Interaction of clozapine with metformin in a schizophrenia rat model.

The low efficacy of antipsychotic drugs (e.g., clozapine) for negative symptoms and cognitive impairment has led to the introduction of adjuvant therapies. Because previous data suggest the procognitive potential of the antidiabetic drug metformin, this study aimed to assess the effects of chronic clozapine and metformin oral administration (alone and in combination) on locomotor and exploratory activities and cognitive function in a reward-based test in control and a schizophrenia-like animal model (Wisket rats). As impaired dopamine D1 receptor (D1R) function might play a role in the cognitive dysfunctions observed in patients with schizophrenia, the second goal of this study was to determine the brain-region-specific D1R-mediated signaling, ligand binding, and mRNA expression. None of the treatments affected the behavior of the control animals significantly; however, the combination treatment enhanced D1R binding and activation in the cerebral cortex. The Wisket rats exhibited impaired motivation, attention, and cognitive function, as well as a lower level of cortical D1R binding, signaling, and gene expression. Clozapine caused further deterioration of the behavioral parameters, without a significant effect on the D1R system. Metformin blunted the clozapine-induced impairments, and, similarly to that observed in the control animals, increased the functional activity of D1R. This study highlights the beneficial effects of metformin (at the behavioral and cellular levels) in blunting clozapine-induced adverse effects.

Caffeine - treat or trigger? Disparate behavioral and long-term dopaminergic changes in control and schizophrenia-like Wisket rats.

The influence of caffeine on behavioral functions in both healthy and schizophrenic subjects is controversial. Here we aimed to reveal the effects of repeated caffeine pre- and post-training treatments on motor and exploratory activities and cognitive functions in a reward-based test (Ambitus) along with a brain region-specific dopamine D2 receptor profile in control and schizophrenia-like WISKET model rats. In the control animals, pre-treatment caused temporary enhancement in motor activity, while permanent improvement in learning function was detected in the WISKET animals. Post-treatment produced significant impairments in both groups. Caffeine caused short-lasting hyperactivity followed by a rebound in the inactive phase determined in undisturbed circumstance. Caffeine treatment substantially enhanced the dopamine D2 receptor mediated G-protein activation in the prefrontal cortex and olfactory bulb of both groups, while it increased in the dorsal striatum and cerebral cortex only in the WISKET animals. Caffeine enhanced the maximal binding capacity in the hippocampus and cerebral cortex of WISKET animals, but it decreased in the prefrontal cortex of the control animals. Regarding the dopamine D2 receptor mRNA expression, caffeine treatment caused significant enhancement in the prefrontal cortex of WISKET animals, while it increased the hippocampal dopamine D2 receptor protein amount in both groups. This study highlights the disparate effects of caffeine pre- versus post-training treatments on behavioral parameters in both control and schizophrenia-like animals and the prolonged changes in the dopaminergic system. It is supposed that the delayed depressive effects of caffeine might be compensated by frequent coffee intake, as observed in schizophrenic patients.

A novel non-opioid binding site for endomorphin-1.

Endomorphins are natural amidated opioid tetrapeptides with the following structure: Tyr-Pro-Trp-Phe-NH2 (endomorphin-1), and Tyr-Pro-Phe-Phe-NH2 (endomorphin-2). Endomorphins interact selectively with the µ-opioid or MOP receptors and exhibit nanomolar or sub-nanomolar receptor binding affinities, therefore they suggested to be endogenous agonists for the µ-opioid receptors. Endomorphins mediate a number of characteristic opioid effects, such as antinociception, however there are several physiological functions in which endomorphins appear to act in a fashion that does not involve binding to and activation of the µ-opioid receptor. Our recent data indicate that a radiolabelled [3H]endomorphin-1 with a specific radioactivity of 2.35 TBq/mmol - prepared by catalytic dehalogenation of the diiodinated peptide precursor in the presence of tritium gas - is able to bind to a second, naloxone insensitive recognition site in rat brain membranes. Binding heterogeneity, i.e., the presence of higher (Kd = 0.4 nM / Bmax = 120 fmol/mg protein) and lower (Kd = 8.2 nM / Bmax = 432 fmol/mg protein) affinity binding components is observed both in saturation binding experiments followed by Schatchard analysis, and in equilibrium competition binding studies. The signs of receptor multiplicity, e.g., curvilinear Schatchard plots or biphasic displacement curves are seen only if the non-specific binding is measured in the presence of excess unlabeled endomorphin-1 and not in the presence of excess unlabeled naloxone. The second, lower affinity non-opioid binding site is not recognized by heterocyclic opioid alkaloid ligands, neither agonists such as morphine, nor antagonists such as naloxone. On the contrary, endomorphin-1 is displaced from its lower affinity, higher capacity binding site by several natural neuropeptides, including methionine-enkephalin-Arg-Phe, nociceptin-orphanin FQ, angiotensin and FMRF-amide. This naloxone-insensitive, consequently non-opioid binding site seems to be present in nervous tissues carrying low density or no µ-opioid receptors, such as rodent cerebellum, or brain of µ-opioid receptor deficient (MOPr-/-) transgenic or 'knock-out' (K.O.) mice. The newly described non-opioid binding component is not coupled to regulatory G-proteins, nor does it affect adenylyl cyclase enzyme activity. Taken together endomorphin-1 carries opioid and, in addition to non-opioid functions that needs to be taken into account when various effects of endomorphin-1 are evaluated in physiological or pathologic conditions.

