Novel serotonin receptors in Fasciola: Characterization by studies on adenylate cyclase activation and [3H]LSD binding

Serotonin (5-HT) receptors coupled to adenylate cyclase ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] in the liver fluke Fasciola hepatica have been characterized by adenylate cyclase activation studies and by direct binding studies using ${}^3\text{H}\text{]-d-}\text{lysergic acid}$ diethylamide (³H]LSD) as a radioligand. Inhibition of 5-HT stimulation of adenylate cyclase by a series of 5-HT antagonists revealed a potency order of LSD = 2-bromo-LSD > methiothepin > metergoline = cyproheptadine > methysergide > spiroperidol. ³H]LSD binding to a cell-free fluke particle preparation was rapid, stereospecific, and proportional to protein concentration. Scatchard analysis indicated multiple binding sites which, when resolved into two components, gave for the high affinity site an apparent dissociation constant of 25 nM and a receptor concentration of 160 fmoles/mg protein. The ability of a series of compounds to compete for ³H]LSD binding sites correlated closely with their ability to inhibit 5-HT stimulation of adenylate cyclase. ³H]LSD binding sites were most concentrated in the anterior region of the fluke which was consistent with the higher levels of 5-HT activated adenylate cyclase found in this region. GTP and 5′-guanylyl imidophosphate, a poorly hydrolyzable GTP analog, decreased the affinity of the agonist 5-HT for the binding sites but had little effect on the affinity of the antagonist 2-bromo-LSD. Calcium at concentrations above 300 μM significantly reduced both ³H]LSD binding and 5-HT activation of adenylate cyclase. The results indicate that ³H]LSD can be used to label the 5-HT receptors coupled to adenylate cyclase activity. The pharmacological specificity and other characteristics of the fluke receptors appear to differ from the properties of reported mammalian 5-HT receptors. As a result, serotonin receptors in the flukes represent sites that may be amenable to selective manipulation by new chemotherapeutic agents useful in the treatment of these parasite infections.