Regulation of intracellular Ca²⁺and gene expression by endothelin-1

In addition to its powerful vasoconstrictive activity, endothelin-1 (ET-1) is a potent agonist for stimulating a multitude of second messenger pathways. In the Rat-1 fibroblastic cell line, ET-1 induces a robust elevation of the intracellular levels of Ca²⁺, diacylglycerols (DGs), and inositol trisphosphate (IP₃). Although low concentrations of ET-1 stimulate a significant increase in the rate of Ca²⁺influx, this Ca²⁺influx is not required for the observed increases in either IP₃or DG levels following ET-1 treatment, as both of these effects are observed even in the absence of extracellular Ca²⁺. The ability of ET-1 to stimulate Ca²⁺influx shows a biphasic pattern, such that Ca²⁺influx is stimulated at low ET-1 concentrations and inhibited at high concentrations. Investigations of the molecular mechanisms underlying this biphasic response indicate that elevated intracellular Ca²⁺levels exert a negative feedback inhibition on Ca²⁺influx, which can be relieved by the chelation of intracellular Ca²⁺. The ability of ET-1 to activate a number of distinct signal transduction pathways appears to have direct functional significance in determining the targeting of ET-1 activation. Short-term effects of ET-1 stimulation such as the induction of gene expression appear to be independent of ET-1's ability to activate protein kinase C (PKC) by elevating DG levels, as depletion of PKC activity has little or no effect on gene expression. In contrast, the ability of ET-1 to induce the rapid expression of the VL30 gene is totally dependent upon the ability of ET-1 to elevate intracellular Ca²⁺levels above a specific threshold. Activation of PKC by ET-1. however, is essential for the long-term effects of ET-1 on cell proliferation and anchorage-independent growth, as the ability of ET-1 to promote DNA synthesis and to synergize with epidermal growth factor in augmenting anchorage-independent growth is significantly inhibited by prior PKC depletion. Thus, in fibroblasts, ET-1 appears to activate at least two bifurcating pathways: A Ca²⁺-sensitive pathway involved in the regulation of gene expression, and a PKC-dependent pathway required for the mitogenic effects of ET-1.