Effects of synthetic analogues of human opiorphin on rat brain opioid receptors.

Human opiorphin (Gln-Arg-Phe-Ser-Arg; QRFSR-peptide) is a physiological inhibitor of enkephalin-inactivating peptidases. We previously demonstrated that opiorphin can substitute for the classic mixture of peptidase inhibitors and greatly improves the specific binding and affinity of the enkephalin-related peptide [(3)H]MERF (Tyr-Gly-Gly-Phe-Met-Arg-Phe; YGGFMRF) for rat brain opioid receptors. To extend the metabolic stability of opiorphin in human plasma two functional derivatives were designed, i.e., Cys-[(CH(2))(6)]-QRF-[Ser-O-octanoyl]-R peptide (monomeric CC6-opiorphin) and its cystine-dipeptide (dimeric CC6-opiorphin) derivative. We found that, in homologous competition experiments, the affinity of [(3)H]MERF for rat brain opioid receptors was significantly increased in the presence of monomeric and dimeric CC6-opiorphin, compared to control-Tris buffer. In addition ten times lower concentrations (5 µM) than those required for native opiorphin (50 µM) were sufficient. In heterologous competition experiments, using unlabeled dynorphin(1-10), affinity increases were also observed: increases in binding were similar with either monomeric or dimeric CC6-opiorphin. Surprisingly, these opiorphin analogues displayed weak competitive effects on [(3)H]MERF binding to rat brain opioid receptors in the absence of unlabeled MERF, effects never observed for the native opiorphin. In conclusion, CC6-opiorphin compounds are certainly more potent than the native opiorphin in increasing the binding and the affinity of homologous and heterologous competition, but the binding enhancement occurs only at temperatures much higher than 0°C, specifically at 24°C.

A novel µ-opioid receptor ligand with high in vitro and in vivo agonist efficacy.

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).

Phylogenetic diversity and functional efficacy of the C-terminally expressed heptapeptide unit in the opioid precursor polypeptide proenkephalin A.

The heptapeptide Met-enkephalin-Arg6-Phe7 (MERF) with the sequence of YGGFMRF is a potent endogenous opioid located at the C-terminus of proenkephalin-A (PENK), the common polypeptide precursor of Met- and Leu-enkephalin. Our systematic bioinformatic survey revealed considerable sequence polymorphism at the heptapeptide region of different PENK prepropeptides among 56 vertebrate animals. Four orthologous heptapeptides with single or double amino acid replacements were identified among 15 animals, such as YGGFMGY (zebrafish), YGGFMRY (newt), YGGFMKF (hedgehog tenrek) and YGGFMRI (mudpuppy). Each novel heptapeptide, together with the mammalian consensus MERF and Met-enkephalin, were chemically synthesized and subjected to functionality studies, using radioligand binding competition and G-protein activation assays in rat brain membranes. Equilibrium binding affinities changed from good to modest as measured by receptor type selective [3H]opioid radioligands. The relative affinities of the heptapeptides reveal slight mu-receptor (MOP) preference over the delta-receptors (DOP). [35S]GTPγS assay, which measures the agonist-mediated G-protein activation, has demonstrated that all the novel heptapeptides were also potent in stimulating the regulatory G-proteins. All peptides were effective in promoting the agonist induced internalization of the green fluorescence protein-tagged human mu-opioid receptor (hMOP-EGFP) stably expressed in HEK293 cells. Thus, the C-terminally processed PENK heptapeptide orthologs exhibited satisfactory bioactivities, moreover they represent further members of the so-called "natural combinatorial neuropeptide library" emerged by evolution.

