Effect of anandamide on cytosolic Ca(2+) levels and proliferation in canine renal tubular cells

Basic Clin Pharmacol Toxicol. 2006 Apr;98(4):416-22. doi: 10.1111/j.1742-7843.2006.pto_350.x.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Arachidonic Acids / toxicity*
  • Calcium / metabolism*
  • Cannabinoid Receptor Modulators / toxicity*
  • Cell Proliferation / drug effects
  • Cells, Cultured
  • Dogs
  • Endocannabinoids
  • Fluorescent Dyes
  • Fura-2
  • Kidney Tubules / cytology
  • Kidney Tubules / drug effects*
  • Kidney Tubules / metabolism
  • Polyunsaturated Alkamides
  • Tetrazolium Salts

Substances

  • 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium
  • Arachidonic Acids
  • Cannabinoid Receptor Modulators
  • Endocannabinoids
  • Fluorescent Dyes
  • Polyunsaturated Alkamides
  • Tetrazolium Salts
  • Calcium
  • Fura-2
  • anandamide