Pharmacological characterization of the nociceptin receptor which mediates reduction of alcohol drinking in rats

Chronic intracerebroventricular (ICV) treatment with nociceptin/orphanin FQ (NC), the endogenous ligand for the opioid receptor-like 1 (ORL1) receptor, reduces ethanol intake in alcohol-preferring rats and abolishes the rewarding properties of ethanol in the place conditioning paradigm. To pharmacologically characterize the receptor involved, the present study evaluated the effect on ethanol drinking in genetically selected Marchigian Sardinian alcohol-preferring (msP) rats of ICV injections for 8 days of NC or of the NC analogs NC(1-17)NH(2), NC(1-13)NH(2), NC(1-12)NH(2) and [Nphe(1)]NC(1-13)NH(2). In vitro studies indicate that NC, NC(1-17)NH(2), NC(1-13)NH(2) and NC(1-12)NH(2) are agonists, while [Nphe(1)]NC(1-13)NH(2) is a selective antagonist at the ORL1 receptor. Freely feeding and drinking rats were offered 10% ethanol 30 min/day at the beginning of the dark phase of the light cycle. NC significantly attenuated ethanol intake at 500 or 1000 ng/rat (210 or 420 pmol/rat). NC(1-17)NH(2), markedly reduced ethanol intake, but its effect was statistically significant at 1000 (420 pmol/rat), not at 500 ng/rat (210 pmol/rat). After the end of treatment ethanol drinking promptly came back to baseline level. Ethanol consumption was also reduced by NC(1-13)NH(2); however, its effect was less potent and pronounced. NC(1-12)NH(2) did not modify ethanol intake at doses up to 4000 ng/rat (2339 pmol/rat). Water and food consumption were not modified. Treatment with [Nphe(1)]NC(1-13)NH(2), 66 or 99 microg/rat, did not modify ethanol intake; however, [Nphe(1)]NC(1-13)NH(2), 66 microg/rat, given just before 1000 ng/rat of NC(1-17)NH(2), abolished the effect of the agonist. The present results show that the 13 amino acid N-terminal sequence of NC is essential for the effect on ethanol intake and indicate that [Nphe(1)]NC(1-13)NH(2) acts as an antagonist to block the effect of NC. These findings provide further evidence that selective agonists at the ORL-1 receptor attenuate ethanol intake in alcohol-preferring rats and suggest that the NC/ORL1 system may represent an interesting target for treatment of alcohol abuse.     

Effects of [Nphe1]nociceptin(1-13)NH2, [Phe1(CH2-NH)Gly2]nociceptin(1- 13)NH2, and nocistatin on nociceptin inhibited constrictions of guinea pig isolated bronchus

Electric field stimulation (EFS) causes excitatory non adrenergic-non cholinergic (eNANC) and cholinergic constrictions in the guinea pig isolated bronchus, the activation of eNANC and cholinergic nerves respectively. We investigated the effects of [Nphe1]nociceptin(1-13)NH2 ([Nphe1]NC(1-13)NH2), [Phe1(CH2-NH)Gly2]nociceptin(1- 13)NH2 ([F/G] NC(1-13)NH2), and nocistatin (NST) on nociceptin (NC) inhibited constrictions in isolated bronchus of guinea pig. The results show that NC (1 micromol/L) inhibited EFS-induced eNANC and cholinergic constrictions compared with the control, in which nociceptin was not applied. After pretreatment with [Nphe1]NC(1-13)NH2, [F/G]NC(1-13)NH2, or NST, the inhibitions of NC were antagonized by [Nphe1]NC(1-13)NH2 and [F/G]NC(1-13)NH2 but not NST. However, [Nphe1]NC(1-13)NH2, [F/G]NC(1-13)NH2, and NST did not affect the inhibitions induced by morphine. Furthermore, [Nphe1]NC(1-13)NH2, [F/G]NC(1-13)NH2 and NST did not cause any appreciable effects on EFS-induced eNANC and cholinergic constrictions in guinea pig bronchi. The results demonstrate that [Nphe1]NC(1-13)NH2 and [F/G]NC(1- 13)NH2 but not NST act as selective antagonists of the NC receptor and the effects of NC on EFS-induced constrictions of guinea pig isolated bronchus.     