Nociceptin inhibits uterine contractions in term-pregnant rats by signaling through multiple pathways.

The actions of the endogenous peptide nociceptin (PNOC; previously abbreviated as N/OFQ) on the myometrium have not been investigated previously. Our aim was to study the presence and functional role of PNOC in the modulation of uterine contractility in pregnant rats at term. The presence of PNOC and its receptors (OPRL1; previously called NOP) in the uterus were detected by radioimmunoassay and radioligand-binding experiments. The PNOC-stimulated G protein activation was assessed by a [(35)S]GTPgammaS-binding technique. The effects of PNOC in uterine rings precontracted with KCl or oxytocin were also tested in vitro. Uterine levels of cAMP were measured by enzyme immunoassay. The K(+) channel blockers tetraethylammonium and paxilline were used to study the role of K(+) channels in mediating the uterine effects of PNOC. Both PNOC and OPRL1 were present in the uterus. PNOC revealed a maximum contraction inhibition of approximately 30%, which was increased to 40% by naloxone. Naloxone and pertussis toxin significantly attenuated the G protein-stimulating effect of PNOC. The uterine cAMP levels were elevated by PNOC and naloxone and after preincubation with pertussis toxin. Tetraethylammonium and paxilline reduced the contraction-inhibiting effect of PNOC and naloxone to approximately 10% and 15%, respectively. We presume that PNOC plays a role in regulating uterine contractility at term. Its effect is mediated partly by stimulatory heterotrimeric G (G(s)) proteins coupled to OPRL1 receptors and elevated cAMP levels, and also by Ca(2+)-dependent K(+) channels. Our results demonstrate a novel action and signaling pathway for PNOC that might be a potential drug target.

Bioinformatic and biochemical studies on the phylogenetic variability of proenkephalin-derived octapeptides.

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.

Binding studies of novel, non-mammalian enkephalins, structures predicted from frog and lungfish brain cDNA sequences.

Leu- and Met-enkephalin were the first endogenous opioid peptides identified in different mammalian species including the human. Comparative biochemical and bioinformatic evidence indicates that enkephalins are not limited to mammals. Various prodynorphin (PDYN) sequences in lower vertebrates revealed the presence of other enkephalin fingerprints in these precursor polypeptides. Among the novel enkephalins Ile-enkephalin (Tyr-Gly-Gly-Phe-Ile) was primarily observed in the African clawed frog (Xenopus laevis) PDYNs, while the structure of Phe-enkephalin (Tyr-Gly-Gly-Phe-Phe) was predicted by analyzing brain cDNA sequences encoding a PDYN of the African lungfish (Protopterus annectens). Ile-enkephalin can also be found in the PDYNs of four other fish species including the eel, bichir, zebrafish and tilapia, but no further occurrence for the Phe-enkephalin motif is available as yet. Based on sequencing data, the biological relevance of Phe- and Ile-enkephalin is suggested, because both of them can arise by regular posttranslational enzymatic processing of the respective neuropeptide precursors. In various receptor binding assays performed on rat brain membrane preparations both of the new peptides turned out to be moderate affinity opioids with a weak preference for the delta-opioid receptor (DOP) sites. Phe-enkephalin of the lungfish displayed rather unexpectedly low affinities toward the mu-opioid receptor (MOP) and DOP, while exhibiting moderate affinity toward the kappa-opioid receptor (KOP). In receptor-mediated G-protein activation assays measured by the stimulation of [(35)S]GTPgammaS binding, Met-enkephalin produced the highest stimulation followed by Leu-enkephalin, Ile-enkephalin and Phe-enkephalin, whereas the least efficacious among these endogenous peptides was still more effective than the prototype opiate agonist morphine in these functional tests.

Altered gene expression and functional activity of opioid receptors in the cerebellum of CB1 cannabinoid receptor knockout mice after acute treatments with cannabinoids.

