (10 mg/kg) at 6 or 8 h. Control rats were treated with saline or 2-BFI or rt-PA. Combined therapy with 2-BFI and rt-PA (6 h) reduced the infarct volume, denatured cell index, BBB permeability, and brain edema. This was associated with increased expression of aquaporin 4 (AQP4) and tight junction proteins (occludin and ZO-1) and downregulation of intercellular adhesion molecule 1 (ICAM-1) and matrix metalloproteinases 2 and 9 (MMP2 and MMP9). We conclude that 2-BFI protects the BBB from damage caused by delayed rt-PA treatment in ischemia. 2-BFI may therefore extend the therapeutic window up to 6 h after stroke onset in rats and may be a promising therapeutic strategy for humans. However, mechanisms to explain the effects oberved in the present study are not yet elucidated.
... At least two different subtypes called imidazoline I 1 and I 2 receptors were identified (Li, 2017). Accumulating studies have shown that 2-(2benzofuranyl)-2-imidazoline (2-BFI), a selective ligand to type 2 IRs, has prominent neuroprotective effects in animal models of many disorders, such as traumatic brain injury, stroke, cerebral ischemia, and Alzheimer's Disease, even in some nociception (Han et al., 2009Jiang et al., 2010;Sampson et al., 2012;Tian et al., 2017;Ni et al., 2019). Moreover, a recent study found that 2-BFI effectively protected against SCI caused by experimental autoimmune encephalomyelitis, a mouse model of multiple sclerosis (Wang et al., 2011). ...
Article
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Previous reports showed that 2-(-2-benzofuranyl)-2-imidazoline (2-BFI) has antioxidant, anti-inflammatory and anti-apoptotic effects on neuroprotection in numerous disorders. However, the precise mechanisms remain elusive. The nuclear factor c factor 2 (Nrf2)/antioxidant response element (ARE) signaling pathway plays an important and essential role in the antioxidant and anti-inflammatory responses of the cell. Therefore, the purpose of this study was to investigate the potential neuroprotective effects of 2-BFI in a rat model of spinal cord injury (SCI) and to determine whether its neuroprotective effects are associated with the activation of Nrf2. To test this hypothesis, we examined the potential roles of 2-BFI in SCI models which were established in rats using a clip-compression injury method. Our results showed that treatment with 2-BFI twice daily improved locomotion recovery from SCI, which increased Nrf2 expression in both neurons and astrocytes, meanwhile, the level of heme oxygenase-1 (HO-1) also significantly enhanced. In addition, after the treatment with 2-BFI increased levels of superoxidase dismutase (SOD) and glutathione peroxidase (GPx) indicated the antioxidant effect of the drug. Furthermore, the upregulation of Bcl-2 and downregulation of Bax and caspase-3 implied antiapoptotic effects on neuroprotection of 2-BFI, which were verified by the Fluoro-Jade B (FJB) staining and TUNEL staining. Collectively, these results add to a growing body of evidence supporting that 2-BFI may attenuate SCI mediated by activation of the Nrf2/HO-1 signaling pathway.
... Recent studies showed more consistent results using a selective I 2 receptor ligand 2-BFI. 2-BFI was found to exert significant neuroprotection in a rat model of cerebral ischemia (middle cerebral artery occlusion model) (Han et al., 2009(Han et al., , 2012. Several cellular and molecular mechanisms appear to be involved in 2-BFI-induced neuroprotection. ...
Article
Imidazoline receptors historically referred to a family of nonadrenergic binding sites that recognize compounds with an imidazoline moiety, although this has proven to be an oversimplification. For example, none of the proposed endogenous ligands for imidazoline receptors contain an imidazoline moiety but they are diverse in their chemical structure. Three receptor subtypes (I1, I2, and I3) have been proposed and the understanding of each has seen differing progress over the decades. I1 receptors partially mediate the central hypotensive effects of clonidine-like drugs. Moxonidine and rilmenidine have better therapeutic profiles (fewer side effects) than clonidine as antihypertensive drugs, thought to be due to their higher I1/α2-adrenoceptor selectivity. Newer I1 receptor agonists such as LNP599 [3-chloro-2-methyl-phenyl)-(4-methyl-4,5-dihydro-3H-pyrrol-2-yl)-amine hydrochloride] have little to no activity on α2-adrenoceptors and demonstrate promising therapeutic potential for hypertension and metabolic syndrome. I2 receptors associate with several distinct proteins, but the identities of these proteins remain elusive. I2 receptor agonists have demonstrated various centrally mediated effects including antinociception and neuroprotection. A new I2 receptor agonist, CR4056 [2-phenyl-6-(1H-imidazol-1yl) quinazoline], demonstrated clear analgesic activity in a recently completed phase II clinical trial and holds great promise as a novel I2 receptor-based first-in-class nonopioid analgesic. The understanding of I3 receptors is relatively limited. Existing data suggest that I3 receptors may represent a binding site at the Kir6.2-subtype ATP-sensitive potassium channels in pancreatic β-cells and may be involved in insulin secretion. Despite the elusive nature of their molecular identities, recent progress on drug discovery targeting imidazoline receptors (I1 and I2) demonstrates the exciting potential of these compounds to elicit neuroprotection and to treat various disorders such as hypertension, metabolic syndrome, and chronic pain.
... The I-1 receptors regulate the sympathetic inhibitory functions of the sympathetic nervous system and regulate the systolic and diastolic blood pressure by reducing the peripheral resistance, and have been well-studied. The functions of the I-2 receptors are yet to be determined, but are suggested to be an important therapeutic target for pain and stroke [61,62]. The I-3 receptors induce insulin secretion from pancreatic β cells. ...
Article
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The central nervous system (CNS) is the most injury-prone part of the mammalian body. Any acute or chronic, central or peripheral neurological disorder is related to abnormal biochemical and electrical signals in the brain cells. As a result, ion channels and receptors that are abundant in the nervous system and control the electrical and biochemical environment of the CNS play a vital role in neurological disease. The N-methyl-d-aspartate receptor, 2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl) propanoic acid receptor, kainate receptor, acetylcholine receptor, serotonin receptor, α2-adrenoreceptor, and acid-sensing ion channels are among the major channels and receptors known to be key components of pathophysiological events in the CNS. The primary amine agmatine, a neuromodulator synthesized in the brain by decarboxylation of l-arginine, can regulate ion channel cascades and receptors that are related to the major CNS disorders. In our previous studies, we established that agmatine was related to the regulation of cell differentiation, nitric oxide synthesis, and murine brain endothelial cell migration, relief of chronic pain, cerebral edema, and apoptotic cell death in experimental CNS disorders. In this review, we will focus on the pathophysiological aspects of the neurological disorders regulated by these ion channels and receptors, and their interaction with agmatine in CNS injury.
... Idazoxan reduced neuron damage in the hippocampus after global ischemia in the rat brain [23] and agmatine, identified as the endogenous I 2 -IR ligand [24], has demonstrated modulatory actions in several neurotransmitters that produce neuroprotection both in vitro and in rodent models [25]. The compelling evidence has demonstrated that other selective I 2 -IR ligands ( Fig. 1) provide benefits such as being neuroprotective against cerebral ischemia in vivo [26,27], inducing beneficial effects in several models of chronic opioid therapy, leading to neuroprotection by direct blocking of N-methyl-D-aspartate receptor (NMDA) mediated intracellular [Ca 2+ ] influx [28], or provoking morphological/ biochemical changes in astroglia that are neuroprotective after neonatal axotomy [22]. ...
Article
As populations increase their life expectancy, age-related neurodegenerative disorders such as Alzheimer’s disease have become more common. I2-Imidazoline receptors (I2-IR) are widely distributed in the central nervous system, and dysregulation of I2-IR in patients with neurodegenerative diseases has been reported, suggesting their implication in cognitive impairment. This evidence indicates that high-affinity selective I2-IR ligands potentially contribute to the delay of neurodegeneration. In vivo studies in the female senescence accelerated mouse-prone 8 mice have shown that treatment with I2-IR ligands, MCR5 and MCR9, produce beneficial effects in behavior and cognition. Changes in molecular pathways implicated in oxidative stress, inflammation, synaptic plasticity, and apoptotic cell death were also studied. Furthermore, treatments with these I2-IR ligands diminished the amyloid precursor protein processing pathway and increased Aβ degrading enzymes in the hippocampus of SAMP8 mice. These results collectively demonstrate the neuroprotective role of these new I2-IR ligands in a mouse model of brain aging through specific pathways and suggest their potential as therapeutic agents in brain disorders and age-related neurodegenerative diseases.
