Effect of anandamide on cytosolic Ca²⁺ levels and proliferation in canine renal tubular cells

The effect of the endogenous cannabinoid anandamide on cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) and proliferation is largely unknown. This study examined whether anandamide altered Ca ²⁺ levels and caused Ca²⁺-dependent cell death in Madin-Darby canine kidney (MDCK) cells. [Ca²⁺]_i and cell death were measured using the fluorescent dyes fura-2 and WST-1 respectively. Anandamide at concentrations above 5 μM increased [Ca²⁺] _i in a concentration-dependent manner. The Ca²⁺ signal was reduced by 78% by removing extracellular Ca²⁺. The anandamide-induced Ca²⁺ 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 Ca²⁺ pump inhibitor), anandamide-induced Ca²⁺ release was inhibited. Inhibition of phospholipase C with U73122 did not change anandamide-induced Ca²⁺ release. At concentrations of 100 μM and 200 μM, anandamide killed 50% and 95% cells, respectively. The cytotoxic effect of 100 μM anandamide was completely reversed by pre-chelating cytosolic Ca²⁺ with BAPTA. Collectively, in MDCK cells, anandamide induced [Ca²⁺]_i rises by causing Ca²⁺ release from endoplasmic reticulum and Ca ²⁺ influx from extracellular space. Furthermore, anandamide can cause Ca²⁺-dependent cytotoxicity in a concentration-dependent manner.