Vol. 55, Issue 4, 684-692, April 1999

Specific G₁₁₃₅ Protein Involvement in Endothelin Receptor-Induced Phosphatidylinositol Hydrolysis and Ca²⁺ Release in Rat Portal Vein Myocytes

Nathalie Macrez,¹ Jean-Luc Morel,¹ and Jean Mironneau

Laboratoire de Physiologie Cellulaire et Pharmacologie Moléculaire, Centre National de la Recherche Scientifique, Enseignement Supérieur Associé 5017, Université de Bordeaux II, 33076 Bordeaux Cedex, France

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_A receptor-mediated Ca²⁺ release was selectively inhibited by antisense oligonucleotides that inhibited the expression of ₁₁, ₃, and ₅ subunits, as checked by immunocytochemistry. Intracellular dialysis of a carboxyl terminal anti-_com antibody and a peptide corresponding to the G binding region of the -adrenergic receptor kinase-1 had no effect on the ET_A receptor-mediated Ca²⁺ release. In contrast, a synthetic peptide corresponding to the carboxyl terminus of the _q/₁₁ 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-_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_A receptor-mediated Ca²⁺ release is selectively transduced by the heterotrimeric G₁₁ protein composed of ₁₁, ₃, and ₅ subunits, and that a delayed stimulation of phospholipase C occurs via the ₁₁ subunit.