Mechanisms underlying Ketoconazole-induced Ca²⁺ mobilization in Madin- Darby canine kidney cells

The effect of ketoconazole on Ca²⁺ signaling in Madin-Darby canine kidney (MDCK) cells was investigated by using fura-2 as a Ca²⁺ probe. Ketoconazole evoked increases in cytosolic free Ca²⁺ concentration ([Ca²⁺](i)) concentration dependently. The response was decreased by external Ca²⁺ removal. In Ca²⁺-free medium, pretreatment with ketoconazole abolished the [Ca²⁺](i) rise induced by thapsigargin, an inhibitor of the endoplasmic reticulum Ca²⁺ pump. Addition of 3 mM Ca²⁺ induced a significant [Ca²⁺](i) rise after preincubation with 150 μM ketoconazole in Ca²⁺-free medium. Pretreatment with aristolochic acid (40 μM) to inhibit phospholipase A₂ inhibited the 150-μM-ketoconazole-induced internal Ca²⁺ release by 37%, but inhibition of phospholipase C with 1-(6- ((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5- dione (U73122) (2 μM) had no effect. Collectively, we found that ketoconazole increases [Ca²⁺](i) in MDCK cells by releasing Ca²⁺ from thapsigargin-sensitive pools in a manner independent of the production of inositol-1,4,5-trisphosphate, followed by Ca²⁺ influx from the external space. (C) 2000 Elsevier Science Inc.