... They induced ischemia for 2 hours brain damage have been elucidated in previous studies and demonstrate that neuronal damage occurring after ischemia and reperfusion might activate apoptosis via oxidative stress or mitochondrial impairment (12,14,17,20). Thus, antioxidant and anti-apoptotic agents are a focal point for the development of neuroprotective drugs in the treatment of stroke (9). ...
Article
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Aim: To investigate the neuroprotective effect of chronic curcumin supplementation on the rat forebrain prior to ischemia and reperfusion Material and Methods: Forebrain ischemia was induced by bilateral common carotid artery occlusion for 1 hour, followed by reperfusion for 72 hours. Older rats were divided into five groups: I, 300 mg/kg oral curcumin for 21 days before ischemia and 300 mg/kg intraperitoneal curcumin after ischemia; II, 300 mg/kg intraperitoneal curcumin after ischemia; III, 300 mg/kg oral curcumin for 21 days before ischemia; IV, only ischemia; V:, sham-operated. The forebrain was rapidly dissected for biochemical parameter assessment and histopathology. Results: In forebrain tissue, enzyme activities of superoxide dismutase, glutathione peroxidase, and catalase were significantly higher in Group I than Groups II or III (p 0.05 while xanthine dehydrogenase and malondialdehyde enzyme activities and concentrations of interleukin-6 and TNF-alpha were significantly lower in Group I compared to Groups II and III (p 0.05). A significant reduction in neurological score was observed after 24 and 72 h in the curcumin-treated groups compared with the ischemic group. We also found a marked reduction in apoptotic index after 72 h in the groups receiving curcumin. Significantly more TUNEL-positive cells were observed in the ischemic group compared to those treated with curcumin. Conclusion: We demonstrated the neuroprotective effect of chronic curcumin supplement on biochemical parameters, neurologic scores and apoptosis following ischemia and reperfusion.
... First, the α 2 adrenergic receptor may still play a part in the neuroprotective effects of dexmedetomidine, although the function and levels of the receptor were possibly altered. Second, in vitro and in vivo studies indicated that dexmedetomidine can exhibit properties against oxygen and glucose deprivation-induced (OGD) injury or I/R injury, independent of the α 2 adrenergic receptor, possibly through imidazoline receptors [52,53]. Furthermore, some studies suggested that the properties of dexmedetomidine against OGD injury are related to the imidazoline I 1 receptors-extracellular-regulated kinases pathways, and this process is likely independent of the α 2 adrenergic receptor response [52,54]. ...
Article
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Background: Transient global cerebral ischemia/reperfusion (I/R) is a major perioperative complication, and diabetes increases the response of oxidative stress and inflammation induced by I/R. The objective of this study was to determine the protective effect of dexmedetomidine against transient global cerebral ischemia/reperfusion induced oxidative stress and inflammation in diabetic rats. Methods: Sixty-four rats were assigned into four experimental groups: normoglycemia, normoglycemia + dexmedetomidine, hyperglycemia, and hyperglycemia + dexmedetomidine and all subsequent neurological examinations were evaluated by a blinded observer. Damage to the brain was histologically assessed using the TUNEL staining method while western blotting was used to investigate changes in the expression levels of apoptosis-related proteins as well as the microglia marker, ionized calcium-binding adapter molecule 1 (Iba1). Water content in the brain was also analyzed. In addition, hippocampal concentrations of malondialdehyde (MDA) and Nox2 (a member of the Nox family of NADPH oxidases), and the activity of superoxide dismutase and catalase were analyzed. Finally, changes in serum concentrations of tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 were detected. Results: Results showed that diabetes increased brain water content, the number of apoptotic neurons, early neurological deficit scores, oxidative stress (MDA and Nox2) and inflammation (pro-inflammatory cytokines including TNF-α and IL-6) levels following transient global I/R injury, but that these symptoms were attenuated following administration of dexmedetomidine. Conclusions: These findings suggest that dexmedetomidine can significantly alleviate damage resulting from I/R, and this mechanism may be related to a reduction in both oxidative stress and inflammation which is normally associated with I/R.
... Reinforcing the neuroprotective actions of I 2 -imidazoline drugs in the brain, the acute and/or chronic treatments with LSL 61122, 2-BFI and BU-224 were not associated with aberrant activation of proapoptotic JNK1/2 (Dhanasekaran and Reddy, 2008) or increased proteolytic cleavage of nuclear PARP-1, a hallmark of apoptosis, which is mainly mediated by caspase-3 (Kumar, 2007;Chaitanya et al., 2010) (Figure 6). In fact, 2-BFI treatment was reported to reduce the number of caspase-3 positive cells in ischemiainduced rat brain injury (Han et al., 2009). ...
Article
Selective I(2)-imidazoline receptor ligands induce neuroprotection through various molecular mechanisms including blockade of N-methyl-D-aspartate (NMDA) receptors. To investigate new neuroprotective mechanisms associated with I(2)-imidazoline receptors, the effects of selective (2-styryl-2-imidazoline (LSL 61122), 2-(2-benzofuranyl)-2-imidazoline (2-BFI), 2-(4,5-dihydroimidazol-2-yl) quinoline hydrochloride (BU-224)) and non-selective (idazoxan) I(2)-drugs on canonical apoptotic pathways were assessed in rat brain cortex. The acute treatment with LSL 61122 (10 mg/kg) reduced the content of mitochondrial (pro-apoptotic) Bax (-33%) and cytochrome c (-31%), which was prevented by idazoxan, an I(2)-receptor antagonist. The sustained stimulation of I(2)-imidazoline receptors with selective drugs (10 mg/kg, every 12 h for seven days) was associated with down-regulation of key components of the extrinsic (Fas receptor: -20%; Fas associated protein with death domain (FADD) adaptor: -47-54%) and/or intrinsic (Bax: -20-23%; cytochrome c: -22-28%) apoptotic signalling and/or up-regulation of survival anti-apoptotic factors (p-Ser194 FADD/FADD ratio: +1.6-2.5-fold; and/or Bcl-2/Bax ratio: +1.5-fold), which in the long-term could dampen cell death in the brain. Similar chronic treatments with LSL 60101 (the imidazole analogue of 2-BFI) and idazoxan (a mixed I(2)/α(2)-ligand) did not induce significant alterations of pro- or anti-apoptotic proteins. The disclosed anti-apoptotic mechanisms of selective I(2)-imidazoline drugs may work in concert with other molecular mechanisms of neuroprotection (e.g. blockade of NMDA receptors) that are engaged by I(2)-ligands.
... Oxidative stress leads to both apoptotic and non-apop-totic pathways. Apoptosis can occur during DM [1,2,99,100], anesthetic exposure [101], tissue ischemia [102][103][104][105], bone fatigue [106], neurodegenerative disorders [17,[107][108][109] and Alzheimer's disease [30][31][32][33]38,[110][111][112][113][114][115], plasticity associated with ischemic preconditioning [116], aging-related diseases [18,117,118], and toxic conditions during development [101,119]. During apoptosis, the cleavage of genomic DNA into fragments [108,120,121] usually occurs after the exposure of membrane phosphatidylserine (PS) residues [122][123][124][125][126][127]. ...
Article
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Diabetes mellitus (DM) impacts a significant portion of the world's population and care for this disorder places an economic burden on the gross domestic product for any particular country. Furthermore, both Type 1 and Type 2 DM are becoming increasingly prevalent and there is increased incidence of impaired glucose tolerance in the young. The complications of DM are protean and can involve multiple systems throughout the body that are susceptible to the detrimental effects of oxidative stress and apoptotic cell injury. For these reasons, innovative strategies are necessary for the implementation of new treatments for DM that are generated through the further understanding of cellular pathways that govern the pathological consequences of DM. In particular, both the precursor for the coenzyme beta-nicotinamide adenine dinucleotide (NAD(+)), nicotinamide, and the growth factor erythropoietin offer novel platforms for drug discovery that involve cellular metabolic homeostasis and inflammatory cell control. Interestingly, these agents and their tightly associated pathways that consist of cell cycle regulation, protein kinase B, forkhead transcription factors, and Wnt signaling also function in a broader sense as biomarkers for disease onset and progression.
