Cardiovascular Research (2011) 89, 193–203 doi:10.1093/cvr/cvq249 G protein-coupled receptor kinase 2 and arrestin2 regulate arterial smooth muscle P2Y-purinoceptor signalling Gavin E. Morris 1, Carl P. Nelson 2, Diane Everitt 2, Paul J. Brighton 1, Nicholas B. Standen 2, R.A. John Challiss 2, and Jonathon M. Willets 1,2* 1Reproductive Sciences Section, Department of Cancer Studies and Molecular Medicine, Clinical Sciences Building, Leicester Royal Inﬁrmary, Leicester LE2 7LX, UK; and 2Department of Cell Physiology and Pharmacology, University of Leicester, Henry Wellcome Building, Lancaster Road, Leicester LE1 9HN, UK Received 29 January 2010; revised 21 June 2010; accepted 14 July 2010; online publish-ahead-of-print 12 August 2010 Time for primary review: 25 days Aims Prolonged P2Y-receptor signalling can cause vasoconstriction leading to hypertension, vascular smooth muscle hypertrophy, and hyperplasia. G protein-coupled receptor signalling is negatively regulated by G protein-coupled receptor kinases (GRKs) and arrestin proteins, preventing prolonged or inappropriate signalling. This study investi- gates whether GRKs and arrestins regulate uridine 5′-triphosphate (UTP)-stimulated contractile signalling in adult Wistar rat mesenteric arterial smooth muscle cells (MSMCs). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Methods Mesenteric arteries contracted in response to UTP challenge: When an EC50 UTP concentration (30 mM, 5 min) was and results added 5 min before (R1) and after (R2) the addition of a maximal UTP concentration (Rmax: 100 mM, 5 min), R2 responses were decreased relative to R1, indicating desensitization. UTP-induced P2Y-receptor desensitization of phospholipase C signalling was studied in isolated MSMCs transfected with an inositol 1,4,5-trisphosphate biosensor and/or loaded with Ca2+-sensitive dyes. A similar protocol (R1/R2 ¼ 10 mM; Rmax ¼ 100 mM, applied for 30 s) revealed markedly reduced R2 when compared with R1 responses. MSMCs were transfected with dominant-negative GRKs or siRNAs targeting speciﬁc GRK/arrestins to probe their respective roles in P2Y-receptor desensitization. GRK2 inhibition, but not GRK3, GRK5, or GRK6, attenuated P2Y-receptor desensitization. siRNA-mediated knock- down of arrestin2 attenuated UTP-stimulated P2Y-receptor desensitization, whereas arrestin3 depletion did not. Speciﬁc siRNA knockdown of the P2Y2-receptor almost completely abolished UTP-stimulated IP3/Ca2+ signalling, strongly suggesting that our study is speciﬁcally characterizing this purinoceptor subtype. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion These new data highlight roles of GRK2 and arrestin2 as important regulators of UTP-stimulated P2Y2-receptor responsiveness in resistance arteries, emphasizing their potential importance in regulating vasoconstrictor signalling pathways implicated in vascular disease. - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Keywords UTP † G protein-coupled receptor kinase † Arrestin † P2Y-purinoceptor † Resistance artery 1. Introduction Extracellular nucleotides play important roles in cardiovascular physi- ology, and in vascular tissues prolonged nucleotide signalling has been implicated in the pathophysiology of restenosis, diabetic microvascular disease, and hypertension.1 Purine/pyrimidine nucleotide release can have different effects on vascular tone depending on the site of release and cell type(s) with which the nucleotide interacts.1 Nucleo- tides released from endothelial cells after shear stress and/or hypoxia can cause smooth muscle relaxation;1,2 contrastingly, nucleotide release from sympathetic neuronal inputs act directly on vascular smooth muscle causing contraction.1,3,4 * Corresponding author. Tel: +44 116 252 5883; fax: +44 116 252 3162, Email: jmw23@le.ac.uk Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2010. For permissions please email: journals.permissions@oup.com. 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