Serotonin 5-HT3 Receptor-Mediated Vomiting Occurs via the Activation of Ca2+/CaMKII-Dependent ERK1/2 Signaling in the Least Shrew (Cryptotis parva)

Abstract

Stimulation of 5-HT3 receptors (5-HT3Rs) by 2-methylserotonin (2-Me-5-HT), a selective 5-HT3 receptor agonist, can induce vomiting. However, downstream signaling pathways for the induced emesis remain unknown. The 5-HT3R channel has high permeability to extracellular calcium (Ca2+) and upon stimulation allows increased Ca2+ influx. We examined the contribution of Ca2+/calmodulin-dependent protein kinase IIα (Ca2+/CaMKIIα), interaction of 5-HT3R 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 Ca2+ levels in brainstem slices and that the selective 5-HT3R antagonist palonosetron, can abolish the induced Ca2+ signaling. Pre-treatment of shrews with either: i) amlodipine, an antagonist of L-type Ca2+ channels present on the cell membrane; ii) dantrolene, an inhibitor of ryanodine receptors (RyRs) Ca2+-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-HT3R 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 Ca2+ mobilization via extracellular Ca2+ influx through 5-HT3Rs/L-type Ca2+ channels, and intracellular Ca2+ release via RyRs on ER, initiate Ca2+-dependent sequential activation of CaMKIIα and ERK1/2, which contribute to the 5-HT3R-mediated, 2-Me-5-HT-evoked emesis.

Intracellular emetic signaling evoked by the L-type Ca2+ channel agonist FPL64176 in the least shrew (Cryptotis parva)

“…To confirm the involvement of emesis-related signaling molecules (Darmani et al, 2013 and 2015; Zhong et al, 2014b and 2016) in FPL64176-evoked vomiting, we determined the effect of FPL64176 (10 mg/kg, i.p.) treatment on the phosphorylation level of some signaling proteins (PKCα/βII, ERK1/2, and AKT) in least shrew brainstems following different exposure periods (5, 10, 15, 20, 25, 30 and 60 min; n = 3 per time point) as well as from corresponding vehicle controls which is referred to as 0 min.…”

“…Our lab has well described the circumventricular organ AP and the different cytoarchitectonic detail such as cell size and packing differentiating DMNX from NTS within the DVC, as part of a stereotaxic atlas of the least shrew brainstem (Ray et al, 2009a). This criterion has been well recognized (Chebolu et al, 2010; Darmani et al, 2008; Ray et al, 2009a and 2009c; Zhong et al, 2014b and 2016). …”

“…The lower half of brainstem, mostly medullary structures, was isolated as described elsewhere (Zhong et al, 2014b) and homogenized in lysis buffer. Protein extracts from brainstem lysates were subjected to Western blot.…”

“…Ca 2+ release from intracellular sarcoplasmic/endoplasmic reticulum (SER) into cytoplasm is accomplished by inositol trisphosphate receptors (IP 3 Rs) and ryanodine receptors (RyRs) (Gomez-Viquez et al, 2003). IP 3 Rs and RyRs are differentially involved in vomiting evoked by different emetogens (Zhong et al, 2014b and 2016). Thapsigargin- and FPL64176-like drugs affecting cytosolic Ca 2+ dynamics are considered as potential targeted apoptotic chemotherapeutics (Cano-Abad et al, 2001; Doan et al, 2015).…”

“…Vomit-associated cytosolic Ca 2+ rise as well as time-dependent increases in phosphorylation of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) occur in the shrew brainstem following 5-HT 3 R-mediated vomiting and emesis caused by thapsigargin (Zhong et al, 2014b and 2016). Moreover, protein kinase Cα/βII (PKCα/βII) and ERK1/2 phosphorylation have been observed during cisplatin-induced emesis (Darmani et al, 2013 and 2015).…”