202 intact vessel contractions are relatively resistant to desensitization when compared with PLC/Ca2+ responses in primary MSMCs. These differences might relate to the differing capacities of intact mesenteric smooth muscle and cultured MSMC preparations to metabolize UTP, and/or ex vivo changes in receptor populations and/or post-receptor components. Nevertheless, using comparable protocols, it is possible to assess the time-course of receptor desen- sitization/resensitization with respect to both UTP-stimulated con- tractile and signalling responses in tissue/cell preparations. Since GRK proteins are known to regulate the signalling of other PLC-coupled GPCRs expressed in MSMCs,14,21,22 we initially utilized dominant-negative (kinase-dead) GRK mutants to disrupt P2Y2- receptor/GRK isoenzyme-speciﬁc interactions in an attempt to attenuate or prevent the reduction in receptor responsiveness observed on re-addition of UTP subsequent to a desensitizing pulse of this agonist. The D110A,K220RGRK2 construct, which has been mutated to prevent both kinase activity and Gaq/11-binding,20 mark- edly attenuated P2Y2-receptor desensitization. Conversely, over- expression of D110A,K220RGRK3, K215RGRK5, or K215RGRK6 mutants affected neither the extent of desensitization, nor the time-course of recovery of P2Y2-receptor responsiveness to UTP. To complement our ﬁndings (and address any potential criticisms associated with the recombinant over-expression of GRK mutants), we also depleted (.75%) endogenous GRK2 expression in MSMCs using isoenzyme- speciﬁc siRNAs, producing near-identical data to those obtained using the D110A,K220RGRK2 construct. Together these ﬁndings indicate that GRK2 is a key endogenous GRK isoenzyme initiating P2Y2-receptor desensitization in MSMCs, with either GRK2 knockdown or disruption of the normal GRK2-receptor interaction causing an 60% attenuation of agonist- stimulated P2Y2-receptor desensitization; a ﬁgure only 15% less than that achievable after full receptor resensitization. It is possible that GRK2 is the only kinase involved in initiating P2Y2-receptor desensitization and that the observed partial effects arise because the experimental ablations of GRK2 activity are incompletely effective. On the other hand, while a predominant GRK isoenzyme can often be identiﬁed as being responsible for initiating receptor desensitization it is rare for this to be the only protein kinase involved.23,24 Therefore, other (minor) mechanisms may yet be shown to be involved in regu- lating P2Y2-receptor responsiveness in MSMCs. GRK2 has previously been reported to be the key GRK isoenzyme regulating angiotensin II type 1 (AT1),25 a1D-adrenergic,22 and ETA14 receptor-mediated contractile responses. The ﬁnding that GRK2 is also key to the regulation of P2Y2-receptor signalling further empha- sizes the importance of this GRK isoenzyme in Gaq/11/PLC-coupled receptor regulation in arterial smooth muscle. GRK-mediated phosphorylation often leads to arrestin recruitment to the receptor, promoting internalization, receptor recycling, and/or downregulation.10,11 When expressed in HEK293 cells, P2Y2- receptors recruit both exogenous arrestin2 and arrestin3,26 however, while suggestive that P2Y2 receptors can interact with arrestin proteins, these studies are not necessarily predictive of how, or if, these receptors are regulated in native tissues. Here, we utilized an RNAi strategy to suppress individual arrestin isoform expression to delineate for the ﬁrst time their role in P2Y2 receptor regulation in resistance artery smooth muscle. Our data indicate a high degree of isoform-selective arrestin interaction with P2Y2, with siRNA-mediated depletion of arrestin2 almost completely attenuating UTP-stimulated P2Y2-receptor desensitization. Conversely, G.E. Morris et al. knockdown of arrestin3 was without effect on P2Y2 signalling. We also assessed whether another important vasoconstrictor GPCR also differentially recruits arrestin2/3 isoforms, and showed that ETA-receptor responses are attenuated by arrestin3, but not arrestin2 knockdown. Moreover, we recently showed that GRK2 is also a criti- cal kinase involved in ETA-receptor desensitization.14 Consequently, our ﬁndings clearly demonstrate that phosphorylation by a common GRK isoenzyme can lead to contrasting arrestin isoform recruitment. A likely explanation that deserves future investigation is that the pattern of GRK2-mediated P2Y2- and ETA-receptor phosphorylation differs sufﬁciently to promote differential recruitment of arrestins. Finally, the potential importance of arrestin recruitment in vascular disease has been highlighted by two recent studies. Data from arrestin knockout mice reveal a role for arrestins in the regulation of vascular smooth muscle migration and proliferation during atherosclerosis and neointimal hyperplasia,27 while arrestin3 regulates angiotensin II-stimulated, ERK-mediated aortic smooth muscle proliferation.28 Since our new ﬁndings highlight a selective arrestin regulation of ETA- and P2Y2-receptors, it is likely that differential recruitment of arrestins to phosphorylated GPCRs has the potential to determine physiological and pathophysiological signalling outcomes activated by UTP and ET-1. Funding This work is supported by Project and Programme funding from the British Heart Foundation (grant nos. PG06/161/22136; RG06/008/ 22062). Funding to pay the Open Access publication charge was provided by the British Heart Foundation. Supplementary material Supplementary material is available at Cardiovascular Research online. Acknowledgements We thank Tobias Meyer (Stanford University, USA) for generously providing the eGFP-PH biosensor, and Robert J. Lefkowitz (Duke Uni- versity, USA) for kindly providing the arrestin (A1CT) antibody. Conﬂict of interest: none declared. References 1. Erlinge D, Burnstock G. P2 receptors in cardiovascular regulation and disease. Puriner- gic Signal 2008;4:1– 20. 2. Winter P, Dora KA. Spreading dilatation to luminal perfusion of ATP and UTP in rat isolated small mesenteric arteries. J Physiol 2007;582:335–347. 3. von Kugelgen I, Haussinger D, Starke K. Evidence for a vasoconstriction-mediating receptor for UTP, distinct from the P2 purinoceptor, in rabbit ear artery. Naunyn Schmiedeberg’s Arch Pharmacol 1987;336:556–560. 4. Malmsjo M, Adner M, Harden TK, Pendergast W, Edvinsson L, Erlinge D. The stable pyrimidines UDPbS and UTPgS discriminate between the P2 receptors that mediate vascular contraction and relaxation of the rat mesenteric artery. Br J Pharmacol 2000; 131:51–56. 5. Abbracchio MP, Burnstock G, Boeynaems JM, Barnard EA, Boyer JL, Kennedy C et al. International Union of Pharmacology LVIII: update on the P2Y G protein-coupled nucleotide receptors: from molecular mechanisms and pathophysiology to therapy. Pharmacol Rev 2006;58:281–341. 6. Kumari R, Goh G, Ng LL, Boarder MR. ATP and UTP responses of cultured rat aortic smooth muscle cells revisited: dominance of P2Y2 receptors. Br J Pharmacol 2003;140: 1169– 1176. 7. Charlton SJ, Brown CA, Weisman GA, Turner JT, Erb L, Boarder MR. 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