Pharmacological profile of hemokinin 1: a novel member of the tachykinin family.

Recently, the cloning of a novel preprotachykinin gene (PPT-C) has been reported. This gene codes for a novel peptide named hemokinin 1 (HK-1). In contrast with the known tachykinins, which are exclusively expressed in neuronal tissues, PPT-C mRNA was detected primarily in hematopoietic cells. In this study, we pharmacologically characterised the effects of HK-1 using three tachykinin monoreceptor systems, namely the rabbit jugular vein (rbJV) for NK(1), the rabbit pulmonary artery (rbPA) for NK(2), and rat portal vein (rPV) for NK(3) receptors. In all these preparations substance P (SP), neurokinin A (NKA) and neurokinin B (NKB) elicited concentration dependent contractions showing similar maximal effects and the following rank order of potency: SP > NKA = NKB in the rbJV, NKA > NKB >> SP in the rbPA, and NKB > NKA > SP in the rPV. In those vessels HK-1 behaved as a full agonist displaying potencies similar (rbPA and rPV) or slightly higher (rbJV) than those of SP. In the rbJV, SR 140333, a selective NK(1) receptor antagonist, antagonised the effects of HK-1 and SP with similar high potencies (pK(B) 9.3 and 9.5, respectively). Similar results were obtained with the pseudopeptide NK(1) antagonist, MEN 11467 (pK(B) 8.8 and 8.6, respectively). Taken together, these data indicate that HK-1 behaves as a NK(1) preferring receptor agonist.

Urodynamic and clinical evidence of acute inhibitory effects of intravesical nociceptin/orphanin FQ on detrusor overactivity in humans: a pilot study.

PURPOSE: Management of neurogenic incontinence is complex and available treatments are not satisfactory. Nociceptin/orphanin FQ, a recently discovered neuropeptide, has been reported to inhibit the voiding reflex in the rat. These experimental results prompted us to investigate the urodynamic and clinical effects of intravesical instillation of nociceptin/orphanin FQ in humans. MATERIAL AND METHODS: Our study involved 5 normal subjects (group 1) with a mean age of 40.4 years (range 21 to 54) and 9 patients (group 2) 40.4 years (24 to 54). All patients in group 2 presented with detrusor hyperreflexia refractory to standard therapy. They were invited to undergo a filling cystometrogram with saline solution and after 30 minutes, a new one with a solution containing 1 microM. nociceptin/orphanin FQ. The urodynamic parameters that were recorded included bladder capacity, volume threshold for the appearance of detrusor hyperreflexia and maximum bladder pressure. Clinical and urodynamic followup was performed after 15 days. The data were statistically analyzed with 1-way analysis of variance followed by the Dunnett test for multiple comparison considered statistically significant with p <0.05. RESULTS: Intravesical instillation of 1 microM. nociceptin/orphanin FQ in group 1 did not produce significant functional changes. This infusion in group 2 produced a statistically significant increase in mean bladder capacity and volume threshold for the appearance of detrusor hyperreflexia from 164 plus or minus standard deviation (SD) 84 to 301 +/- 118 and 93 plus or minus SD 41 to 231 +/- 104 ml. (p <0.05, respectively). Mean maximum bladder pressure decreased from 79 plus or minus SD 25 to 54 +/- 44 cm. water but was not statistically significant (p = 0.19). After 15 days an absence of clinical improvement was noticed in group 2, and the urodynamic control did not show any significant changes compared to the values before nociceptin/orphanin FQ treatment. No severe symptomatic reactions were observed during infusion of 1 microM. nociceptin/orphanin FQ. CONCLUSIONS: Our results demonstrate that nociceptin/orphanin FQ is able to elicit a robust inhibitory effect on voiding reflex in group 2 but not 1. The ideal dosage, route of administration of nociceptin/orphanin FQ and treatment interval are not yet established.

Structure-activity studies of the Phe(4) residue of nociceptin(1-13)-NH(2): identification of highly potent agonists of the nociceptin/orphanin FQ receptor.

