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 (ETA)-mediated increase in cytosolic Ca2+ concentration was mainly dependent on Ca2+ release from the intracellular store. ETAreceptor-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-βcom antibody and a peptide corresponding to the Gβγ binding region of the β-adrenergic receptor kinase-1 had no effect on the ETAreceptor-mediated Ca2+ release. In contrast, a synthetic peptide corresponding to the carboxyl terminus of the αq/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 ETA 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-αq 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 ETAreceptor-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.
Footnotes
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Send reprint requests to: Dr J. Mironneau, Laboratoire de Physiologie Cellulaire et Pharmacologie Moléculaire, CNRS ESA 5017, Université de Bordeaux II, 146 rue Léo Saignat, 33076 Bordeaux Cedex, France. Email: jean.mironneau{at}esa5017.u-bordeaux2.fr
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↵1 Contributed equally to the work.
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This work was supported by a grant from the Centre National de la Recherche Scientifique (France).
- Abbreviations:
- ET-1
- endothelin-1
- AII
- angiotensin II
- βARK1
- β-adrenergic receptor kinase-1
- [Ca2+]i
- cytosolic Ca2+concentration
- InsP
- inositol phosphate
- InsP1
- inositol monophosphate
- InsP2
- inositol bisphosphate
- InsP3
- inositol trisphosphate
- NE
- norepinephrine
- PLC
- phospholipase C
- ETA
- endothelin receptor subtype A
- ETB
- endothelin receptor subtype B
- U73122
- 1-(6-((17β-3 methoxystra 1,3,5 (10)-trien-17-yl) amino) hexyl)-1H-pyrrole-2,5-dione
- PTX
- pertussis toxin
- AM
- indo-1-acetoxymethylester
- Received June 22, 1998.
- Accepted January 9, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
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