Solubilization of a 5-HT3 binding site from rabbit small bowel muscularis membranes.

A 5-HT3 binding site, with high affinity for (S-)[3H]zacopride, was solubilized from rabbit small bowel muscularis membranes utilizing 0.5% sodium cholate and 400 mM (NH4)2SO4. Approximately 72% of the (S-)[3H]zacopride binding activity was recovered in a form that retained the high affinity (Kd = 0.7 nM) and specificity for this radioligand that is characteristic of the membrane-bound receptor. ICS 205-930 and other 5-HT3 compounds were effective inhibitors and exhibited the same rank order of potency in the solubilized and membrane-bound preparations. The receptor-detergent complex did not sediment after centrifugation for 1 h at 150,000 x g and eluted between thyroglobulin (MW = 669,000) and apoferritin (MW = 443,000) when fractionated by high-performance liquid chromatography gel filtration. This is the first report of the solubilization of a 5-HT3 binding site.

Antagonism of [3H]zacopride binding to 5-HT3 recognition sites by its (R) and (S) enantiomers.

[3H]Zacopride exhibits high affinity (Kd less than or equal to 1 nM) for 5-HT3 binding sites (inhibited by ICS 205-930) in rabbit intestinal muscularis and vagus nerve, human jejunum, rat intestinal muscularis and rat brain cortex. Its binding was inhibited by several 5-HT3 antagonists that displayed similar rank orders of potency in the tissues examined. Zacopride's (S) enantiomer was significantly more potent than its (R) enantiomer (21- to 42-fold in rabbit and human; 8- to 12-fold in rat) as an inhibitor of [3H]zacopride binding. These studies indicate that the utility of [3H]zacopride as a high affinity 5-HT3 ligand resides with the (S) enantiomer.

[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.

Association of [3H]zacopride with 5-HT3 binding sites.

An assay was developed for [3H]zacopride binding to 5-HT3 specific sites in membranes from rabbit ileum muscularis. The binding was rapid, saturable, reversible, salt-insensitive, unaffected by pH between 6.5 and 9.5, and of high affinity (apparent KD = 0.65 +/- 0.15 nM). ICS 205-930, a potent 5-HT3 antagonist that inhibited competitively, was utilized to define 5-HT3 specific binding. Other 5-HT3 antagonists and agonists, although exhibiting marked differences in potency, were also effective inhibitors; whereas, antagonists of other classes of serotonin receptors, guanyl nucleotides and numerous receptor-specific ligands, including peptide hormones, were inactive. Vagus nerve exhibited the greatest amount of 5-HT3 specific binding amongst rabbit tissues and virtually all of the [3H]zacopride was bound to 5-HT3 binding sites. In rabbit, rat and ferret a fairly uniform distribution of 5-HT3 binding sites was observed along the muscularis of the small bowel. [3H]Zacopride is a high-affinity ligand for detecting 5-HT3 binding sites and rabbit small bowel muscularis membranes are a sensitive system for evaluating the potency of 5-HT3 antagonists or agonists.