Calcium responses induced by acetylcholine in submucosal arterioles of the guinea-pig small intestine

1. Calcium responses induced by brief stimulation with acetylcholine (ACh) were assessed from the fluorescence changes in fura-2 loaded submucosal arterioles of the guinea-pig small intestine. 2. Initially, 1-1.5 h after loading with fura-2 (fresh tissues), ACh increased [Ca2+](i) in a concentration-dependent manner. This response diminished with time, and finally disappeared in 2-3 h (old tissues). 3. Ba2+ elevated [Ca2+](i) to a similar extent in both fresh and old tissues. ACh further increased the Ba2+-elevated [Ca2+](i) in fresh tissues, but reduced it in old tissues. Responses were not affected by either indomethacin or nitroarginine. 4. In fresh mesenteric arteries, mechanical removal of endothelial cells abolished the ACh-induced increase in [Ca2+](i), with no alteration of [Ca2+](i) at rest and during elevation with Ba2+. 5. In the presence of indomethacin and nitroarginine, high-K+ solution elevated [Ca2+](i) in both fresh and old tissues. Subsequent addition of ACh further increased [Ca2+](i) in fresh tissues without changing it in old tissues. 6. Proadifen, an inhibitor of the enzyme cytochrome P450 mono-oxygenase, inhibited the ACh-induced changes in [Ca2+](i) in both fresh and Ba2+-stimulated old tissues. It also inhibited the ACh-induced hyperpolarization. 7. In fresh tissues, the ACh-induced Ca2+ response was not changed by apamin, charybdotoxin (CTX), 4-aminopyridine (4-AP) or glibenclamide. In old tissues in which [Ca2+](i) had previously been elevated with Ba2+, the ACh-induced Ca2+ response was inhibited by CTX but not by apamin, 4-AP or glibenclamide. 8. It is concluded that in submucosal arterioles, ACh elevates endothelial [Ca2+](i) and reduces muscular [Ca2+](i), probably through the hyperpolarization of endothelial or smooth muscle membrane by activating CTX-sensitive K+ channels.