Tachykinin NK3 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 microM), guanethidine (3 microM) and of the tachykinin NK1 (SR 140,333 0.1 microM) and NK2 (GR 94,800 3 microM) receptor antagonists, the application of the tachykinin NK3 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 microM isoprenaline. Senktide (EC50 0.16 nM) was about 64-fold more potent than neurokinin B (EC50 10.3 nM). When tested in the presence of peptidase inhibitors (thiorphan 1 microM, captopril 1 microM and amastatin 10 microM), neurokinin B (EC50 0.24 nM) was equipotent to senktide (EC50 0.19 nM). At 1 nM, substance P and neurokinin A were ineffective in producing a NANC relaxation of the colon. At 1 microM 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 microM, 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 microM tetrodotoxin and largely (> 90%) inhibited by 100 microM L-nitroarginine (L-NOARG). The inhibition by L-NOARG was partially reversed by L-arginine (3 mM) but not D-arginine. Apamin (1 microM) produced a slight (about 20%) inhibition of the response to senktide. The peptide blocker of N-type calcium channels, omega-conotoxin (0.1 microM) was ineffective. In sucrose gap electrophysiological experiments, superfusion with senktide (0.1 microM 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 NK3 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 NK3 receptor stimulation does not require calcium influx through N-type calcium channels.