L-type calcium channels contribute to 5-HT₃-receptor-evoked CaMKIIα and ERK activation and induction of emesis in the least shrew (Cryptotis parva)

Activation of serotonergic 5-HT₃ receptors by its selective agonist 2-methyl serotonin (2-Me-5-HT) induces vomiting, which is sensitive to selective antagonists of both 5-HT₃ receptors (palonosetron) and L-type calcium channels (LTCC) (amlodipine or nifedipine). Previously we demonstrated that 5-HT₃ receptor activation also causes increases in a palonosetron-sensitive manner in: i) intracellular Ca²⁺ concentration, ii) attachment of calmodulin (CaM) to 5-HT₃ receptor, and iii) phosphorylation of Ca²⁺/calmodulin-dependent protein kinase IIα (CaMKIIα) and extracellular-signal-regulated kinase 1/2 (ERK1/2). Here, we investigate the role of the short-acting LTCC blocker nifedipine on 2-Me-5-HT-evoked intracellular Ca²⁺ increase and on downstream intracellular emetic signaling, which have been shown to be coupled with 2-Me-5-HT's emetic effects in the least shrew. Using the cell-permeant Ca²⁺ indicator fluo-4 AM, here we present evidence for the contribution of Ca²⁺ influx through LTCCs (sensitive to nifedipine) in 2-Me-5-HT (1 μM) -evoked rise in cytosolic Ca²⁺ levels in least shrew brainstem slices. Nifedipine pretreatment (10 mg/kg, s.c.) also suppressed 2-Me-5-HT-evoked interaction of 5-HT₃ receptors with CaM as well as phosphorylation of CaMKIIα and ERK1/2 in the least shrew brainstem, and 5-HT₃ receptors -CaM colocalization in jejunum of the small intestine. In vitro exposure of isolated enterochromaffin cells of the small intestine to 2-Me-5-HT (1 μM) caused CaMKIIα phosphorylation, which was also abrogated by nifedipine pretreatment (0.1 μM). In addition, pretreatment with the CaMKII inhibitor KN62 (10 mg/kg, i.p.) suppressed emesis and also the activation of CaMKIIα, and ERK in brainstem caused by 2-Me-5-HT (5 mg/kg, i.p.). This study provides further mechanistic explanation for our published findings that nifedipine can dose-dependently protect shrews from 2-Me-5-HT-induced vomiting.