-
[show abstract]
[hide abstract]
ABSTRACT: The opioid/nociceptin receptors are involved in many neurological disorders such as Alzheimer's disease, Parkinson's disease and epilepsy. Kainic acid (KA) is an analog of the excitatory amino acid transmitter glutamate and the systemic administration of KA induces status epilepticus (SE) in rodents. In this study, we examined the alterations in the G-protein activity and the gene expression levels of mu, kappa, delta opioid and nociceptin receptors (MOPr, KOPr, DOPr and NOPr) as well as PNOC, the precursor polypeptide of nociceptin-OFQ (N/OFQ) in KA-induced seizures in the rat brain cortex. KA was used to create seizures with the dose of 10mg/kg body weight i.p. Following the KA administration, the rats were observed for 3h to assess seizure activity. Seizures occurred approximately 45min after the KA injection. Only rats exhibiting full limbic seizures, forelimb clonus with rearing, were used in this study. All animals were decapitated 4h after the administration of KA. Our [(35)S]GTPγS binding results showed that there was a significant difference in both the affinity and efficacy particularly one of NOPr stimulation following KA treatment. Slight, but significant increase was observed for MOPr. Moreover PNOC, NOPr and MOPr mRNA levels were increased by KA treatment but there were no significant changes in the levels of DOPr and KOPr mRNAs. These results show that the activities of opioid/nociceptin receptors can be modified by KA-treatment, and MOPr, PNOC and NOPr are the most responsive to KA-induced seizures in the rat brain cortex.
Epilepsy research 01/2013; · 2.48 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Endomorphin-1 (EM-1) and endomorphin-2 (EM-2) are two endogenous tetrapeptides with very high affinities for the μ-opioid receptor. Until recently the precise neuroanatomical localization of the binding sites for these peptides was unknown. However, the recent synthesis of tritiated forms of these molecules has permitted these binding sites to be analysed with a very high degree of neuroanatomical specificity. Preliminary studies demonstrated a superior binding profile for EM-2, with less non-specific binding than EM-1. As the endogenous cannabinoid and opioid systems interact at several levels, we investigated how deletion of the CNR1 gene, which encodes the cannabinoid receptor 1 (CB(1)R) protein, affects the brain distribution of EM-2 binding sites. Our results revealed no differences in the average density of EM-2 binding sites in CB(1) receptor knockout (CB(1)R KO) and WT mice. However, when both hemispheres were analysed separately, we detected specific alterations in the distribution of EM-2 binding sites in the right hemisphere of CB(1)R KO mice. While, the density of EM-2 binding sites in CB(1)R KO mice was higher in the CA3 hippocampal field and in the pontine tegmental nuclei, it was lower in the superior colliculus and ventral tegmental area than in WT controls. No differences were observed in the left hemisphere for any of the regions analysed. For the first time these findings demonstrate a lateralization effect on cerebral opioid binding sites that may be mediated by the central cannabinoid system.
European journal of pharmacology 11/2012; · 2.59 Impact Factor
-
E Lacko,
A Varadi,
R Rapavi,
F Zador,
P Riba,
S Benyhe, A Borsodi,
S Hosztafi,
J Timar,
B Noszal,
S Furst,
M Al-Khrasani
[show abstract]
[hide abstract]
ABSTRACT: The aims of this study were to synthesize 14-O-Methylmorphine-6-O-sulfate (14-O-MeM6SU) and examine its opioid properties (potency, affinity, efficacy) in receptor ligand binding and isolated tissues (mouse vas deferens, MVD and rat vas deferens, RVD bioassays). The results were then compared to the parent compounds morphine-6-O-sulfate (M6SU) and morphine, as well as the �- opioid receptor (MOR) selective agonist peptide [D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin (DAMGO). An additional objective was to compare the effect of subcutaneously (s.c.) or intracerebroventricularly (i.c.v.) administered 14-O-MeM6SU, M6SU and morphine in thermal nociception, rat tail-flick (RTF) test. In MVD, the EC50 (nM) value was 4.38 for 14-O-MeM6SU, 102.81 for M6SU, 346.63 for morphine and 238.47 for DAMGO. The effect of 14-O-MeM6SU and DAMGO was antagonized by naloxone (NAL) with Ke value 1-2.00 nM. The Emax values (%) were 99.10, 36.87, 42.51 and 96.99 for 14-O-MeM6SU, M6SU, morphine and DAMGO, respectively. In RVD 14-O-MeM6SU and DAMGO but not M6SU or morphine showed agonist activity. In binding experiments the affinity of 14-OMeM6SU, M6SU, morphine and DAMGO for MOR was 1.12, 11.48, 4.37 and 3.24 nM, respectively. The selectivity of 14-O-MeM6SU was κ/μ= 269 and δ/μ= 9. In G-protein activation experiments, 14-O-MeM6SU and DAMGO showed higher Emax values than M6SU or morphine. S.c. or i.c.v-injected 14-O-MeM6SU, M6SU and morphine produced a dose and time-dependent increase in RTF response latency. 14-O-MeM6SU was the most potent. Our results showed that introduction of 14-O-Me in M6SU increased the binding affinity, agonist potency, and most importantly, the intrinsic efficacy (Emax).
