Endothelin signalling in arterial smooth muscle is tightly regulated by G protein-coupled receptor kinase 2

Abstract

Prolonged endothelin (ET) receptor signalling causes vasoconstriction and can lead to hypertension, vascular smooth muscle hypertrophy, and hyperplasia. Usually, G protein-coupled receptor signalling is negatively regulated by G protein-coupled receptor kinases (GRKs), preventing prolonged or inappropriate signalling. This study investigated whether GRKs regulate ET receptor contractile signalling in adult Wistar rat mesenteric arterial smooth muscle cells (MSMCs). ET-1-stimulated phospholipase C (PLC) activity and changes in [Ca²⁺]_i were assessed using confocal microscopy in rat MSMCs transfected with the pleckstrin-homology domain of PLCδ1 (eGFP-PH) and loaded with Fura-Red. ET-1 applications (30 s) stimulated transient concentration-dependent eGFP-PH translocations from plasma membrane to cytoplasm and graded [Ca²⁺]_i increases. ET-1-mediated PLC signalling was blocked by the type A endothelin receptor (ET_AR) antagonist, BQ123. To characterize ET_AR desensitization, cells were stimulated with a maximally effective concentration of ET-1 (50 nM, 30 s) followed by a variable washout period and a second identical application of ET-1. This brief exposure to ET-1 markedly decreased ET_AR responsiveness to re-challenge, and reversal was incomplete even after increasing the time period between agonist challenges to 60 min. To assess GRK involvement in ET_AR desensitization, MSMCs were co-transfected with eGFP-PH and catalytically inactive ^D110A,K220RGRK2, ^D110A,K220RGRK3, ^K215RGRK5, or ^K215RGRK6 constructs. ^D110A,K220RGRK2 expression significantly attenuated ET_AR desensitization, whereas other constructs were ineffective. Small interfering RNA-targeted GRK2 depletion equally attenuated ET_AR desensitization. Finally, immunocyotchemical data showed that ET_AR activation recruited endogenous GRK2 from cytoplasm to membrane. These studies identify GRK2 as a key regulator of ET_AR responsiveness in resistance arteries, highlighting the potential importance of this GRK isoenzyme in regulating vasoconstrictor signalling pathways implicated in vascular disease.