A total of 32 compounds was prepared to investigate the functional role of Phe(4) in NC(1-13)-NH(2), the minimal sequence maintaining the same activity as the natural peptide nociceptin. These compounds could be divided into three series in which Phe(4) was replaced with residues that would (i) alter aromaticity or side chain length, (ii) introduce steric constraint, and (iii) modify the phenyl ring. Compounds were tested for biological activity as (a) inhibitors of the electrically stimulated contraction of the mouse vas deferens; (b) competitors of the binding of [(3)H]-NC-NH(2) to mouse brain membranes; and (c) inhibitors of forskolin-stimulated cAMP accumulation in CHO cells expressing the recombinant human OP(4) receptor. Results indicate that all compounds of the first and second series were inactive or very weak with the exception of [N(CH(3))Phe(4)]NC(1-13)-NH(2), which was only 3-fold less potent than NC(1-13)-NH(2). Compounds of the third series showed higher, equal, or lower potencies than NC(1-13)-NH(2). In particular, [(pF)Phe(4)]NC(1-13)-NH(2) (pF) and [(pNO(2))Phe(4)]NC(1-13)-NH(2) (pNO(2)) were more active than NC(1-13)-NH(2) by a factor of 5. In the mVD, these compounds showed the following order of potency: (pF) = (pNO(2)) > or = (pCN) > (pCl) > (pBr) > (pI) = (pCF(3)) = (pOCH(3)) > (pCH(3)) > (pNH(2)) = (pOH). (oF) and especially (mF) maintained high potencies but were less active than (pF). Similar orders of potency were observed in binding competition and cAMP accumulation studies. There was a strong (r(2) > or = 0.66) correlation between data observed in these assays. Biological activity data of compounds of the third series were plotted against some Hansch parameters that are currently used to quantify physicochemical features of the substituents. In the three biological assays agonist potency/affinity positively correlates with the electron withdrawal properties of the groups in the p-position of Phe(4) and inversely with their size.

Classification of kinin receptors.

This minireview is divided into three parts: the first part refers to the characterization and classification of kinin receptors using agonists and antagonists in isolated tissues (classical pharmacology). Two kinin receptors have been considered on the basis of their distinct pharmacology, namely the B1 receptor of the rabbit aorta (rank order of potency of agonists: LysdesArg9BK > desArg9BK > or = LysBK > BK; apparent affinities of antagonists Lys[Leu8]desArg9BK (pIC50 8.4) > [Leu8]desArg9BK (pIC50 7.4) >>> HOE 140, a B2 receptor antagonist, pIC50<5.0), and the B2 receptor of the rabbit jugular vein (potency of agonists: LysBK = BK >>> LysdesArg9BK = desArg9BK and HOE 140 (pIC50 9.0) >>> Lys[Leu8]desArg9BK, pIC50<5.0). The second part describes species-related B1 receptor subtypes, demonstrated by different pharmacological profiles of agonists and antagonists: human, rabbit and pig subtypes (LysdesArg9BK >> desArg9BK and Lys[Leu8]desArg9BK > [Leu8]desArg9BK) and dog, rat, mouse and hamster B1 receptors (desArg9BK = LysdesArg9BK and [Leus]desArg9BK = Lys[Leu8]desArg9BK). Affinities of agonists and antagonists in some species (man, rabbit, pig) are significantly increased (at least 10-fold) by the presence of a Lys at their N-terminus. The last part describes species-related B2 receptor subtypes supported by results obtained with non-peptide receptor agonists (FR 190997) and antagonists (FR 173657). While BK acts as a full agonist in man, rabbit and pig, FR 190997 behaves as a full agonist on human, as partial agonist on rabbit, and as pure antagonist on pig B2 receptors. Various hypotheses are considered to interpret these findings.

Permeability of endothelial monolayers to albumin is increased by bradykinin and inhibited by prostaglandins.

Using monolayers of bovine aortic endothelial cells (BAEC) in modified Boyden chambers, we examined the role of prostaglandins (PGs) in the bradykinin (BK)-induced increase of albumin permeability. BK induced a concentration-dependent increase of the permeability of BAEC, which reached 49.9 +/- 1% at the concentration of 10(-8) M. Two inhibitors of the prostaglandin G/H synthase, indomethacin (2.88 microM) and ibuprofen (10 microM), potentiated BK-induced permeability 1.8- and 3.9-fold, respectively. Exogenously administered PGE2 and iloprost, a stable analog of prostacyclin, attenuated the effect of BK in a concentration-dependent manner. Butaprost equally reduced the effect of BK, suggesting the participation of the EP2 receptor in this phenomenon. However, the EP4-selective antagonist AH-23848 did not significantly inhibit the protective effect of PGE2. The inhibitory effect of PGE2 was reversed by the adenylate cyclase inhibitor MDL-12330A (10 microM). These results suggest that BK-induced increase of permeability of BAEC monolayer to (125)I-labeled albumin is negatively regulated by PGs. This postulated autocrine activity of PGs may involve an increase in the intracellular level of cAMP.

