Fendiline increases [Ca 2+] i in Madin Darby canine kidney (MDCK) cells by releasing internal Ca 2+ followed by capacitative Ca 2+ entry

Abstract

The effect of fendiline, a documented inhibitor of L-type Ca 2+ channels and calmodulin, on Ca 2+ signaling in Madin Darby canine kidney (MDCK) cells was investigated using fura-2 as a Ca 2+ probe. Fendiline at 5–100 μM significantly increased [Ca 2+] i concentration-dependently. The [Ca 2+] i rise consisted of an initial rise and a slow decay. External Ca 2+ removal partly inhibited the Ca 2+ signals induced by 25–100 μM fendiline by reducing both the initial rise and the decay phase. This suggests that fendiline triggered external Ca 2+ influx and internal Ca 2+ release. In Ca 2+-free medium, pretreatment with 50 μM fendiline nearly abolished the [Ca 2+] i rise induced by 1 μM-thapsigargin, an endoplasmic reticulum Ca 2+ pump inhibitor, and vice versa, pretreatment with thapsigargin prevented fendiline from releasing internal Ca 2+. This indicates that the internal Ca 2+ source for fendiline overlaps with that for thapsigargin. At a concentration of 50 μM, fendiline caused Mn 2+ quench of fura-2 fluorescence at the 360 nm excitation wavelenghth, which was inhibited by 0.1 mM La 3+ by 50%, implying that fendiline-induced Ca 2+ influx has two components separable by La 3+. Consistently, 0.1 mM La 3+ pretreatment suppressed fendiline-induced [Ca 2+] i rise, and adding La 3+ during the rising phase immediately inhibited the signal. Addition of 3 mM Ca 2+ increased [Ca 2+] i after preincubation with 50–100 μM fendiline in Ca 2+-free medium. However, 50–100 μM fendiline inhibited 1 μM thapsigargin-induced capacitative Ca 2+ entry. Pretreatment with 40 μM aristolochic acid to inhibit phospholipase A 2 inhibited 50 μM fendiline-induced internal Ca 2+ 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 [Ca 2+] i in MDCK cells by releasing internal Ca 2+ in a manner independent of inositol-l,4,5-trisphosphate (IP 3), followed by external Ca 2+ influx.