Current Medicinal Chemistry 08/2012; 19(27):4699-707. · 4.86 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Excitotoxicity is a contributing factor to the pathogenesis of acute or chronic neurodegenerative disease states. Kainic acid (KA) is an excitotoxic substance and the administration of it to rodents induces seizure activity (status epilepticus, SE) and leads to neurodegeneration. In this study the effect of KA-induced excitotoxicity on the G-protein activations and the gene expression levels of the opioid/nociceptin system receptors as MOPr, KOPr, DOPr, ORL-1, and PNOC (N/OFQ) were investigated, and the regulator effect of naloxone (Nal) on the gene expressions of the opioid system receptors against KA-induced seizures in the rat hippocampus was tested. In addition, the expression levels of stress-toxicity genes were assessed in the hippocampus following KA-induced excitotoxicity in order to determine the potential genetic targets which can be helpful for neuroprotective interventions. Our results indicate that the KA-induced excitotoxicity increased the mRNA levels of MOPr, DOPr, KOPr, PNOC, and ORL-1. However, G-protein activations of MOPr, DOPr, and KOPr remained relatively unchanged while both the potency and efficacy of N/OFQ were significantly increased. The PCR array data showed that KA-induced excitotoxicity altered the expression levels of genes in the cellular stress or toxicity pathways. Our data suggests that the induction of the opioid/nociceptin system may be involved in the cellular stress response following a neurodegenerative insult and that the genes modulated by the KA-treatment in the stress-toxicity pathways may be evaluated as targets of potential neuroprotective interventions.
Neurochemistry International 02/2012; 60(6):555-64. · 2.86 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: It has been known for centuries that exogenous cannabinoids, such as tetrahydrocannabinol have anticonvulsant activity. Recent studies have advanced our understanding of the endogenous cannabinoid system and renewed the interest in cannabinoids as a potential treatment for epilepsy. The endogenous cannabinoid system is rapidly activated after seizure activity but still little is known about the molecular mechanisms underlying the role of the cannabinoid system in epilepsy. In this study epileptiform activity was induced by kainic acid (KA) and effects of the CB1 receptor agonists N-(2-Chloroethyl)-5Z,8Z,11Z,14Z-eicosatetraenamide (ACEA) on G-protein signaling using the agonist-stimulated [(35)S]GTPγS binding assay were evaluated. Control and KA treated rat hippocampus and cortex membranes were used. Our results showed that the ACEA displayed a high potency and efficacy in stimulating the G-proteins and when compared to the control animals, significant enhancements were observed in tissues from the KA treated animals. Potency and efficacy values were in particular increased in the hippocampus tissues. Furthermore, gene expression levels of the cannabinoid receptor 1 (CB1) receptor and cannabinoid receptor interacting protein 1 (CRIP1) were measured by RT-PCR, where both CB1 and CRIP1 expressions were found to be elevated in the KA treated animals.
