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

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), N^G-nitro-l-arginine (l-NNA, 100 M) and their combination did not inhibit the relaxations induced by VIP (up to 0.3 M) or 0.3–3 M ATP but reduced those by 10 M ATP. Suramin (300 M) 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 M), suramin did not significantly inhibit the relaxations to 5-HT. Suramin did not affect the relaxations to isoprenaline, nitroglycerin or exogenous NO (1 M), 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 M), NO (1 ,M) and to nitroglycerin were not inhibit ed. In the presence of l-NNA (100 M), apamin inhibited the relaxations to 5-HT only at 30 M. ,\-methylene-ATP (,-Me-ATP; 100 M) 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 M onwards). Thus, it was not possible to test either ,-Me-ATP or reactive blue 2 against the relaxations to 5-HT. -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 M) and VIP_10–28 (1 and 3 M) did not affect the concentration-response curve to VIP and were hence not tested against 5-HT. Phosphoramidon (1 M) had no effect on the relaxations to VIP or 5-HT.It can be concluded that in the guinea-pig colon longitudinal muscle, VIP and ATP induce relaxation via a direct effect on the smooth muscle, not involving NO. 5-HT-induced relaxations are mediated by NO as well as by a substance which is sensitive to inhibition by suramin but not apamin. It is suggested that this substance is ATP and not a peptide.