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.
Highlights
Chemotherapy-induced nausea and vomiting (CINV) is mediated via neurochemical circuits that involve braingut interactions [1]
We addressed the relevance of 5-HT3 receptors (5-HT3Rs)-mediated Ca2+ influx in the anti-emetic potential of L-type Ca2+ channel blockers on vomiting caused by the selective 5-HT3R agonist 2-Me-5-HT
To further validate a role for CaMKIIa activation in 5-HT3R-mediated emesis, we examined the antiemetic potential of the calmodulindependent kinase II (CaMKII) inhibitor, KN93 (Figure 6)
Summary
Chemotherapy (e.g. cisplatin)-induced nausea and vomiting (CINV) is mediated via neurochemical circuits that involve braingut interactions [1]. The DVC emetic nuclei consists of the nucleus tractus solitarius (NTS), the dorsal motor nucleus of the vagus (DMNX) and the area postrema (AP) [1] These brainstem emetic loci can be activated by emetogens, such as serotonin, either directly or indirectly through gastrointestinal signaling [4]. Cisplatin-like drugs cause vomiting via release of 5-HT from the gastrointestinal EC cells which subsequently activates local 5-HT3Rs present on the GIT vagal afferents [1,9,10]. This activation results in vagal nerve depolarization which subsequently triggers the brainstem DVC emetic nuclei to initiate the vomiting reflex
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