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Cardiovascular Research Advance Access first published online on September 11, 2009
This version [Corrected Proof] published online on October 6, 2009
Cardiovascular Research, doi:10.1093/cvr/cvp310
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Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2009. For permissions please email: journals.permissions@oxfordjournals.org.
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Endothelin signalling in arterial smooth muscle is tightly regulated by G protein-coupled receptor kinase 2

Gavin E. Morris1, Carl P. Nelson2, Nicholas B. Standen2, R.A. John Challiss2 and Jonathon M. Willets1,2,*

1 Reproductive Sciences Section, Department of Cancer Studies and Molecular Medicine, Clinical Sciences Building, Leicester Royal Infirmary, Leicester LE2 7LX, UK
2 Department of Cell Physiology and Pharmacology, University of Leicester, Henry Wellcome Building, Lancaster Road, Leicester LE1 9HN, UK

* Corresponding author. Tel: +44 116 252 5883, Fax: +44 116 252 5883, Email: jmw23{at}le.ac.uk

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 [Ca2+]i were assessed using confocal microscopy in rat MSMCs transfected with the pleckstrin-homology domain of PLC{delta}1 (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 (ETAR) antagonist, BQ123. To characterize ETAR 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 ETAR 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 ETAR desensitization, MSMCs were co-transfected with eGFP-PH and catalytically inactive D110A,K220RGRK2, D110A,K220RGRK3, K215RGRK5, or K215RGRK6 constructs. D110A,K220RGRK2 expression significantly attenuated ETAR desensitization, whereas other constructs were ineffective. Small interfering RNA-targeted GRK2 depletion equally attenuated ETAR desensitization. Finally, immunocyotchemical data showed that ETAR activation recruited endogenous GRK2 from cytoplasm to membrane.

Conclusion: These studies identify GRK2 as a key regulator of ETAR responsiveness in resistance arteries, highlighting the potential importance of this GRK isoenzyme in regulating vasoconstrictor signalling pathways implicated in vascular disease.

KEYWORDS Endothelin-1; Endothelin-A receptor; G protein-coupled receptor kinase; Receptor desensitization; Vasoconstriction; Resistance artery; Mesenteric

Time for primary review: 23 days


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