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.

Nociceptin/orphanin FQ receptor ligands.

Nociceptin (NC), alias Orphanin FQ (OFQ) is a heptadecapeptide structurally related to opioid peptides, especially Dynorphin A, which, however, does not interact with classic opioid receptors. NC selectively activates its own receptor (OP(4)), which has been shown to be insensitive to the naturally occurring opioid peptides as well as to a large number of non-peptide opioid receptor ligands, including naloxone. Thus, the NC/OP(4) system represents a new peptide-based signaling pathway, which is pharmacologically distinct from the opioid systems. The pharmacological tools available for investigating NC actions are at present rather limited and include: 1) peptide ligands obtained from structure activity studies performed using NC(1-13)NH(2) as a template or discovered by screening peptide combinatorial libraries; 2) nonpeptide ligands that are either molecules already known to interact with classic opioid receptors or novel molecules designed and synthesized as selective ligands of the OP(4) receptor. In the present paper the functional data obtained from both in vitro and in vivo studies with each relevant OP(4) receptor ligand will be analyzed and discussed comparing the advantages and disadvantages of each molecule. We hope that the present work will aid investigators, working in the NC/OP(4) field, in the choice of the pharmacological tools suitable for their experiments.

Receptors for NPY in peripheral tissues bioassays.

Neuropeptide Y (NPY) and its congeners, peptide YY (PYY) and the pancreatic polypeptide (PP), have a large spectrum of peripheral actions. NPY is found in peripheral neurons, co-localized or not with noradrenaline; PYY and PP are expressed in endocrine cells of the pancreas and in the intestine of vertebrates. NPY is the most abundant peptide in the brain and is involved in the regulation of food intake and of circadian rhythm. It intervenes also in the process of anxiety and memory. NPY is a potent vasoconstrictor, a cardiac stimulant, and may affect the gut through enteric neurons. PYY and PP act mainly on the gastrointestinal system; however, when in blood, they can cross-react with functional sites elsewhere and replace NPY in some parts of the brain (e.g. regions involved in feeding behavior). These peptides act through G protein coupled receptors (GPCR) of which five different types are known and have been cloned (1,2); functional sites (receptors) for NPY have been found in vessels, the gut, and in vasa deferentia (3-6).

Further studies on nociceptin-related peptides: discovery of a new chemical template with antagonist activity on the nociceptin receptor.

Three series of nociceptin (NC)-related peptides were synthesized and their abilities (i) to bind to the NC sites expressed in mouse forebrain membranes, (ii) to inhibit the electrically evoked contraction of the mouse vas deferens, and (iii) to inhibit forskolin-stimulated cAMP accumulation in Chinese hamster ovary cells expressing the human recombinant NC receptor (CHONCR) were investigated. The compounds of the first series (a series) have an ordinary Xaa1-Gly2 bond, those of the second series (b series) have a Xaa1psi(CH2-NH)Gly2 pseudopeptide bond, and those of the third series (c series) have a peptoid (Nxaa1-Gly2) structure. The affinity values measured in the binding assay and in the two functional assays with the compounds of the three series showed high levels of correlation. Thus, (I) the compounds of the a series in which Phe1 was substituted with Tyr, Cha, or Leu acted as potent NC receptor agonists; (II) the b series compounds behaved as NC receptor antagonists in the mouse vas deferens and as full agonists in CHO(NCR) cells with different potencies depending on the first amino acid residue, [Phe1psi(CH2-NH)Gly2]NC(1-17)NH2 and [Phe1psi(CH2-NH)Gly2]NC(1-13)NH2 being the most potent compounds; (III) the compounds of the third series were all inactive both as agonists and as antagonists with the exception of [Nphe1]NC(1-17)NH2 and [Nphe1]NC(1-13)NH2, which behaved as NC receptor antagonists both in the isolated tissue and in CHO(NCR) cells (pKB 6.1-6.4). In conclusion, this study demonstrates that chemical requirements for NC receptor agonists are different from those of antagonists. Moreover, modifications of the steric orientation of the aromatic residue Phe1 in the NC sequence as obtained with the pseudopeptide bond between Phe1 and Gly2 or with the displacement of the benzyl side chain by one atom, as in Nphe1, lead respectively to reduction or elimination of efficacy. Indeed, in contrast to [Phe1psi(CH2-NH)Gly2]NC(1-13)NH2 which has been reported to exhibit agonist activity in several assays involving either central or recombinant NC receptors, [Nphe1]NC(1-13)NH2 antagonizes the effect of NC at human recombinant NC receptors and in the mouse tail withdrawal assay.