Study in vitro and in vivo of nociceptin/orphanin FQ(1-13)NH2 analogues substituting N-Me-Gly for Gly2 or Gly3

In the present study, two analogues containing N-Me-Gly (Sarcosine, Sar) were synthesized to further investigate the structural-activity relationships of orphanin FQ/nociceptin (OFQ/NC, NC). The replacement of Gly(2) or Gly(3) with Sar increased the flexibility and decreased the hydrophobicity of the N-terminal tetrapeptide. The activity of the analogues was investigated in a series of assays in vivo and in vitro. [Sar(2)]NC(1-13)NH(2) was found to (1) produce dose-dependent inhibition of the electrically induced contraction in MVD assay (pEC(50) = 6.14); (2) produce significant hyperalgesia effects in a dose-dependent manner when intracerebroventricularly (i.c.v.) injected in mice. The inhibitive effects of [Sar(2)]NC(1-13)NH(2) in MVD assay could be significantly antagonized by [Nphe(1)]NC(1-13)NH(2), and partially antagonized by naloxone; the hyperalgesic effect of [Sar(2)]NC(1-13)NH(2) could be significantly antagonized by naloxone, and partially antagonized by [Nphe(1)]NC(1-13)NH(2). On the contrary, [Sar(3)]NC(1-13)NH(2) showed no effects in these assays. All the findings suggest that the flexibility of the peptide bond between Phe(1) and Gly(2) and between Gly(2) and Gly(3) play an important role in NC-OP(4) receptor interaction, and the hydrophobicity of the N-terminal tetrapeptide showed no significant effect on this interaction. The present work also helps to provide a novel method to elucidate structural and conformational requirements of the opioid peptide-receptor interaction.     

Involvement of ORL1 receptor and ERK kinase in the orphanin FQ-induced nociception in the nucleus accumbens of rats

Nociceptin/orphanin FQ (N/OFQ) is a heptadecapeptide, which has been identified as an endogenous ligand of the opioid receptor-like (ORL1) receptor. The present study investigated the nociceptive effect of intra-nucleus accumbens (intra-NAc) injection of OFQ, and the involvement of ERK pathway in such effect. Intra-NAc injection of OFQ (0.1, 0.5, 1 nmol) dose-dependently decreased the nociceptive thresholds on the hindpaw withdrawal response to thermal and mechanical stimulation in rats. Moreover, the intra-NAc injection of OFQ-induced decreases in HWLs were antagonized by intra-NAc injection of (Nphe(1))nociceptin(1-13)NH(2), an antagonist of ORL1 receptor, in a dose-dependent way. Furthermore, the OFQ-induced nociception could be attenuated by pretreatment with the mitogen-activated protein kinase/ERK kinase (MEK) inhibitor 1,4-diamino-2,3-dicyano-1,4-bis(2-aminopheylthio)butadiene (U0126). Our results demonstrate that OFQ induces nociceptive effects in NAc. The effect was blocked by the antagonist (Nphe(1))nociceptin(1-13)NH(2) and attenuated by U0126, suggesting that the activation of ERK pathways is involved in the OFQ-induced nociceptive effect in the NAc of rats.     

Intrathecal [Nphe1]nociceptin( 1-13)NH2 selectively reduces the spinal inhibitory effect of nociceptin

The effects of intrathecal (i.t.) application of the proposed nociceptin receptor antagonist [Nphe1]nociceptin(1-13)NH2 on the flexor reflex was evaluated in spinalized rats. I.t. [Nphe1]nociceptin (1-13)NH2 dose-dependently facilitated the flexor reflex with no depression. Pretreatment with 16.5 nmol of [Nphe1]nociceptin(1-13)NH2 prevented the development of reflex depression following 0.55 nmol i.t. nociceptin, but strongly enhance the initial excitatory effect of nociceptin. The reflex depressive effect of i.t. endomorphine-2 was not blocked by [Nphe1]nociceptin(1-13)NH2 pretreatment. It is concluded that [Nphe1]nociceptin(1-13)NH2 is a selective antagonist of the spinal receptor mediating the inhibitory action of nociceptin. It can be further suggested that the spinal inhibitory effect of nociceptin may be tonically active.     

