title: Thapsigargin-induced activation of Ca(2+)-CaMKII-ERK in brainstem contributes to substance P release and induction of emesis in the least shrew.
abstract: Cytoplasmic calcium (Ca(2+)) mobilization has been proposed to be an important factor in the induction of emesis. The selective sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA) inhibitor thapsigargin, is known to deplete intracellular Ca(2+) stores, which consequently evokes extracellular Ca(2+) entry through cell membrane-associated channels, accompanied by a prominent rise in cytosolic Ca(2+). A pro-drug form of thapsigargin is currently under clinical trial as a targeted cancer chemotherapeutic. We envisioned that the intracellular effects of thapsigargin could cause emesis and planned to investigate its mechanisms of emetic action. Indeed, thapsigargin did induce vomiting in the least shrew in a dose-dependent and bell-shaped manner, with maximal efficacy (100%) at 0.5 mg/kg (i.p.). Thapsigargin (0.5 mg/kg) also caused increases in c-Fos immunoreactivity in the brainstem emetic nuclei including the area postrema (AP), nucleus tractus solitarius (NTS) and dorsal motor nucleus of the vagus (DMNX), as well as enhancement of substance P (SP) immunoreactivity in DMNX. In addition, thapsigargin (0.5 mg/kg, i.p.) led to vomit-associated and time-dependent increases in phosphorylation of Ca(2+)/calmodulin kinase IIα (CaMKIIα) and extracellular signal-regulated protein kinase 1/2 (ERK1/2) in the brainstem. We then explored the suppressive potential of diverse chemicals against thapsigargin-evoked emesis including antagonists of: i) neurokinin-1 receptors (netupitant), ii) the type 3 serotonin receptors (palonosetron), iii) store-operated Ca(2+) entry (YM-58483), iv) L-type Ca(2+) channels (nifedipine), and v) SER Ca(2+)-release channels inositol trisphosphate (IP3Rs) (2-APB)-, and ryanodine (RyRs) (dantrolene)-receptors. In addition, the antiemetic potential of inhibitors of CaMKII (KN93) and ERK1/2 (PD98059) were investigated. All tested antagonists/blockers attenuated emetic parameters to varying degrees except palonosetron, however a combination of non-effective doses of netupitant and palonosetron exhibited additive antiemetic efficacy. A low-dose combination of nifedipine and 2-APB plus dantrolene mixture completely abolished thapsigargin-evoked vomiting, CaMKII-ERK1/2 activation and SP elevation. In addition, pretreatment with KN93 or PD98059 suppressed thapsigargin-induced increases in SP and ERK1/2 activation. Intracerebroventricular injection of netupitant suppressed vomiting caused by thapsigargin which suggests that the principal site of evoked emesis is the brainstem. In sum, this is the first study to demonstrate that thapsigargin causes vomiting via the activation of the Ca(2+)-CaMKII-ERK1/2 cascade, which is associated with an increase in the brainstem tissue content of SP, and the evoked emesis occurs through SP-induced activation of neurokinin-1 receptors.
authors.affiliation: Department of Basic Medical Sciences, College of Osteopathic Medicine of the Pacific, Western University of Health Sciences, Pomona CA 91766, USA.
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, Pomona CA 91766, USA.
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, Pomona CA 91766, USA. Electronic address: ndarmani@westernu.edu.
authors.forename: Nissar A
authors.initials: NA
authors.lastname: Darmani
mesh_terms: D000818:Animals; D001933:Brain Stem / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000378:metabolism*; D005260:Female; D020935:MAP Kinase Signaling System; D008297:Male; D010766:Phosphorylation; D018040:Receptors, Neurokinin-1 / Q000378:metabolism; D012788:Shrews; D013373:Substance P / Q000378:metabolism*; D019284:Thapsigargin / Q000008:administration & dosage*; D014839:Vomiting / Q000139:chemically induced* / Q000378:metabolism*
chemical_list: D018040:Receptors, Neurokinin-1; D013373:Substance P; D019284:Thapsigargin; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D002118:Calcium
keywords: Brainstem; ERK; Emesis; LTCC; Substance P; Thapsigargin