Abstract

The whole-cell patch-clamp technique was used to analyze the effects of 5-hydroxytryptamine (5-HT) and alosetron on cultured myenteric neurons from newborn guinea pigs. All neurons responded to 5-HT (EC₅₀ ∼ 38.7 μM) with a concentration-dependent inward current (reversal potential = 7.1 ± 1.7 mV) with a short latency and rapid decay. Because the 5-HT-induced inward current was mimicked by 2-methyl-5-hydroxytryptamine (50 μM) and blocked by ondansetron (5.0 μM) and MDL 72222 (0.05 μM), it was 5-HT₃-mediated. Alosetron blocked (IC₅₀ ∼ 0.05 μM; Hill coefficient ∼ 1.24) the 5-HT- and 2-methyl-5-hydroxytryptamine-induced inward currents. This effect was independent of membrane potential and was not seen when alosetron was delivered to the inside of cells. Alosetron-sensitive sites are, thus, accessible only on the ectodomain of the plasmalemma. The effect of alosetron was reversible, but not surmountable. Although nicotine (100 μM) mimicked the 5-HT-induced inward current, the response was antagonized by hexamethonium (100 μM), but not by alosetron, implying its potential to be a selective 5-HT₃antagonist. Hexamethonium did not affect responses to 5-HT. Most neurons in the cultures were 5-HT-immunoreactive and immunostained with an antibody raised against 5-HT₃ receptors. The 5-HT-selective uptake inhibitor, fluoxetine (30 μM), gradually reduced the amplitude of the current induced by 5-HT; the residual response was abolished by alosetron (0.2 μM). The effect of fluoxetine could have been caused by either the desensitization of 5-HT₃ receptors or by a nonspecific 5-HT₃antagonistic effect of fluoxetine. It is concluded that alosetron is a potent and noncompetitive 5-HT₃ antagonist on myenteric neurons.