Effect of diethylstilbestrol (DES) on intracellular Ca2+ levels in bovine lens epithelial cells

Abstract

Abstract: : Purpose: Estrogens modulate the intracellular calcium concentration ([Ca²⁺]_i). Previous work in this laboratory indicated that progesterone rapidly increased Ca²⁺ influx and [Ca²⁺]_i in bovine lens epithelial cells (Samadi A, et al., Pflugers Arch – Eur J Physiol (2002) 444:700–709). The effect of the synthetic estrogen diethylstilbestrol (DES) on intracellular calcium concentrations [Ca²⁺]_i in lens epithelial cells was investigated. Methods: Bovine lens epithelial cells (BLEC) were treated with varying concentrations (10^–6 – 10^–4 M) of diethylstilbestrol. Lens epithelial cells were attached to a glass coverslip and loaded with 10µM fura–2/AM in AAH media at 35^oC. Fluorescence of individual cells was monitored using conventional techniques Results: DES at 10–100µM increased [Ca²⁺]_i in a concentration–dependent manner. At 10µM, the increase from basal levels was more rapid. However, the [Ca²⁺]_i did not decline to basal levels. At 100µM, the cellular calcium response was slower; however, in the majority of cells the [Ca²⁺]_i eventually reached R_max levels. Mn²⁺ quench assays of resting Ca influx indicated that 10µM DES did not increase influx in 90% of cells. However, in some cells, a delayed increase in quench rate was observed. This is in contrast to previous results in which all lens epithelial cells showed pronounced increases in Ca²⁺ influx following the addition of progesterone. Depletion of extracellular Ca²⁺ reduced [Ca²⁺]_i, but did not abolish the effect of 10µM DES on [Ca²⁺]i. Pretreatment of cells with thapsigargin and CPA reduced the effect of 10µM DES on [Ca²⁺]_i. Conclusion: Unlike progesterone, DES induced a rapid and significant increase in [Ca²⁺]_i, probably by releasing Ca²⁺ from intracellular stores. We are currently investigating the potential role of calcium influx in DES–mediated increases in [Ca²⁺]_i.

Keywords: calcium • signal transduction