endothelin-1 and carboxyamido-triazole

Agents such as thapsigargin and endothelin elevate intracellular calcium levels by a combination of calcium release from intracellular stores and calcium influx across the plasma membrane; however, the relative contribution of influx vs. release in modulating calcium-dependent gene expression is not as well understood in nonexcitable cells as in excitable cells. In this report we have been able to separate thapsigargin-induced elevation of intracellular calcium into release and influx components, using carboxyamido-triazole (CAI), a known inhibitor of calcium influx with antiproliferative activity against a number of human carcinomas, to selectively inhibit influx without affecting release. The results of these experiments indicate that the ability of thapsigargin to induce calcium-dependent gene expression in nonexcitable cells is dependent on the induction of calcium influx, presumably through store-operated calcium channels. CAI treatment specifically inhibited thapsigargin- or endothelin-stimulated expression from the c-fos promoter in Rat-1 cells and in epithelial cell lines derived from ovary and breast. Use of the VL30 model system confirmed the ability of CAI to inhibit calcium-dependent gene expression and further demonstrated that the ability of elevated calcium to synergize with other signaling pathways required close temporal coupling. In addition to inhibiting endothelin-induced calcium influx, CAI treatment also resulted in a partial inhibition of IP3 production and calcium release. CAI treatment also blocked the increase in ERK1 kinase activity observed in response to either endothelin or thapsigargin, suggesting a role for calcium influx in the activation of mitogen-activated protein kinase pathways.

Topics: Animals; Calcium; Calcium Channel Blockers; Calcium-Calmodulin-Dependent Protein Kinases; Cell Line; Chloramphenicol O-Acetyltransferase; Endothelin-1; Enzyme Activation; Gene Expression Regulation; Genes, fos; Genes, Reporter; Imidazoles; Rats; Thapsigargin; Triazoles