Effect of diethylstilbestrol (DES) on intracellular Ca ²⁺ levels in renal tubular cells

The effect of the estrogen diethylstilbestrol (DES) on intracellular Ca ²⁺ concentrations ([Ca ²⁺] _i) in Madin Darby canine kidney (MDCK) cells was investigated, using the fluorescent dye fura-2 as a Ca ²⁺ indicator. DES (10-50 μM) evoked [Ca ²⁺] _i increases in a concentration-dependent manner. Extracellular Ca ²⁺ removal inhibited 45 ± 5% of the Ca ²⁺ response. In Ca ²⁺-free medium, pretreatment with 50 μM DES abolished the [Ca ²⁺] _i increases induced by 2 μM carbonylcyanide m-chlorophenylhydrazone (CCCP; a mitochondrial uncoupler) and 1 μM thapsigargin (an endoplasmic reticulum Ca ²⁺ pump inhibitor); and pretreatment with CCCP and thapsigargin partly inhibited DES-induced [Ca ²⁺] _i signals. Adding 3 mM Ca ²⁺ increased [Ca ²⁺] _i in cells pretreated with 50 μM DES in Ca ²⁺-free medium, suggesting that DES may induce capacitative Ca ²⁺ entry. 17β-Estradiol (2-20 μM) increased [Ca ²⁺] _i, but 100 μM diethylstilbestrol dipropionate had no effect. Pretreatment with the phospholipase C inhibitor U73122 (1 μM) to abolish inositol 1,4,5-trisphosphate formation inhibited 30% of DES-induced Ca ²⁺ release. DES (20 μM) also increased [Ca ²⁺] _i in human normal hepatocytes and osteosarcoma cells. Cumulatively, this study shows that DES induced rapid and sustained [Ca ²⁺] _i increases by releasing intracellular Ca ²⁺ and triggering extracellular Ca ²⁺ entry in renal tubular cells.