Pharmacological characterization of the nociceptin receptor mediating hyperphagia: identification of a selective antagonist.

RATIONALE: Central injections of nociceptin (NC) stimulate feeding in rats. OBJECTIVE: The present study evaluated the effect of N-terminal partial sequences or analogues of NC on food intake in male Wistar rats, to characterize pharmacologically the NC receptor mediating the hyperphagic effect. METHODS: NC and related peptides were injected into the lateral (LV) or third (3V) cerebroventricle in freely feeding rats. RESULTS: In the LV, NC stimulated feeding. The N-terminal fragment NC(1-13)NH2 proved to be the least active sequence with hyperphagic activity; NC(1-12)NH2 and NC(1-9)NH2 were inactive. [Phe(1)psi(CH2-NH)Gly(2)]NC(1-13)NH2 ([F/G)]NC(1-13)NH2), an analogue of NC(1-13)NH2, markedly stimulated feeding and, coadministered in the LV with NC, never reduced the hyperphagic effect of the natural sequence. These findings suggest that [F/G)]NC(1-13)NH2, which has been reported to act as a NC receptor antagonist in peripheral tissues, behaves as a full agonist at the central NC receptors controlling feeding. The hyperphagic potencies of NC and [F/G)]NC(1-13)NH2 were much higher following injection into the 3V than in the LV. Another analogue of NC(1-13)NH2, namely [Nphe(1)]NC(1-13)NH2, injected into the 3V did not stimulate feeding, but reduced the effect of NC. [Nphe(1)]NC(1-13)NH2 at a dose of 16.8 nmol/rat significantly reduced, and at 168 nmol/rat almost completely abolished the effect of NC (1.68 nmol/rat). The latter dose of [Nphe(1)]NC(1-13)NH2 significantly reduced also feeding induced by food deprivation, but did not modify the hyperphagic effect of neuropeptide Y (0.3 nmol/rat). CONCLUSIONS: The present results confirm the orexigenic effect of NC in freely feeding rats and indicate that [Nphe(1)]NC(1-13)NH2 may represent a selective NC receptor antagonist to study the physiological and pathophysiological role of NC in feeding behaviour.

[Nphe(1)]nociceptin-(1-13)NH(2) selectively antagonizes nociceptin effects in the rabbit isolated ileum.

When suspended in vitro in isolated organ baths, segments of the rabbit ileum show a fairly strong and stable spontaneous activity, which derives from the continuous release of acetylcholine and the activation of muscarinic receptors, since the activity is completely eliminated by atropine. Dynorphin A (pEC(50): 8.6+/-0.07), neuropeptide Y and its congener human pancreatic polypeptide (pEC(50): 9.40+/-0.10), and nociceptin (pEC(50): 8.08+/-0.12) dose-dependently inhibit the spontaneous activity through the activation of receptors, which are specifically antagonised respectively by naloxone (pA(2): 7.17+/-0.12), 2-(naphtalen-1-ylamino)-3-phenylpropionitrile (JCF 104; pA(2): 5. 80+/-0.10), and [Nphe(1)]nociceptin-(1-13)NH(2) (pA(2): 6.17+/-0.19). This last compound, a selective nociceptin-receptor (OP(4)) antagonist, inhibits the effect of nociceptin in a competitive manner, as demonstrated by Schild analysis. [Nphe(1)]nociceptin-(1-13)NH(2) also antagonizes the effects of other OP(4) receptor ligands such as the full agonist, nociceptin-(1-13)-NH(2), and the partial agonists, [Phe(1)psi(CH(2)-NH)Gly(2)]nociceptin-(1-13)-NH(2) (intrinsic activity (alpha(E))=0.5) and Ac-RYYWK-NH(2) (alpha(E)=0.5), with pA(2) values ranged from 5.8 to 6.2. These results indicate that the functional site mediating the inhibitory effect of nociceptin in the rabbit ileum, is pharmacologically identical to the OP(4) sites of other species (mouse, rat, guinea pig, man), since the potencies (pA(2) values) of the pure and competitive antagonist [Nphe(1)]nociceptin-(1-13)NH(2) is very similar to the values obtained in the other species. Moreover, the rabbit ileum is one of the few isolated organs that allow classifying compounds, which interact with OP(4) receptors as full agonists, partial agonists, or pure antagonists.

