The orphan opioid receptor and its endogenous ligand--nociceptin/orphanin FQ.

Following the elucidation of the amino acid sequences of the mu-, delta- and kappa-opioid receptors, a new 'orphan opioid receptor' was cloned with a high degree of homology to the 'classical' opioid receptors. The endogenous opioid peptides show little or no activity at this new receptor; however, a novel endogenous peptide for the orphan opioid receptor has been isolated and sequenced. Here, Graeme Henderson and Sandy McKnight review recent findings on this new receptor and its endogenous ligand, and address the contentious issue of whether activation of this receptor results in hyperalgesia or analgesia.

Evidence that cholecystokinin induces immediate early gene expression in the brainstem, hypothalamus and amygdala of the rat by a CCKA receptor mechanism.

The effect of sulphated cholecystokinin octapeptide (CCK-8S) on immediate early gene expression in the rat CNS was investigated using the technique of in situ hybridization. A rapid and transient induction of c-fos, NGFI-A and NGFI-B (nerve growth factor-induced genes A and B) mRNA was demonstrated in the nucleus tractus solitarius (NTS), area postrema (AP), hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei, and central nucleus of the amygdala, following peripheral administration of CCK-8S (1-100 micrograms/kg i.p.). In contrast, levels of c-jun, junB and junD mRNA were unaffected by the peptide. The closely related decapeptide, caerulein (50 micrograms/kg i.p.), induced the same pattern of IEG expression as CCK-8S, whereas the desulphated octapeptide, CCK-8DS (50 micrograms/kg i.p.), had no effect on levels of mRNA for any IEG studied. Expression of IEG mRNA in these areas was suppressed by bilateral subdiaphragmatic vagotomy, and by pretreatment with the selective CCKA receptor antagonist, devazepide (0.1 and 1 mg/kg i.p.). In contrast, CCK-8S induction of IEG mRNA was not blocked by pretreatment with the selective CCKB receptor antagonist, CI-988 (1 or 10 mg/kg i.p.). In addition, the selective CCKB receptor agonists, CCK-4 (50 micrograms/kg i.p.) or pentagastrin (2 mg/kg i.p.), failed to induce IEG expression in any of the areas studied. These results suggest that systemic CCK-8S primarily acts via CCKA receptors on vagal afferents to stimulate IEG mRNA expression in the rat CNS.

The opioid receptors in the hamster vas deferens are of the delta-type.

The motor responses of the isolated vas deferens of the hamster were unaffected by opioid-receptor agonists which are selective for the mu- or kappa-receptor, while agonists which show degrees of selectivity for the delta-opioid receptor caused dose-related inhibition of the stimulation-evoked contractions. The agonist action of the enkephalins and their congeners was only apparent when various inhibitors of tissue peptidases were present. Responses to opioid agonists were antagonised in a competitive manner by naloxone and by the selective delta-receptor antagonist, ICI 174864. It is noteworthy that the benzomorphans bremazocine, ethylketocyclazocine and Mr 2034, which are agonists at kappa-receptors in other tissues, are antagonists at the delta-receptor of the hamster vas deferens. Thus, the vas deferens of the hamster contains opioid receptors only of the delta-type and may therefore provide a simple and specific test for the assay of activity at the delta-opioid receptor.

Cyclic peptides as selective tachykinin antagonists.

Twenty homodetic cyclic peptides based on the C-terminal sequence of substance P were prepared (Table I) by a combination of solid-phase techniques and cyclizations using azide coupling procedures. Incorporation of dipeptide mimics based on substituted gamma-lactams were used in some cases to restrict their conformational mobility. Five of these cyclic peptides were shown to have high tachykinin antagonist activity (pA2 > 6) at NK-2 receptors (rat vas deferens). The two most potent of this series, XVII, cyclo(Gln-Trp-Phe-Gly-Leu-Met) (pA2 = 8.1), and I cyclo(Gln-Trp-Phe(R)Gly[ANC-2]Leu-Met) (pA2 = 6.7), were selective for NK-2 receptors compared with the other tachykinin receptors (Table II).

10-Ketonaltrexone and 10-ketooxymorphone.

