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[Sar9, Met(O2)11]-substance P-induced chromodacryorrhea in gerbils; A model of NK1 receptor function
238 P120 Functional Antagonism of Substance P and Related Peptides by RP 67580 and CP96,345, two Novel Non-peptide NKl-receptor Antagonists, in the Rat Urinary Bladder in vitro and in vivo F. Montier, A. Carruette, S. Moussaoui, D. Boccio and C. Garret Rhone-Poulenc Rorer S. A., Biology Department, Centre de Recherche de Vitry-Alfortville, 94403 Vittysur-Seine, Cedex, France Tonic contraction of rat urinary bladder was elicited in vitro and in vivo (urethane anaesthesia and chlorisondamine pretreatment) by substance P (SP), two selective NKI receptor agonists (septide, [S&,Met (O#l] SP) and an NK2 agonist [LysS,MeLeu9,Nle10]NKA+,01, but not by se&tide, an m agonist. SP only stimulated the NKI receptors of smooth muscle in this organ. The non-peptide selective NKI antagonists, RP 67580 and CP-96,345, both inhibited SP-induced contraction (p& values = 6.7 and 5.7, respectively; EDSO= 1.4 and 5.0 m&g i.v., respectively) and septide-induced contraction @KB values = 7.5 and 6.5; EDso = 0.076 and 0.250 mgkg i.v., respectively). Both antagonists also blocked plasma extravasation produced either by SP or by septide in urinary bladders of pentobarbital-anaesthetized rats. RP 67580 was always more potent than CP-96,345. That both antagonists blocked the effects of septide from 7 to 21 times more strongly than the effects of SP in the rat suggests the existence of NK1 receptor subtypes which are not species-dependent. Selective NKI antagonists, by blocking both spasm and plasma extravasation in the urinary bladder, would be useful in treating SP-related disorders and cystitis.
P121 Miotic Effects of CGRP(32-37) and CGRP(f!L37) in the Rabbit Eye; Interactions with Tachyhinin Receptors B. Almegiird and S. E. Andersson Department of Physiology and Medical Biophysics, Uppsala University, Sweden We have previously reported that CGRP(32-37) is a miotic in the monkey eye, interacting with CCK-A receptors. The present experiments were designed to investigate the effects of intracameml injections of CGRP(32-37) and CGRP(8-37) on pupil diameter and blood-aqueous barrier in the rabbit eye. The experiments were performed on anaesthetized New Zealand White rabbits, which were indomethacin pretreated and under muscarinic blockade. The animals responded with miosis to both CGRP fragments. CGRl?(8-37) was more potent than CGRP(32-37), but one order of magnitude less potent than substance P. The effect was not inhibited by tetrodotoxin, indicating a direct effect on the muscle. Pretreatment with the classical tachykinin receptor antagonist spantide, or the NK-1 selective antagonist GR82334 caused a rightward shift of
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the dose-response curves for both fragments, while the CCK-receptor antagonist loxighunide had no inhibitory effect on the miosis. Neither of the fragments induced any marked leakage of Evans blue into the aqueous humor, indicating that there was no agonistic interaction with CGRP receptors in the eye. We conclude that CGRP(8-37) and CGRP(32-37) are miotic agents in the rabbit eye, possibly by acting as neurokinin receptor agonists.
