Mechanisms of miconazole-induced rise in cytoplasmic calcium concentrations in Madin Darby canine kidney (MDCK) cells

Abstract

The effect of miconazole on intracellular calcium levels ([Ca 2+] i) in Madin Darby canine kidney (MDCK) cells was studied using fura-2 as the Ca 2+ indicator. Miconazole increased [Ca 2+] i dose-dependently at concentrations of 5–100 μM. The [Ca 2+] i transient consisted of an initial rise, a gradual decay and an elevated plateau (220 s after addition of the drug). Removal of extracellular Ca 2+ partly reduced the miconazole response. Mn 2+ quench of fura-2 fluorescence confirmed that miconazole induced Ca 2+ influx. The miconazole-sensitive intracellular Ca 2+ store overlaped with that sensntive to thapsigargin, an inhibitor of the endoplasmic reticulum Ca 2+ pump, because 20 μM miconazole depleted the thapsigargin (1 μM)-sensitive store and conversely, thapsigargin abolished miconazole-induced internal Ca 2+ release. Miconazole (20–50 μM) partly inhibited the capacitative Ca 2+ entry induced by 1 μM thapsigargin, measured by depleting intracellular Ca 2+ store in Ca 2+-free medium followed by addition of 10 mM CaCl 2. Miconazole induced capacitative Ca 2+ entry on its own. Pretreatment with 0.1 mM La 3+ partly inhibited 20 μM miconazole-induced Mn 2+ quench of fura-2 fluorescence and [Ca 2+] i rise, suggesting that miconazole induced Ca 2+ influx via two pathways separable by 0.1 mM La 3+. Miconazole-induced internal Ca 2+ release was not altered when the cytosolic level of inositol 1,4,5-trisphosphate (IP 3) was substantially inhibited by the phospholipase C inhibitor U73122.