Ethylketocyclazocine (1) has greater kappa/mu selectivity than cyclazocine in brain binding assays. 10-Ketonaltrexone (11) and 10-ketooxymorphone (10) were prepared from naltrexone 3-methyl ether and oxycodone, respectively. Bioassays in the myenteric plexus longitudinal muscle preparation of the guinea pig ileum and in the mouse vas deferens, in addition to brain binding assays, demonstrated that 10 and 11 were far less potent than naltrexone (2) and oxymorphone (3) at mu sites and also had little affinity for kappa and delta sites. It is concluded that introduction of the 10-keto group in naltrexone and oxymorphone diminished opioid effects at all binding sites.

Use of a dipeptide chemical library in the development of non-peptide tachykinin NK3 receptor selective antagonists.

The use of a dipeptide library as the source of a micromolar chemical lead compound for the human tachykinin NK3 receptor is described. The screening of a dipeptide library through a cloned human NK3 receptor binding assay resulted in the identification of Boc(S)Phe(S)PheNH2 (1), which has subsequently been developed, following a 'peptoid' design strategy, into a series of high-affinity NK3 receptor selective antagonists. The structure-activity relationship of the C-terminal portion of this dipeptide lead was first explored and led to the identification of the urea derivative Boc(S)Phe(R)alphaMePheNH(CH2)7NHCONH2 (41, PD157672). This modified dipeptide has a Ke of 7 nM in blocking senktide-induced increases in intracellular calcium levels in human NK3 receptors stably expressed in CHO cells. Subsequent optimization of the N-terminal BocPhe group and the alphaMePhe residue side chain of 41 led to the identification of [S-(R*,S*)]-[2-(2,3-difluorophenyl)-1-methyl-1-[(7-ureidoheptyl)ca r bamoyl]ethyl]carbamic acid 2-methyl-1-phenylpropyl ester (60, PD161182), a non-peptide NK3 receptor selective antagonist. Compound 60 blocks the senktide-evoked increases in intracellular calcium levels in cloned human NK3 receptors stably expressed in CHO cells with Ke of 0.9 nM.

The actions of some vasoactive polypeptides and their antagonists on the anococcygeus muscle.

1 The action of three polypeptides, bradykinin, substance P and eledoisin known to inhibit vascular smooth muscle has been examined on the anococcygeus muscle of the rat, cat and rabbit.2 In the atonic rat muscle, bradykinin and substance P had little or no effect on tone but eledoisin produced a sustained dose-related contraction which could be abolished by phentolamine (1 muM) and is, therefore, probably an indirect sympathomimetic effect. On the motor response to field stimulation of adrenergic nerves, bradykinin had no effect whereas both substance P and eledoisin reduced this response. The mechanism of action was further analysed with eledoisin by examining its effect on the response to noradrenaline. Eledoisin did not alter the dose-response curve to noradrenaline and its inhibitory action is likely, therefore, to be presynaptic.3 In the rat anococcygeus muscle in which the tone was raised by guanethidine or carbachol, bradykinin and substance P reduced this tone whereas eledoisin continued to exert a motor action. Compared with substance P the inhibitory effect of bradykinin appeared at lower concentrations (threshold 0.01 mug/ml), developed more rapidly and the size of the response was greater.4 The effect of bradykinin on the tonically contracted cat and rabbit anococcygeus muscles was examined in addition to that of the rat. In all three species bradykinin caused inhibition and the magnitude of the response was equal to the maximum effect of inhibitory nerve stimulation. None of the peptides affected the inhibitory response to nerve stimulation itself.5 The effects of three substances, hesperitin, khellin and apiin, reported in other tissues to antagonize the action of bradykinin were examined both on the inhibitory response to bradykinin and to field stimulation. None of them was able to inhibit either response, although they reduced tone when given by themselves. During these experiments it was found that ethanol antagonized the inhibitory response to field stimulation.6 The possibility that bradykinin or some related peptide might play a part in the inhibitory response to nerve stimulation in the anococcygeus is discussed.

Block by gabapentin of the facilitation of glutamate release from rat trigeminal nucleus following activation of protein kinase C or adenylyl cyclase.

