Use of stereoisomers of zacopride to analyze actions of 5-hydroxytryptamine on enteric neurons

Two subtypes of excitatory 5-hydroxytryptamine (5-HT) receptor, 5-HT1P and 5-HT3, are found on type 2-AH neurons of the guinea pig myenteric plexus. The 5-HT1P receptor mediates a slow and the 5-HT3 receptor a fast depolarization of these cells, however, the role of these receptors in the physiology of the gut is unknown. Renzapride (BRL 24924), a substituted benzamide, has previously been found to antagonize responses of myenteric neurons mediated by both 5-HT1P and 5-HT3 receptors. The effects on myenteric type 2-AH neurons of a structurally similar benzamide, zacopride, which unlike renzapride has S and R stereoisomers, were investigated to gain further insight into 5-HT receptor function. In contrast to renzapride, S-, but not R-zacopride, was found to mimic the 5-HT1P receptor-mediated slow response to 5-HT. Desensitization of 5-HT1P receptors with 5-HT inhibited slow depolarizing responses to S-zacopride, and desensitization with S-zacopride antagonized slow responses to 5-HT. Responses to S-zacopride were also inhibited by renzapride and the 5-HT1P receptor antagonist N-acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide (5-HTP-DP). S-zacopride, like renzapride and 5-HT, presynaptically inhibited nicotinic fast excitatory postsynaptic potentials, an effect that can be mediated by 5-HT1P or 5-HT1A receptors. Both S and R stereoisomers of zacopride antagonized 5-HT3 receptor-mediated fast responses to 5-HT. Unlike 5-HTP-DP, neither zacopride or its stereoisomers nor renzapride inhibited the binding of 5-3HHT to 5-HT1P receptors. 3Hzacopride (5-10 nM) was found to bind to a site in the gut from which it could be displaced by a 1,000-fold excess of renzapride and S-zacopride (but not R-zacopride) greater than 5-HTP-DP much greater than the 5-HT3 receptor antagonist ICS 205-930. These observations suggest that, in addition to 5-HT3 receptors, there is a benzamide binding site on myenteric neurons that interacts with, but is distinct from, the 5-HT recognition site of 5-HT1P receptors. Benzamides may affect coupling of the 5-HT1P receptor to its effector.