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).

[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.

BIIE0246, a potent and highly selective non-peptide neuropeptide Y Y(2) receptor antagonist.

1. BIIE0246, a newly synthesized non-peptide neuropeptide Y (NPY) Y(2) receptor antagonist, was able to compete with high affinity (8 to 15 nM) for specific [(125)I]PYY(3 - 36) binding sites in HEK293 cells transfected with the rat Y(2) receptor cDNA, and in rat brain and human frontal cortex membrane homogenates. 2. Interestingly, in rat brain homogenates while NPY, C2-NPY and PYY(3 - 36) inhibited all specific [(125)I]PYY(3 - 36) labelling, BIIE0246 failed to compete for all specific binding suggesting that [(125)I]PYY(3 - 36) recognized, in addition to the Y(2) subtype, another population of specific NPY binding sites, most likely the Y(5) receptor. 3. Quantitative receptor autoradiographic data confirmed the presence of [(125)I]PYY(3 - 36)/BIIE0246-sensitive (Y(2)) and-insensitive (Y(5)) binding sites in the rat brain as well as in the marmoset monkey and human hippocampal formation. 4. In the rat vas deferens and dog saphenous vein (two prototypical Y(2) bioassays), BIIE0246 induced parallel shifts to the right of NPY concentration-response curves with pA(2) values of 8.1 and 8.6, respectively. In the rat colon (a Y(2)/Y(4) bioassay), BIIE0246 (1 microM) completely blocked the contraction induced by PYY(3 - 36), but not that of [Leu(31), Pro(34)]NPY (a Y(1), Y(4) and Y(5) agonist) and hPP (a Y(4) and Y(5) agonist). Additionally, BIIE0246 failed to alter the contractile effects of NPY in prototypical Y(1) in vitro bioassays. 5. Taken together, these results demonstrate that BIIE0246 is a highly potent, high affinity antagonist selective for the Y(2) receptor subtype. It should prove most useful to establish further the functional role of the Y(2) receptor in the organism.

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.

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.

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.

Pharmacological heterogeneity of neurotensin receptors: an in vitro study.

Neurotensin (NT), a linear tridecapeptide, has been shown to exert a variety of biological effects in the periphery and in the central nervous system. The aim of the present study was to characterize the NT receptors mediating the contractions of two isolated organs, the rat stomach strip and the guinea pig ileum. More than 20 compounds, peptides, nonpeptides, or pseudopeptides, were tested for their agonistic and antagonistic effects against NT and a series of potent analogs or fragments. Receptors were characterized using the two classical criteria suggested by Schild, the order of potency of agonists and the affinity of antagonists. The results shown in this study indicate that the contractions of the guinea pig ileum in response to NT are mediated by acetylcholine and prostaglandins because they are blocked by atropine and indomethacin. The contractions induced by NT in the rat stomach are not influenced by atropine, indomethacin, methysergide, and diphenhydramine and may result from the direct activation of smooth muscle receptors. Differences in the order of potency of agonists were also found between the two preparations: in the rat stomach strip, the order of potency was AcNT(8-13) > Arg-NT(8-13) > Lys-NT(8-13) > NT = NT(8-13) and in the guinea pig ileum was Arg-NT(8-13) > AcNT(8-13) > NT = NT(8-13) > Lys-NT(8-13). The nonpeptide antagonist SR 48692 was shown to possess higher apparent affinity for the rat stomach functional sites (pA2 8.0) than for those of the guinea pig ileum (pA2 6.45). The results presented in this paper suggest that two different pharmacological entities may subserve the myotropic effect of NT and some analogs and fragments in the gastrointestinal tract of the guinea pig and the rat.

Neurokinin receptors (NK1, NK2) in the mouse: a pharmacological study.

