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

Abstract

Aims

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).

Methods and results

ET-1-stimulated phospholipase C (PLC) activity and changes in [Ca ²⁺] _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 [Ca ²⁺] _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.

Conclusion

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.