Epilepsy research 11/2011; 99(1-2):64-8. · 2.48 Impact Factor
-
Benjamin Aguila,
Laurent Coulbault,
Audrey Davis,
Nicolas Marie,
Ahmed Hasbi,
Florian Le bras,
Géza Tóth, Anna Borsodi,
Vsevolod V Gurevich,
Philippe Jauzac,
Stéphane Allouche
[show abstract]
[hide abstract]
ABSTRACT: We have previously reported on the differential regulation of the human δ-opioid receptor (hDOR) by alkaloid (etorphine) and peptidic (DPDPE and deltorphin I) ligands, in terms of both receptor desensitization and post-endocytic sorting. Since ßarrestins are well known to regulate G protein-coupled receptors (GPCRs) signaling and trafficking, we therefore investigated the role of ßarrestin1 (the only isoform expressed in our cellular model) in the context of the hDOR. We established clonal cell lines of SK-N-BE cells over-expressing ßarrestin1, its dominant negative mutant (ßarrestin1(319-418)), and shRNA directed against endogenous ßarrestin1. Interestingly, both binding and confocal microscopy approaches demonstrated that ßarrestin1 is required for hDOR endocytosis only when activated by etorphine. Conversely, functional experiments revealed that ßarrestin1 is exclusively involved in hDOR desensitization promoted by the peptides. Taken together, these results provide substantial evidence for a ßarrestin1-biased agonism at hDOR, where ßarrestin1 is differentially involved during receptor desensitization and endocytosis depending on the ligand.
Cellular signalling 11/2011; 24(3):699-707. · 4.09 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Experiments were designed to evaluate different variables of the dopaminergic system in the temporal cortex of surgically treated patients with temporal lobe epilepsy (TLE) associated with mesial sclerosis (MTLE, n=12) or with cerebral tumor or lesion (n=8). In addition, we sought to identify dopaminergic abnormalities in those patients with epilepsy that had comorbid anxiety and depression. Specifically, we investigated changes in dopamine and its metabolites, D1 and D2 receptors, tyrosine hydroxylase (TH) and dopamine transporter. Results obtained from patients with epilepsy were compared with those found in experiments using autopsy material. The neocortex of patients with MTLE demonstrated high D1 expression (1680%, p<0.05) and binding (layers I-II, 31%, p<0.05; layers V-VI, 28%, p<0.05), and decreased D2 expression (77%, p<0.05). The neocortex of patients with TLE secondary to cerebral tumor or lesion showed high expression of D1 receptors (1100%, p<0.05), and D2-like induced activation of G proteins (layers I-II, 503%; layers III-IV, 557%; layers V-VI, 964%, p<0.05). Both epileptic groups presented elevated binding to the dopamine transporter and low tissue content of dopamine and its metabolites. Analysis revealed the following correlations: a) D1 receptor binding correlated negatively with seizure onset age and seizure frequency, and positively with duration of epilepsy; b) D2 receptor binding correlated positively with age of seizure onset and negatively with duration of epilepsy; c) dopamine transporter binding correlated positively with duration of epilepsy and frequency of seizures; d) D2-like induced activation of G proteins correlated positively with the age of patients. When compared with autopsies and patients with anxiety and depression, patients without neuropsychiatric disorders showed high D2-like induced activation of G proteins, an effect that correlated positively with age of patient and seizure onset age, and negatively with duration of epilepsy. The present study suggests that alterations of the dopaminergic system result from epileptic activity and could be involved in the physiopathology of TLE and the comorbid anxiety and depression.
Neurobiology of Disease 09/2011; 45(1):499-507. · 5.40 Impact Factor
-
Manola Cuellar-Herrera,
Ana Luisa Velasco,
Francisco Velasco,
Laura Chavez,
Sandra Orozco-Suarez,
Guliz Armagan,
Ezgi Turunc,
Engin Bojnik,
Ayfer Yalcin,
Sandor Benyhe, Anna Borsodi,
Mario Alonso-Vanegas,
Luisa Rocha
[show abstract]
[hide abstract]
ABSTRACT: Mu opioid receptors (MOR) are known to be involved in seizure activity. The main goal of the present study was to characterize the MOR mRNA expression, binding, as well as G protein activation mediated by these receptors in epileptic hippocampus of patients with pharmacoresistant mesial temporal lobe epilepsy (TLE). In contrast with autopsy samples, hippocampus obtained from patients with mesial TLE demonstrated enhanced MOR mRNA expression (116%). Saturation binding experiments revealed significantly higher (60%) B(max) values for the mesial TLE group, whereas the K(d) values were not statistically different. Although mesial TLE group demonstrated high levels of basal binding for the G proteins (136%), DAMGO-stimulated [(35)S]GTPγS binding did not demonstrate significant alterations. In conclusion, our present data provide strong evidence that the epileptic hippocampus of patients with pharmacoresistant mesial TLE presents significant alterations in MOR. Such changes may represent adaptive mechanisms to compensate for other as yet unknown alterations.
