Abstract

In this study, we identified the receptor subtype activated by endothelin-1 (ET-1) and the subunit composition of the G protein coupling this receptor to increase in cytosolic Ca²⁺concentration in rat portal vein myocytes. We used intranuclear antisense oligonucleotide injection to selectively inhibit the expression of G protein subunits. We show here that the endothelin receptor subtype A (ET_A)-mediated increase in cytosolic Ca²⁺ concentration was mainly dependent on Ca²⁺ release from the intracellular store. ET_Areceptor-mediated Ca²⁺ release was selectively inhibited by antisense oligonucleotides that inhibited the expression of α₁₁, β₃, and γ₅ subunits, as checked by immunocytochemistry. Intracellular dialysis of a carboxyl terminal anti-β_com antibody and a peptide corresponding to the Gβγ binding region of the β-adrenergic receptor kinase-1 had no effect on the ET_Areceptor-mediated Ca²⁺ release. In contrast, a synthetic peptide corresponding to the carboxyl terminus of the α_q/α₁₁ subunit, heparin (an inhibitor of inositol 1,4,5-trisphosphate receptors), and U73122 (an inhibitor of phosphatidylinositol-phospholipase C) inhibited, in a concentration-dependent manner, the ET_A receptor-mediated Ca²⁺ responses. Accumulation of [³H]inositol trisphosphate evoked by norepinephrine peaked at ∼15 s, whereas that evoked by ET-1 progressively increased within 2 min. In myocytes injected with anti-α_q antisense oligonucleotides, both amplitude and time course of the norepinephrine-induced Ca²⁺ release became similar to those of the ET-1-induced Ca²⁺ response. We conclude that the ET_Areceptor-mediated Ca²⁺ release is selectively transduced by the heterotrimeric G₁₁ protein composed of α₁₁, β₃, and γ₅ subunits, and that a delayed stimulation of phospholipase C occurs via the α₁₁ subunit.