The effect of activation of protein kinase C (PKC) or adenylyl cyclase on release of glutamate has been investigated in a perfused slice preparation from the rat caudal trigeminal nucleus. Stimulation of PKC by phorbol 12-myristate 13-acetate (PMA) produced a concentration-dependent increase in K(+)-evoked release of [(2)H]-glutamate (maximum increase 45%, EC(50) 11.8 nM), but in the presence of gabapentin (30 microM) the facilitation of release was blocked. The adenylyl cyclase activator forskolin (FSK) also induced a concentration-dependent increase in K(+)-evoked release of [(3)H]-glutamate (maximum increase 36%, EC(50) 2.4 microM), and again this facilitatory effect was blocked by gabapentin (30 microM). We suggest that these results may be of relevance to the antihyperalgesic properties of gabapentin, in conditions where concomitant release of substance P and CGRP produces activation of PKC and adenylyl cyclase respectively.

Characterization of the ORL(1) receptor on adrenergic nerves in the rat anococcygeus muscle.

1. Nociceptin, the endogenous ORL(1) receptor agonist inhibited the motor response to electrical-field stimulation in the rat anococcygeus muscle. This effect was characterized using the peptide ligands acetyl-Arg-Tyr-Tyr-Arg-Trp-Lys-NH(2) (Ac-RYYRWK-NH(2)), acetyl-Arg-Tyr-Tyr-Arg-Ile-Lys-NH(2) (Ac-RYYRIK-NH(2)) and [Phe(1)psi(CH(2)-NH)Gly(2)]nociceptin(1-13)NH(2) ([F/G]NC(1-13)NH(2)), and the non-selective opioid antagonist naloxone benzoylhydrazone (NalBzOH). 2. Nociceptin produced a concentration-dependent inhibition of the adrenergic motor response to electrical-field stimulation (EC(50) 19 nM, pEC(50) 7.7+/-0.1, n=8), but the response to exogenous noradrenaline (0.2 - 1 microM) was unaffected. The inhibitory nerve response was not affected by up to 1 microM nociceptin. 3. After inhibition of nitric oxide synthase (N(omega)-nitro-L-arginine 100 microM), and in the presence of peptidase inhibitors, nociceptin produced full inhibition of the pure adrenergic motor response (EC(50) 4 nM; pEC(50) 8.4+/-0.1, E(max) 98.3+/-1.2%, n=12). Ac-RYYRWK-NH(2) was a potent partial-agonist (pEC(50) 9.0+/-0.1, E(max) 66.4+/-5.2; n=11) but the efficacy of Ac-RYYRIK-NH(2) (pEC(50) 8.0+/-0.2, E(max) 36.7+/-3.5; n=12) was lower and the peptide could be tested as an antagonist (pA(2) 9.01). 4. [F/G]NC(1-13)NH(2) and NalBzOH had little or no efficacy and were competitive antagonists with pK(B) values of 7.4 (95% c.l. 7.1, 7.7) and 6.9 (95% c.l. 6.7, 7.1) respectively. Both increased the response to field stimulation at high concentrations, suggesting the release of an endogenous agonist for the ORL(1) receptor during stimulation. 5. Rat nocistatin did not affect the response to electrical-field stimulation, nor did it modify the inhibitory action of nociceptin. 6. Our findings suggest there is a significant endowment of ORL(1) receptors on sympathetic terminals of the rat anococcygeus, where nociceptin mediates a powerful inhibitory effect on adrenergic neuromuscular transmission.

Reduction by gabapentin of K+-evoked release of [3H]-glutamate from the caudal trigeminal nucleus of the streptozotocin-treated rat.

Recently, we showed that gabapentin can inhibit a facilitatory effect of substance P (SP) on K(+)-evoked glutamate release in rat trigeminal slices (Maneuf et al., 2001), and we have now examined the effect of gabapentin on glutamate release in the trigeminal slice from the streptozotocin (STZ)-treated rat. 1. At 4 weeks following STZ treatment (50 mg kg(-1) i.p.), blood glucose was increased in the majority of cases, compared to the control level. All the treated animals showed a significant degree (P<0.001) of tactile allodynia (assessed using von Frey filaments) that did not appear to correlate with blood glucose levels. 2. In this study, we demonstrated that, after STZ treatment, 30 microM gabapentin reduced K(+)-evoked release of [(3)H]-glutamate in either normal (11 mM) or high (30 mM) glucose conditions by 24 and 22%, respectively. In the normal rat, gabapentin (up to 100 microM) is ordinarily unable to affect release of glutamate from the trigeminal slice. 3. The uptake of glutamate in Sp5C punches from streptozotocin-treated rats was reduced under normal glucose conditions (41.7% of control), whereas high glucose restored uptake to normal (84.7% of control). 4. The addition of 1 microm substance P potentiated the evoked release of glutamate in both normal (40% increase) and high glucose (28%), and this was blocked by gabapentin (30 microM) in both conditions. It is interesting to speculate that this ability of gabapentin to reduce the release of glutamate in the trigeminal nucleus after streptozotocin treatment may be of relevance to the antihyperalgesic-allodynic actions of the drug.