Hippocampus 11/2010; 22(2):122-7. · 5.18 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Besides generally accepted lower analgesic potencies of opioids in neuropathic pain, our recent pharmacological reports have demonstrated that the effectiveness of the micro-opioid receptor (MOR) agonists in neuropathy might depends upon the chemical/structural property of these compounds (alkaloid vs. peptides). Such findings prompted us to investigate the changes in MOR mRNA expression (estimated by PCR) as well as MOR functional activity (examined by [(35)S]GTPgammaS binding) in the dorsal horn of the spinal cord and the dorsal root ganglia (DRG) of neuropathic rats at different time points after sciatic nerve ligation. We found that the spinal MOR mRNA level and agonist-stimulated [(35)S]GTPgammaS binding were not affected by nerve injury. In contrast, down-regulation of MOR mRNA in the ipsilateral side of DRG developed 3 (approximately 63% reduction) and 14 (approximately 89% reduction) days after the ligation. The decrease was paralleled with pronounced reduction in the stimulation of [(35)S]GTPgammaS binding by morphine (approximately 37-39%). Thus, neuropathy-induced specific dysfunction of MOR to activate G-protein together with changes in the MOR synthesis might be related, at least in part, to diminish analgesic efficacy of morphine in neuropathic pain. Interesting observations from current studies are linked to endomorphins (EMs), which do not affect the G protein stimulation of MOR after nerve ligation. This intriguing property of EMs, together with previously reported high analgesic efficacy of these compounds indicate that chemically/structurally different MOR agonists, particularly morphine versus EMs, may differentially interact with receptors causing distinct pharmacological effects in chronic pain.
Acta Neurovegetativa 03/2010; 117(4):421-9. · 2.73 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Leu- and Met-enkephalins are proenkephalin-derived endogenous pentapeptides with opioid (morphine) activity. Among the seven enkephalin units found in the human proenkephalin (PENK), the fourth copy being an octapeptide: Tyr-Gly-Gly-Phe-Met-Arg-Gly-Leu ((Hs)YGGFMRGL). Bioinformatic analysis of the available PENK sequences revealed the presence of other octapeptide orthologues in these precursor polypeptides. Four types of the elongated Met-enkephalins we identified by searching protein databases, (Xl)YGGFMRGY (three frog species and platypus), (Gg)YGGFMRSV (chicken and one fish species), (Hp)YGGFMNGF (shark) and (Mm)YGGFMRSL (mouse and two lungfish species) were chemically synthesized and studied in receptor binding and G-protein activation assays performed on rat brain membranes. All peptides have also been prepared containing oxidized methionine (M(O)). The overall binding and signalling profile of the novel octapeptides revealed moderate opioid agonist activities and a rank order of potencies for the mu approximately delta>kappa receptor binding sites. Peptides with the oxidized M(O) residue were found to be less potent in both receptor binding and G-protein stimulation studies. Phylogenetic neuropeptide libraries, defined here as a collection of mutationally different species variants of orthologous and paralogous peptide sequences, represent the natural molecular diversity of the neuropeptides. Such libraries can provide a wide range of structural information establishing comparative functional analyses. Since DNA sequencing data are rapidly increasing, more development in the natural peptide library approach is expected.
Neuroscience 10/2009; 165(2):542-52. · 3.38 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Nociceptin/orphanin FQ (N/OFQ) is an endogenous neuropeptide, which is widely distributed in central and peripheral nervous system. Some N/OFQ sequence unrelated hexapeptides can effectively bind to the N/OFQ peptide (NOP) receptor and they were used as template for structure-activity studies that lead to discovery of the new NOP selective ligands. In the present study, the pharmacological profile of the novel hexapeptide Ac-RYYRIR-ol was investigated using various in vitro assays including receptor binding and G-protein activation in rat brain membranes, mouse and rat vas deferens, guinea pig ileum, mouse colon and Ca(2+) mobilization assay in chinese hamster ovary (CHO) cells co-expressing the human recombinant NOP receptor and the C-terminally modified Galpha(qi5) protein. In rat brain membranes Ac-RYYRIR-ol displaced both [(3)H]nociceptin/OFQ and [(3)H]Ac-RYYRIK-ol with high affinity (pK(i) 9.35 and 8.81, respectively) and stimulated [(35)S]GTPgammaS binding showing however lower maximal effects than N/OFQ (alpha=0.28). The stimulatory effect of Ac-RYYRIR-ol was antagonized by the selective NOP receptor antagonist UFP-101. In the electrically stimulated mouse vas deferens Ac-RYYRIR-ol displayed negligible agonist activity while antagonizing in a competitive manner (pA(2) 7.99) the inhibitory effects of N/OFQ. Similar results were obtained in the rat vas deferens. In the mouse colon Ac-RYYRIR-ol produced concentration dependent contractile effects with similar potency and maximal effects as N/OFQ. Finally, in the Ca(2+) mobilization assay performed with CHO-hNOP-Galpha(qi5) cells Ac-RYYRIR-ol displayed lower potency and maximal effects (alpha=0.87) compared with N/OFQ. In conclusion, the novel NOP receptor selective hexapeptide Ac-RYYRIR-ol has been shown to have fine selectivity, high potency, furthermore agonist and antagonist effects toward the NOP receptors were measured in various assays; this is likely due to its partial agonist pharmacological activity.
