Ca2 mobilization induced by δ‐hexachlorocyclohexane in Madin Darby canine kidney cells

Abstract

The effect of the pesticide δ-hexachlorocyclohexane (δ-HCH) were examined on Ca2+ signaling in Madin Darby canine kidney (MDCK) using fura-2 as a Ca2+ probe. δ-HCH at concentrations of 5–200 mM increased intracellular free Ca2+ concentration ([Ca2+]i) concentration-dependently. The [Ca2+]i increase comprised an immediate rise followed by a sustained phase within 5 min of measurement. External Ca2+ removal slightly reduced the [Ca2+]i increase. In Ca2+-free medium, 150 μM δ-HCH did not increase [Ca2+]i after pretreatment with carbonylcyanide m-chlorophenylhydrazone (CCCP; 2 μM), a mitochondrial uncoupler, and two endoplasmic reticulum (ER) Ca2+ pump inhibitors, thapsigargin (1 μM) and cyclopiazonic acid (100 μM). Conversely, pretreatment with δ-HCH prevented thapsigargin, cyclopiazonic acid, and CCCP from releasing more Ca2+, suggesting 150 μM δ-HCH released Ca2+ from the ER and mitochondria. δ-HCH (150 μM) activated Mn2+ quench of fura-2 fluorescence, confirming that δ-HCH induced Ca2+ influx. Addition of 3 mM Ca2+ induced a concentration-dependent [Ca2+]i increase after pretreatment with 100–200 μM δ-HCH for 870 sec in Ca2+-free medium. The δ-HCH (150 μM)-induced Ca2+ release was decreased by inhibiting phospholipase C with 1 μM U73122. Collectively, we have found that δ-HCH increased [Ca2+]i in MDCK cells by releasing Ca2+ from the ER and mitochondria, followed by capacitative Ca2+ entry. Drug Dev. Res. 50:186–192, 2000. © 2000 Wiley-Liss, Inc.