P122 Characteristics of [3H]-[Sar9,Met(O#1]substance P Binding to Astrocytoma Ceiis; Dependence Upon Peptide used to Define Nonspecific Binding C. J. Fowler and G. Briinnstrijm Preclinical Research, Astra Pain Control AE, S- 15 1 85 Siidertlilje, Sweden Substance P (SP)- sensitive [3H]-[S&,Met(O#‘]-SP binding has been shown in membrane preparations to label selectively the NK-1 receptor.’ In intact cells, however, such SP-sensitive binding will overestimate the number of NK- 1 receptors, since the cell culture wells contain SPsensitive binding sites2 The aim of the present study was therefore to find an alternative to SP for the definition of non-specific binding. The binding of 0.5 nM [3H]-[S&, Met(O$+SP to 24 well culture plates was inhibited by 70-90% by 1 pM SP (I& 100 nM). Physalaemin, on the other hand, did not inhibit this binding even at a concentration of 10 pM. SP-sensitive and physalaemin-sensitive [‘HI-[Sar9, Met(O#l]-SP binding were compared using UC1 1MG cells (which express NK-1 receptors, ‘). At low cell densities and in the absence of Ca2+ and Mg2+, a significantly higher Ko (1.57 vs 0.77 nM) andB, (244~s 106 fmoV106 cells) was found with SP than with physalaemin. (i)CP96345 (5 nM) inhibited physalaemin-sensitive binding by 78%, whereas the SP-sensitive binding was inhibited by 49%. Although the differences in SP- andphysalaemin-sensitive binding site densities can be minimised by increasing the cell density and by the presence of Ca2+ and MgZ in the assay medium, the data indicate that physalaemin should be used to define non-specific binding. 1. Dam, T.-V., Martinelli, B. and Quirion, R. (1990). Brain Res, 531: 333-337. 2. Fowler, C. J. and Martinsson, K. (1992). Regul. Peptides Suppl. 1: S58. 3. Johnson, C. L. and Johnson, C. G. (1992). J Neurochem, 58: 471-477.
P123 [Sarg, Met(Oz)“]-Substance P-induced Chromodacryorrhea in Gerbils; A model of NKl receptor function L. J. Bristow and L. Young Merck, Sharp and Dohme Research Laboratories, Neuroscience Research Centre, Terlings Park, Eastwick Rd., Harlow, Essex, CM20 2QR, UK
NEUROPEPTIDES:
239
FUNCTION AND PHARMACOLOGY
Recent studies have demonstrated the existence of species homologues of the tachykinin NK1 receptor. The present study has investigated the effects of the selective NKI receptor agonist [S&, Met(O$+Substance P in gerbils which have a similar NK1 receptor pharmacology to man. Male Mongolian gerbils (55-75 g) were anaesthetised with sodium pentobarbitone (60 mgkg, i.p.) and the jugular vein exposed. Intravenous injection of [S&, Met(O$l]-Substance P induced a dose dependent chromodacryorrhea (EDTO= 0.017 nmoles/gerbil) which was significantly antagonised following 15 min pretreatment with the NKI receptor antagonists (+CP 96,345 (ED50 = 0.48 mg/kg, iv.) or CP-99,994 (ED50= 0.15 mg/kg, i.v.). In contrast, CP-10,263 (inactive enantiomer of CP99,994; l-10 mgkg, i.v.) and the rodent selective NKI receptor antagonist, R&67,580 (0.3-l 0 mgkg, i.v.) failed to attenuate this response. In contrast to [S&, Met(O#‘]-Substance P, the selective NK receptor agonist, senktide (0.1-100 nmoles/gerbil, i.v.) did not induce chromodacryorrhea. However, intravenous administration of GR-64349, reported to have > 300-fold selectivity for NIC receptors compared to NKI receptors, also induced significant chromodacryorrhea (EDJO= 0.13 nmoles/gerbil). This response, however, was dose-dependently antagonised by pretreatment with the selective NK, receptor antagonist CP-99,994 (EDso = 0.09 mgkg, i.v.). In conclusion, the present studies suggest that [Sar’, Met(O$+Substance P-induced chromodacryorrhea in gerbils is an NK1 receptor mediated response. Furthermore, the ability of selective NK, receptor antag-
onists to attenuate GR-64349~induced chromodacryorrhea suggests that, in the gerbil at least, GR-64349 has significant NKI agonist activity in vivo. P124 Baculovirus Expression of Human NIG
Receptor
J. M. Guillaurne, F. Bellot, A. Jolly, N. Couteault, A. Fallourd, H. M. Soria, I. Gravier, S. Benoist, V. Fardin and A. Crespo Rhone-Poulenc Rorer, 13 quai Jules Guesde, 94403 Vitry sur Seine, France The cDNA encoding human NIG tachykinin receptor (receptor for neurokinin A) was cloned in a baculovirus transfer vector. Upon infection with the resulting NIG recombinant baculovirus, Sf 2 1 insect cells displayed lzsI NKA ligand binding activity, over 95% specific. Expression is maximum 4 days post-infection. The density of binding sites (Eimax) is about 1200 fmol/mg membrane protein as assessed by 125INKA radioligand binding on membrane preparations. Competition studies with selective NIG takykinin related agonist and antagonist compounds exhibit characteristic pharmacological profiles of human NK, receptor expressed in mammalian cell line (see Fardin et al., this meeting). The baculovirus-insect cell expression system offers significant advantages in terms of ease of use and high level of expression; therefore it may provide a powerful tool for large-scale drug screening procedures.
