title: 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 (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-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 Ca(2+) levels in brainstem slices and that the selective 5-HT3R 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) 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 Ca(2+) mobilization via extracellular Ca(2+) influx through 5-HT3Rs/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-HT3R-mediated, 2-Me-5-HT-evoked emesis.
authors.affiliation: Department of Basic Medical Sciences, College of Osteopathic Medicine of the Pacific, Western University of Health Sciences, California, United States of America.
authors.forename: Weixia
authors.initials: W
authors.lastname: Zhong
authors.affiliation: Department of Basic Medical Sciences, College of Osteopathic Medicine of the Pacific, Western University of Health Sciences, California, United States of America.
authors.forename: Tarun E
authors.initials: TE
authors.lastname: Hutchinson
authors.affiliation: Department of Basic Medical Sciences, College of Osteopathic Medicine of the Pacific, Western University of Health Sciences, California, United States of America.
authors.forename: Seetha
authors.initials: S
authors.lastname: Chebolu
authors.affiliation: Department of Basic Medical Sciences, College of Osteopathic Medicine of the Pacific, Western University of Health Sciences, California, United States of America.
authors.forename: Nissar A
authors.initials: NA
authors.lastname: Darmani
mesh_terms: D000818:Animals; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000378:metabolism*; D004789:Enzyme Activation; D005260:Female; D020935:MAP Kinase Signaling System; D008297:Male; D044406:Receptors, Serotonin, 5-HT3 / Q000502:physiology*; D012788:Shrews; D015398:Signal Transduction; D014839:Vomiting / Q000503:physiopathology*
chemical_list: D044406:Receptors, Serotonin, 5-HT3; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2