Structure-activity relationship of [Nphe1]-NC-(1-13)-NH2, a pure and selective nociceptin/orphanin FQ receptor antagonist.

A series of analogs of the ORL1 receptor antagonist [Nphe1]-NC(1-13)-NH2 was prepared and tested for agonistic and antagonistic activities in the mouse vas deferens, a preparation that shows high sensitivity to nociceptin and related peptides. The purpose of this study was to determine the role of the aromatic residue at the N-terminal for antagonism and eventually identify compounds with improved potency. Results indicated that all 23 compounds are inactive as agonists, and the antagonistic potency of the initial template [Nphe1]-NC(1-13)-NH2 is high (pKB 6.43) compared with those of all other compounds except [(S)(betaMe)Nphe1]NC(1-13)-NH2 (pK(B) 6.48). The other 22 compounds can be divided into two groups: 10 show antagonistic potencies (pK(B)) ranging from 5.30 to 5.86, whereas the other 12 compounds are inactive. This study clearly shows that the aromatic ring of Nphe is very critical for the interaction with the ORL1 receptor and can not be enlarged or sterically modified without significant loss of antagonistic potency.     

Nociceptin receptor activation inhibits tachykinergic non adrenergic non cholinergic contraction of guinea pig isolated bronchus

We studied the action of nociceptin (NC) on the atropine-resistant contractions of the guinea pig isolated bronchus evoked by the electrical field stimulation (EFS), an effect that is mediated by the activation of excitatory non adrenergic-non cholinergic (eNANC) nerves and the subsequent release of tachykinins. The functional site by which NC acts in this preparation was investigated using few different NC receptor agonists and the newly discovered NC receptor antagonist, [Phe1psi(CH2-NH)Gly2]NC(1-13)NH2 ([F/G]NC(1-13)NH2). NC inhibited in a concentration dependent manner (pEC50 7.14; Em - 87 +/- 3% of control values) EFS induced contractions. NC effect was mimicked by the NC analogues, NCNH2 and NC(1-13)NH2, but not by NC(1-9)NH2. NC (1 microM) did not affect the contractile effects of exogenously applied neurokinin A (1 microM). [F/G]NC(1-13)NH2 (10 microM) completely prevented the inhibition induced by NC (1 microM), whereas naloxone (1 microM) was found inactive. Both naloxone and ([F/G]NC(1-13)NH2 were per se inactive on basal resting tone as well as on the electrically induced contractions. The present findings show that NC inhibits the atropine-resistant EFS-induced contraction in the guinea pig bronchus by inhibiting eNANC nerves, and suggest the presence of NC receptors, distinct from opioid receptors, on the nerves of the guinea pig bronchus.     

Blockade effects of (Nphe1)Nociceptin(1-13)-NH(2) on anti-nociception induced by intrathecal administration of nociceptin in rats

The present study investigated the roles of the opioid-receptor-like (ORL1) receptor and its endogenous ligand nociceptin on nociception in the spinal cord of rats. Intrathecal administration of 10 nmol of nociceptin produced significant increases in hindpaw withdrawal latencies (HWLs) to thermal and mechanical stimulation. There were no significant changes of average maximum angles in inclined plane tests after intrathecal injection of 10 nmol of nociceptin in rats. The intrathecal nociceptin-induced increases in HWL were antagonized by intrathecal administration of (Nphe1)Nociceptin(1-13)-NH(2), a selective antagonist of ORL1 receptor, in a dose-dependent manner. The results demonstrated that ORL1 receptor is involved in the nociceptin-induced anti-nociceptive effect in the spinal cord of rats.     