P121 Miotic Effects of CGRP(32-37) and CGRP(f!L37) in the Rabbit Eye; Interactions with Tachyhinin Receptors B. Almegiird and S. E. Andersson Department of Physiology and Medical Biophysics, Uppsala University, Sweden We have previously reported that CGRP(32-37) is a miotic in the monkey eye, interacting with CCK-A receptors. The present experiments were designed to investigate the effects of intracameml injections of CGRP(32-37) and CGRP(8-37) on pupil diameter and blood-aqueous barrier in the rabbit eye. The experiments were performed on anaesthetized New Zealand White rabbits, which were indomethacin pretreated and under muscarinic blockade. The animals responded with miosis to both CGRP fragments. CGRl?(8-37) was more potent than CGRP(32-37), but one order of magnitude less potent than substance P. The effect was not inhibited by tetrodotoxin, indicating a direct effect on the muscle. Pretreatment with the classical tachykinin receptor antagonist spantide, or the NK-1 selective antagonist GR82334 caused a rightward shift of
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the dose-response curves for both fragments, while the CCK-receptor antagonist loxighunide had no inhibitory effect on the miosis. Neither of the fragments induced any marked leakage of Evans blue into the aqueous humor, indicating that there was no agonistic interaction with CGRP receptors in the eye. We conclude that CGRP(8-37) and CGRP(32-37) are miotic agents in the rabbit eye, possibly by acting as neurokinin receptor agonists.
P122 Characteristics of [3H]-[Sar9,Met(O#1]substance P Binding to Astrocytoma Ceiis; Dependence Upon Peptide used to Define Nonspecific Binding C. J. Fowler and G. Briinnstrijm Preclinical Research, Astra Pain Control AE, S- 15 1 85 Siidertlilje, Sweden Substance P (SP)- sensitive [3H]-[S&,Met(O#‘]-SP binding has been shown in membrane preparations to label selectively the NK-1 receptor.’ In intact cells, however, such SP-sensitive binding will overestimate the number of NK- 1 receptors, since the cell culture wells contain SPsensitive binding sites2 The aim of the present study was therefore to find an alternative to SP for the definition of non-specific binding. The binding of 0.5 nM [3H]-[S&, Met(O$+SP to 24 well culture plates was inhibited by 70-90% by 1 pM SP (I& 100 nM). Physalaemin, on the other hand, did not inhibit this binding even at a concentration of 10 pM. SP-sensitive and physalaemin-sensitive [‘HI-[Sar9, Met(O#l]-SP binding were compared using UC1 1MG cells (which express NK-1 receptors, ‘). At low cell densities and in the absence of Ca2+ and Mg2+, a significantly higher Ko (1.57 vs 0.77 nM) andB, (244~s 106 fmoV106 cells) was found with SP than with physalaemin. (i)CP96345 (5 nM) inhibited physalaemin-sensitive binding by 78%, whereas the SP-sensitive binding was inhibited by 49%. Although the differences in SP- andphysalaemin-sensitive binding site densities can be minimised by increasing the cell density and by the presence of Ca2+ and MgZ in the assay medium, the data indicate that physalaemin should be used to define non-specific binding. 1. Dam, T.-V., Martinelli, B. and Quirion, R. (1990). Brain Res, 531: 333-337. 2. Fowler, C. J. and Martinsson, K. (1992). Regul. Peptides Suppl. 1: S58. 3. Johnson, C. L. and Johnson, C. G. (1992). J Neurochem, 58: 471-477.
