[3H]zacopride binding to 5-hydroxytryptamine3 sites on partially purified rabbit enteric neuronal membranes

5-Hydroxytryptamine3 (5-HT3) receptors are present in both central and peripheral neuronal tissues but radioligand binding studies have thus far been limited to crude membranes from brain and vagus nerve. The present studies describe the isolation and characterization from the rabbit small bowel of neuronal membranes enriched in binding sites for the potent 5-HT3 ligand, [3H]zacopride. The number of specific [3H]zacopride binding sites per milligram of protein was increased 6-fold in a 10,000 to 100,000 x g membrane fraction as compared to the homogenate. [3H]Zacopride bound to these membranes with high specificity (greater than 90%), exhibited high affinity for a homogeneous population of binding sites (Kd = 0.3 nM) and its binding was inhibited competitively by other 5-HT3 compounds with the following rank order of potency: ICS 205-930 greater than GR 38032F greater than or equal to quipazine greater than BRL 24924 approximately MDL 72222 much greater than metoclopramide greater than 2-CH3-5-HT3. On a discontinuous sucrose gradient, specific [3H]zacopride binding was increased an additional 3.5-fold and copurified with three plasma membrane markers. Fractionation on a continuous sucrose gradient demonstrated that specific [3H]zacopride binding was associated with the enteric neuronal plasma membranes. Comparative studies in rabbit vagus nerve also demonstrated a large number (maximum binding = 148 fmol/mg of protein) of high affinity [3H]zacopride binding sites (Kd = 0.4 nM), in membranes that exhibited a density and binding characteristics similar to those from enteric neurons. Thus, membranes enriched in 5-HT3 binding sites can be isolated from both enteric and vagus neurons and [3H]zacopride is a potent ligand useful for characterization of these sites.