Fendiline increases [Ca²⁺](i) in Madin Darby canine kidney (MDCK) cells by releasing internal Ca²⁺ followed by capacitative Ca²⁺ entry

The effect of fendiline, a documented inhibitor of L-type Ca²⁺ channels and calmodulin, on Ca²⁺ signaling in Madin Darby canine kidney (MDCK) cells was investigated using fura-2 as a Ca²⁺ probe. Fendiline at 5- 100 μM significantly increased [ Ca²⁺](i) concentration-dependently. The [ Ca²⁺](i) rise consisted of an initial rise and a slow decay. External Ca²⁺ removal partly inhibited the Ca²⁺ signals induced by 25-100 μM fendiline by reducing both the initial rise and the decay phase. This suggests that fendiline triggered external Ca²⁺ influx and internal Ca²⁺ release. In Ca²⁺-free medium, pretreatment with 50 μM fendiline nearly abolished the [Ca²⁺](i) rise induced by 1 μM-thapsigargin, an endoplasmic reticulum Ca²⁺ pump inhibitor, and vice versa, pretreatment with thapsigargin prevented fendiline from releasing internal Ca²⁺. This indicates that the internal Ca²⁺ source for fendiline overlaps with that for thapsigargin. At a concentration of 50 μM, fendiline caused Mn²⁺ quench of tiara-2 fluorescence at the 360 nm excitation wavelength, which was inhibited by 0.1 mM La³⁺ by 50%, implying that fendiline-induced Ca²⁺ influx has two components separable by La³⁺. Consistently, 0.1 mM La³⁺ pretreatment suppressed fendiline-induced [Ca²⁺](i) rise, and adding La³⁺ during the rising phase immediately inhibited the signal. Addition of 3 mM Ca²⁺ increased [Ca²⁺](i) after preincubation with 50-100 μM fendiline in Ca²⁺-free medium. However, 50-100 μM fendiline inhibited 1 μM thapsigargin-induced capacitative Ca²⁺ entry. Pretreatment with 40 μM aristolochic acid to inhibit phospholipase A₂ inhibited 50 μM fendiline- induced internal Ca²⁺ release by 48%, but inhibition of phospholipase C with 2 μM U73122 or inhibition of phospholipase D with 0.1 mM propranolol had no effect. Collectively, we have found that fendiline increased [Ca²⁺](i) in MDCK cells by releasing internal Ca²⁺ in a manner independent of inositol-1,4,5-trisphosphate (IP₃), followed by external Ca²⁺ influx.