Brain research bulletin 10/2009; 81(4-5):477-83. · 2.18 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: There is no information concerning signal transduction mechanisms downstream of the opioid/nociceptin receptors in the human epileptic brain. The aim of this work was to evaluate the level of G-proteins activation mediated by DAMGO (a mu receptor selective peptide) and nociceptin, and the binding to mu and nociceptin (NOP) receptors and adenylyl cyclase (AC) in neocortex of patients with pharmacoresistant temporal lobe epilepsy. Patients with temporal lobe epilepsy associated with mesial sclerosis (MTLE) or secondary to tumor or vascular lesion showed enhanced [3H]DAMGO and [3H]forskolin binding, lower DAMGO-stimulated [35S]GTPgammaS binding and no significant changes in nociceptin-stimulated G-protein. [3H]Nociceptin binding was lower in patients with MTLE. Age of seizure onset correlated positively with [3H]DAMGO binding and DAMGO-stimulated [35S]GTPgammaS binding, whereas epilepsy duration correlated negatively with [3H]DAMGO and [3H]nociceptin binding, and positively with [3H]forskolin binding. In conclusion, our present data obtained from neocortex of epileptic patients provide strong evidence that a) temporal lobe epilepsy is associated with alterations in mu opioid and NOP receptor binding and signal transduction mechanisms downstream of these receptors, and b) clinical aspects may play an important role on these receptor changes.
Neurobiology of Disease 07/2009; 35(3):466-73. · 5.40 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The synthetic hexapeptide Ac-Arg-Tyr-Tyr-Arg-Ile-Lys-ol (Ac-RYYRIK-ol) represents a highly potent and selective partial agonist ligand for the nociceptin/orphanin FQ (N/OFQ) peptide receptor (nociceptin receptor, NOPr). Ac-RYYRIK-ol has been labeled with tritium yielding [(3)H]Ac-RYYRIK-ol with exceptionally high specific radioactivity of 94Ci/mmol. The radioprobe is chemically stable even at 24 degrees C in ethanol solution for at least 4 days. No significant decomposition of the [(3)H]ligand occurred under the condition of the binding experiments indicating a fine enzymatic stability of the peptide. Radioreceptor binding studies were conducted using native neuronal NOPr preparation of rat brain membrane fractions and recombinant human nociceptin receptor ((h)NOPr) preparations from cultured Chinese Hamster Ovary (CHO) cells stably expressing (h)NOPr. Specific binding of the compound was reversible, saturable and of high affinity. No cross-reaction with the opioid receptors was observed suggesting superior NOPr selectivity of the ligand. Monophasic isotherm curves obtained in radioligand binding saturation and homologous displacement experiments indicated the presence of single binding sites in both preparations. Average densities of the [(3)H]Ac-RYYRIK-ol recognition sites were 237 and 749fmol/mg protein in rat brain and transfected cells, respectively. Equilibrium affinity values (K(d)s) were determined by three independent way providing identical results. In rat brain membranes K(d)s of 0.3-1.3nM were found depending upon the assay type. In homologous competition studies performed on (h)NOP-CHO cell membranes almost the same binding affinities were measured for Ac-RYYRIK-ol either with [(3)H]Ac-RYYRIK-ol (K(i) 2.8nM) or with [(3)H](Leu(14))nociceptin (2.3nM). A number of NOPr and opioid ligands were screened in heterologous displacement experiments and displayed a rank order of affinity profile being consistent with fairly good NOPr selectivity of the sites labeled by [(3)H]Ac-RYYRIK-ol. Taken together, the high molar activity, improved chemical and biological stability and the capability of the selective and high affinity labeling make this novel radioprobe available for further exploring the biochemical pharmacology and receptor-ligand interaction of the NOP receptor.