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A labeling technique was developed for the imidazoline I 2 receptor ligand 2‐(3‐fluoro‐tolyl)‐4, 5‐dihydro‐1H‐imidazole (FTIMD) using Pd(0)‐mediated ¹¹ C‐carbomethoxylation with [ ¹¹ C]CO, followed by imidazoline ring formation with ethylenediamine‐trimethylaluminium (EDA‐AlMe 3 ). To achieve this, [ ¹¹ C]CO was passed through a methanol (MeOH) solution containing 3‐fluoro‐4‐methylphenylboronic acid ( 1 ), palladium (II) acetate (Pd [OAc] 2 ), triphenylphosphine (PPh 3 ), and p ‐benzoquinone (PBQ). The mixture was then heated at 65°C for 5 min. EDA was introduced into the reaction mixture, and MeOH was completely evaporated at temperatures exceeding 100°C. The dried reaction mixture was combined with an EDA‐AlMe (1:1) toluene solution and heated at 145°C for 10 min. Portions of the reaction mixture were analyzed through high‐performance liquid chromatography, resulting in [ ¹¹ C]FTIMD with 26% ( n = 2) decay‐corrected radiochemical yield (RCY). This method could be utilized for various arylborons to produce [2‐ ¹¹ C]imidazolines 4a – h with RCYs ranging from low to moderate. Notably, [2‐ ¹¹ C]benazoline was obtained with a moderate RCY of 65%. The proposed technique serves as an alternative to the Grignard method, which uses [ ¹¹ C]CO to generate a [2‐ ¹¹ C]‐labeled imidazoline ring.
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Increased microglial NADPH oxidase (NOX2) production may make an important contribution to the increased incidence and severity of ischemic stroke associated with diabetes. Imidazoline receptors are closely associated with neuroprotection, but the neuroprotective effects of the selective I2-imidazoline receptor ligand 2-(2-benzofuranyl)-2-imidazoline (2BFI) in diabetes has not been established. The effect of 2BFI on microglial NOX2 production was investigated using a co-culture of neurons and microglia, and the effect on cerebral ischemia-reperfusion (IR) injury was determined in diabetic rats. Garcia neurological scores, brain infarct volumes, brain water content, TUNEL staining, blood-brain barrier, and immunofluorescent labeling for microglia were evaluated. Western blots were used to determine gp91phox and Tyr1472 expression. Anti-inflammatory cytokine (IL-10) and inflammatory cytokine secretion was determined using ELISA kits. The brain infarct volumes, TUNEL-positive neurons, expression of microglia, brain water content, blood-brain barrier structure damage, and gp91phox and Tyr1472 expression were increased, the Garcia neurological scores were significantly decreased in the IR group, and 2BFI relieved these alterations. The IL-10 concentration was increased in the IR group; 2BFI significantly improved this increase. The neuron apoptosis and necrosis rates, and production of reactive oxygen species (ROS) and inflammatory cytokines, including IL-6, IL-8, TNF-α, and 8-iso-PGF2α, were significantly increased by high glucose stimulation combined with oxygen-glucose deprivation treatment, which were inhibited by 2BFI. The 2BFI ameliorated cerebral ischemia-reperfusion injury in diabetes and decreased neuron death in an in vitro model. The mechanism underlying these findings may be related to the decreased production of inflammatory factors and reactive oxygen species from microglia.
Article
Recent findings unveil the pharmacological modulation of imidazoline I2 receptors (I2-IR) as a novel strategy to face unmet medical neurodegenerative diseases. In this work, we report the chemical characterization, three-dimensional quantitative structure-activity relationship (3D-QSAR) and ADMET in silico of a family of benzofuranyl-2-imidazoles that exhibit affinity against human brain I2-IR and most of them have been predicted to be brain permeable. Acute treatment in mice with 2-(2-benzofuranyl)-2-imidazole, known as LSL60101 (garsevil), embodying in vitro ADMET non-warning properties and an optimal pharmacokinetic profile, decreased the body temperature and the pro-apoptotic FADD protein in hippocampus. In vivo studies in the familial Alzheimer’s disease 5xFAD murine model with the representative compound, revealed significant decreases in the protein expression levels of antioxidant enzymes superoxide dismutase and glutathione peroxidase in hippocampus. Overall, LSL60101 plays a neuroprotective role by reducing apoptosis and oxidative stress.
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Stroke is the third leading cause of death and disability in developing countries. The effective therapy for acute ischemic stroke is thrombolysis with recombinant tissue plasminogen activator (rt-PA) within 4.5 h of stroke onset. An effective post-ischemic neuroprotectant would extend the advantages of rt-PA, and protect against complications of thrombolysis. We previously reported that 2-(2-benzofuranyl)-2-imidazoline (2-BFI), a newly discovered ligand for high-affinity type 2 imidazoline receptor (I2R), provides neuroprotection against ischemic stroke in rats. Here we investigated the protective effects of 2-BFI in combination with delayed intravenous rt-PA after stroke induced by embolic middle cerebral artery occlusion (eMCAO) in rats. Infarct size was determined using 2, 3, 5-triphenyltrazolium chloride staining, while neurological deficit was assessed based on neurological score. Numbers of apoptotic cells in vivo were estimated using TUNEL stain, and expression of the pro-apoptotic protein BAX and anti-apoptotic protein BCL-2 were quantified by Western blotting. The results showed that 2-BFI (3 mg/kg) administered at 0.5 h after embolic MCAO combined with rt-PA (10 mg/kg) administered at 6 h reduced brain infarct size, mitigated neurological deficit, decreased the number of TUNEL-positive cells, down-regulated BAX expression, and up-regulated BCL-2 expression. These findings suggest that 2-BFI may extend the therapeutic window of rt-PA to 6 h after embolic stroke onset in rats.
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Astrocytes perform a variety of functions that are important for normal neuronal activity and recovery after brain injury. Because astrocytes are very vulnerable to H2O2, protection of astrocytes from oxidative damage in various neurological diseases is important in maintaining brain function and preventing brain damage. In this study, we investigated the characteristics and mechanisms of a specific imidazoline I2 receptor agonist 2-BFI-mediated cytoprotection using a rat astrocyte cultures of H2O2-exposed oxidative stress. Here we show that 2-BFI in H2O2-exposed astrocytes protects cell death through increased lysosomal membrane stability, LC3-II conversion, and subsequently suppresses accumulation of p62. These effects of 2-BFI were significantly reversed after treatment with the lysozyme activity inhibitor Bafilomycin A1. These results suggest that the cytoprotective effects of 2-BFI, which increases lysosomal stability in oxidative stress, may involve regulation of lysosomal-associated membrane protein-dependent autophagy and autolysosome degradation in astrocytes.
Article
Background: We showed previously that 2-(2-benzofuranyl)-2-imidazoline (2-BFI), a ligand to type 2 imidazoline receptor (I2R) exerts neuroprotective effects in ischemia stroke via an unknown mechanism. The present study was to investigate whether 2-BFI can protect the neurovascular unit (NVU) using a rat model of 90 min focal cerebral ischemia. Methods: Rats were randomly divided into three groups: thesham-operated group; the vehicle control group and the 2-BFI group which received 2-BFI (3 mg/kg) immediately after the start of middle cerebralartery occlusion (MCAO). Neurological deficit score, infarct size, apoptosis level, brain water content and Evans Blue extravasation were assessed at 24 h after stroke. Expressions of occludin and zonula occludens 1 (ZO-1), collagen IV, aquaporin-4 (AQP-4), matrix metalloproteinase-9 (MMP-9) and MMP-2 were assessed by Western blotting. Results: 2-BFI treatment was associated with significant improvement of neurological performance and decreased infarct volume at 24 h after stroke. Apoptosis level reduced significantly by 2-BFI compared to the vehicle group (34.3 ± 5.4% vs 56.1 ± 7.9%, p < 0.05). Significant decreased of brain water content (79.5 ± 2.6% vs 84.62 ± 2%, p < 0.05) and Evans Blue extravasation (1.2 ± 0.5 vs 2.5 ± 0.41 µg/g, p < 0.05) of ipsilateral hemisphere was observed in 2-BFI group compared to vehicle group. Expressions of occludin, ZO-1 and collagen IV were significantly higher while MMP-9 level significantly lower in 2-BFI group. AQP-4 and MMP-2 showed no difference between 2-BFI and the vehicle groups. Conclusions: These results suggest that the neuroprotective effects of 2-BFI in acute ischemic brain damage are at least partly due to the drug's ability to improve the functions of NVU.
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Astrocytes play a pivotal role in neuronal survival in the setting of post-ischemic brain inflammation, but the astrocyte-derived mediators of ischemic brain injury remain to be defined. 2-(2-Benzofu-ranyl)-2-imidazoline (2-BFI) is a newly discovered ligand for high-affinity imidazoline I2 receptors (I2Rs) mainly located on the mitochondrial outer membrane in astrocytes. We previously reported that in a rat model of cerebral ischemia-reperfusion injury, 2-BFI limits infarct volume, reduces neurological impairment scores, and inhibits neuronal apoptosis in the ischemic penumbra. This study was performed to clarify the underlying mechanism in an astrocyte oxygen-glucose deprivation (OGD) model. The results show that 2-BFI reduces lipid peroxidation and inhibits mitochondria apoptotic pathways.