Experiments were performed on strips of mouse stomach and urinary bladder to characterize the receptors involved in the contractile responses of these tissues to neurokinins (substance P (SP), neurokinin A (NKA), neurokinin B (NKB), and neuropeptide gamma (NP gamma). The neurokinin receptors were characterized by using assays with selective agonists as well as peptide and nonpeptide antagonists and by applying the two Schild criteria for receptor classification, namely, the order of potency of agonists and the apparent affinity of competitive antagonists. The mouse stomach contains primarily NK1 and NK2 functional sites and possibly some NK3 receptors, whereas the urinary bladder possesses only the NK2 receptor. The rank order of potency of agonists in the stomach is Ac[Arg6,Sar9,Met(O2)11]SP-(6-11) > NKA > SP > [beta-Ala8]NKA-(4-10) > NKB > [MePhe7]NKB. Among the selective agonists, Ac[Arg6,Sar9,Met(O2)11]SP-(6-11) is more active than SP and [Sar9,Met(O2)11]SP on the NK1 receptor, whereas the order of potency on the NK2 receptor is NKA > NP gamma > or = [beta-Ala8]NKA-(4-10) > [Nle10]NKA-(4-10). The order of potency of agonists in the bladder is NP gamma > NKA > [beta-Ala8]NKA-(4-10). The myotropic responses mediated by NK1 selective agonists are blocked by SR 140333 (pA2 8.57) and those mediated by the NK2 selective agonists are inhibited by SR 48968 (pA2 9.05). RP 67580 (pA2 8.41) is more active than CP 99994 (pA2 6.06) on the mouse NK1 receptor. The NK1 receptor of the mouse shows, therefore, a pharmacological profile similar to that of the NK1 receptor of the rat. Similarly, MEN 10627 (pA2 9.20) is more active than R 396 (pA2 6.21), suggesting that the mouse NK2 receptor is similar to that of the rabbit. The mouse NK2 receptor of the urinary bladder shows similarity with that of the stomach, but is less sensitive to [beta-Ala8]NKA-(4-10).

Antagonists for kinin B1 and B2 receptors in the mouse.

Contractile responses to B1 and B2 receptor agonists have been demonstrated in the mouse stomach; the mouse urinary bladder responds only to B2 receptor agonists. These tissues were used in this study to investigate the antagonistic effect of four B2 receptor antagonists, namely, DArg[Hyp3,DPhe7,Leu8]BK (BK, bradykinin), HOE-140, WIN 64338, and FR-173657 (B2 receptor antagonists), as well as three B1 kinin receptor antagonists; [Leu8]desArg9BK, Lys[Leu8]desArg9BK, and AcLys[D beta Nal7,Ile8]desArg9BK, were investigated. Results shown indicate that DArg[Hyp3,DPhe7,Leu8]BK is a partial agonist, while HOE-140 and FR-173657 are pure antagonists, devoid of direct myotropic effects, and quite selective for the B2 receptor. WIN 64338 was essentially inactive on both B1 and B2 receptors. The myotropic effect of DArg[Hyp3,DPhe7,Leu8]BK is blocked by HOE-140. Similarly, Lys[Leu8]desArg9BK and [Leu8]desArg9BK are B1 receptor partial agonists whose activities are blocked by AcLys[D beta Nal7,Ile8]desArg9BK (code name R 715), a fairly pure B1 receptor antagonist. Both HOE-140 and FR-173657 are long-acting, slowly reversible compounds that exert a noncompetitive type of antagonism, while R 715 is rapidly reversible and, thus, possibly competitive. Data presented in this paper provide a pharmacological characterization of B1 and B2 receptor antagonists in the mouse and underline the positive features of FR-173657 as a potent and selective B2 receptor antagonist, as well as the potency and purity of R 715 as a B1 receptor antagonist in the mouse.

Pharmacological profiles of the human and rabbit B1 receptors.

Twenty-two peptides related to kinins were used (i) to examine some chemical features required for the human and rabbit B1 receptor activation or blockade and (ii) to establish the existence of a correlation between the pharmacological spectrum of the B1 receptor obtained on the rabbit aorta (rbA) and the human umbilical vein (hUV). The apparent affinities of these peptides were measured in vitro using classical bioassays and are expressed in terms of pD2 (for agonists) or pA2 values (for antagonists). Selectivity for the B1 receptor was demonstrated by testing the peptides against the effect of bradykinin (BK) on the hUV and the rabbit jugular vein (rbJV), two preparations containing B2 receptor-mediating vasoconstriction. The results show that (i) lysyl-peptide agonists and antagonists demonstrate higher affinities than nonlysyl compounds on human and rabbit B1 receptors, (ii) peptides containing hydrophobic D-residues (e.g., Tic, beta Nal, Hyp(trans-propyl), Igl) in position 7 are suitable for B1 receptor antagonism, and (iii) the additive substitution of an Oic residue in position 8 leads to nonselective kinin receptor antagonists. Moreover, a high (r = 0.92) and positive (regression slope = 0.99 +/- 0.09) correlation between the affinities measured for the kinin analogues in two B1 receptor bioassay systems has been revealed. Based on the similarity of pharmacological profiles observed in the rabbit and human B1 receptors, we suggest that the B1 receptor domain in which peptide agonists and antagonists interact may be similar in these two species.

