CHARACTERIZATION OF THE NON-5-HT(3) HIGH-AFFINITY R-BINDING SITE FOR (R)-ZACOPRIDE IN BRAIN AND OTHER TISSUES

Previous studies showed that whereas the potent 5-HT3 receptor antagon ist (S)-[H-3]zacopride only labels 5-HT3 receptor binding sites, the ( R)-enantiomer, (R)-[H-3]zacopride, labels these receptors and another class of high-affinity binding sites, named the R sites, in membranes from the rat cerebral cortex and NG 108-15 clonal cells (Kidd et al., Eur. J. Pharmacol. 211, 133, 1992). Further studies of R sites reveale d that they existed not only in the cerebral cortex but also in variou s other areas of the rat brain and spinal cord. In addition, R sites w ere also found in post-mortem human brain tissues. Both in the rat and in man, the regional distribution of central R sites was markedly dif ferent from that of 5-HT3 receptors specifically labelled with (S)-[H- 3]zacopride. Under appropriate conditions for the specific labelling o f R sites (with (R)-[H-3]zacopride in the presence of 1.0 muM ondanset ron to saturate 5-HT3 receptor binding sites - and 0.1 mM mianserin fo r the determination of non-specific binding), these R sites were also found in rat peripheral tissues (intestine > spleen > kidney > testicl es = liver > adrenals > lung > heart). At least in the kidney and the liver, the pharmacological profile of R sites corresponded exactly to that found in NG 108-15 cells. R sites were also detected in membranes from C6 glioma cells and glial cells cultured from the whole cortex o f new born rats. In contrast, no specific binding of (R)-[H-3]zacoprid e to R sites could be found in membranes from N1E-115 neuroblastoma ce lls. Conversely, 5-HT3 receptors could be labelled by (S)-[H-3]zacopri de in the latter cells but not in C6 glioma and cultured glial cells. As expected from their glial location, the density of R sites increase d in the rat hippocampus lesioned with kainic or ibotenic acid to indu ce local gliosis. In contrast, the density of hippocampal 5-HT3 recept ors was unchanged in lesioned rats. Finally, the determination of the apparent molecular size of R sites by radiation inactivation gave a va lue (almost-equal-to 30 kDa) which was significantly lower than that o f 5-HT3 receptor binding sites in the rat entorhinal cortex (40 kDa) a nd NG 108-15 cells (57 kDa). All these data clearly showed that R site s and 5-HT3 receptors are different molecular species. Whether R sites mediate the 5-HT3 receptor-unrelated actions of (R)-zacopride deserve s further investigations.