Calmidazolium-induced rises in cytosolic calcium concentrations in Madin Darby canine kidney cells

The effect of calmidazolium on Ca²⁺ signaling in Madin Darby canine kidney (MDCK) cells was investigated using fura-2 as a Ca²⁺ probe. Calmidazolium at 2-5 μM increased [Ca²⁺](i) concentration dependently. The [Ca²⁺](i) rise induced by 2-5 μM calmidazolium comprised an immediate rise and a slow decay. External Ca²⁺ removal partly inhibited the Ca²⁺ signals, suggesting that calmidazolium activated external Ca²⁺ influx and internal Ca²⁺ release. In Ca²⁺-free medium, pretreatment with 3 μM calmidazolium abolished the Ca²⁺ release induced by 1 μM thapsigargin, an endoplasmic reticulum Ca²⁺ pump inhibitor, and, vice versa, pretreatment with thapsigargin inhibited calmidazolium-induced Ca²⁺ release. This indicates that thapsigargin-sensitive Ca²⁺ store was the source of calmidazolium-induced Ca²⁺ release. Calmidazolium (3 μM) induced Mn²⁺ quench of fura-2 fluorescence at 360 nm excitation wavelength, which was suppressed by 0.1 mM La³⁺. Addition of 3 mM Ca²⁺ increased [Ca²⁺](i) after pretreatment with 3-5 μM calmidazolium in Ca²⁺-free medium. This implies that calmidazolium activated concentration-dependent capacitative Ca²⁺ entry. Calmidazolium (3 μM) augmented the capacitative Ca²⁺ entry induced by 1 μM thapsigargin or 0.1 mM ATP by 38%. Calmidazolium (3 μM)- induced Ca²⁺ release was blocked by pretreatment with 40 μM aristolochic acid and 2 μM U73122 (2 μM) to inhibit phospholipase A₂ and phospholipase, respectively, but pretreatment with 0.1 mM propranolol to inhibit phospholipase D had no effect. This suggests that calmidazolium induced internal Ca²⁺ release in a manner dependent on phospholipases C- and A₂- coupled events. (C) 2000 Academic Press.