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Since first introduced more than two decades ago, the research in imidazoline I2 receptors has been steadily increasing. This review provides an update on the current status of I2 receptor pharmacology. Imidazoline I2 receptors or I2 binding sites refer to several (at least four) different proteins that bind to [³H]-idazoxan and [³H]-2-BFI with high affinity. The molecular identities of the proteins remain elusive. One of the proteins (45 kD) seems to be consistent with the identity of brain creatine kinase. The biological functions of I2 receptors have been primarily unveiled by the studies of selective I2 receptor ligands. Accumulating evidence suggests that I2 receptor ligands are effective analgesics for persistent and chronic painful conditions such as inflammatory, neuropathic and postoperative pain. One selective I2 receptor ligand, CR4056, has been advanced to phase II clinical trial with the therapeutic indication of chronic inflammatory pain (osteoarthritis). The expansion to the treatment of other chronic pain conditions should be expected if CR4056 could eventually be approved as a new drug. I2 receptor ligands also demonstrate robust discriminative stimulus activity and induce a characteristic discriminative cue in animals. Biochemical and preclinical in vivo investigations also suggest that I2 receptor ligands have neuroprotective activity and modulate body temperature. The emerging discrepancies of a range of purported selective I2 receptor ligands suggest different pharmacological effects mediated by discrete I2 receptor components which likely attribute to the I2 receptor-related proteins. It is proposed that the I2 receptors represent an emerging drug target for the treatment of neurological disorders such as pain and stroke, and deserve more research attention to translate preclinical findings to pharmacotherapies.
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Objectives: To investigate whether curcumin regulates Notch signaling to cause neuroprotection and neurogenesis after focal ischemia reperfusion injury. Method: Focal ischemia reperfusion injury was modeled in rats by occluding the middle cerebral artery. These animals were given either curcumin (300 mg/kg) or corn oil (vehicle) by intraperitoneal injection starting 1 h after stroke and continuing for 7 d. In parallel, sham-operated control animals received vehicle. All animals were killed on day 12. The different treatment groups were compared in terms of neurobehavioral deficits, BrdU incorporation, and levels of doublecortin (DCX) and Notch intracellular domain (NICD) using immunohistochemistry, immunofluorescence and Western blotting. Results: Animals treated with curcumin showed significantly smaller neurobehavioral deficits than vehicle-treated animals after 3, 7, and 12 d of reperfusion (all p < 0.05). Tissue sections from curcumin-treated animals contained significantly greater numbers of BrdU-positive cells (p < 0.05) and BrdU/DCX-positive cells (p < 0.01), as well as significantly higher NICD levels (p < 0.01). Conclusion: Curcumin may protect from focal cerebral ischemia reperfusion injury as well as stimulate neurogenesis by activating the Notch signaling pathway.
Article
Objective: To investigate the effect of 2-(2-benzofuranyl)-2-imidazoline (2-BFI) at various doses on ischemic brain injury and Bcl-2 expression in rats. Methods: Focal cerebral ischemia was induced by middle cerebral artery occlusion in rats. Rats were treated with 2-BFI (1.5, 3, 6 mg·kg -1) or vehicle. Neurological deficits, infarct volume, number of apoptotic cells, Bcl-2 expression and neuronal ultrastructures were examined. Results: 2-BFI 3 mg·kg -1 significantly improved neurological deficits, reduced infarct volume and the number of apoptotic cells, and activated Bcl-2 expression in comparison with vehicle and 2-BFI at 1.5, 6 mg·kg -1. Conclusion: 2-BFI at 3 mg·kg -1 has a beneficial neuroprotective effect, and can increase Bcl-2 expression.
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Stroke is caused by vascular dysfunction and currently there are no effective therapeutics to stroke induced brain damage. In contrast to an intense emphasis on neuroprotection, relatively few studies have addressed means of vascular protection in cerebral ischemia. Here we discovered that the ligand to immidazolin receptor, 2-BFI, not only provided potent neuroprotection during middle cerebral artery occlusion in rat, which confirmed our previous reports, but also protected the integrity of the cerebral vasculature. Treatment with 2-BFI twice daily after the occlusion of the middle cerebral artery for 14 d significantly improved the neurological deficits, reduced brain infarction, and importantly, protected the cerebral vasculature as evidenced by the increased expression of an endothelial marker, von Willebrand factor, and better preservation of the cerebral vasculature, as viewed under a confocal microscope on rat brain perfused with FITC-labeled dextran. These results indicated that 2-BFI contributes to protection of neurovasculature. Understanding the molecular mechanisms could eventually lead to development of more effective therapies for stroke.
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Stroke is the third leading cause of death and disability in North America and is becoming the most frequent cause of death in the rapid developing China. Protecting neurons in order to minimize brain damage represents an effective approach towards stroke therapeutics. Our recent study demonstrated that 2-(-2-benzofuranyl)-2-imidazoline (2-BFI), a ligand for imidazoline I(2) receptors, is potently neuroprotective against stroke, possibly through transiently antagonizing NMDA receptor activities. In this study, we further investigated the characteristics and mechanisms of 2-BFI-mediated neuroprotection using a rat stroke model of transient occlusion of the middle cerebral artery. Here, we show that 2-BFI was most effective at the dose of 3mg/kg in vivo, with significantly reduced brain infarct size and improved neurological deficits. Lower doses of 2-BFI at 1.5mg/kg, or higher dose of 2-BFI at 6 mg/kg, were either not effective, or toxic to the brain, respectively. Treating stroke rats with 3mg/kg 2-BFI significantly reduced the number of TUNEL positive cells and preserved the integrity of subcellular structures such as nuclear membranes and mitochondria as shown under the electron microscope, confirming neuroprotection. Most interestingly, 2-BFI-treated brains exhibited significant expression of Bcl-2, a gene with a known function in neuroprotection. Taken together, these studies not only demonstrated that 2-BFI at 3mg/kg was effective in neuroprotection, but also, for the first time, showed that 2-BFI provided neuroprotection through up-regulating the neuroprotective gene Bcl-2. 2-BFI can be further developed as a therapeutic drug for stroke treatment.
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Intracellular calcium ([Ca(2+)]i) influx through N-methyl-d-aspartic acid (NMDA) receptors in cortical neurons is central to NMDA receptor-mediated excitotoxicity. Drugs that uncompetitively modulate NMDA receptor-mediated [Ca(2+)]i influx are potential leads for development to treat NMDA receptor-mediated neuronal damage since these drugs spare NMDA receptor normal functions. Ligands to alpha(2)-adrenoceptors and imidazoline I(2) receptors confer neuroprotection possibility through modulating NMDA receptor-mediated [Ca(2+)]i influx. Here, we investigated the characteristics of several ligands to alpha(2)-adrenoceptors and imidazoline I(2) receptor, in inhibiting NMDA receptor-mediated [Ca(2+)]i influx in cultured cortical neurons using a ratiometric calcium imaging technique. In contrast to MK801, which non-reversibly blocks NMDA receptor-mediated [Ca(2+)]i influx, imidazoline I(2) receptor antagonists, Idazoxan, and 2-(2-benzofuranyl)-2-imidazoline (2-BFI)-mediated inhibition of [Ca(2+)]i influx can be rapidly reversed when removed, in a manner similar to that of memantine, an uncompetitive antagonist to NMDA receptors. Interestingly, ligands to alpha(2)-adrenoceptors, including agmatine sulfate and yohimbine, and a ligand to the nicotinic receptor, levamisol, neither inhibited NMDA receptor-mediated [Ca(2+)]i influx, nor provided neuroprotection against glutamate toxicity, suggesting selective inhibition of NMDA receptor activities. The inhibition of NMDA receptor by Idazoxan and 2-BFI also led to the suppression of NMDA receptor-mediated calpain activity as a result of blocking NMDA receptor activity, rather than through direct inhibition of calpain activity. Collectively, these studies demonstrated that imidazoline I(2) receptor antagonists transiently and reversibly block NMDA receptor-mediated [Ca(2+)]i influx. These compounds are leads for further development as uncompetitive antagonists to NMDA receptor-mediated excitotoxicity.