Nphe1]nociceptin(1-13)-NH2 antagonizes nociceptin-induced hypotension, bradycardia, and hindquarters vasodilation in the anesthetized rat

The purpose of this study was to investigate the effects of [Nphe1]nociceptin(1-13)-NH2 on nociceptin-induced decreases in mean arterial pressure (MAP), heart rate (HR), and hindquarters vascular bed resistance (HVBR) of the anesthetized rat. The results showed that i.c.v. or i.v. [Nphe1]nociceptin(1-13)-NH2 (1.5-12 nmol/kg and 5-120 nmol/kg, respectively) could antagonize the depressor effects of i.c.v. or i.v. nociceptin (3 and 30 nmol/kg, respectively) on MAP and HR. Furthermore, [Nphe1]nociceptin(1-13)-NH2 (5-120 nmol/kg) could reverse nociceptin (30 nmol/kg)-induced decrease of HVBR. However, [Nphe1]nociceptin(1-13)-NH2 had no significant effects on similar effects induced by morphine. Our results suggest that [Nphe1]nociceptin(1-13)-NH2 acts as a selective antagonist of the nociceptin receptor in the cardiovascular system of the rat.     

Inhibitory activity of nociceptin/orphanin FQ on capsaicin-induced bronchoconstriction in the guinea-pig

In vivo studies were conducted in the guinea-pig to investigate the activity of the selective ORL1 receptor agonist nociceptin/orphanin FQ against capsaicin-induced bronchoconstriction, a response mediated by the release of tachykinins from pulmonary sensory nerves. Anesthetized guinea-pigs were ventilated with a rodent ventilator and placed in a whole-body plethysmograph, and pulmonary resistance (R(L)) and dynamic lung compliance (C(Dyn)) were monitored. Intravenous administration of nociceptin/orphanin FQ (0.3 mg/kg) inhibited the capsaicin-induced bronchoconstriction. The new nonpeptide ORL1 receptor antagonist 1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one (J-113397) administered intravenously (1 mg/kg) produced a significant blockade of the inhibitory effect of nociceptin/orphanin FQ (0.3 mg/kg) on capsaicin-induced bronchoconstriction, whereas the nonselective opioid receptor antagonist naloxone (1 mg/kg) had no effect. Nociceptin/orphanin FQ (0.3 mg/kg) did not affect the bronchoconstriction induced exogenously by the tachykinin NK2 receptor agonist [beta-ala8]-neurokinin A (4-10). We conclude that nociceptin inhibits in vivo capsaicin-evoked tachykinin release from sensory nerve terminals in the guinea-pig by a prejunctional mechanism. This inhibitory action does not involve activation of opioid receptors.     

Neuropeptide FF receptors antagonist, RF9, attenuates opioid-evoked hypothermia in mice

The present study used the endpoint of hypothermia to investigate opioid and neuropeptide FF (NPFF) interactions in conscious animals. Both opioid and NPFF systems played important roles in thermoregulation, which suggested a link between opioid receptors and NPFF receptors in the production of hypothermia. Therefore, we designed a study to investigate the relationship between opioid and NPFF in control of thermoregulation in mice. The selective NPFF receptors antagonist RF9 (30nmol) injected into the third ventricle failed to induce significant effect, but it completely antagonized the hypothermia of NPFF (45 nmol) after cerebral administration in mice. In addition, RF9 (30 nmol) co-injected i.c.v. in the third ventricle reduced the hypothermia induced by morphine (5nmol,) or nociceptin/orphanin FQ (N/OFQ) (2 nmol). Neither the classical opioid receptors antagonist naloxone (10 nmol) nor NOP receptor antagonist [Nphe(1)]NC(1-13)NH(2) (7.5 nmol) reduced the hypothermia induced by the central injection of NPFF at dose of 45 nmol. Co-injected with a low dose of NPFF (5 nmol), the hypothermia of morphine (5 nmol) or N/OFQ (2 nmol) was not modified. These results suggest that NPFF receptors activation is required for opioid to produce hypothermia. In contrast, NPFF-induced hypothermia is mainly mediated by its own receptors, independent of opioid receptors in the mouse brain. This interaction, quantitated in the present study, is the first evidence that NPFF receptors mediate opioid-induced hypothermia in conscious animals.     

