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Notes: Abstract We have determined the ability of the novel nonpeptide tachykinin (TK) NK3 receptor antagonist, SR 142801, [(S) -(N)-(1-(3-(1-benzoyl-3-(3,4-dichlorophenyl) piperidin-3-yl) propyl)-4-phenylpiperidin-4-yl)-N-methylacetamide] in inhibiting the nitric oxide (NO)-independent prejunctional inhibition of cholinergic twitches and the NO-dependent relaxation produced by the NK3 receptor selective agonist, senktide, in the circular muscle of the guinea-pig proximal colon. Under moderate load (10 mN) and isometric recording of mechanical activity, single pulse electrical field stimulation (EFS) produced atropine- and tetrodotoxin-sensitive twitch contractions of mucosa-free circular muscle strips from the guinea-pig proximal colon. In the presence of NK1 and NK2 receptor antagonists (SR 140333 0.01 μM and GR 94800 0.1 μM, respectively) the NK3 receptor selective agonist, senktide (EC50 33 pM) and the NK3 receptor preferring natural TK, neurokinin B (NKB, EC50 13 pM) produced a concentration-dependent slowly developing inhibition of cholinergic twitches. Senktide (1 nM) did not affect the contractile response to acetylcholine (1 gM) indicating that depression of evoked twitches occurs prejunctionally. The inhibitory effect of senktide was 'unaffected when evoked in the presence of the cyclooxygenase inhibitor (S)-ketoprofen (10 μM), guanethidine (10 μM), naloxone (0.3 μM), the GABAB receptor antagonist 2-hydroxysaclofen (10 μM) or the combined application of the adenosine A1 and A2 receptor antagonists, 8-cyclopentyl-1,3-dipropylxanthine (10 μM) and 3,7-dimethyl-l-propargylxanthine (30 μM) respectively. In the presence of NK1 and NK2 receptor antagonists, the NO-synthase inhibitor l-nitroarginine (L-NOARG 30-100 μM) did not affect twitch inhibition induced by senktide (EC50 33 pM). The response to NKB (EC50 95 pM) was slightly reduced by L-NOARG, yet the bulk of the inhibitory effect of both agonists on cholinergic twitches was substantially independent of NO generation. SR 142801 (0.1–0.3 μM) produced a moderate rightward shift of the concentration-response curve to senktide without depression of the Emax to the agonist, yielding an apparent pKB value of 7.65. Under low resting tone (3 mN) and isotonic recording of mechanical activity, mucosa-free circular muscle strips from the guinea-pig proximal colon gained a high intrinsic tone suitable for testing the response to relaxant agents. In the presence of atropine (1 μM), guanethidine (3 μM), SR 140333 (0.01 [M) and GR 94800 (0.1 μLM), senktide (EC50 50 pM) produced a concentration-dependent relaxation of the strips, which was blocked by L-NOARG. SR 142801 (0.01–0.1 μM) produced a large rightward shift of the L-NOARG-sensitive concentration-response curve to senktide yielding an apparent pKB value of 8.62. Under isometric recording condition, SR 142801 (0.1 μM) did not affect twitch inhibition produced by 3 nM clonidine. Under isotonic recording condition, SR 142801 did not affect the L-NOARG-sensitive relaxation produced by EFS. The present results indicate that NK3 receptor stimulation produces a NO-dependent relaxation of the guinea-pig colon and a substantially NO-independent prejunctional inhibition of cholinergic twitches. The variable affinities of SR 142801 in antagonizing various senktide-induced neuromodulatory effects in the guinea-pig intestine suggest a possible intraspecies heterogeneity of NK3 receptors in the enteric nervous system.