P123 [Sarg, Met(Oz)“]-Substance P-induced Chromodacryorrhea in Gerbils; A model of NKl receptor function L. J. Bristow and L. Young Merck, Sharp and Dohme Research Laboratories, Neuroscience Research Centre, Terlings Park, Eastwick Rd., Harlow, Essex, CM20 2QR, UK
NEUROPEPTIDES:
239
FUNCTION AND PHARMACOLOGY
Recent studies have demonstrated the existence of species homologues of the tachykinin NK1 receptor. The present study has investigated the effects of the selective NKI receptor agonist [S&, Met(O$+Substance P in gerbils which have a similar NK1 receptor pharmacology to man. Male Mongolian gerbils (55-75 g) were anaesthetised with sodium pentobarbitone (60 mgkg, i.p.) and the jugular vein exposed. Intravenous injection of [S&, Met(O$l]-Substance P induced a dose dependent chromodacryorrhea (EDTO= 0.017 nmoles/gerbil) which was significantly antagonised following 15 min pretreatment with the NKI receptor antagonists (+CP 96,345 (ED50 = 0.48 mg/kg, iv.) or CP-99,994 (ED50= 0.15 mg/kg, i.v.). In contrast, CP-10,263 (inactive enantiomer of CP99,994; l-10 mgkg, i.v.) and the rodent selective NKI receptor antagonist, R&67,580 (0.3-l 0 mgkg, i.v.) failed to attenuate this response. In contrast to [S&, Met(O#‘]-Substance P, the selective NK receptor agonist, senktide (0.1-100 nmoles/gerbil, i.v.) did not induce chromodacryorrhea. However, intravenous administration of GR-64349, reported to have > 300-fold selectivity for NIC receptors compared to NKI receptors, also induced significant chromodacryorrhea (EDJO= 0.13 nmoles/gerbil). This response, however, was dose-dependently antagonised by pretreatment with the selective NK, receptor antagonist CP-99,994 (EDso = 0.09 mgkg, i.v.). In conclusion, the present studies suggest that [Sar’, Met(O$+Substance P-induced chromodacryorrhea in gerbils is an NK1 receptor mediated response. Furthermore, the ability of selective NK, receptor antag-
onists to attenuate GR-64349~induced chromodacryorrhea suggests that, in the gerbil at least, GR-64349 has significant NKI agonist activity in vivo. P124 Baculovirus Expression of Human NIG
Receptor
J. M. Guillaurne, F. Bellot, A. Jolly, N. Couteault, A. Fallourd, H. M. Soria, I. Gravier, S. Benoist, V. Fardin and A. Crespo Rhone-Poulenc Rorer, 13 quai Jules Guesde, 94403 Vitry sur Seine, France The cDNA encoding human NIG tachykinin receptor (receptor for neurokinin A) was cloned in a baculovirus transfer vector. Upon infection with the resulting NIG recombinant baculovirus, Sf 2 1 insect cells displayed lzsI NKA ligand binding activity, over 95% specific. Expression is maximum 4 days post-infection. The density of binding sites (Eimax) is about 1200 fmol/mg membrane protein as assessed by 125INKA radioligand binding on membrane preparations. Competition studies with selective NIG takykinin related agonist and antagonist compounds exhibit characteristic pharmacological profiles of human NK, receptor expressed in mammalian cell line (see Fardin et al., this meeting). The baculovirus-insect cell expression system offers significant advantages in terms of ease of use and high level of expression; therefore it may provide a powerful tool for large-scale drug screening procedures.