Novel effects of clotrimazole on Ca²⁺ signaling in madin darby canine kidney cells

The effect of clotrimazole on Ca²⁺ signaling in Madin Darby canine kidney (MDCK) cells was investigated by using fura-2 as a Ca²⁺ indicator. Clotrimazole (1-30 μM) induced a concentration-dependent [Ca²⁺](i) increase. The [Ca²⁺](i) increase comprised an initial rise and a slow decay. External Ca²⁺ removal partly inhibited the Ca²⁺ signals by reducing both the initial rise and the decay phase, indicating that clotrimazole triggered both Ca²⁺ influx and Ca²⁺ release. Pretreatment with 30 μM clotrimazole in Ca²⁺ -free medium abolished the Ca²⁺ release induced by thapsigargin (1 μM), an endoplasmic reticulum Ca²⁺ pump inhibitor, and conversely, pretreatment with thapsigargin prevented clotrimazole from releasing more Ca²⁺. This suggests that the thapsigargin- sensitive Ca²⁺ store is the source of clotrimazole-induced Ca²⁺ release. Clotrimazole (10 μM) triggered Mn²⁺ quench of fura-2 fluorescence which was partly inhibited by 1 mM La³⁺. Addition of 3 mM Ca²⁺ induced a [Ca²⁺](i) increase after preincubation with 10 μM clotrimazole in Ca²⁺ - free medium, indicating that clotrimazole activated capacitative Ca²⁺ entry. However, 10 and 30 μM clotrimazole inhibited 1 μM thapsigargin- induced capacitative Ca²⁺ entry by 21% and 74%, respectively. Pretreatment with 40 μM aristolochic acid to inhibit phospholipase A₂ reduced 30 μM clotrimazole-induced Ca²⁺ release by 51%, but inhibiting phospholipase C with 2 μM U73122 had little effect. This implies that clotrimazole induces Ca²⁺ release in an IP₃-independent manner, which could be modulated by phospholipase A₂-coupled events.