Tachykinin NK 3 receptor mediates NANC hyperpolarization and relaxation via nitric oxide release in the circular muscle of the guinea-pig colon

Abstract

In the presence of atropine (1 μM), guanethidine (3 μM) and of the tachykinin NK 1 (SR 140,333 0.1 μM) and NK 2 (GR 94,800 3 μM) receptor antagonists, the application of the tachykinin NK 3 receptor selective agonist senktide, or that of neurokinin B, produced concentration-dependent sustained nonadrenergic noncholinergic (NANC) relaxation of mucosa-free circular muscle strips from the guinea-pig proximal colon. The maximal relaxant responses to senktide and neurokinin B were similar, approaching about 70% of the relaxation to 1 μM isoprenaline. Senktide (EC 50 0.16 nM) was about 64-fold more potent than neurokinin B (EC 50 10.3 nM). When tested in the presence of peptidase inhibitors (thiorphan 1 μM, captopril 1 μM and amastatin 10 μM), neurokinin B (EC 50 0.24 nM) was equipotent to senktide (EC 50 0.19 nM). At 1 nM, substance P and neurokinin A were ineffective in producing a NANC relaxation of the colon. At 1 μM substance P, neurokinin A and neurokinin B produced a NANC relaxation, which averaged 23, 40 and 79% of the maximal response to isoprenaline, respectively. In the presence of peptidase inhibitors, 1 nM substance P and neurokinin A produced threshold relaxant responses and, at 1 μM, the three natural tachykinins were equieffective ( 66 ± 8, 72 ± 5 and 75 ± 5% of the relaxation to isoprenaline for substance P, neurokinin A and neurokinin B, respectively). The relaxant response to 1 nM senktide (producing about 70–80% of its maximal effect) was totally abolished by 1 μM tetrodotoxin and largely (>90%) inhibited by 100 μM l-nitroarginine ( l-NOARG). The inhibition by l-NOARG was partially reversed by l-arginine (3 mM) but not by d-arginine. Apamin (1 μM) produced a slight (about 20%) inhibition of the response to senktide. The peptide blocker of N-type calcium channels, ω-conotoxin (0.1 μM) was ineffective. In sucrose gap electrophysiological experiments, superfusion with senktide (0.1 μM for 10 s) produced a slowly developing and prolonged hyperpolarization of the membrane and relaxation. Both effects were inhibited by l-NOARG while apamin had no effect. These findings indicate that a neuronal NK 3 receptor mediates NANC hyperpolarization and relaxation of the circular muscle of the guinea-pig proximal colon, principally through the release of NO. NO generation/release in response to NK 3 receptor stimulation does not require calcium influx through N-type calcium channels.