Kinin receptors in the diabetic mouse.

It has been proposed that kinins are important inflammatory mediators involved in the pathogenesis of several diseases. In the present study, we attempted to determine the effects of kinins in a type I diabetic mouse model, using in vitro assays. Injection of streptozotocin (STZ) to the C57BL/Ks mdb mice causes an insulitis (inflammation of Langerhans islets) that leads to the diabetic condition. Ten days following the STZ treatment, the mice showed increased glycemia. We examined the effect of kinins and other agents (substance P, neurokinin A, acetylcholine) on the stomach fundus and urinary bladder of control and diabetic mice. Our results show that the sensitivity of the stomach fundus to bradykinin (BK) and desArg9BK (DBK), but not to other contractile agents, was substantially increased in the tissues of diabetic mice. The maximal contractions induced by BK and DBK were increased 1.5- to 2-fold in the stomachs from diabetic mice compared with those from normal mice. BK induced similar maximal contractions of urinary bladder strips from normal or STZ-treated mice, while DBK did not show any effect on this preparation. Interestingly, the apparent affinities of all agonists are similar in the two groups, normal and diabetic. These results suggest that B1 and B2 receptors are overexpressed in the stomach fundus but not in the urinary bladder of diabetic mice.

The dog saphenous vein: a sensitive and selective preparation for the Y2 receptor of neuropeptide Y.

The dog saphenous vein responds to neuropeptide Y with a dose-dependent contraction and this vasopressor effect is not altered by the removal of the endothelium nor by the neuropeptide Y Y1 receptor antagonist, BIBP 3226 ((R)-N2-(diphenylacetyl)-N-[(n-hydroxyphenyl)methyl]-argininami de). The dose-response curves obtained with neuropeptide Y, peptide YY and with C-terminal fragments such as neuropeptide Y-(2-26), neuropeptide Y-(13-36) and peptide YY-(3-36) have similar slopes and maxima. EC50 values of these compounds vary between 30 +/- 10 and 89 +/- 47 nM. The neuropeptide Y Y1 receptor-selective agonist [Leu31,Pro34]neuropeptide Y and human pancreatic polypeptide are inactive up to 1 microM. This pharmacological profile suggests that the contraction of the dog saphenous vein induced by neuropeptide Y and its homologues or fragments is mediated by a neuropeptide Y Y2 receptor type. Moreover, this neuropeptide Y Y2 receptor appears to be localized in the venous smooth muscle, where it exerts a direct myotropic effect that may be useful for the pharmacological characterization of new compounds acting as agonists or antagonists of the neuropeptide Y Y2 receptor.

Structure-activity studies of B1 receptor-related peptides. Antagonists.

We tested several peptides related to des-Arg9-bradykinin as stimulants or inhibitors of B1 (rabbit aorta, human umbilical vein) and B2 (rabbit jugular vein, guinea pig ileum, human umbilical vein) receptors. We also incubated the compounds with purified angiotensin-converting enzyme from rabbit lung to test their resistance to degradation. We evaluated apparent affinities (in terms of the affinity constant pA2) of compounds and their potential residual agonistic activities (alpha E). Bradykinin and des-Arg9-bradykinin were used as agonists for the B2 and B1 receptors, respectively. Degradation of peptides by the angiotensin-converting enzyme was prevented in the presence of a D-residue in position 7 of des-Arg9-bradykinin. Replacement of Pro7 with D-Tic combined with Leu, Ile, Ala, or D-Tic in position 8 led to weak B1 receptor antagonists, some of which had strong residual agonistic activities on the B2 receptor preparations. The use of D-beta Nal in position 7, combined with Ile in position 8 and AcLys at the N-terminal (eg, AcLys[D-beta Nal7, Ile8]des-Arg9-bradykinin) gave the most active B1 receptor antagonist (pA2 of 8.5 on rabbit aorta and human umbilical vein), which is also partially resistant to enzymatic degradation. Extension of the N-terminal end by Sar-Tyr-epsilon Ahx (used for labeling purposes) and even cold-labeling of Tyr with iodine were compatible with high, selective, and specific antagonism of the B1 receptors. We compared some compounds with some already known B1 receptor antagonists to underline the novelty of new peptidic compounds.