Neuropeptide Y-induced contraction is mediated by neuropeptide Y Y2 and Y4 receptors in the rat colon.

Ascending and descending segments of the rat colon were studied to analyze their contractile responses to neuropeptide Y and related peptides. These responses are (a) completely eliminated by tetrodotoxin (1 microM), (b) reduced to a variable extent (20 to 60%) by atropine (1 microM) and (c) not modified by indomethacin, diphenhydramine or methysergide. The order of potency of agonists for peptides related to neuropeptide Y was as follows: human pancreatic polypeptide = rat pancreatic polypeptide > peptide YY = peptide YY-(3-36) = [Leu31,Pro34]neuropeptide Y > neuropeptide Y-(2-36) > C2-neuropeptide Y = neuropeptide Y > neuropeptide Y-(13-36), with minor differences observed between the two parts of the colon. This selectivity pattern does not correspond to the profile of any known cloned neuropeptide Y receptors. BIBP3226, a selective antagonist for the neuropeptide Y Y1 receptor sub-type, was found to be inactive, while a neuropeptide Y Y2 receptor antagonist, T4-[NPY-(33-36)]4, reduced the effects of neuropeptide Y, peptide YY, peptide YY-(3-36) and C2-neuropeptide Y without affecting those of human pancreatic polypeptide, rat pancreatic polypeptide and [Leu31,Pro34]neuropeptide Y. JCF 104 (compound 28), a putative neuropeptide Y Y5 receptor antagonist, showed no effect or a weak inhibition of human pancreatic polypeptide or [Leu31,Pro34]neuropeptide Y-induced contraction. Taken together, these data suggest that: (1) at least two neuropeptide Y receptor types are present in the rat colon autonomic nerve terminals and modulate the release of acetylcholine and possibly other transmitters; (2) a proportion of the receptors mediating the contractile response of the rat colon (especially descending part) to neuropeptide Y and related peptides appears to be of the Y2 type and (3) the significant portion of the response is mediated by a receptor which is insensitive to neuropeptide Y Y1, Y2 and to neuropeptide Y Y5 receptor antagonists. This receptor behaves as a neuropeptide Y Y4 receptor sub-type and appears to be located on enteric nerves.

Characterization of nociceptin receptors in the periphery: in vitro and in vivo studies.

Nociceptin (NC), a series of NC fragments, naloxone as well as the pseudopeptide [Phe1psi(CH2-NH)Gly2]NC(1-13)NH2 ([F/G]NC(1-13)NH2) were used to characterize NC receptors in peripheral isolated organs and in vivo. Experiments on isolated organs were performed in the mouse (mVD) and rat (rVD) vas deferens (noradrenergic nerve terminals), in the guinea pig ileum (gpI; cholinergic nerves) and in the renal pelvis (gpRP; sensory nerves), and, in vivo, by measuring the blood pressure (BP) and heart rate (HR) in anaesthetised rats. NC, NCNH2 and NC(1-13)NH2 acted as full agonists with similar affinities, while shorter fragments (e.g. NC(1-12)NH2, NC(1-9)NH2, NC(1-5)NH2) were much weaker or inactive. The inhibitory effects of NC were not modified by naloxone. [F/G]NC(1-13)NH2 acted as an antagonist with similar pA2-values (6.75 mVD, 6.83 rVD, 7.26 gpI) in the three species. In addition, it blocked NC actions in the rat in vivo. Linear Schild plots with slopes near to unity indicated that [F/G]NC(1-13)NH2 is a competitive antagonist, specific for NC receptors both in vitro (since it was inactive on opioid receptors) and in vivo (since it was inactive against carbachol). [F/G]NC(1-13)NH2 showed a residual agonistic activity in vitro (alpha = 0.2-0.3 in the rVD and gpI) and especially in vivo (alpha = 0.4 BP, 0.2 HR). These pharmacological data indicate that NC and related peptides exert their inhibitory effects in peripheral organs of various species by activating the same receptor type. Moreover, [F/G]NC(1-13)NH2 appears to be a useful tool for receptor characterization and classification.