The effect of ethanol on inhibitory and motor responses in the rat and rabbit anococcygeus and the bovine retractor penis muscles.

1 Ethanol (200 mM) reduced the response to inhibitory nerve stimulation in the rat and rabbit anococcygeus and the bovine retractor penis (BRP) muscles. Ethanol also reduced the response to the inhibitory extract from the BRP consistent with the inhibitory factor in these extracts playing some part in the response to inhibitory nerve stimulation. 2 Ethanol's effect on the response to other inhibitory stimuli was examined in the rabbit anococcygeus and the BRP. In the anococcygeus the response to carbachol was reduced, to bradykinin and isobutylmethylxanthine (IBMX) unaltered, and to isoprenaline and adenosine 5'-triphosphate (ATP) potentiated. In the BRP responses to IBMX and sodium nitroprusside were unaltered but in this tissue the response to isoprenaline was reduced. Ethanol's ability to reduce inhibitory responses is, therefore, selective and confined to inhibitory nerve stimulation, inhibitory extract, carbachol, and, in the BRP, isoprenaline. 3 Ethanol reduced the rate of development of inhibition even where the magnitude of the inhibitory response was unaltered. 4 In the rat anococcygeus, ethanol (200 mM) potentiated the response to motor nerve stimulation and to noradrenaline (NA) at low frequencies and low concentrations respectively. Higher ethanol concentrations (400 mM) reduced the response to both motor nerve stimulation and NA. The motor response to carbachol was also reduced. 5 Ethanol (200 mM) itself caused an easily reversible contraction in all three tissues. This was not due to the release of NA but was highly sensitive to the removal of external calcium from the medium. 6 A unified explanation of these varied effects of ethanol based on a reduction in membrane binding of calcium and a reduced efficiency of receptor coupling is suggested.

Characterization of CCK receptors in a novel smooth muscle preparation from the guinea-pig stomach by use of the selective antagonists CI-988, L-365,260 and devazepide.

1. The cholecystokinin receptors mediating motor responses in a novel smooth muscle preparation from the corpus region of the guinea-pig stomach have been characterized by use of five agonist peptides and the antagonists CI-988, L-365,260 and devazepide. 2. Mucosa-denuded strips of circular muscle were contracted in a concentration-dependent manner by the five cholecystokinin (CCK)-related peptides CCK-8S, pentagastrin, gastrin-I, CCK-8US and CCK-4. 3. CI-988 was a powerful antagonist of the response to pentagastrin with an affinity (pKB = 9.49) similar to that obtained in CCKB receptor binding assays. With CCK-8S as the agonist, CI-988 was approximately 1000 fold less powerful as an antagonist. 4. Devazepide powerfully blocked responses to CCK-8S with an affinity (pKB = 9.54) that was in agreement with reported functional data obtained in pancreatic amylase secretion studies, a system exhibiting CCKA receptor activity. Devazepide displayed lower affinity against pentagastrin than against CCK-8S. 5. CI-988 blocked responses to pentagastrin in an insurmountable manner in the presence of 3 nM devazepide; a concentration previously shown to block the CCKA receptor. The nature of the antagonism observed with L-365,260 was unaltered by the presence of devazepide. 6. The guinea-pig stomach corpus smooth muscle preparation contains both subtypes of CCK receptor and will be useful as a pharmacological tool for investigating the functional effects of novel CCK ligands.

The effect of CCKB/gastrin antagonists on stimulated gastric acid secretion in the anaesthetized rat.