Receptors for kinins: from classical pharmacology to molecular biology.

In the past twenty years, we have focused our efforts on the study of kinin receptors involved in contraction or relaxation of vascular smooth muscle. Initial studies on rabbit vessels led to the discovery of two kinin receptors, B1 and B2, mediating contraction of the rabbit aorta (B1) and the rabbit jugular vein (B2). Studies on dog vessels contributed to the identification of B2 receptors in arterial endothelium promoting the release of NO and the relaxation of arterial smooth muscles; further studies on dog renal vessels led to the demonstration of B2 receptors in endothelia and in the smooth muscle, mediating relaxation through NO (endothelia) and prostanoids (smooth muscle). B1 receptors that relax renal arterial smooth muscle through the release of prostanoids were also identified. In other vessels, B2 receptors may also mediate smooth muscle contraction. Recent studies in human vessels (umbilical vein) have confirmed the existence of contractile B1 and B2 receptors in venous smooth muscles. B1 and B2 receptors have been cloned; molecular biology has provided the reference data for comparison with findings of classical pharmacology and binding assays. Similarities and differences in B1 and B2 receptors between human and animal tissues demonstrate the heterogeneity (related to species) of kinin B2 and B1 receptors and confirm the findings of early classical pharmacological experiments.

Receptors for kinins: from classical pharmacology to molecular biology.

In the past 20 years, we have focused our efforts on the study of kinin receptors involved in contraction or relaxation of vascular smooth muscle. Initial studies on rabbit vessels led to the discovery of two kinin receptors, B1 and B2, mediating contraction of the rabbit aorta (B1) and the rabbit jugular vein (B2). Studies on dog vessels contributed to the identification of B2 receptors in arterial endothelium promoting the release of NO and the relaxation of arterial smooth muscles; further studies on dog renal vessels led to the demonstration of B2 receptors in endothelia and in the smooth muscle, mediating relaxation through NO (endothelia) and prostanoids (smooth muscle). B1 receptors that relax renal arterial smooth muscle through the release of prostanoids were also identified. In other vessels, B2 receptors may also mediate smooth muscle contraction. Recent studies in human vessels (umbilical vein) have confirmed the existence of contractile B1 and B2 receptors in venous smooth muscles. B1 and B2 receptors have been cloned; molecular biology has provided the reference data for comparison with findings of classical pharmacology and binding assays. Similarities and differences in B1 and B2 receptors between human and animal tissues demonstrate the heterogeneity (related to species) of kinin B2 and B1 receptors and confirm the findings of early classical pharmacological experiments.

Pharmacological characterization of SR 142801: a new non-peptide antagonist of the neurokinin NK-3 receptor.

Pharmacological and biochemical assays were performed to characterize SR 142801, a new NK-3 receptor antagonist, and its [R]-enantiomer, SR 142806. The compounds were tested (1) in the guinea pig isolated ileum stimulated with [MePhe7]NKB (NK-3 system) in order to evaluate onset and duration of action and to estimate the apparent affinity of the antagonist in terms of pA2 at 140 min after application; (2) in 6 selected monoreceptor systems, the rabbit (rb) vena cava for the NK-1rb receptor, the rabbit pulmonary artery and the hamster (hs) urinary bladder for the NK-2rb and NK-2hs receptors, the rat (r) portal vein for the NK-3r receptor, and in two multireceptor systems adequately treated with NK-1 or NK-2 receptor antagonists to obtain monoreceptor-mediated biological responses (the rat urinary bladder treated with SR 48968 for evaluating the NK-1r and the guinea pig-gp-ileum treated with CP-99994 for measuring the antagonist affinity on the NK-3gp receptor), in order to evaluate the antagonist selectivity, and (3) in various plasma membrane preparations containing NK-3-binding sites from rats, guinea pigs, and man. The data presented indicate that SR 142801 is a potent, fairly selective non-peptide antagonist of the functional (pA2 9.4) and binding (Ki 0.11 nmol/l) site of the guinea pig and human (Ki 0.21 nmol/l) NK-3 receptors, while being much less active on the Nk-3 receptors of other species, particularly the rat (pA2 7.0; Ki 15 nmol/l). SR 142801 shows a slow onset of action and acts as a long-lasting irreversible antagonist, specific for neurokinin receptors, especially the NK-3 sites of guinea pigs and man.