Numerous studies have shown functional links between the cannabinoid and opioid systems. The goal of this study was to evaluate whether acute treatments by endogenous cannabinoid agonist, selective CB1 or CB2 receptor antagonists modulate the expression of mu- (MOR) and delta- (DOR) opioid receptor mRNA levels and functional activity in the cerebellum of transgenic mice deficient in the CB1 type of cannabis receptors. We examined the effect of noladin ether (endogenous cannabinoid agonist) pretreatment on MOR and DOR mRNA expression by using reverse transcription and real-time polimerase chain reaction (PCR) and the ability of subsequent application of the opioid agonists to activate G-proteins, as measured by [35S]GTPgammaS binding, in wild-type (CB1+/+) and CB1 cannabinoid receptor deficient (CB1-/-, 'knockout', K.O.) mice. The acute administration of noladin ether markedly reduced MOR-mediated G-protein activation and caused a significant increase in the level of MOR mRNAs in the cerebella of wildtype, but not in the CB1-/- mice. No significant differences were observed in DOR functional activity and mRNA expression in wild-type animals. In CB1-/- mice the expression of DOR mRNA increased after noladin ether treatment, but no changes were found in DOR functional activity. In addition, Rimonabant (selective central cannabinoid CB1 receptor antagonist) and SR144528 (selective peripheral cannabinoid CB2 receptor antagonist) caused significant potentiation in MOR functional activity in the wild-type animals, whereas DOR mediated G-protein activation was increased in the CB1-/- mice. In contrast, Rimonabant and SR144528 decreased the MOR and DOR mRNA expressions in both CB1+/+ and CB1-/- mice. Taken together, these results indicate that acute treatment with cannabinoids causes alterations in MOR and DOR mRNA expression and functional activity in the cerebella of wild-type and CB1 knockout mice indicating indirect interactions between these two signaling systems.

Kappa-receptor selective binding of opioid ligands with a heterocyclic bicyclo[3.3.1]nonan-9-one structure.

Previous pharmacological results have suggested that members of the heterocyclic bicyclo[3.3.1]nonan-9-one-like compounds are potent kappa-opioid receptor specific agonists. One lead molecule of this series. called compound 1 (dimethyl 7-methyl-2,4-di-2-pyridyl-3.7-diazabicyclo[3.3.1]nonan-9-one-1,5-dicarboxylate) exhibited high affinity for [3H]ethylketocyclazocine and [3H]U-69.593 binding sites in guinea pig cerebellar membranes which known to be a good source for kappa1 receptors. It was shown by molecular modelling that heterocyclic bicyclo[3.3.1]nonan-9-ones fit very well with the structure of ketazocine, a prototypic kappa-selective benzomorphan compound; when compared to the arylacetamide structure of U-69.593, a specific kappa1-receptor agonist, a similar geometry was found with a slightly different distribution of the charges. It is postulated, that the essential structural skeleton involved in the opioid activity is an aryl-propyl-amine element distributed along the N7-C6-C5-C4-aryl bonds.

Restraint stress and anti-tumor immune response in mice.

Psychological stress modulates the immune system through the hypothalamic-pituitary-adrenal axis, the sympatho-adrenomedullary axis and the opioid system. According to literature data, restraint stress increases the immune cell apoptosis, decreases the spleen and thymus cell content, the natural killer (NK) activity in the spleen, and it compromises the anti-tumor immune response in mice. We immobilized mice in two consecutive nights, and then determined the cell number, apoptosis, NK cell content, NK activity and the level of cytokine mRNAs (TNF-beta, TNF-alpha, IL-4, IL-5, IL-1alpha, IFN-gamma, IL-2, IL-6, IL-1beta and IL-3) in the thymus and spleen. No consistent changes were detected in any of the immune parameters either in C57Bl/6 or in DBA/2 mice. Stressed or control B6 mice were injected with B16 melanoma cells immediately after the immobilization or one week later. No significant differences were found in the growth of primary tumors and lung metastases in stressed and control animals. Taken together, our mice, kept in a general-purpose non-SPF animal house, seemed to be refractory to the stress-induced immunomodulation. Our interpretation is that stress-induced immunomodulation can occur only in mice isolated from any background stressors, or rather natural stimuli, throughout their life.

Receptor constants for endomorphin-1 and endomorphin-1-ol indicate differences in efficacy and receptor occupancy.