1. The urethane-anaesthetized, vagotomised rat preparation was used to investigate the effects of the histamine H2-antagonist ranitidine, the proton pump inhibitor omeprazole and the CCKB/gastrin antagonists CI-988, PD 136450 and L-365,260 on pentagastrin-, histamine- and bethanechol-induced gastric acid secretion. 2. The novel CCKB/gastrin antagonists CI-988 and PD 136450, and L-365,260 dose-dependently inhibited pentagastrin-induced secretion. The ED50 value for PD 136450 was 0.05 mumol kg-1, the same following intravenous or subcutaneous administration. 3. CI-988 and PD 136450 administered subcutaneously at dose levels highly effective for antagonism of pentagastrin responses had no effect on basal acid secretion. 4. Ranitidine inhibited pentagastrin-, bethanechol-, and histamine-induced acid secretion, whereas the CCKB/gastrin antagonists inhibited only the secretory response to pentagastrin. 5. The selective CCKA antagonist, devazepide, was inactive at up to 300 mumol kg-1 i.p. against the three stimulants of acid secretion. 6. CI-988 and PD 136450 will be useful research tools with which to investigate the role of CCKB/gastrin receptors in gastric acid secretion and the trophic activities of gastrin and cholecystokinin (CCK) on the gastrointestinal tract.

Pre-incubation of guinea-pig myenteric plexus with beta-funaltrexamine: discrepancy between binding assays and bioassays.

The acute effects of beta-funaltrexamine and the effects of pre-incubation with this compound were examined in five in vitro assay tissues and in selective binding assays in homogenates of guinea-pig brain and myenteric plexus. In competitive displacement assays with selective ligands, beta-funaltrexamine had highest affinity for the mu-binding site in the myenteric plexus and brain of guinea-pig. Its affinity for the kappa-site was about 15% of that for the mu-site. Pre-incubation of the assay tissues with beta-funaltrexamine caused an increase in the IC50 values of mu- and delta-receptor agonists but not of kappa-agonists. Although in bioassays on the myenteric plexus-longitudinal muscle preparation of the guinea-pig, the IC50 value of the mu-receptor ligand [D-Ala2, MePhe4, Gly-ol5] enkephalin was increased up to 124 fold, its binding at the mu-site in homogenates of the preparation was not affected by this treatment. These findings indicate that the effects of pre-incubation with beta-funaltrexamine on agonist potency of the mu-receptor ligand are due to an interference with the coupling mechanism between the mu-binding site and the effector system.

Activation of the cloned human NK3 receptor in Chinese Hamster Ovary cells characterized by the cellular acidification response using the Cytosensor microphysiometer.

1. The aim of the present study was to validate the Cytosensor microphysiometer, a novel system that measures the extracellular acidification rate as a reliable index of the integrated functional response to receptor activation, as a method for studying NK3 receptor pharmacology, and then to use this system to assess the functional activity of novel compounds at this receptor. 2. The selective NK3 agonist senktide caused reproducible, concentration-related increases in acidification ratein CHO-NK3 cells, with a pEC50 value of 8.72+/-0.11 (n=15). [Beta-Ala8]NKA(4-10), the selective NK2 agonist, elicited a much weaker response (pEC50=6.68+/-0.08, n=4), while the NK1-selective agonist substance P methylester only caused a very weak response at concentrations > or =3 microM (n=2). The rank order of potency for the endogenous tachykinins NKB>NKA>substance P (n=3) confirmed the response was mediated by the NK3 receptor. Moreover, the actual potencies obtained were consistent with affinities measured in radioligand binding studies. 3. The novel compounds PD156319-121 (0.3-1 microM), PD161182 (10-300 nM), PD168001 (10-100 nM) and PD168073 (10-100 nM) all acted as surmountable antagonists of the senktide-induced acidification response, with pA2 values of 7.49, 8.67, 9.17 and 9.25 respectively (n=3-5). In comparison the known NK3 antagonist SR142801 (10-100 nM) had a pA2 value of 8.83 (n=8) for the interaction with senktide. Again, these values are consistent with the radioligand binding data. 4. Amiloride (1 mM) inhibited the senktide-induced acidification response by 68.3+/-3.3 (n=4), indicating that the Na+/H+ antiporter plays an important role in this response, and this is consistent with the importance of this antiporter in other acidification responses. 5. Inhibition of protein kinase C with staurosporine (0.1 microM), or depletion of the intracellular Ca2+ stores with thapsigargin (1 microM), both resulted in a reduction in the maximum response to senktide (63.3+/-1.7 and 68.9+/-3.2% respectively, n=3-5), and co-application of these inhibitors abolished the response (n=3). This strongly suggested that the NK3 receptor was coupling via phospholipase C (PLC), as would be expected, although this could not be confirmed by the use of the putative PLC/PLA2 inhibitor U73122. 6. In conclusion, we have demonstrated the utility of the Cytosensor in the characterization of functional responses to agonists, and assessment of the affinities of antagonists in CHO cells expressing the human NK3, and have shown that our series of novel compounds are non-peptide NK3 antagonists of high affinity, as exemplified by PD168073.