Pharmacological characterization of the nociceptin receptor, ORL1

A novel opioid receptor-like orphan receptor (ORL1) was cloned and identified to be homologous to classical opioid receptors but insensitive to traditional opioids. A heptadecapeptide, termed orphanin FQ or nociceptin (OFQ/N), was identified as its endogenous ligand. OFQ/N shares overlapping distribution sites in pain-processing areas and common cellular mechanisms with opioids but exerts diverse effects on nociceptive responses. Of the two reported ORL1 antagonists, [Phe(1)psi(CH(2)-NH)- Gly(2)] nociceptin-(1-13)-NH(2) (Phepsi) and naloxone benzoylhydrazone (NBZ), antagonisms were validated in the activation of inward rectifying K channels induced by OFQ/N, using the patch clamp technique in ventrolateral periaqueductal gray slices. Results showed that Phepsi acted as a partial agonist and NBZ was a weak nonselective antagonist of ORL1. It is comparable with most but not all of the findings from other tissues. Comparing all the reports supports the above inference for these two antagonists. The possible causes for the discrepancy were discussed. A brief review on the putative ORL1 antagonists, acetyl-RYYRIK-NH2, some sigma-ligands and the functional antagonist, nocistatin, is also included. It indicates that a potent and selective ORL1 antagonist is expecting to elucidate the physiological role of OFQ/N.     

The alpha adrenoceptors on endothelial cells

Endothelial cells release a powerful factor (endothelium-derived relaxing factor [EDRF]) that relaxes smooth muscle cells in response to some vasodilating agents such as acetylcholine. Contraction curves to norepinephrine (NE) in greyhound, mongrel dog, and pig coronary artery rings were studied in vitro in the presence of propranolol. Removal of endothelium increased the sensitivity and maximum contraction in response to NE. In other experiments pig coronary rings were precontracted with a thromboxane mimetic U 46619 in the presence of propranolol. NE relaxed these arteries only if endothelium was present. Methoxamine was without effect but the relaxation response to NE was antagonized by phentolamine, idazoxan, and yohimbine, which suggests that there are alpha 2 adrenoceptors on endothelial cells that mediate the release of EDRF. Greyhound and mongrel dog large coronary arteries relaxed to NE only if prazosin was present, which suggests that alpha 1-adrenoceptor stimulation on the vascular smooth muscle can override the relaxation response to EDRF. Comparison of NE responses in carotid, mesenteric, renal, and femoral large arteries of the pig, greyhound, and mongrel dog indicate the nonuniformity of distribution of alpha 2 adrenoceptors on endothelium and alpha 1 and alpha 2 adrenoceptors on vascular smooth muscle. The integrity of the endothelium must now be considered in interpreting the vascular responses to alpha-adrenoceptor agonists.     

Exogenous, but not endogenous nociceptin modulates mesolimbic dopamine release in mice

The effect of nociceptin (an endogenous ligand of the ORL1 receptor) on mesolimbic dopamine release and simultaneous horizontal locomotion was studied in freely moving mice undergoing microdialysis of the nucleus accumbens. Intracerebroventricular (i.c.v.) administration of nociceptin (7 nmol) induced a long-lasting suppression of mesolimbic dopamine release and horizontal locomotion in wild-type but not ORL1 knockout mice. I.c.v. administration of the recently reported peptide nociceptin antagonist [Nphe1, Arg14, Lys15] nociceptin-NH(2) (known also as UFP-101, 5 nmol) completely abolished the suppressive effect of nociceptin on mesolimbic dopamine release. However, UFP-101 administration alone induced a mild and lasting suppression of mesolimbic dopamine release in both wild-type and ORL1 knockout mice that was magnified in ORL1 knockout mice by coadministration of nociceptin. UFP-101 administration alone suppressed locomotion in both genotypes. These results confirm that the suppressive action of nociceptin on mesolimbic dopamine release is mediated entirely by the ORL1 receptor, and that UFP-101 effectively antagonizes this action. However, the lack of a stimulatory effect of UFP-101 in wild-type mice indicates that despite being sensitive to exogenous nociceptin action, basal mesolimbic dopaminergic activity is not determined by endogenous nociceptin in mice.     

Structural requirements of nociceptin antagonist Ac-RYYRIK-NH2 for receptor binding.