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Nicotinamide, the amide form of vitamin B(3) (niacin), is changed to its mononucleotide compound with the enzyme nicotinic acide/nicotinamide adenylyltransferase, and participates in the cellular energy metabolism that directly impacts normal physiology. However, nicotinamide also influences oxidative stress and modulates multiple pathways tied to both cellular survival and death. During disorders that include immune system dysfunction, diabetes, and aging-related diseases, nicotinamide is a robust cytoprotectant that blocks cellular inflammatory cell activation, early apoptotic phosphatidylserine exposure, and late nuclear DNA degradation. Nicotinamide relies upon unique cellular pathways that involve forkhead transcription factors, sirtuins, protein kinase B (Akt), Bad, caspases, and poly (ADP-ribose) polymerase that may offer a fine line with determining cellular longevity, cell survival, and unwanted cancer progression. If one is cognizant of the these considerations, it becomes evident that nicotinamide holds great potential for multiple disease entities, but the development of new therapeutic strategies rests heavily upon the elucidation of the novel cellular pathways that nicotinamide closely governs.
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Imidazoline binding sites (IBS) were proposed to be responsible for some of the pharmacological and therapeutic activities of imidazoline and related compounds and have been classified into two subtypes, I1BS and I2BS. Convergent studies attribute a role in central blood pressure regulation to the I1BS. In contrast, the function of I2BS remains unknown. In the present study, by combining biochemical and molecular biology approaches, we show that 1) microsequencing of I2BS purified from rabbit kidney mitochondria allowed the recovery of four peptide sequence stretches displaying up to 85.7% similarity with human, rat, and bovine monoamine oxidases (MAO)-A and -B; 2) I2BS and MAO displayed identical biophysical characteristics as their activities, measured by [³H]idazoxan binding and [¹⁴C]tyramine oxidation, respectively, could not be separated using various chromatographic procedures; and 3) heterologous expression of human placenta MAO-A and human liver MAO-B in yeast, inherently devoid of I2BS and MAO activities, led to the co-expression of [³H]idazoxan binding sites displaying ligand-recognition properties typical of I2BS. These results show definitely that I2BS is located on both MAO-A and -B. The fact that I2BS ligands inhibited MAO activity independently of the interaction with the catalytic region suggests that I2BS might be a previously unknown MAO regulatory site.
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An accurate, reproducible method for determining the infarct volumes of gray matter structures is presented for use with presently available image analysis systems. Areas of stained sections with optical densities above that of a threshold value are automatically recognized and measured. This eliminates the potential error and bias inherent in manually delineating infarcted regions. Moreover, the volume of surviving normal gray matter is determined rather than that of the infarct. This approach minimizes the error that is introduced by edema, which distorts and enlarges the infarcted tissue and surrounding white matter.Keywords: Brain; Infarction; Nissl stain; Succinate dehydrogenase
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Treatment with the alpha 2-adrenergic antagonist idazoxan (IDA) can provide protection from global cerebral ischemia. However, IDA also recognizes another class of receptors, termed imidazole (IM) receptors, which differ from alpha 2-adrenergic receptors and are responsible for the hypotensive actions of some centrally acting agents such as the oxazole rilmenidine (RIL). We therefore sought to determine whether RIL, an agent highly selective for IM receptors, offered protection from focal cerebral ischemia elicited in rat by ligation of the middle cerebral artery (MCA). We compared the effects of RIL with the effects of IDA and the selective non-IM alpha 2-antagonist SKF 86466 (SKF). In addition, we examined whether the neuroprotective effects of RIL and IDA could be attributed to changes in local CBF (LCBF). The MCA was occluded and animals either received immediate administration of drug while arterial pressure was maintained for 1 h or had local CBF increased to 200% of control for 1 h by hypercapnia or hypertension. RIL elicited a significant dose-dependent preservation of tissue to 33% of control at optimal dose (0.75 mg/kg). IDA (3 mg/kg) significantly reduced the size of ischemic infarction by 22%. In contrast, SKF (15 mg/kg) as well as doubling of LCBF did not preserve ischemic tissue. We conclude that both RIL and IDA can reduce focal ischemic infarction but that the mechanism does not appear secondary to antagonism of alpha 2-adrenergic receptors or elevation of LCBF. Occupation of IM receptors, either in the ischemic zone or at remote brain sites, may be responsible for neuroprotection of RIL and IDA.
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Imidazoline-guanidinium-receptive site (IGRS) is a membrane protein that, even if recognized by a series of imidazoline and guanidinium alpha 2-adrenergic compounds, is insensitive to catecholamine and physically distinct from alpha 2 receptors (Parini, A., Coupry, I., Graham, R. M., Uzielli, I., Atlas, D., and Lanier, S. M. (1989) J. Biol. Chem. 264, 11874-11878). In the present report, we defined the subcellular localization of IGRS by performing binding studies with the imidazoline radioligand [3H]idazoxan. Binding studies on subcellular fractions of homogenates from human and rabbit liver showed a significant increase in [3H]idazoxan binding in a membrane fraction enriched in cytochrome oxidase activity, a specific marker for mitochondria. The enrichment in [3H]idazoxan binding sites correlates closely with cytochrome oxidase activity in the nuclear, mitochondrial, plasma membrane, microsomal, and soluble fractions (r = 0.966, p less than 0.002) but not with the specific markers for other cell compartments, suggesting a major localization of IGRS in mitochondria. Separation of inner and outer mitochondrial membranes by digitonin treatment showed that [3H]idazoxan binding correlates positively with monoamine oxidase (r = 0.960) and negatively with cytochrome oxidase (r = -0.950) activities. In addition, in highly purified preparations of outer mitochondrial membranes obtained by hypotonic shock, [3H]idazoxan binding activity was 12.5-fold enriched with respect to intact mitochondria. Taken together, these data show, for the first time, that IGRS in human and rabbit liver are mainly associated with the outer mitochondrial membranes. This demonstration of the major mitochondrial localization of IGRS will facilitate the characterization of its functional activity in liver.
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Pharmacologically active compounds with an imidazoline and/or guanidinium moiety are recognized with high affinity by a family of membrane-bound proteins collectively known as imidazoline binding sites or imidazoline/guanidinium receptive sites. Two such receptive sites may correspond to imidazoline binding domains identified on the A and B isoforms of monoamine oxidase (MAO), but the detection of monoamine oxidase isoforms in multiple tissues contrasts with the restricted expression of imidazoline-binding proteins. To address these issues, we determined the relationship between monoamine oxidase isoforms and subtypes of imidazoline-binding proteins in human tissues known to express one or both isoforms of MAO. 2-(3-Azido-4-[¹²⁵I]iodophenoxy)methylimidazoline ([¹²⁵I]AZIPI), a photoaffinity adduct that selectively labels imidazoline-binding proteins, photolabeled an Mr = ∼59,000 peptide in liver and an Mr = ∼63,000 peptide in placenta, consistent with the Mr of the MAO isoforms identified by immunoblots in these tissues. The photolabeled species in liver was immunoprecipitated with MAO-B selective antibodies, whereas the photolabeled species in placenta was immunoprecipitated by MAO-A selective antibodies consistent with the isoform of MAO predominantly expressed in these tissues. The imidazoline/guanidinium ligands interact with the enzyme at a site distinct from the substrate recognition domain, and the immunoprecipitated peptides in liver and placenta display distinct ligand recognition properties consistent with those reported for subtypes of imidazoline binding sites. However, the imidazoline binding domain was not detected in platelet membrane preparations containing amounts of MAO-B equivalent to those in the photolabeled liver membranes indicating that recognition of this domain is tissue-restricted. Restricted access to the imidazoline binding domain on platelet MAO-B was not altered by membrane washing with 500 mM KCl or by solubilization and partial purification of the enzyme suggesting that there are distinct subpopulations of MAO. Identification of a binding domain on MAO that recognizes this class of pharmacologically active compounds suggests a novel mechanism for regulation of substrate oxidation/selectivity or that the enzyme may subserve an as yet undefined function.