Receptors for kinins in the human isolated umbilical vein.

1. The human umbilical vein has been found to contract in response to bradykinin (BK) and desArg9BK. 2. The rank order of potency of agonists, in the presence of the B1 receptor antagonist Lys[Leu8]desArg9BK, is as follows: [Hyp3, Tyr(Me)8]BK (pD2 8.88) = [Hyp3]BK (pD2 8.86) = LysBK (pD2 8.81) > or = BK (pD2 8.60) >> [Aib7]BK (pD2 6.38) >> desArg9BK and LysdesArg9BK (inactive). 3. Hoe 140 (pA2 8.42) inhibits the effects of BK while other B2 receptor peptide antagonists are very weak and WIN 64338 is practically inactive. 4. Venoconstrictor responses to desArg9BK of fresh tissues increase with time during the in vitro incubation and reach a maximum after 4-6 h. The activity of Hoe 140 (pA2 5.48) is negligible against B1 receptor agonists. 5. When measured in the presence of the selective B2 receptor antagonist Hoe 140 (400 nM), the order of potency of kinin related peptides on the B1 receptor is Lys[desArg9]BK (pD2 8.60) > desArg9BK (pD2 6.69). BK, LysBK, [Hyp3]BK and other B2 receptor agonists are inactive. 6. The B1 receptor antagonist, Lys[Leu8]desArg9BK (pA2 7.99), inhibits the response of the human vein to B1 receptor agonists (LysdesArg9BK or desArg9BK), but do not alter the effect of BK. 7. The results summarized in this paper indicate that the human isolated umbilical vein is a sensitive preparation containing both B1 and B2 receptors. The human B2 receptor shows some similarity with that of the rabbit (at least for agonist potencies) and differs from the B2 receptor of the guinea-pig. Compared to the rabbit B1 receptor, the human B1 receptor shows low sensitivity to peptides that lack the N-terminal Lys.

In vitro and in vivo characterization of bradykinin B2 receptors in the rabbit and the guinea pig.

A comparative study has been performed in isolated organs and in anesthetized animals, rabbits, and guinea pigs, to evaluated the myotropic responses (in the organs) and the blood pressure changes (in the animals) induced by bradykinin (BK) and related peptides. Antagonist affinities have also been estimated in vitro in terms of PA2 and in vivo in terms of ID50, to characterize the kinin B2 receptors in the two species. Differences have been found both in the order of potency of agonists and in the affinity of antagonists: in fact, in the rabbit, [Hyp3]BK > [Aib7]BK, is the opposite order of what is found in the guinea pig, namely, [Aib7]BK < [Hyp3]BK, both in vitro and in vivo. Results obtained with antagonists also show important differences between the two species, since DArg[Hyp3, DPhe7, Leu8]BK is more active in the rabbit than in the guinea pig, while WIN-64338 is fairly active in the guinea pig and almost inactive in the rabbit. HOE-140, the long-acting antagonist of the B2 receptor, shows similar affinities in vitro in the two species. In another series of experiments, peptide degradation by angiotensin converting enzyme (ACE) has been investigated to see whether the differences of potency observed between certain peptides interacting with the B2 receptor were due to metabolic degradation. When incubated in the presence of pure ACE from rabbit lung, BK,[Hyp3]BK, and des Arg9BK are readily degraded, while [Aib7]BK, HOE-140, and DArg[Hyp3, DPhe7, Leu8]BK are not. When applied intravenously (i.v.), to obtain degradation by the lung, and intraarterially (i.a.), to avoid such degradation, the effect of BK (i.v.) is markedly reduced (compared with the effect i.a.), while no difference is observed for [Aib7]BK. Thus, despite its resistance to degradation by ACE, [Aib7]BK shows very little activity in the rabbit, suggesting that the major cause in the variation of affinities observed between kinin analogs is related to their pharmacodynamic properties. Taken together, the results speak strongly in favor of the existence of B2 receptor subtypes in the peripheral circulation of the rabbit and the guinea pig. Results obtained in vivo, both in pharmacological and biochemical experiments, are in accord with the findings obtained in isolated organs and with purified ACE enzyme.