Kinin B1 receptor antagonists containing alpha-methyl-L-phenylalanine: in vitro and in vivo antagonistic activities.

-To protect from metabolism and to improve potency of the AcLys-[D-betaNal7,Ile8]desArg9-bradykinin (BK) (R 715), we prepared and tested 3 analogues containing alpha-methyl-L-Phe ([alphaMe]Phe) in position 5: these are the AcLys-[(alphaMe)Phe5,D-betaNal7, Ile8]desArg9BK (R 892), Lys-Lys-[(alphaMe)Phe5,D-betaNal7, Ile8]desArg9BK (R 913), and AcLys-Lys-[(alphaMe)Phe5,D-betaNal7, Ile8]desArg9BK (R 914). The new compounds were tested against the contractile effect induced by desArg9BK on 2 B1 receptor bioassays, the human umbilical vein, and the rabbit aorta. Their antagonistic activities were compared with those of the early prototypes (Lys-[Leu8]desArg9BK and [Leu8]desArg9BK) and with other recently described peptide antagonists. The 3 (alphaMe)Phe analogues showed high antagonistic potencies (pA2) at both the human (8.8, 7.7, and 8. 7, respectively) and rabbit (8.6, 7.8, and 8.6, respectively) B1 receptors. No antagonistic effects (pA2<5) were observed on the B2 receptors that mediate the contractile effects of BK on the human umbilical vein, the rabbit jugular vein, and the guinea pig ileum. Moreover, these new B1 antagonists were found to be resistant to in vitro degradation by purified angiotensin-converting enzyme from rabbit lung. The Nalpha-acetylated forms, R 892 and R 914, were resistant to aminopeptidases from human plasma. In vivo antagonistic potencies (ID50) of B1 receptor antagonists were evaluated in anesthetized lipopolysaccharide-treated (for B1 receptor) and nontreated (for B2 receptor) rabbits against the hypotensive effects of exogenous desArg9BK and BK. R 892 efficiently inhibited (ID50 2.8 nmol/kg IV) hypotension induced by desArg9BK without affecting that evoked by BK (ID50 >600 nmol/kg IV). Conversely, the peptide antagonists Lys-Lys-[Hyp3,Igl5,D-Igl7,Oic8]desArg9BK (B 9858) and DArg-[Hyp3,Thi5,D-Tic7,Oic8] desArg9BK (S 0765) showed dual B1/B2 receptor antagonism in vitro and in vivo. It is concluded that R 892 and congeners provide selective, highly potent, and metabolically stable B1 kinin receptor antagonists that can be useful for the assessment of the physiological and pathological roles of kinin B1 receptors.

Effects of vasoactive agents in healthy and diseased human saphenous veins.