Nociceptin induced inhibition of K+ evoked glutamate release from rat cerebrocortical slices.

Nociceptin, an endogenous ligand for the orphan receptor ORL1, has recently been described. In this study we have shown that nociception inhibits 46 mM K(+)-stimulated glutamate release from rat perfused cerebrocortical slices with an IC50 of 51 nM. At 100 nM the inhibition amounted to 68 +/- 14% and was naloxone (10 microM)-insensitive excluding an activation of mu, delta and kappa opioid receptors. These data demonstrate the functional coupling of ORL1 in glutamatergic neurones and implicates a role for nociceptin in glutamatergic neurotransmission.

Inhibition by nociceptin of the light-evoked release of ACh from retinal cholinergic neurones.

The retina possesses cholinergic amacrine cells which release acetylcholine (ACh) in response to flickering light. Using an eye-cup preparation in anaesthetized rabbits we found that when the retina was exposed to nociceptin, the light-evoked release of ACh was reduced in a concentration-dependent manner (IC50 = 100 nM), the maximum effect being 60% inhibition. Opioid receptors were not involved in the inhibitory effect of nociceptin because its action was not blocked by naloxone (1 microM) and furthermore mu-opioids enhanced the light-evoked release of ACh. Using rabbit retina homogenates we found that the retina possessed a substantial number of high-affinity binding sites for [3H]-nociceptin indicating the presence of ORL1-receptors. Since [des-Phe1]-nociceptin, which has no affinity for the ORL1-receptor, had no effect on the light-evoked release of ACh it is unlikely that the action of nociceptin was simply non-specific. We conclude that the inhibitory effect of nociceptin on retinal ACh release involves activation of the ORL1 receptors.

Orphanin FQ inhibits capsaicin-induced thermal nociception in monkeys by activation of peripheral ORL1 receptors.

1. Orphanin FQ (OFQ), an endogenous peptide for ORL1 receptors, has been identified. Although the actions of OFQ have much in common with those of opioid peptides at the cellular level, behavioral studies in rodents seem conflicting. 2. The aim of this study was to investigate the potential pronociceptive or antinociceptive function of peripheral ORL1 receptors in primates. Experiments were conducted to verify whether local administration of OFQ can attenuate capsaicin-induced nociception and whether peripheral ORL1 receptors selectively mediate the local action of OFQ in monkeys. 3. Capsaicin (100 microg) was administered subcutaneously in the tail to locally evoke a nociceptive response (thermal allodynia/hyperalgesia), which was manifested as a reduced tail-withdrawal latency in normally innocuous 46 degreeC warm water. 4. Co-administration of OFQ (1--30 microg) with capsaicin in the tail dose-dependently inhibited thermal nociception. However, a locally effective dose of OFQ (30 microg), when applied in the back, did not inhibit capsaicin-induced nociception. 5. OFQ-induced local antinociception was antagonized by a small dose (10 microg) of J-113397, a selective ORL1 receptor antagonist, in the tail. Similarly, s.c. administration of 10 microg of J-113397 in the back did not antagonize local antinociception of OFQ. 6. In addition, s.c. administration of either OFQ or J-113397 in the tail alone did not change its thermal nociceptive threshold. Local administration of opioid receptor antagonists selective for mu, kappa, and delta opioid receptors did not antagonize OFQ-induced local antinociception. Local administration of J-113397 also did not interfere with the local actions of mu, kappa, and delta opioid agonists in the tail. 7. These results provide the first functional evidence that activation of peripheral ORL1 receptors produces thermal antinociception in primates and this action is independent of antinociception produced at classical opioid receptors.

