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.

[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.

Neurotransmitter release from bradykinin-stimulated PC12 cells. Stimulation of cytosolic calcium and neurotransmitter release.

The effect of bradykinin on intracellular free Ca2+ and neurotransmitter secretion was investigated in the rat pheochromocytoma cell line PC12. Bradykinin was shown to induce a rapid, but transient, increase in intracellular free Ca2+ which could be separated into an intracellular Ca2+ release component and an extracellular Ca2+ influx component. The bradykinin-induced stimulation of intracellular free Ca2+ displayed a similar time course, concentration dependencies and extracellular Ca2+ dependence as that found for neurotransmitter release, indicating an association between intracellular free Ca2+ levels and neurotransmitter secretion. The selective BK1-receptor antagonist des-Arg9,[Leu8]BK (where BK is bradykinin) did not significantly affect the stimulation of intracellular free Ca2+ or neurotransmitter release. In contrast, these effects of bradykinin were effectively blocked by the selective BK2-receptor antagonist [Thi5,8,D-Phe7]BK, and mimicked by the BK2 partial agonist [D-Phe7]BK in a concentration-dependent manner. The stimulation of intracellular free Ca2+ and neurotransmitter release induced by bradykinin was shown not to involve voltage-sensitive Ca2+ channels, since calcium antagonists had no effect on either response at concentrations which effectively inhibit depolarization-induced responses. These results indicate that bradykinin, acting through the interaction with the BK2 receptor, stimulates an increase in intracellular free Ca2+ leading to neurotransmitter secretion. Furthermore, bradykinin-induced responses involve the release of intracellular Ca2+ and the influx of extracellular Ca2+ that is not associated with the activation of voltage-sensitive Ca2+ channels.

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.