PURPOSE: Smooth muscle reactivity is one of the factors involved in the pathogenesis of varicose veins. We investigated the myotropic effects of the 3 main vasoconstrictor agents norepinephrine (NE), angiotensin II (Ang II), and endothelin-1 (ET-1) in isolated human saphenous veins. METHODS: Human saphenous veins were collected from 23 patients with primary chronic venous insufficiency who underwent elective varicose vein resections and who were stratified into the following 3 groups: group 1, 7 patients in clinical class 2; group 2, 9 patients in clinical classes 3 and 4; and group 3, 7 patients in clinical classes 5 and 6. Moreover, 6 patients who underwent arterial bypass grafting procedures represented the control group. The tissues were suspended in organ baths that contained Krebs solution, and their mechanical responses were measured isometrically. The cumulative concentration-response curves to Ang II, NE, and ET-1 were performed at 90-minute intervals in each tissue. RESULTS: In the control tissues, NE, Ang II, and ET-1 induced concentration-dependent contractions with apparent affinities (pEC50, the negative logarithm to base 10 of the molar concentration of the agonist, which produces the 50% of the maximal effect) and maximal effects (maximum effect, g of contraction) that were equal to 7.06 +/- 0.23, 8.53 +/- 0.34, 7.63 +/- 0.10, and 2.21 +/- 0.33, 1.65 +/- 0.31, 2.60 +/- 0.77, respectively. Two main findings were evident in comparison of varicose veins with control tissues. First, the maximum effect that was evoked by all of the stimulants was reduced progressively with the increasing severity of the disease, which raised the third group to statistical significance for both NE and Ang II (P <.05). Second, a marked reduction of Ang II apparent affinity was already evident in tissues that were taken from patients in an early stage of the disease (P <.05). CONCLUSION: The demonstration of a significant reduction in Ang II and NE contractile activities and the important reduction of that of ET-1 in the diseased veins as compared with the control tissues extends the previous observations regarding the impairment of smooth muscle contractility in primary chronic venous insufficiency. Moreover, the dramatic reduction of Ang II affinity, which appears in an early stage of the disease, supports the hypothesis that such abnormality within the venous wall could play a role in the pathogenesis of primary varicose vein disease.

Bioassays for NPY receptors: old and new.

Classical pharmacology performed on isolated organ preparations is an essential tool for receptor characterization and classification. Basic pharmacological parameters (e.g. ED50, ID50, pD2, pA2 as measures of apparent affinities) obtained by relating the agent concentration with the biological effect are the final results of the various steps required for drug action and necessarily reflect the complex mechanisms of cell function. Results obtained with bioassays are therefore a useful and essential part in the assessment of endogenous systems, in the present case, the NPY family of peptides and their receptors. An attempt has been made, in the present review, to present a choice of isolated organs that may provide a starting point towards the construction of a solid classical pharmacology of receptors for NPY and congeners. Some of these organs appear to be 'monoreceptor systems' (e.g. the rabbit saphenous vein) whose response is contributed by a single receptor type, others (e.g. the rat colon) are 'multiple receptor systems' and their pharmacology is much too complex and requires the use of a variety of compounds from the naturally occuring peptides, to some selective agonists and when available, specific and selective antagonists. Such compounds have been utilised by us and other workers to detect specific biological responses to NPY and congeners in peripheral tissues: such responses have been quantified, carefully analysed in pharmacological terms and characterized as biological effects mediated by Y1 (the rabbit saphenous vein), Y2 (dog saphenous vein, rat vas deferens, rat colon), Y4 (rat colon) and Y5 (rabbit ileum) receptors. Compared to findings obtained with binding assays and molecular biology experiments, the results of the bioassays show very interesting similarities. Much remains however to be done in view of providing the classical pharmacological bases that are needed in the field of NPY.

Nociceptin receptor binding in mouse forebrain membranes: thermodynamic characteristics and structure activity relationships.

The present study describes the labelling of the nociceptin (NC) receptor, ORL1, in mouse forebrain membranes with a new ligand partially protected from metabolic degradation at the C-terminal; the ligand, [3H]-NC-NH2, has a specific activity of 24.5 Ci mmol(-1). Saturation experiments revealed a single class of binding sites with a KD value of 0.55 nM and Bmax of 94 fmol mg(-1) of protein. Non specific binding was 30% of total binding. Kinetic binding studies yielded the following rate constants: Kobs = 0.104 min(-1); K1 =0.034 min(-1): T1/2=20 min; K(+1)=0.07 min nM(-1). Thermodynamic analyses indicated that [3H]-NC-NH2 binding to the mouse ORL1 is totally entropy driven, similar to what has been observed for the labelled agonists to the opioid receptors OP1(delta), OP2(kappa) and OP3(mu). Receptor affinities of several NC fragments and analogues, including the newly discovered ORL-1 receptor antagonist [Phe1psi(CH2-NH)Gly2]NC(1-13)-NH2([F/G]NC(1-13)-NH2), were also evaluated in displacement experiments. The competition curves for these compounds were found to be parallel to that of NC and the following order of potency was determined for NC fragments: NC-OH = NC-NH2-NC(1-13)-NH2 > > NC(1-12)-NH2 > NC(1-13)-OH > > NC(1-11)-NH2, and for NC and NC(1-13)-NH2 analogues: [Tyr1]NC-NH2 > or = [Leu1]NC(1-13)-NH2 > or = [Tyr1]NC(1-13)-NH2 > or = [F/G]NC(1-13)-NH2 > > [Phe3]NC(1-13)-NH2 > [DF/G]NC(1-13)-NH2. Standard opioid receptor ligands (either agonists or antagonists) were unable to displace [3H]-NC-NH2 binding when applied at concentrations up to 10 microM indicating that this new radioligand interacts with a non opioid site, probably the ORL1 receptor.