Pharmacological specificity of novel, synthetic, cyclic peptides as antagonists at tachykinin receptors.

1. The interaction at tachykinin receptors of a series of novel cyclic hexapeptides has been examined by use of radioligand binding assays (NK1 and NK3 sites in rat cortex, NK2 sites in hamster urinary bladder) and functional pharmacological assays (guinea-pig ileum, rat vas deferens and rat portal vein for NK1, NK2 and NK3 receptors, respectively). 2. The compounds cyclo(GlnTrpPhe(R)Gly[ANC-2]LeuMet) (L-659,837) and cyclo(GlnTrpPheGly-LeuMet) (L-659,877) were powerful and selective displacers of NK2 binding (pIC50 6.9 and 8.0, respectively), and were competitive antagonists of responses to stimulation of NK2 receptors in rat vas deferens (pKB for antagonism of responses to eledoisin 6.7 and 8.1, respectively). Responses in the NK1 and NK3 pharmacological assays were blocked only weakly, if at all. 3. In the longitudinal muscle of the small intestine of the rat, responses to stimulation of the putative NK2 receptor by eledoisin, neurokinin A or neurokinin B were antagonized by both cyclo(GlnTrpPhe(R)-Gly[ANC-2]LeuMet) and cyclo (GlnTrpPheGlyLeuMet) in a manner consistent with the presence in this tissue of a uniform population of receptors, indistinguishable from the NK2 receptor of the rat vas deferens. 4. The compounds cyclo(GlnTrpPheGlyLeuMet) and the lactam-containing analogue are among the most selective antagonists for the NK2 receptor that have been described; their availability should be of value in the characterization of the receptors mediating responses to tachykinins, and in elucidating the physiological functions of the tachykinin receptors.

Comparison of the effects of [Phe1psi(CH2-NH)Gly2]nociceptin(1-13)NH2 in rat brain, rat vas deferens and CHO cells expressing recombinant human nociceptin receptors.

Nociceptin(NC) is the endogenous ligand for the opioid receptor like-1 receptor (NC-receptor). [Phe1(psi)(CH2-NH)Gly2]Nociceptin(1-13)NH2 ([F/G]NC(1-13)NH2) has been reported to antagonize NC actions in peripheral guinea-pig and mouse tissues. In this study, we investigated the effects of a range of NC C-terminal truncated fragments and [F/G]NC(1-13)NH2 on NC receptor binding, glutamate release from rat cerebrocortical slices (rCX), inhibition of cyclic AMP accumulation in CHO cells expressing the NC receptor (CHO(NCR)) and electrically evoked contractions of the rat vas deferens (rVD). In radioligand binding assays, a range of ligands inhibited [125I]-Tyr14-NC binding in membranes from rCX and CHO(NCR) cells. As the peptide was truncated there was a general decline in pKi. [F/G]NC(1-13)NH2 was as potent as NC(1-13)NH2. The order of potency for NC fragments to inhibit cyclic AMP accumulation in whole CHO(NCR) cells was NCNH2> or =NC=NC(1-13)NH2>NC(1-12)NH2> >NC(1-11)NH2. [F/G]NC(1-13)NH2 was a full agonist with a pEC50 value of 8.65. NCNH2 and [F/G]NC(1-13)NH2 both inhibited K+ evoked glutamate release from rCX with pEC50 and maximum inhibition of 8.16, 48.5+/-4.9% and 7.39, 58.9+/-6.8% respectively. In rVD NC inhibited electrically evoked contractions with a pEC50 of 6.63. Although [F/G]NC(1-13)NH2, displayed a small (instrinsic activity alpha = 0.19) but consistent residual agonist activity, it acted as a competitive antagonist (pA2 6.76) in the rVD. The differences between [F/G]NC(1-13)NH2 action on central and peripheral NC signalling could be explained if [F/G]NC(1-13)NH2 was a partial agonist with high strength of coupling in the CNS and low in the periphery. An alternative explanation could be the existence of central and peripheral receptor isoforms.