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I2-Imidazoline sites ([3H]-idazoxan binding) have been identified on monoamine oxidase (MAO) and proposed to modulate the activity of the enzyme through an allosteric inhibitory mechanism (Tesson et al., 1995). The main aim of this study was to assess the inhibitory effects and nature of the inhibition of imidazol(ine)/guanidine drugs on rat liver MAO-A and MAO-B isoforms and to compare their inhibitory potencies with their affinities for the sites labelled by [3H]-clonidine in the same tissue. Competition for [3H]-clonidine binding in rat liver mitochondrial fractions by imidazol(ine)/guanidine compounds revealed that the pharmacological profile of the interaction (2 - styryl - 2 - imidazoline, LSL 61112>idazoxan>2 - benzofuranyl - 2 - imidazoline, 2-BFI=cirazoline>guanabenz>oxymetazoline>>clonidine) was typical of that for I2-sites. Clonidine inhibited rat liver MAO-A and MAO-B activities with very low potency (IC50s: 700 μM and 6 mM, respectively) and displayed the typical pattern of competitive enzyme inhibition (Lineweaver-Burk plots: increased Km and unchanged Vmax values). Other imidazol(ine)/guanidine drugs also were weak MAO inhibitors with the exception of guanabenz, 2-BFI and cirazoline on MAO-A (IC50s: 4–11 μM) and 2-benzofuranyl-2-imidazol (LSL 60101) on MAO-B (IC50: 16 μM). Idazoxan was a full inhibitor, although with rather low potency, on both MAO-A and MAO-B isoenzymes (IC50s: 280 μM and 624 μM, respectively). Kinetic analyses of MAO-A inhibition by these drugs revealed that the interactions were competitive. For the same drugs acting on MAO-B the interactions were of the mixed type inhibition (increased Km and decreased Vmax values), although the greater inhibitory effects on the apparent value of Vmax/Km than on the Vmax value indicated that the competitive element of the MAO-B inhibition predominated. Competition for [3H]-Ro 41-1049 binding to MAO-A or [3H]-Ro 19-6327 binding to MAO-B in rat liver mitochondrial fractions by imidazol(ine)/guanidine compounds revealed that the drug inhibition constants (Ki values) were similar to the IC50 values displayed for the inhibition of MAO-A or MAO-B activities. In fact, very good correlations were obtained when the affinities of drugs at MAO-A or MAO-B catalytic sites were correlated with their potencies in inhibiting MAO-A (r=0.92) or MAO-B (r=0.99) activity. This further suggested a direct drug interaction with the catalytic sites of MAO-A and MAO-B isoforms. No significant correlations were found when the potencies of imidazol(ine)/guanidine drugs at the high affinity site (pKiH, nanomolar range) or the low-affinity site (pKiL, micromolar range) of I2-imidazoline receptors labelled with [3H]-clonidine were correlated with the pIC50 values of the same drugs for inhibition of MAO-A or MAO-B activity. These discrepancies indicated that I2-imidazoline receptors are not directly related to the site of action of these drugs on MAO activity in rat liver mitochondrial fractions. Although these studies cannot exclude the presence of additional binding sites on MAO that do not affect the activity of the enzyme, they would suggest that I2-imidazoline receptors represent molecular species that are distinct from MAO.
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This is the first study characterising the binding of the new imidazoline I2 receptor selective radioligand [3H]2-(2-benzofuranyl)-2-imidazoline (2-BFI) to rabbit brain membranes. [3H]2-BFI binding was found to be saturable and of high affinity identifying two binding sites with KD1 = 0.27 nM, Bmax = 111.2 fmol mg−1 protein and KD2 = 8.97 nM, Bmax = 268 fmol mg−1 protein. Specific binding represented greater than 90% of total binding. Kinetic studies revealed that the binding was rapid and reversible and also showed [3H]2-BFI interacted with these two sites or two affinity states. In competition binding studies against [3H]2-BFI (0.3-lnM) idazoxan, 2-BFI, cirazoline, guanabenz, naphazoline, amiloride and BU224 (2-(4,5-dihydroimidaz-2-yl-quinoline) displaced with high affinity. In contrast the α2-adrenoceptor antagonists efaroxan and rauwolscine, the I1 site selective drug moxonidine, the monoamine oxidase-A inhibitor clorgyline and the proposed endogenous imidazoline receptor ligand, agmatine, were weak at displacing [3H]2-BFI binding. These findings are consistent with [3H]2-BFI recognising imidazoline receptors of the I2 subtype in rabbit brain.
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Clonidine and related imidazoline compounds bind to alpha 2-adrenergic as well as to newly described non-adrenergic imidazole/imidazoline receptors in brain and peripheral tissues. The present study was undertaken to identify the signal transduction mechanism coupled to this new class of receptors (imidazole receptors) using bovine adrenal chromaffin cells. Clonidine did not modify the basal or forskolin-stimulated production of cyclic AMP (cAMP), suggesting the absence of functionally active alpha 2-adrenergic receptors in adrenal chromaffin cells. Clonidine also failed to modify the basal and GTP gamma S- or carbachol-stimulated increase in phosphoinositide hydrolysis. However, clonidine increased significantly the production of cyclic GMP (cGMP) as well as the uptake of 45Ca2+. The cGMP response to clonidine was slower (peak at 15 min) and smaller (only about 50% over control) than the response to acetylcholine and was not shared by other agents that bind to imidazole receptors. In contrast, all agents that bind to imidazole receptors increased the influx of 45Ca2+ into chromaffin cells. It is concluded that (a) alpha 2-adrenergic and imidazole receptors are functionally distinct and linked to different signal transduction mechanisms; (b) the classical G-protein coupled soluble second messenger systems are not coupled to imidazole receptors; (c) clonidine may increase cGMP by a non-receptor-mediated intracellular action; and (d) imidazole receptors may regulate intracellular calcium levels through an ion regulating system that may be different from calcium channels.
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To develop a simple, relatively noninvasive small-animal model of reversible regional cerebral ischemia, we tested various methods of inducing infarction in the territory of the right middle cerebral artery (MCA) by extracranial vascular occlusion in rats. In preliminary studies, 60 rats were anesthetized with ketamine and different combinations of vessels were occluded; blood pressure and arterial blood gases were monitored. Neurologic deficit, mortality rate, gross pathology, and in some instances, electroencephalogram and histochemical staining results were evaluated in all surviving rats. The principal procedure consisted of introducing a 4-0 nylon intraluminal suture into the cervical internal carotid artery (ICA) and advancing it intracranially to block blood flow into the MCA; collateral blood flow was reduced by interrupting all branches of the external carotid artery (ECA) and all extracranial branches of the ICA. In some groups of rats, bilateral vertebral or contralateral carotid artery occlusion was also performed. India ink perfusion studies in 20 rats documented blockage of MCA blood flow in 14 rats subjected to permanent occlusion and the restoration of blood flow to the MCA territory in six rats after withdrawal of the suture from the ICA. The best method of MCA occlusion was then selected for further confirmatory studies, including histologic examination, in five additional groups of rats anesthetized with halothane. Seven of eight rats that underwent permanent occlusion of the MCA had resolving moderately severe neurologic deficits (Grade 2 of 4) and unilateral infarcts averaging 37.6 +/- 5.5% of the coronal sectional area at 72 hours after the onset of occlusion.(ABSTRACT TRUNCATED AT 250 WORDS)
Article
The effects of clorgyline, the MAO-A inhibitor, and lazabemide, the MAO-B inhibitor, on the levels of the hydroxyl radicals appearing in the cerebral ventricles of mice during brain ischemia/reperfusion were examined by using a salicylate trapping method. The amount of hydroxyl radicals formed peaked at 20 min after reperfusion (approximately 150% vs. basal level). The dopamine level markedly increased shortly after the initiation of an ischemic insult and thereafter waned. By contrast, the concentration of 3,4-dihydroxyphenylacetic acid (DOPAC) level decreased during a 40-min period of ischemia, gradually returning to the preischemic basal level upon reperfusion. The ischemia reperfusion-induced hydroxyl radical generation was attenuated by 3 mg/kg of clorgyline and lazabemide. Furthermore, mice pretreated with these MAO inhibitors showed decreased DOPAC levels in comparison with those of their respective vehicle-treated control groups; recovery of the reduced DOPAC level was also delayed. In conclusion, it is likely that both type A and type B MAOs participate in the generation of hydroxyl radicals during brain ischemia/reperfusion. This finding suggests the possible use of MAO inhibitors as neuroprotective agents for treating ischemic injury.
Article
The concentration of the astrocytic marker, glial fibrillary acidic protein (GFAP) was quantitated by immunoblotting (Western blotting) in the rat brain after treatment with the novel ligand for imidazoline I2 receptors LSL 60101 [2-(2-benzofuranyl)imidazole] and its 6-methoxy derivative LSL 60125. Chronic (7-21 days), but not acute (1 day) or short-term (3 days), treatment with LSL 60101 (10 mg/kg i.p.) markedly increased (44-49%) GFAP immunoreactivity in the rat cerebral cortex. In contrast, chronic (7 days) treatment with LSL 60125 (10 mg/kg i.p.) did not significantly modify GFAP concentrations. In vitro, both drugs displayed moderate high affinity and high selectivity for imidazoline I2 receptors versus alpha 2-adrenoceptors; however, only chronic treatment with LSL 60101 (10 mg/kg i.p.) but not with LSL 60125 (10 mg/kg i.p.) was associated with an up-regulation of imidazoline I2 receptors. These data indicate that glial imidazoline I2 receptors may have a direct physiological function related to GFAP expression and that LSL 60101 could be a good tool for the study of the implication of these receptors on astrocyte activation and neuronal regeneration.
