CHARACTERISTICS OF [C-14] GUANIDINIUM ACCUMULATION IN NG 108-15 CELLS EXPOSED TO SEROTONIN 5-HT3 RECEPTOR LIGANDS AND SUBSTANCE-P

In the presence of substance P (SP: 10 muM), serotonin (5-HT; 1 muM) triggered a cation permeability in cells of the hybridoma (mouse neuroblastoma x rat glioma) clone NG 108-15 that could be assessed by measuring the cell capacity to accumulate [C-14]guanidinium for 10-15 min at 37-degrees-C. In addition to 5-HT (EC50 0.33 muM), the potent 5-HT3 receptor agonists 2-methyl-serotonin, phenylbiguanide, and m-chlorophenylbiguanide, and quipazine, markedly increased [C-14]guanidinium uptake in NG 108-15 cells exposed to 10 muM SP. In contrast. 5-HT3 receptor antagonists prevented the effect of 5-HT. The correlation (r = 0.97) between the potencies of 16 different ligands to mimic or prevent the effects of 5-HT on [C-14]guanidinium uptake, on the one hand, and to displace [H-3]zacopride specifically bound to 5-HT3 receptors on NG 108-15 cells, on the other hand, clearly demonstrated that [C-14]guanidinium uptake was directly controlled by 5-HT3 receptors. Various compounds such as inorganic cations (La3+, Mn2+, Ba2+, Ni2+, and Zn2+), D-tubocurarine, and memantine inhibited [C-14]guanidinium uptake in NG 108-15 cells exposed to 5-HT and SP, as expected from their noncompetitive antagonistic properties at 5-HT3 receptors. However, ethanol (100 mM), which has been reported to potentiate the electrophysiological response to 5-HT3 receptor stimulation, prevented the effects of 5-HT plus SP on [C-14]guanidinium uptake. The cooperative effect of SP on this 5-HT3-evoked response resulted neither from an interaction of the peptide with the 5-HT3 receptor binding site nor from a possible direct activation of G proteins in NG 108-15 cells. Among SP derivatiVeS, [D-Pro9]SP, a compound inactive at the various neurokinin receptor classes, was the most potent to mimic the stimulatory effect of SP on [C-14]guanidinium uptake in NG 108-15 cells exposed to 5-HT. Although the cellular mechanisms involved deserve further investigations, the 5-HT-evoked [C-14]guanidinium uptake appears to be a rapid and reliable response for assessing the functional state of 5-HT3 receptors in NG 108-15 cells.