Serotonin 5-HT ₃ Receptor-Mediated Vomiting Occurs via the Activation of Ca ²⁺/CaMKII-Dependent ERK1/2 Signaling in the Least Shrew ( Cryptotis parva)

Abstract

Stimulation of 5-HT ₃ receptors (5-HT ₃Rs) by 2-methylserotonin (2-Me-5-HT), a selective 5-HT ₃ receptor agonist, can induce vomiting. However, downstream signaling pathways for the induced emesis remain unknown. The 5-HT ₃R channel has high permeability to extracellular calcium (Ca ²⁺) and upon stimulation allows increased Ca ²⁺ influx. We examined the contribution of Ca ²⁺/calmodulin-dependent protein kinase IIα (Ca ²⁺/CaMKIIα), interaction of 5-HT ₃R with calmodulin, and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling to 2-Me-5-HT-induced emesis in the least shrew. Using fluo-4 AM dye, we found that 2-Me-5-HT augments intracellular Ca ²⁺ levels in brainstem slices and that the selective 5-HT ₃R antagonist palonosetron, can abolish the induced Ca ²⁺ signaling. Pre-treatment of shrews with either: i) amlodipine, an antagonist of L-type Ca ²⁺ channels present on the cell membrane; ii) dantrolene, an inhibitor of ryanodine receptors (RyRs) Ca ²⁺-release channels located on the endoplasmic reticulum (ER); iii) a combination of their less-effective doses; or iv) inhibitors of CaMKII (KN93) and ERK1/2 (PD98059); dose-dependently suppressed emesis caused by 2-Me-5-HT. Administration of 2-Me-5-HT also significantly: i) enhanced the interaction of 5-HT ₃R with calmodulin in the brainstem as revealed by immunoprecipitation, as well as their colocalization in the area postrema (brainstem) and small intestine by immunohistochemistry; and ii) activated CaMKIIα in brainstem and in isolated enterochromaffin cells of the small intestine as shown by Western blot and immunocytochemistry. These effects were suppressed by palonosetron. 2-Me-5-HT also activated ERK1/2 in brainstem, which was abrogated by palonosetron, KN93, PD98059, amlodipine, dantrolene, or a combination of amlodipine plus dantrolene. However, blockade of ER inositol-1, 4, 5-triphosphate receptors by 2-APB, had no significant effect on the discussed behavioral and biochemical parameters. This study demonstrates that Ca ²⁺ mobilization via extracellular Ca ²⁺ influx through 5-HT ₃Rs/L-type Ca ²⁺ channels, and intracellular Ca ²⁺ release via RyRs on ER, initiate Ca ²⁺-dependent sequential activation of CaMKIIα and ERK1/2, which contribute to the 5-HT ₃R-mediated, 2-Me-5-HT-evoked emesis.