5-HT-induced neurogenic relaxations of the guinea-pig proximal colon: investigation into the role of ATP and VIP in addition to nitric oxide

Abstract

In the guinea-pig proximal colon, 5-hydroxytryptamine (5-HT) relaxes the longitudinal muscle by stimulating neuronal 5-HT receptors, which induces the release of nitric oxide (NO). It was investigated whether the inhibitory neurotransmitters adenosine 5'-triphosphate (ATP) and/or vasoactive intestinal polypeptide (VIP) could be involved as well. Antagonists to block the contractile response to 5-HT via 5-HT2, 5-HT3 or 5-HT4 receptors were present throughout the experiments and methacholine was administered to precontract the strips. ATP, VIP and 5-HT induced concentration-dependent relaxations, in the case of 5-HT yielding a non-monophasic concentration-response curve. Tetrodotoxin (TTX; 300 nM), NG-nitro-L-arginine (L-NNA, 100 microM) and their combination did not inhibit the relaxations induced by VIP (up to 0.3 microM) or 0.3-3 microM ATP but reduced those by 10 microM ATP. Suramin (300 microM) strongly inhibited the relaxations to ATP and VIP. L-NNA and suramin also inhibited the relaxations to 5-HT. In the presence of L-NNA (100 microM), suramin did not significantly inhibit the relaxations to 5-HT. Suramin did not affect the relaxations to isoprenaline, nitroglycerin or exogenous NO (1 microM), demonstrating its specificity. Apamin (30 nM) inhibited both the relaxations to ATP (by 70-100%) and to 5-HT; relaxations to isoprenaline were partially inhibited, indicating a non-specific component in the inhibitory action of apamin. However, relaxations to exogenous VIP (up to 0.3 microM), NO (1 microM) and to nitroglycerin were not inhibited. In the presence of L-NNA (100 microM), apamin inhibited the relaxations to 5-HT only at 30 microM. alpha, beta-methylene-ATP (alpha, beta-Me-ATP; 100 microM) did not desensitize the responses to ATP. Reactive blue 2 affected the relaxations to isoprenaline at concentrations necessary to significantly inhibit the relaxations to ATP (i.e. from 10 microM onwards). Thus, it was not possible to test either alpha, beta-Me-ATP or reactive blue 2 against the relaxations to 5-HT. alpha-Chymotrypsin (0.015 mg.ml-1) and trypsin (0.005 mg.ml-1) almost abolished the relaxations to VIP, but did not affect those to isoprenaline and 5-HT. The VIP receptor antagonists [p-Cl-D-Phe6, Leu17]VIP (1 microM) and VIP10-28 (1 and 3 microM) did not affect the concentration-response curve to VIP and were hence not tested against 5-HT. Phosphoramidon (1 microM) had no effect on the relaxations to VIP or 5-HT.(ABSTRACT TRUNCATED AT 400 WORDS)