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


RESULTS
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 PLCdelta1 (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(A)R) antagonist, BQ123. To characterize ET(A)R 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(A)R 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(A)R desensitization, MSMCs were co-transfected with eGFP-PH and catalytically inactive (D110A,K220R)GRK2, (D110A,K220R)GRK3, (K215R)GRK5, or (K215R)GRK6 constructs. (D110A,K220R)GRK2 expression significantly attenuated ET(A)R desensitization, whereas other constructs were ineffective. Small interfering RNA-targeted GRK2 depletion equally attenuated ET(A)R desensitization. Finally, immunocyotchemical data showed that ET(A)R activation recruited endogenous GRK2 from cytoplasm to membrane.


CONCLUSIONS
These studies identify GRK2 as a key regulator of ET(A)R responsiveness in resistance arteries, highlighting the potential importance of this GRK isoenzyme in regulating vasoconstrictor signalling pathways implicated in vascular disease.
Morris
Gavin E
GE
Reproductive Sciences Section, Department of Cancer Studies and Molecular Medicine, Clinical Sciences Building, Leicester Royal Infirmary, Leicester LE2 7LX, UK.
Nelson
Carl P
CP
Standen
Nicholas B
NB
Challiss
R A John
RA
Willets
Jonathon M
JM
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D019332:Endothelin-1 / Q000494:pharmacology; D054769:G-Protein-Coupled Receptor Kinase 2 / Q000037:antagonists & inhibitors / Q000502:physiology*; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000502:physiology; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000201:enzymology / Q000502:physiology*; D011493:Protein Kinase C / Q000502:physiology; D051381:Rats; D017208:Rats, Wistar; D044022:Receptor, Endothelin A / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology*; D014661:Vasoconstriction
D019332:Endothelin-1; D044022:Receptor, Endothelin A; D011493:Protein Kinase C; C496569:Grk2 protein, rat; D054769:G-Protein-Coupled Receptor Kinase 2; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D002118:Calcium