Ac-RYYRIK-NH2 is a peptide isolated from the peptide library as an antagonist that inhibits the biological activities of nociceptin, a hyperalgesic neuropeptide. In order to clarify the structural requirements of this peptide for binding to the nociceptin receptor ORL1, systematic structure-activity studies were carried out. The result of Ala-scanning indicated that the N-terminal tripeptide RYY(= Arg-Tyr-Tyr) is crucially important for binding to the ORL1 receptor. Residual truncations from the N- or C-terminus revealed the special importance of the N-terminal Arg residue. The removal of protecting groups indicated that the N-terminal acetyl group is essential, but the C-terminal amide group is insignificant. These results indicated the conspicuous importance of acetyl-Arg at position 1 of Ac-RYYRIK-NH2 as a key structure allowing binding to the receptor. To investigate the binding site of this peptide in the ORL1 receptor, we synthesized and assayed a series of analogues of the nociceptin dibasic repeat region, residues 8-13 of RKSARK. None of the derivatives were active. Ac-RYYRIK-NH2 was inactive for the mu opioid receptor to which nociceptin binds with considerable strength. All the results suggested that the mode of binding between Ac-RYYRIK-NH2 and the ORL1 receptor is different to that between the ORL1 receptor and nociceptin, and that it may consist of interaction with the receptor site to which nociceptin(1-7) or -(14-17) binds.     

A study of mechanisms involved in vasodilatation induced by resveratrol in isolated porcine coronary artery

The present study was designed to investigate the acute relaxing effect of phytoestrogen resveratrol on isolated porcine coronary arteries and to determine the mechanisms underlying its vasodilatation. Rings of porcine coronary arteries were suspended in organ baths containing Krebs-Henseleit solution, and then isometric tension was measured. Resveratrol concentration-dependently relaxed arterial rings precontracted with 30 mM KCl. The IC(50) value of resveratrol was 38.67+/-3.21 microM. Incubation with N(omega)-L-nitro-arginine (L-NNA), endothelium removal or the presence of a potent inhibitor of protein tyrosine phosphatase sodium orthovanadate partly decreased the relaxation induced by resveratrol. However, the relaxation induced by resveratrol was unaffected by the estrogen receptor antagonist tamoxifen, the inhibitor of prostanoid synthesis indomethacin, the antagonist of beta-adrenoceptors propranolol or the protein synthesis inhibitor, cycloheximide. In addition, resveratrol significantly decreased the contractile responses of 5-HT, KCl and CaCl(2), and shifted their cumulative concentration-response curves to the right. These results suggest that the mechanisms of vasorelaxation induced by resveratrol are heterogeneous, two mechanisms participating partially in the relaxation of porcine coronary artery were detected in the study, one being the nitric oxide released from the endothelium, the other causing inhibition of Ca(2+) influx, but estrogen receptors were not involved in resveratrol-induced relaxation.     

Heterodimerization of ORL1 and Opioid Receptors and Its Consequences for N-type Calcium Channel Regulation

We have investigated the heterodimerization of ORL1 receptors and classical members of the opioid receptor family. All three classes of opioid receptors could be co-immunoprecipitated with ORL1 receptors from both transfected tsA-201 cell lysate and rat dorsal root ganglia lysate, suggesting that these receptors can form heterodimers. Consistent with this hypothesis, in cells expressing either one of the opioid receptors together with ORL1, prolonged ORL1 receptor activation via nociceptin application resulted in internalization of the opioid receptors. Conversely, μ-, δ-, and κ-opioid receptor activation with the appropriate ligands triggered the internalization of ORL1. The μ-opioid receptor/ORL1 receptor heterodimers were shown to associate with N-type calcium channels, with activation of μ-opioid receptors triggering N-type channel internalization, but only in the presence of ORL1. Furthermore, the formation of opioid receptor/ORL1 receptor heterodimers attenuated the ORL1 receptor-mediated inhibition of N-type channels, in part because of constitutive opioid receptor activity. Collectively, our data support the existence of heterodimers between ORL1 and classical opioid receptors, with profound implications for effectors such as N-type calcium channels.     

孤啡肽受体的配体研究进展

孤啡肽(nociceptin或orphanin FQ)发现于1995年底, 它是阿片受体样受体(ORL1或LC 132) 的内源性配体,在痛觉调节、心血管系统、离子通道、依赖和耐受、学习和记忆等方面具有广泛的生物学活性. 最近几年, 对孤啡肽受体与相关配体构效关系的研究成为一个新的热点.对在研究构效关系过程中所发现的孤啡肽受体相关配体（片段、拮抗剂、激动剂、部分激动剂和阻断剂）的研究情况进行了介绍.     

