G Protein βγ Subunits Mediate Upregulated ETB- but not ETA-Receptor Stimulated Arterial Contractile Responses

JGR De Mey1,2, KR Hughes1, MG Compeer1. 1Dept. of Pharmacology, Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, The Netherlands, 2Dept. of Cardiovascular and Renal Research, Institute of Molecular Medicine, University of Southern Denmark, Odense, Denmark

Endothelins (ET) cause arterial contractions and vasospasm through ETA-receptors but in stroke, ischemia and in arterial organ culture, arterial smooth muscle stimulation by ETB-receptors is upregulated. We tested the hypotheses that ET-receptor subtypes act through different G proteins and that upregulation of ETB-responses potentiates vasoconstrictor responses to the endogenous mixed ETA-/ETB-agonist ET1. Isolated rat mesenteric resistance arteries were incubated ex vivo during 18 hours and their vasomotor responses were analyzed. Incubated arteries displayed potent contractile responses to the ETB selective agonist sarafotoxin 6c (S6c) and to the ETB-preferring agonist ET3 which were not sustained. Next, ET1 still caused potent contraction and vasospasm. Presence of pertussis toxin (PTX) during the incubation did not alter relaxing responses to isoproterenol but abolished their reversal by neuropeptide Y (NPY) indicating inhibition of Gi proteins. PTX (100 ng/ml during 18 hours or 500 ng/ml during the last 2 hours of incubation) reduced the potency and efficacy of S6c but did not affect subsequent responses to ET1. Presence of gallein during the analysis abolished relaxing responses to calcitonin gene-related peptide and abolished reversal of β-adrenergic relaxations by NPY indicating inhibition of G protein βγ subunits. Gallein abolished upregulated responses to S6c but did not affect subsequent responses to ET1. Incubation with PTX and presence of gallein or BQ788 (ETB-antagonist) during analysis did not significantly alter contractile responses to ET1. Also, transient exposure of incubated arteries to S6c or ET3 did not significantly alter contractile responses to ET1. We conclude that upregulated arterial ETB-mediated contractions are brought about by G protein βγ subunits that are partly derived from Gi proteins and that are not involved in ETA-mediated contractions. Despite ETB upregulation, ET1-induced responses were not modified by ETB-antagonism, ETB-desensitization and inhibition of ETB-signaling. This suggests that ETA-stimulation might inhibit ETB-signaling in arterial smooth muscle.

Work performed in the frame of Top Institute Pharma project T2-301.