Article
The concentration of the astrocytic marker, glial fibrillary acidic protein (GFAP) was quantitated by immunoblotting (western blotting) in the rat brain after treatment with various imidazoline drugs and other agents. Chronic (7 days) but not acute (1 day) treatment with the imidazoline drugs, cirazoline (1 mg kg ⁻¹ , i.p.) and idazoxan (10 mg kg ⁻¹ , i.p.), but not with the structurally related α 2 ‐adrenoceptor antagonists, RX821002 (2‐methoxy idazoxan) (10 mg kg ⁻¹ , i.p.) and efaroxan (10 mg kg ⁻¹ , i.p.), markedly increased (45%) GFAP immunoreactivity in the rat cerebral cortex. Chronic treatment (7 days) with yohimbine (10 mg kg ⁻¹ , i.p.), a non‐imidazoline α 2 ‐adrenoceptor antagonist, did not significantly modify GFAP immunoreactivity in the cerebral cortex. Chronic treatment (7 days) with cirazoline and idazoxan did not alter the density of brain monoamine oxidase (MAO)‐B sites labelled by [ ³ H]‐Ro 19–6327 (lazabemide), another relevant astroglial marker. Moreover, these imidazoline drug treatments did not modify the levels of α‐tubulin in the cerebral cortex. These negative results reinforced the specificity of the effects of imidazoline drugs on GFAP. Irreversible inactivation of brain α 2 ‐adrenoceptors (and other neurotransmitters receptors) after treatment with an optimal dose of the peptide‐coupling agent EEDQ (1.6 mg kg ⁻¹ , i.p., for 6–24 h) did not alter GFAP immunoreactivity in the cerebral cortex. These results further disproved the involvement of these receptors on astroglial cells in the tonic control of GFAP levels. The binding of [ ³ H]‐idazoxan in the presence of 10 ⁻⁶ m (–)‐adrenaline was used to quantitate in parallel I 2 ‐imidazoline preferring sites in the rat brain after the same treatments. Chronic treatment (7 days) with cirazoline and idazoxan, but not with RX821002, efaroxan or yohimbine, significantly increased (25%) the density of I 2 ‐sites in the cerebral cortex. The up‐regulation of I 2 ‐sites induced by cirazoline was not observed in the liver, a tissue that also expresses I 2 ‐sites but lacks glial cells. A strong correlation ( r = 0.97) was found when the mean percentage changes in GFAP immunoreactivity were related to the mean percentage changes in I 2 imidazoline sites after the various drug treatments. Together the results suggest a direct physiological function of glial I 2 ‐imidazoline preferring sites in the regulation of GFAP levels.
Article
Clonidine and related imidazoline agents, beside binding to alpha 2-adrenergic receptors, have been shown to bind to a non-adrenergic site (imidazoline sites) in brain and peripheral tissues. However, which cell types in brain, namely neurons or glia, express this binding site and the cellular effects of activation of this site are not known. We investigated the cellular localization of imidazoline binding sites in cultured rat cortical astrocytes and neurons. Membranes prepared from cultured astrocytes showed specific, high affinity binding (KD: 4 nM) for 3H-idazoxan with about tenfold higher number of binding sites than alpha 2-adrenergic sites (Bmax: 220 vs. 20 fmol/mg protein). Displacement studies exhibited the rank order of potency: cirazoline > idazoxan > amiloride > clonidine > epinephrine = ruawolscine defining this site as I-2a subtype of imidazoline binding sites. Moreover, the binding was inhibited by K+ but not by Na+, another characteristic of imidazoline binding sites. In contrast, membranes prepared from cultured neurons showed fewer binding sites for 3H-idazoxan that were completely displayed by adrenergic agents. Incubation of astrocytes with idazoxan, but not rauwolscine, resulted in a concentration-dependent increase in the levels of mRNA for the astrocyte specific molecule glial fibrillary acidic protein. We conclude that (a) the non-adrenergic imidazoline binding sites are expressed in astrocytes but not in neurons in rat cerebral cortex and (b) these "receptors" may influence astrocyte physiology by regulating the levels of GFAP.
Article
Imidazoline (I) receptors constitute a family of nonadrenergic high-affinity binding sites for clonidine, idazoxan, and allied drugs. One major subclass, the I1 receptors, whose subcellular distribution and signal transduction mechanisms are uncertain, partly mediates the central hypotensive actions of clonidine-like drugs. The I2 receptors, another subclass, are mitochondrial, not G protein coupled, and have diversified functions. Several endogenous ligands for I receptors, collectively termed clonidine-displacing substances (CDSs), have been detected in tissues and serum, but the structure of only one, agmatine (decarboxylated arginine), is known. Agmatine, widely distributed and bioactive, binds, like clonidine, to alpha 2-adrenergic and I receptors of all subclasses. The presence of agmatine and its biosynthetic enzyme in synaptosomes and specific neuronal pathways as well as serum suggests that it may be a novel neurotransmitter/hormone. Another CDS that binds to I receptors and to antibodies to imidazoline drugs has been detected, but its structure is unknown.
Article
2-(2-Benzofuranyl)-2-imidazoline (2-BFI) has recently been characterised as a selective ligand for the I2-type of imidazoline-receptor binding site(s) (I2-RBS). The present studies determined the relative levels of specific [3H]2-BFI binding to membrane homogenates of brain and kidney from rat, guinea pig and rabbit and identified the pharmacological characteristics of [3H]2-BFI binding sites in rabbit kidney membranes. Rabbit kidney membranes had the highest relative density of specific [3H]2-BFI binding of all tissues studied (2000 fmol/mg protein). Rabbit brain and guinea pig kidney had moderate levels of specific [3H]2-BFI binding (350–500 fmol/mg protein), while rat kidney and guinea pig and rat brain displayed much lower densities of binding (40–65 fmol/mg protein). Studies of [3H]2-BFI binding kinetics in rabbit kidney homogenates revealed binding to two distinct sites with K d values of 0.10 ± 0.01 nmol/l and 1.00 ± 0.36 nmol/l respectively. Equilibrium saturation studies were also consistent with the presence of two binding sites – [3H]2-BFI (0.01–20 nmol/l) bound to sites with affinities of 0.10 ± 0.01 nmol/l and 0.92 ± 0.13 nmol/l and binding densities of 470 ± 80 and 840 ± 60 fmol/mg protein (n=3), representing 36 and 64% respectively. Drug inhibition studies revealed that l-adrenaline; α1-adrenoceptor drugs (prazosin, l-phenylephrine) and α2-adrenoceptor drugs (rauwolscine, methoxyidazoxan, 2-(2,4-(O-methoxyphenyl)-piperazin-1-yl)-ethyl-4,4-dimethyl-1,3-(2H,4H)-isoquinolindione (ARC-239) had extremely low affinities for [3H]2-BFI binding sites (IC50 ≥ 10 μmol/l). Putative I1-RBS compounds, p-aminoclonidine, moxonidine, imidazole-4-acetic acid and cimetidine, inhibited [3H]2-BFI binding to rabbit renal membranes with low to very low affinities (K i values 3 to ≥100 μmol/l), suggesting [3H]2-BFI does not label I1-RBS in rabbit kidney membranes. I2-RBS compounds – 2-(4,5-dihydroimidaz-2-yl)-quinoline (BU224), 2-(4,5-dihydroimidaz-2-yl)-quinoxaline (BU239), idazoxan and cirazoline – potently inhibited [3H]2-BFI binding (K i values 0.37–1.6 nmol/l), confirming the labelling of I2-RBS. Inhibition of [3H]2-BFI binding by certain compounds was consistent with their interaction with two binding site populations – for example (drug, K i values) guanabenz, 0.65 nmol/l and 0.17 μmol/l; naphazoline, 0.94 nmol/l and 2.8 μmol/l; amiloride, 76 nmol/l and 26 μmol/l rilmenidine, 150 nmol/l and 50 μmol/l; and clonidine, 230 nmol/l and 70 μmol/l. The high affinity of amiloride for a high proportion (85%) of the binding is consistent with the presence of the I2A-subtype of I-RBS in rabbit kidney. These results demonstrate that [3H]2-BFI is a highly selective and high affinity radioligand for I2-RBS which should be useful for the further characterisation of these sites in mammalian tissues.