Neurochemistry International 06/2009; 55(7):458-66. · 2.86 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The recently discovered endomorphin 1 (Tyr-Pro-Trp-Phe-NH2) and endomorphin 2 (Tyr-Pro-Phe-Phe-NH2) were investigated with respect to their direct receptor-binding properties, and to their ability to activate G proteins and to inhibit adenylyl cyclase in both cellular and animal models. Both tetrapeptides activated G proteins and inhibited adenylyl cyclase activity in membrane preparations from cells stably expressing the μ opioid receptor, an effect reversed by the μ receptor antagonist CTAP ( d-Phe-Cys-Tyr- d-Trp-Arg-Thr-Pen-Thr-NH2), but they had no influence on cells stably expressing the δ opioid receptor. To further establish the selectivity of these peptides for the μ opioid receptor, brain preparations of mice lacking the μ opioid receptor gene were used to study their binding and signalling properties. Endomorphin 2, tritiated by a dehalotritiation method resulting in a specific radioactivity of 1.98 TBq/mmol (53.4 Ci/mmol), labelled the brain membranes of wild-type mice with a Kd value of 1.77 n m and a Bmax of 63.33 fmol/mg protein. In membranes of mice lacking the μreceptor gene, no binding was observed, and both endomorphins failed to stimulate [35S]guanosine-5′-O-(3-thio)triphosphate ([35S]GTPγS) binding and to inhibit adenylyl cyclase. These data show that endomorphins are capable of activating G proteins and inhibiting adenylyl cyclase activity, and all these effects are mediated by the μ opioid receptors.
European Journal of Neuroscience 10/2008; 12(2):577 - 584. · 3.63 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Formerly considered as an exclusively peripheral receptor, it is now accepted that CB(2) cannabinoid receptor is also present in limited amounts and distinct locations in the brain of several animal species, including mice. However, the possible roles of CB(2) receptors in the brain need to be clarified. The aim of our work was to study the mu-opioid receptor (MOR) mRNA expression level and functional activity after acute in vivo and in vitro treatments with the endocannabinoid noladin ether (NE) and with the CB(2) receptor antagonist SR144528 in brainstem of mice deficient in either CB(1) or CB(2) receptors. This study is based on our previous observations that noladin ether (NE) produces decrease in the activity of MOR in forebrain and this attenuation can be antagonized by the CB(2) cannabinoid antagonist SR144528, suggesting a CB(2) receptor mediated effect. We used quantitative real-time PCR to examine the changes of MOR mRNA levels, [(35)S]GTPgammaS binding assay to analyze the capability of mu-opioid agonist DAMGO to activate G-proteins and competition binding assays to directly measure the ligand binding to MOR in mice brainstem. After acute NE administration no significant changes were observed on MOR signaling. Nevertheless pretreatment of mice with SR144528 prior to the administration of NE significantly decreased MOR signaling suggesting the involvement of SR144528 in mediating the effect of MOR. mRNA expression of MORs significantly decreased both in CB(1) wild-type and CB(1) knockout mice after a single injection of SR144528 at 0.1mg/kg when compared to the vehicle treated controls. Consequently, MOR-mediated signaling was attenuated after acute in vivo treatment with SR144528 in both CB(1) wild-type and CB(1) knockout mice. In vitro addition of 1microM SR144528 caused a decrease in the maximal stimulation of DAMGO in [(35)S]GTPgammaS binding assays in CB(2) wild-type brainstem membranes whereas no significant changes were observed in CB(2) receptor knockouts. Radioligand binding competition studies showed that the noticed effect of SR144528 on MOR signaling is not mediated through MORs. Our data demonstrate that the SR144528 caused pronounced decrease in the activity of MOR is mediated via CB(2) cannabinoid receptors.
