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 mu M 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 mu M 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 mu M fendiline nearly abolished the [Ca2+](i) rise induced by 1 mu M-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 mu M, fendiline caused Mn2+ quench of fura-2 fluorescence at the 360 nm excitation wavelenghth, 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 mu M fendiline in Ca2+-free medium. However, 50-100 mu M fendiline inhibited 1 mu M thapsigargin-induced capacitative Ca2+ entry. Pretreatment with 40 mu M aristolochic acid to inhibit phospholipase A(2) inhibited 50 mu M fendiline-induced internal Ca2+ release by 48%, but inhibition of phospholipase C with 2 mu M 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.