The opioid properties of endomorphin derivatives containing a C-terminal alcoholic(-ol) function were compared to the parent amidated compounds in isolated organs (longitudinal muscle strip of guinea-pig ileum and mouse vas deferens). Similar data were also generated for the mu-opioid receptor selective agonist synthetic peptide (D-Ala2, MePhe4, Gly5-ol)-enkephalin (DAMGO) and its Gly5-NH2 congener (DAMGA). Endomorphin-1-ol (Tyr-Pro-Trp-Phe-ol) had an IC50 of 80.6 nM in mouse vas deferens and 61.2 nM in guinea-pig ileum; the corresponding values for endomorphin-2-ol (Tyr-Pro-Phe-Phe-ol) were 49.6 and 48.2 nM, for DAMGO 59.8 and 29.2 nM, respectively. As it was indicated by the antagonism by naltrexone, the agonist actions were exerted exclusively at mu-opioid receptors in both organs. The -ol derivatives were slightly (2.3-4.3 times) less potent than the parent amides in the bioassays: all peptides had, apparently, full agonist properties in intact preparations. With the aim of revealing potential partial agonist properties among the investigated peptides, we partially inactivated the mu-opioid receptor pool in mouse vas deferens by 5x10(-7) M beta-funaltrexamine. The calculated receptor constants indicated a "high-affinity, low intrinsic efficacy" profile (i.e. a potential partial agonist property) for endomorphin-1, an intermediate character for endomorpin-1-ol and full agonism for DAMGA and DAMGO. Apparently, a higher receptor fraction remained accessible for endomorphin-1 (42.8%) than for the -ol congener (14.0%), DAMGO (20.2%) and DAMGA (14.1%) after partial inactivation.

Radioligand binding properties of VV-hemorphin 7, an atypical opioid peptide.

Receptor binding properties of the hemoglobin-derived nonapeptide VV-hemorphin 7 (Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg-Phe-OH) were studied using both the unlabelled form and tritium-labelled derivative of the peptide. In binding studies using selective opioid radioligands, VV-hemorphin 7 exhibited a rank order of potency of mu > kappa >> delta. VV-hemorphin 7 was tritiated resulting in a compound with 1.03 TBq/mmol (27.8 Ci/mmol) specific radioactivity. The maximal number of binding sites was found to be 66.5 pmol/mg protein with an affinity of 82.1 nM in rat brain membranes. In competition studies, marked similarity was observed to the binding profile of the naturally occurring opioid heptapeptide Met-enkephalin-Arg-Phe (MERF) and its analogues to their naloxone-insensitive binding site. The common -Arg-Phe sequence at the carboxyl terminal end, which is similar to those of other endogenous peptides (-Arg-Phe-NH(2) in neuropeptide FF and FMRF-NH(2)) brings attention to the C-terminal end of the molecule and points to the possible existence of a common nonopioid binding site in mammals.

Promitogenic effects of ethanol, methanol, and ethanolamine in insulin-treated fibroblasts.

The zinc-dependent potentiating effect of ethanol (EtOH) on insulin-stimulated DNA synthesis was studied with a focus on the possible site of EtOH action and the ability of other alcohols to elicit similar promitogenic effects. In serum-starved (27 hr) NIH 3T3 fibroblasts, 200-300 mM methanol (MeOH) and 0.1-1.5 mM ethanolamine (Etn), but not 3- to 9-carbon normal alcohols, enhanced the effect of insulin on DNA synthesis to varying extents. The promitogenic effects of EtOH and MeOH, but not that of Etn, required the presence of 15-25 microM zinc. The potentiating effects of Etn were enhanced by 5 mM choline (Cho) and inhibited by 1-3 mM hemicholinium-3 (HC-3), an inhibitor of Cho transporter and Cho kinase. In the presence of 15 microM zinc, 40 mM EtOH, which had no effect on its own, inhibited the potentiating effects of Cho and enhanced the inhibitory effects of HC-3 on synergistic stimulation of DNA synthesis by Etn and insulin. On the other hand, both Cho and HC-3 partially inhibited the promitogenic effect of 80 mM EtOH in the presence of 25 microM zinc. After a 10-min incubation, EtOH decreased the amount of cell-associated [(14)C]Cho in the absence but not in the presence of HC-3. After a 40-min incubation, Cho (5 mM) partially inhibited the cellular uptake as well as the metabolism of [(14)C]Etn. Whereas after the 40-min incubation 80 mM EtOH had no effects on Etn metabolism, in the absence of Cho it decreased the amount of cell-associated [(14)C]Etn. However, EtOH had no detectable effects on cell association of [(14)C]Etn after the 10-min incubation. The results suggest that in NIH 3T3 fibroblasts EtOH is a remarkably specific promitogen, and that it may act via a cell membrane site(s), also regulated by Cho (agonist) and HC-3 (antagonist), which can influence membrane binding and the promitogenic activity of Etn.