Article
Previous studies have shown that a subpopulation of the catecholamine-degrading enzymes monoamine oxidase (MAO) A and B holds a previously unknown regulatory site, the I2-imidazoline binding site (I2BS). In the present work, we characterized the isoforms of monoamine oxidases expressed in the rabbit renal proximal tubule, defined their relationship with I2BS, and investigated the ability of I2BS ligands to inhibit enzyme activity in intact cells. Two findings indicate that MAO-B is the predominant isoform expressed in the renal proximal tubule cells: 1) Western blot performed with an anti-MAO-A/MAO-B polyclonal antiserum revealed a single 55-kDa band corresponding to MAO-B; 2) enzyme assays showed an elevated MAO-B activity ([14C]beta-phenylethylamine oxidation: Vmax = 1.31 +/- 0.41 nmol/min/mg protein), whereas MAO-A activity was only detectable ([14C]5-HT oxidation: Vmax = 80.3 +/- 19 pmol/min/mg protein). Photoaffinity labeling with the I2BS ligand [125I]2-(3-azido-4-iodophenoxy)-methylimidazoline revealed a single 55-kDa band, which indicates that MAO-B of the renal proximal tubule cells holds the I2 imidazoline binding site. [3H]Idazoxan binding studies and enzyme assays showed that, in intact cells, I2BS ligands bind to and inhibit MAO-B. Indeed, the increase in the accessibility of intracellular compartment by cell permeabilization did not enhance [3H]idazoxan binding, which indicates that, in intact cells, intracellular I2BS are fully occupied by imidazoline ligands. In addition, enzyme assays showed that incubation of proximal tubule cells with imidazoline ligands leads to a complete, dose-dependent inhibition of MAO activity. These data show the predominant expression of MAO-B in rabbit renal proximal tubule and its regulation by imidazoline ligands in intact cells.
Article
Rilmenidine, a ligand for imidazoline and alpha2-adrenergic receptors, is neuroprotective following focal cerebral ischemia. We investigated the effects of rilmenidine on cytosolic free Ca2+ concentration ([Ca2+]i) in rat astrocytes. Rilmenidine caused concentration-dependent elevation of [Ca2+]i, consisting of a transient increase (1-100 microM rilmenidine) or a transient increase followed by sustained elevation above basal levels (1-10 mM rilmenidine). A similar elevation in [Ca2+]i was induced by the imidazoline ligand cirazoline. The transient response to rilmenidine was observed in Ca2+-free medium, indicating that rilmenidine evokes release of Ca2+ from intracellular stores. However, the sustained elevation of Ca2+ was completely dependent on extracellular Ca2+, consistent with rilmenidine activating Ca2+ influx. Pretreatment with thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+-ATPase, abolished the response to rilmenidine, confirming the involvement of intracellular stores and suggesting that rilmenidine and thapsigargin activate a common Ca2+ influx pathway. The alpha2-adrenergic antagonist rauwolscine attenuated the increase in [Ca2+]i induced by clonidine (a selective alpha2 agonist), but not the response to rilmenidine. These results indicate that rilmenidine stimulates both Ca2+ release from intracellular stores and Ca2+ influx by a mechanism independent of alpha2-adrenergic receptors. In vivo, rilmenidine may enhance uptake of Ca2+ from the extracellular fluid by astrocytes, a process that may contribute to the neuroprotective effects of this agent.
Article
I2-binding sites (I2-BS) are attributed to be a regulative site on monoamine oxidase (MAO). The in vivo and in vitro effects of various imidazoline and guanidine derivatives on MAO activity and on mitochondrial respiration were studied. Substances with high affinity for I2-BS (antazoline, idazoxan, and cirazoline: IC50 = 20.3, 33.8, and 43.4 microM) had a stronger inhibitory effect on MAO activity than did I1-ligands (efaroxan, rilmenidine, clonidine, and moxonidine: IC50 = 277, 801, 1,224, and > 10,000 microM). Substances with the highest inhibitory effects were BDF8082 (IC50 = 1.7 microM) and 2-(2-benzofuranyl)-2-imidazoline (BFI; IC50 = 4.0 microM). The enzyme is inhibited noncompetitively and is reversible, because its activity is completely or partially restored after dialysis. Agmatine, the putative endogenous ligand for IBS, also decreased MAO activity (IC50 = 168 microM), whereas its precursor, L-arginine, and its metabolite, putrescine, had no effects. In vitro inhibition of MAO and mitochondrial respiration by the IBS-ligands tested could not be correlated, suggesting no link between the function of the inner and outer mitochondrial membrane. MAO activity in vivo was significantly reduced only by pargyline (-95%), BDF8082 (-68%), BFI (-43%), and 1-(m-chlorophenyl)-biguanide (-28%). Catecholamine content of livers obtained from animals treated with different IBS-ligands was consequently increased. In conclusion, the strong inhibitory effects of I2 selective imidazoline ligands confirm the existence of I2-BS as a regulatory site on MAO.
Article
I2-imidazoline receptors are mainly expressed on glial cells in the rat brain. This study was designed to test the effect of treatment with the I2-imidazoline selective receptor ligand LSL 60101 [2-(2-benzofuranyl)imidazole] on the morphology of astrocytes in the neonate and adult rat brain, and to explore the putative neuroprotective effects of this glial response. Short-term (3 days) or chronic (7–10 days) treatment with LSL 60101 (1 mg kg−1, i.p. every 12 h) enhanced the area covered by astroglial cells in sections of facial motor nucleus from neonate rats processed for glial fibrillary acidic protein (GFAP) immunostaining. Facial motoneurons surrounded by positive glial cell processes were frequently observed in sections of LSL 60101-treated rats. A similar glial response was observed in the parietal cortex of adult rats after chronic (10 days) treatment with LSL 60101 (10 mg kg−1, i.p. every 12 h). Western-blot detection of the specific astroglial glutamate transporter GLT-1, indicated increased immunoreactivity after LSL 60101 treatment in the pons of neonate and in the parietoccipital cortex of adult rats. In the facial motor nucleus of neonate rats, the glial response after LSL 60101 treatment was associated to a redistribution of the immunofluorescence of the basic fibroblast growth factor (FGF-2) from the perinuclear area of motoneurons to cover most of their cytoplasm, suggesting a translocation of this mitogenic and neurotrophic factor towards secretion pathways. The neuroprotective potential of the above effects of LSL 60101 treatment was tested after neonatal axotomy of facial motor nucleus. Treatment with LSL 60101 (1 mg kg−1, i.p. every 12 h from day 0 to day 10 after birth) significantly reduced (38%) motoneuron death rate 7 days after facial nerve axotomy performed on day 3 after birth. It is concluded that treatment with the I2-imidazoline selective receptor ligand LSL 60101 provokes morphological/biochemical changes in astroglia that are neuroprotective after neonatal axotomy. British Journal of Pharmacology (2000) 130, 1767–1776; doi:10.1038/sj.bjp.0703485
Article
An increasing number of reports suggest the involvement of oxidative stress in neurodegenerative diseases where the increased formation of reactive oxygen species (ROS) leads to neuronal damage and cell death. Dopamine may contribute to neurodegenerative disorders such as Parkinson's disease and ischemia/reperfusion-induced damage. Monoamine oxidase (MAO) enzyme (particularly MAO-B) is responsible for metabolizing dopamine and plays an important role in oxidative stress through altering the redox state of neuronal and glial cells. MAO participates in the generation of hydroxyl radicals during ischemia/reperfusion. This suggests the possible use of MAO inhibitors as neuroprotective agents for treating ischemic injury. The protective effect of deprenyl (N-methyl-N-(1-methyl-2-phenyl-ethyl)-prop-2-yn-1-amine, CAS 14611-51-9) (2 and 10 mg/kg), a MAO-B inhibitor, and beta-carotene (10 and 20 mg/kg), a natural antioxidant, was examined in a rat model of cerebral ischemia. Ischemia was induced in rats by bilateral carotid artery occlusion for 1 h followed by declamping for another hour. The effect of the drugs on the brain activity of lactate dehydrogenase (LDH) and some of the oxidative stress biomarkers such as brain activity of superoxide dismutase (SOD) and catalase (CAT) enzymes and brain malondialdehyde (MDA) content was determined. In addition, the content of catecholamines such as noradrenaline (NA) and dopamine (DA) was determined. Deprenyl decreased the ischemia-induced elevation of LDH activity and MDA content and normalized the SOD activity. In addition, deprenyl increased the CAT activity back to normal, and increased the noradrenaline and dopamine content in the brain of rats. Beta-carotene administration ameliorated the effect of ischemia followed by reperfusion (I/R) demonstrated as decreasing the LDH activity and MDA content and by increasing the SOD activity. The drug also increased CAT activity in the brain of rats. However, beta-carotene did not alter the NA and DA content. These results indicate that deprenyl protected the rat brains against the ischemia-induced oxidative damage, an effect which might be explained through multiple mechanisms, possibly due to reduction of dopamine catabolism with a subsequent increased activity on dopaminergic D2 receptors and suppressing the action of ROS as well.