Pharmacology of kinins in the arterial and venous mesenteric bed of normal and B2 knockout transgenic mice.

We have tested the vasoactive effects of kinins in addition to various other endothelium-dependent or independent agonists in the arterial and venous perfused mesenteric circuits of the mouse. Bradykinin (0.1 pmol-100 nmol), but not des-Arg9-bradykinin (10 nmol) induced a dose-dependent vasodilation of the precontracted arterial and venous mesenteric vasculature of the mouse. Furthermore, acetylcholine (2.5 nmol) also induced a marked arterial vasodilation but was without effect on the venous side. Other endothelium-dependent vasodilators, such as platelet-activating factor (PAF) (1 nmol), tachykinin NK1 selective agonist ([Sar9,Met(O2)(l1) ]substance P) (0.5 nmol) and adenosine diphosphate (5 nmol), were without effect on either side of the mesenteric bed of the mouse. The bradykinin B2 receptor selective antagonist (HOE 140) abolished the arterial and venous vasodilation induced by bradykinin without affecting that of acetylcholine or sodium nitroprusside. In addition, the bradykinin B1 receptor antagonist des-Arg9-[Leu8]bradykinin was without effect on the responses induced by bradykinin. A nitric oxide synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) markedly reduced, whereas removal of the endothelium with 3-[3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) abolished dilatation to bradykinin and acetylcholine (arterial side only) without affecting that induced by sodium nitroprusside in the mouse arterial and venous mesenteric circuits. In the same two circuits of transgenic B2 knockout mice, the vasodilatory responses to bradykinin were absent, whereas the arterial circuit still responded to acetylcholine by a L-NAME-sensitive vasodilation. Our results suggest the exclusive contribution of B2 receptors located on the endothelium in the vasodilatory effects of bradykinin in the arterial and venous mesenteric circuits of the mouse.

The rabbit ileum: a sensitive and selective preparation for the neuropeptide Y Y5 receptor.

The rabbit ileum shows high sensitivity to neuropeptide Y. Relaxations are obtained in this tissue with human pancreatic polypeptide > peptide YY > > [Leu31,Pro34]neuropeptide Y > rat pancreatic polypeptide > human neuropeptide Y in this order of potency that is indicative of a Y5 receptor. Effects of neuropeptide Y and congeners are not affected by neuropeptide Y Y1 receptor antagonist (BIBP 3226), but are reduced by the neuropeptide Y Y5 receptor antagonist JCF 104 (2-(naphtalen-1-yl)-3-phenylpropane-1,2-diamine). Rabbit ilea provide sensitive and selective neuropeptide Y Y5 receptor preparations.

DNA antisense strategies in the study of receptors for vasoactive peptides, and of growth and wound-healing factors.

Antisense oligodeoxynucleotide technology has contributed greatly to the overall understanding of both mRNA stability as well as translational processes leading to protein synthesis. Arrest of translational processes by DNA antisense strands usually reduces maximal effects of agonists without affecting their apparent affinities in treated isolated vascular or nonvascular preparations. In the present study, examples are given of DNA antisense oligonucleotide-induced repression of receptors for endothelins, kinins as well as of the platelet-derived growth factor. Furthermore, the efficiency of this technology illustrates the roles of protooncogenes (c-myc and c-myb) in wound-healing mechanisms. The overall mechanism of action of these oligomers is described and the relevance of size, chemical alterations and mode of delivery are illustrated. Release of oligophosphorothioates from polymer matrices and gels can produce a prolonged effect in vivo. Antisense oligonucleotides remain essential in experimental models for which receptor antagonists or selective inhibitors of intracellular components are currently unavailable.