Effect of anandamide on cytosolic Ca(2+) levels and proliferation in canine renal tubular cells.
The effect of the endogenous cannabinoid anandamide on cytosolic free Ca(2+) concentration ([Ca(2+)](i)) and proliferation is largely unknown. This study examined whether anandamide altered Ca(2+) levels and caused Ca(2+)-dependent cell death in Madin-Darby canine kidney (MDCK) cells. [Ca(2+)](i) and cell death were measured using the fluorescent dyes fura-2 and WST-1 respectively. Anandamide at concentrations above 5 muM increased [Ca(2+)](i) in a concentration-dependent manner. The Ca(2+) signal was reduced by 78% by removing extracellular Ca(2+). The anandamide-induced Ca(2+) influx was insensitive to L-type Ca(2+) channel blockers and the cannabinoid receptor antagonist AM 251, but was inhibited differently by aristolochic acid, WIN 55,212-2 (a cannabinoid receptor agonist), phorbol ester, GF 109203X and forskolin. After pretreatment with thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor), anandamide-induced Ca(2+) release was inhibited. Inhibition of phospholipase C with U73122 did not change anandamide-induced Ca(2+) release. At concentrations of 100 muM and 200 muM, anandamide killed 50% and 95% cells, respectively. The cytotoxic effect of 100 muM anandamide was completely reversed by pre-chelating cytosolic Ca(2+) with BAPTA. Collectively, in MDCK cells, anandamide induced [Ca(2+)](i) rises by causing Ca(2+) release from endoplasmic reticulum and Ca(2+) influx from extracellular space. Furthermore, anandamide can cause Ca(2+)-dependent cytotoxicity in a concentration-dependent manner.
Yeh
Jeng-Hsien
JH
Pathology and Laboratory Medicine Department, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan 813.
Cheng
He-Hsiung
HH
Huang
Chun-Jen
CJ
Chung
Hsiao-Min
HM
Chiu
Hui-Fen
HF
Yang
Yu-Lin
YL
Yeh
Mei-Yin
MY
Chen
Wei-Chuan
WC
Kao
Cheng-Hsing
CH
Chou
Chiang-Ting
CT
Jan
Chung-Ren
CR
D000818:Animals; D001095:Arachidonic Acids / Q000633:toxicity*; D002118:Calcium / Q000378:metabolism*; D063385:Cannabinoid Receptor Modulators / Q000633:toxicity*; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D004285:Dogs; D063388:Endocannabinoids; D005456:Fluorescent Dyes; D016257:Fura-2; D007684:Kidney Tubules / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D053284:Polyunsaturated Alkamides; D013778:Tetrazolium Salts
C110656:2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium; D001095:Arachidonic Acids; D063385:Cannabinoid Receptor Modulators; D063388:Endocannabinoids; D005456:Fluorescent Dyes; D053284:Polyunsaturated Alkamides; D013778:Tetrazolium Salts; D002118:Calcium; D016257:Fura-2; C078814:anandamide