Neurochemistry International 09/2008; 53(6-8):309-16. · 2.86 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We examined the occurrence of possible changes in mRNA expression and the functional activity of opioid receptors after acute in vivo and in vitro treatment with the putative endogenous cannabinoid noladin ether. While noladin ether (NE) demonstrates agonist activity at CB1 cannabinoid receptors, recent data indicate that NE acts as a full agonist at CB2 cannabinoid receptors too. Considering the functional interactions between opioids and cannabinoids, it is of interest to examine whether NE affects the opioid system. To that end, we studied the influence of NE on mu-opioid receptor (MOR) mRNA expression and MOR mediated G-protein signaling. We used real-time PCR and [35S]GTPgammaS binding assays to examine the changes of MOR mRNA levels and the capability of the mu-opioid agonist peptide ([D-Ala2,(NMe)Phe4,Gly5-ol]enkephalin (DAMGO) in activating regulatory G-proteins via MORs in forebrain membrane fractions of wild-type (w.t., CB1+/+) and CB1 receptor deficient transgenic mice (knockout, CB1-/-). We found, that the expression of MOR mRNAs significantly decreased both in CB1+/+ and CB1-/- forebrain after a single injection of NE at 1 mg/kg when compared to control. Consequently, MOR-mediated signaling is attenuated after acute in vivo treatment with NE in both CB1+/+ and CB1-/- mice. Inhibition on MOR mediated activation is observed after in vitro NE administration as well. Radioligand binding competition studies showed that the noticed effect of NE on MOR signaling is not mediated through MORs. Both in vivo and in vitro attenuations of NE can be antagonized by the CB2 selective antagonist SR144528. Taken together, our data suggest that the NE caused pronounced decrease in the activity of MOR is mediated via CB2 cannabinoid receptors.
Neurochemistry International 02/2008; 52(1-2):321-8. · 2.86 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The effects with pretreatment with nociceptin (0.03-30nmol, i.c.v.) were evaluated on the threshold for eliciting afterdischarge (ADT), generation and spread of seizure activity and postictal depression in rats with kindling stimulation. Nociceptin produced a decrease in ADT (32-45%) in rats with partial seizures (PS, stage II-III), and an increase (61-92%) in rats with generalized seizures (GS, kindled state). Nociceptin did not modify the behavioral changes, spike frequency and duration of afterdischarge elicited at ADT in both experimental groups. In rats with GS, nociceptin enhanced postictal depression (34-44%) evaluated with a recycling paradigm. Autoradiography experiments revealed enhanced nociceptin opioid receptor (NOP) binding in medial amygdala (22-26%), frontal (21-23%) and entorhinal (27-32%) cortices, and reduced binding in the substantia nigra pars compacta (28%) and medial central gray (29%) of rats with PS. The GS group displayed significant decreased NOP binding (40-70%) in most of the brain areas evaluated. These results suggest that nociceptin facilitates ictal activity in rats with PS, whereas in animals with GS, it induces inhibitory effects on ADT and enhances the postictal period. These effects correlate with significant changes in NOP binding.
Epilepsy Research 01/2008; 77(2-3):75-84. · 2.29 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Opioid receptor binding was evaluated in parahippocampal cortex (PHC) obtained from patients with intractable mesial temporal lobe epilepsy (MTLE) with and without subacute high frequency electrical stimulation (HFS) in this brain area. Mu, delta and nociceptin receptor binding was determined by autoradiography in PHC of five patients (ESAE group) with MTLE history of 14.8 +/- 2.5 years and seizure frequency of 11 +/- 2.9 per month, two of them (40%) with mesial sclerosis. This group demonstrated antiepileptic effects following subacute HFS (130 Hz, 450 micros, 200-400 microA), applied continuously during 16-20 days in PHC. Values were compared with those obtained from patients with severe MTLE (history of 21.7 +/- 2.8 years and seizure frequency of 28.2 +/- 14 per month) in whom electrical stimulation did not induce antiepileptic effects (ESWAE group, n = 4), patients with MTLE in whom no electrical stimulation was applied (MTLE group, n = 4) and autopsy material acquired from subjects without epilepsy (n = 4 obtained from three subjects). Enhanced 3H-DAMGO (MTLE, 755%; ESAE, 375%; ESWAE, 693%), 3H-DPDPE (MTLE, 242%; ESAE, 80%; ESWAE, 346%) and 3H-nociceptin (MTLE, 424%; ESAE, 217%; ESWAE, 451%) binding was detected in the PHC of all epileptic groups. However, tissue obtained from ESAE group demonstrated lower opioid receptor binding (3H-DAMGO, 44.5%, p < 0.05; 3H-DPDPE, 47%, p < 0.05; 3H-nociceptin, 39.3%, p < 0.5) when compared with MTLE group. The present results indicate that a high effectiveness to the antiepileptic effects induced by HFS is associated with reduced opioid peptide binding.
