Characteristics of [14C]Guanidinium Accumulation in NG 108‐15 Cell Exposed to Serotonin 5‐HT3 Receptor Ligands and Substance P

Abstract: In the presence of substance P (SP; 10 μM), serotonin (5‐HT; 1 μM) 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 [14C]guanidinium for 10‐15 min at 37°C. In addition to 5‐HT (EC50, 0.33 μM), the potent 5‐HT3 receptor agonists 2‐methyl‐serotonin, phenylbiguanide, and m‐chlorophenylbiguanide, and quipazine, markedly increased [14C]guanidinium uptake in NG 108‐15 cells exposed to 10 μM 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 [14C]guanidinium uptake, on the one hand, and to displace [3H]zacopride specifically bound to 5‐HT3 receptors on NG 108‐15 cells, on the other hand, clearly demonstrated that [14C]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 [14C]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 [14C]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 [14C]guanidinium uptake in NG 108‐15 cells exposed to 5‐HT. Although the cellular mechanisms involved deserve further investigations, the 5‐HT‐evoked [14C]guanidinium uptake appears to be a rapid and reliable response for assessing the functional state of 5‐HT3 receptors in NG 108‐15 cells.