Receptor binding and G-protein activation by new Met5-enkephalin-Arg6-Phe7 derived peptides.

Met5-enkephalin-Arg6-Phe7 (Tyr-Gly-Gly-Phe-Met-Arg-Phe, MERF) is a naturally occurring heptapeptide that binds to opioid and non-opioid recognition sites in the central nervous system. Four synthetic analogs with single or double amino acid substitutions were prepared by solid phase peptide synthesis to achieve proteolytically more stable structures: Tyr-D-Ala-Gly-Phe-Met-Arg-Phe (I), Tyr-D-Ala-Gly-Phe-D-Nle-Arg-Phe (II), Tyr-D-Ala-Gly-Phe-L-Nle-Arg-Phe (III) and Tyr-Gly-Gly-Phe-L-Nle-Arg-Phe (IV). In this study receptor binding characteristics and G-protein activation of MERF and its derivatives were compared in crude membrane fractions of frog and rat brain. Synthetic MERF-derived peptides were potent competitors for [3H]MERF and [3H]naloxone binding sites with the exception of analog (II) which turned to be substantially less active. The presence of 100 mM NaCl or 100 microM 5'-guanylylimidodiphosphate, Gpp(NH)p, decreased the affinity of the peptides in [3H]naloxone binding assays, suggesting that these ligands might act as agonists at the opioid receptors. Some of the compounds were also used to stimulate guanosine-5'-O-(3-[gamma-[35S]thio)triphosphate ([35S]GTPgammaS) binding in rat and frog brain membranes at concentrations of 10(-9)-10(-5) M. The EC50 values of analog (II) were the highest in both tissues. Analog (I) was as effective as MERF in rat brain membranes, but showed lower maximal stimulation in frog brain preparation. Again, analog (II) seemed to be the least efficacious peptide that stimulated [35S]GTPgammaS binding only by 59%. Specificity of the peptides was further investigated by the inhibition of agonist-stimulated [35S]GTPgammaS binding in the presence of selective antagonists for the opioid receptor types. The mu-selective antagonist cyprodime displayed the lowest potency in inhibiting the effects of the peptides, whereas norbinaltorphimine (kappa-selective antagonist) and naltrindole (delta-selective antagonist) were quite potent in both tissues. We concluded that MERF and its derivatives are able to activate G-proteins mainly via kappa- and delta-opioid receptors.

Comparison of the endogenous heptapeptide Met-enkephalin-Arg6-Phe7 binding in amphibian and mammalian brain.

In previous communications [4, 38] we published that [3H]Met-enkephalin-Arg6-Phe7 (MERF) binds to opioid (kappa2 and delta) and sigma2 sites in frog and rat brain membrane preparations, however no binding to kappa1 sites could be established. In the present paper we compare the frog, rat and guinea pig brain membrane fractions with respect to their MERF binding data. No qualitative differences were found between the three species but specific binding of labelled MERF was maximal in frog brain and lowest in guinea pig brain, which corresponds to their kappa2 opioid receptor distribution. The naloxone resistant binding was also present in all investigated species and varied from 25% in frog and guinea pig cerebrum, to 50% in rat cerebrum and cerebellum, but no naloxone inhibition was found in guinea pig cerebellum where no kappa2 opioid receptors have been found. The presence of sigma2-like receptor was demonstrated in each investigated membrane fraction with displacement experiments using (-)N-allyl-normetazocine as competitor of tritiated MERF. It was shown that this site was responsible for 60-80% of [3H]MERF binding. The remaining part of the naloxone resistant labelled MERF binding could be displaced only with endogenous opioid peptides as met-enkephalin, dynorphin and beta-endorphin. The eventual physiological role of multiple MERF receptors is discussed.

Irreversible labelling of the opioid receptors by a melphalan-substituted [Met5]enkephalin-Arg-Phe derivative.