Seizure 10/2007; 16(7):645-52. · 1.80 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: A novel opioid peptide antagonist analogue, [3H]Dmt-Tic-(2S,3R)betaMePhe-Phe, derived from the potent, delta-receptor selective TIPP tetrapeptide (Tyr-Tic-Phe-Phe) series was synthesized and radiolabeled by catalytic tritiation of its iodinated precursor peptide. The purified radioprobe exhibited a specific activity of 2.15 TBq/mmol (58 Ci/mmol). The novelty of this compound is that it contains structurally modified tyrosine residue (2',6'-dimethyltyrosine, Dmt1) replacing tyrosine (Tyr1) at the N-terminus, and beta-methyl substituted phenylalanine (betaMePhe3) at the third position. As the configuration of betaMePhe3 side-chain might be different due to diastereomerism, and accordingly can alter the biological activity, both unlabeled threo (2S,3R and 2R,3S) diastereomeric analogues were also prepared and included in this study. The affinity and selectivity (delta-opioid versus mu-opioid receptor) were evaluated by radioreceptor binding assays. Agonist or antagonist potencies were determined in [35S]GTPgammaS binding experiments using Chinese Hamster Ovary (CHO) cells selectively expressing delta- or mu-opioid receptors. The equilibrium binding of the radiolabeled peptide derivative [3H]Dmt-Tic-(2S,3R)betaMePhe-Phe to rat brain membranes was saturable and the Scatchard analysis indicated a single binding site with a Kd of 0.3 nM and a Bmax of 127 fmol/mg protein. A study of [3H]Dmt-Tic-(2S,3R)betaMePhe-Phe binding displacement by various receptor-type specific opioid ligands showed the rank order of competitor's potency delta > mu > kappa, suggesting selective labeling of opioid delta-sites. In the functional tests, the (2S,3R) and (2R,3S) peptides exhibited partial agonist behaviour by weakly stimulating regulatory G-proteins in CHO cell membranes transfected with different receptors. Both isomers were quite weak partial agonists at the delta-receptor and reasonable partial agonists at the mu-receptor, with a prevalence of (2S,3R) over (2R,3S) for the mu-receptor. Consistent with these observations both stereomers competitively inhibited the stimulation of [35S]GTPgammaS binding induced by the prototype delta-agonist peptide (pClPhe4)-DPDPE in delta(m) CHO cell membranes, and still the (2S,3R) compound exerted more potent delta-antagonist effect. [3H]Dmt-Tic-(2S,3R)betaMePhe-Phe represents a high affinity new radioligand and also constitute further example of the influence of beta-methyl substitution on the potency and selectivity of TIPP analogues, thus becoming a valuable biochemical and pharmacological tool in opioid research.
Brain Research Bulletin 09/2007; 74(1-3):119-29. · 2.82 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Prodynorphins (PDYNs) from the African clawed frog (Xenopus laevis), originally described as 'proxendorphins', are novel members of the family of opioid-like precursor polypeptides and were recently discovered based on polymerase chain reaction (PCR) isolates from a Xenopus brain cDNA library. This amphibian prodynorphin was found in two isoforms, (Xen)PDYN-A and (Xen)PDYN-B, consisting of 247 and 279 amino acids, respectively. Each prepropeptide contains five potential opioid-like peptides, collectively named xendorphins. One of these, xendorphin B1 ((Xen)PDYN-B sequence 96-111: YGGFIRKPDKYKFLNA), is a hexadecapeptide that displaced [3H]naloxone and the radiolabelled kappa opioid, [3H]dynorphin A (1-17), with nanomolar affinity from rat brain membranes. Using the acetic acid pain test, the present study examined the antinociceptive effects of spinally administered xendorphin B1 in amphibians. Xendorphin B1 produced a long-lasting and dose-dependent antinociceptive effect in the Northern grass frog (Rana pipiens) with an ED50 value of 44.5 nmol/frog. The antinociceptive effects of xendorphin B1 were significantly blocked by pretreatment with the non-selective opioid antagonist, naltrexone. This is the first report of the in vivo characterization of a non-mammalian prodynorphin-derived peptide and suggests that xendorphin peptides may play a role in the modulation of noxious information in vertebrates.
Brain Research Bulletin 04/2007; 71(6):628-32. · 2.82 Impact Factor