NF-kappaB activates fibronectin gene expression in rat hepatocytes

Resveratrol (RSVL), an edible polyphenolic stilbene, claims a myriad of cardiovascular benefits. However, the molecular underpinnings of such actions are poorly understood. Currently, in sheep coronary arteries (SCA), RSVL markedly (threefold) enhanced cGMP formation (t(1/2): 6.5 min; EC(50): 3 microM). This response was not abrogated by the phosphodiesterase inhibitor (IBMX, 0.5 mM), but was partly sensitive (20-30%) to either removal of the endothelium, treatment with the nitric oxide synthase-inhibitor (L-NMMA, 10 microM), or with the soluble GC (sGC)-inhibitor (ODQ, 10 microM). In membrane preparations from denuded SCA, either RSVL or the pGC agonist atrial natriuretic peptide (ANP, 0.1-1 microM) activated GC in the particulate, but not in the soluble, membrane fraction. By contrast, the nitric oxide donor, sodium nitroprusside (SNP, 1-10 microM), stimulated GC only in the soluble fraction. Further, pretreatment with RSVL partly desensitized the ANP response, but was additive to that of SNP. In arterial tension studies, RSVL relaxed PGF(2alpha)-precontracted denuded rings in a concentration-dependent manner, a response that was markedly enhanced (approximately 18 fold) in the presence of IBMX. Conversely, precontraction with phorbol ester, which also desensitizes pGC, blunted relaxations to RSVL but not to forskolin or SNP. These findings demonstrate that RSVL increases cGMP in coronary arteries, mostly by activation of pGC. This pathway triggers vasorelaxant responses that remain effective in endothelium-disrupted arteries.     

Structure-activity relationships of nociceptin and related peptides: comparison with dynorphin A

Nociceptin and its receptor (OP(4)) share sequence homologies with the opioid peptide ligand dynorphin A and its receptor OP(2). Cationic residues in the C-terminal sequence of both peptides seem to be required for selective receptor occupation, but the number and the distribution of these basic residues are different and quite critical. Both receptors are presumably activated by the peptides N-terminal sequence (Xaa-Gly Gly-Phe, where Xaa = Phe or Tyr); however, although OP(4) requires Phe(4) as a determinant pharmacophore, OP(2) requires Tyr(1) as do the other opioid receptors. An extensive structure-activity analysis of the N-terminal tetrapeptide has led to conclude that the presence of aromatic residues in position one and four, preferably Phe, as well as the distance between Phe(1) and Phe(4) are extremely critical for occupation and activation of OP(4) in contrast with other opioid receptors (e.g. OP(1), OP(3), OP(2)). Modification of distance between the side chains of Phe(1) and Phe(4) (as obtained with Nphe(1) substitution in both NC and NC(1-13)-NH(2)) and/or conformational orientation of Phe(1) (as in Phe(1)psi(CH(2)-NH)-Gly(2)) has brought to discovery of pure antagonist ([Nphe(1)]-NC(1-13)-NH(2)) and a partial agonist ([Phe(1) psi(CH(2)-NH)-Gly(2)]-NC(1-13)-NH(2)), which have allowed us to characterize and classify the OP(4) receptor in several species. Thus, although antagonist activities at the OP(4) receptor are obtained by chemical modification of Phe(1)-Gly(2) peptide bond or by a shift of Phe(1) side chain of NC peptides, antagonism at the OP(2) receptor requires the diallylation of the N-terminal amino function, for instance, of dynorphin A. These considerations support the interpretation that the two systems nociceptin/OP(4) and dynorphin A/OP(2) are distinct pharmacological entities that differs in both their active sites (Tyr(1) for Dyn A and Phe(4) for NC) and the number and position of cationic residues in the C-terminal portions of the molecules. The chemical features of novel OP(4) receptor ligands either pseudopeptides obtained by combinatorial library screening or molecules of nonpeptide structure are reported and discussed in comparison with NC and NC related peptides.