[Met5]enkephalin-Arg-Phe (Tyr-Gly-Gly-Phe-Met-Arg-Phe) was modified with the methyl esther of melphalan (Mel; 4-bis(2-chloroethyl)amino-L-phenylalanine) and the resulting compounds were studied for their opioid binding properties in guinea pig and rat brain membranes. Three new peptides, with a substitution of a single amino acid, were synthesized (Mel-Gly-Gly-Phe-Met-Arg-Phe, Tyr-Gly-Gly-Mel-Met-Arg-Phe and Tyr-Gly-Gly-Phe-Met-Arg-Mel). In the rat brain, none of these ligands displayed any type specificity, whereas in guinea pig brain membranes the C-terminally modified peptide, Tyr-Gly-Gly-Phe-Met-Arg-Mel ([Mel7]peptide), displayed a kappa-binding profile and was a weak kappa-opioid-receptor agonist in isolated guinea pig ileum. The effect of sodium ions on [Mel7]peptide competition against [3H]naloxone binding indicated a weak agonist nature of the compound. When guinea pig brain membranes were preincubated with 1-10 microM of [Mel7]peptide, an apparently irreversible inhibition of [3H]naloxone ligand binding was observed. These results suggest that the heptapeptide containing melphalan at the C-terminus can be used as a relatively high-affinity irreversible label for the kappa-opioid receptor.

Nociceptin binding sites in frog (Rana esculenta) brain membranes.

The recently discovered natural heptadecapeptide nociceptin (orphanin FQ) shares some homology with the opioid peptides but it binds to a distinct receptor type, termed nociceptin receptor. This study demonstrates the presence of specific nociceptin recognition sites in brain membrane fractions of an amphibian, Rana esculenta. Para-iodo-Phe(1)-nociceptin-amide was radiolabelled by catalytic dehalotritiation, resulting in p[(3)H]Phe(1)-nociceptin-amide of 25 Ci/mmol specific radioactivity. Specific binding of [(3)H]nociceptin-amide to frog brain membranes was found to be saturable and of high affinity with equilibrium K(d) values in the low nanomolar range. A single set of binding sites with about 180 fmol/mg protein maximal binding capacity was obtained in saturation and competition experiments. [(3)H]Nociceptin-amide binding could easily be inhibited by synthetic nociceptin compounds but not by opioid ligands. Both sodium ions and 5'-guanylylimidodiphosphate decreased the binding of the radioligand by transferring the receptor to a lower affinity state. Nociceptin dose-dependently stimulated the binding of the nonhydrolysable, radiolabeled GTP-analogue guanosine-5'-O-(3-thio)triphosphate ([(35)S]GTPgammaS) to G-proteins in frog brain membranes. Addition of 1 microM naloxone caused no significant change in the curves, indicating that nociceptin-mediated activation of G-proteins occurred through nonopioid mechanism.

Characterization of [3H]Met-enkephalin-Arg6-Phe7 binding to multiple sites in rat and guinea pig cerebellum.

[3H]Met-enkephalin-Arg6-Phe7 (MERF) has been shown to label opioid (kappa2 and delta) and sigma2 sites in rat and frog brain membrane preparations, and no specific binding to kappa1 opioid receptors could be established (refs. 6 and 8). In this study the binding was examined in rat cerebellar membranes which are relatively rich in kappa2-sites, and in guinea pig cerebellar preparations where kappa1 opioid receptors are almost exclusively present. In accordance with our previous results, [3H]MERF binding could not be displaced in guinea pig cerebellar membranes neither with U-69,593 nor with naloxone or levorphanol suggesting no interaction with opioid sites, nevertheless a Kd of 2.8 nM was calculated in cold saturation experiments. In rat cerebellar membrane fractions about the half of the specific [3H]MERF binding sites was inhibited by opiate alkaloids such as naloxone, ethylketocyclazocine, or bremazocine. This portion of the heptapeptide binding sites was stereoselective as demonstrated by the difference in the affinities of the enantiomeric compounds levorphanol and dextrorphan, therefore it would represent an opioid site. In both tissues (-)N-allyl-normetazocine (SKF-10,047), which is also considered as sigma2 ligand, displayed the highest affinities. Among opioid peptides beta-endorphin and dynorphin(1-13) showed the highest potencies, displacing [3H]MERF also from its non-opioid sites. It was concluded therefore that [3H]MERF does not bind to kappa1 sites, and besides kappa2-opioid sites substantial binding to peptide preferring non-opioid sites, and/or sigma2 receptors also occurs.