Identification of lymphocyte 5-HT3 receptor subtype and its implication in fish T-cell proliferation

In the present study, we identified the serotonergic receptor of type 3 (5-HT3) on the lymphocytes of a teleost fish, Oncorhynchus mykiss. In the pharmacological studies on the binding of [3H]serotonin to membrane receptor sites, 2-methyl-5-HT, an agonist of 5-HT3 receptors, displaced the binding of [3H]serotonin to fish lymphocytes, indicating the presence of 5-HT3 receptors on these cells. The known antagonists of the mammalian 5-HT3 receptor, ICS-205-930 and metoclopramide, failed to displace [3H]serotonin binding to lymphocytes during the period of association equilibrium (8 min); however, these antagonists progressively displaced [3H]serotonin binding from 10 to 40 min of incubation. These results suggest that fish 5-HT3 lymphocyte receptors may differ pharmacologically from mammalian receptors. As mammalian 5-HT3 receptors are coupled with Na+ inward movements, we undertook a study on Na+ influx by using SBFI/AM, a fluorescent probe. In SBFI/AM loaded fish lymphocytes, 2-methyl-5-HT leaked Na+ inward movements. Prior incubation of lymphocytes for 30 min in the presence of 5-HT3 antagonists, ICS-205-930, metoclopramide and MDL-72222, curtailed significantly the Na+ influx evoked by 2-methyl-5-HT, demonstrating that Na+ is leaked into fish lymphocytes via the 5-HT3 receptor-channel whose functioning is blocked by these antagonists. Furthermore, 2-methyl-5-HT exerted immunosuppressive effects in a dose dependent manner on fish T-lymphocytes stimulated by phytohaemagglutinin (PHA). Serotonin and 2-methyl-5-HT blocked the cell cycle progression of PHA-stimulated T-cells from G0/G1 to S phase. The immunosuppressive effects of 2-methyl-5-HT on T-cells were partially reversed by the antagonists, metoclopramide and ICS-205-930; however, the latter antagonist at high concentrations synergized with the immunosuppressive effects of 2-methyl-5-HT. These results demonstrate that the fish lymphocyte 5-HT3 receptor, which may be pharmacologically different from mammalian receptor subtype, is functionally implicated in fish T-cell proliferation.