200 Figure 5 Depletion of endogenous GRK2 attenuates P2Y2- receptor desensitization. MSMCs were nucleofected with 0.5 mg eGFP-PH and either negative-control (NC) or anti-GRK2 (10 nM) siRNAs. Cells were loaded with Fura-Red and subjected to the stan- dard R1/Rmax/R2 desensitization protocol (see Section 2). Represen- tative traces are shown for single cells transfected with NC siRNA (A) or anti-GRK2 siRNA (B). P2Y receptor desensitization was determined as the relative (%) change in R2 response compared with R1. Cumulative data (C ) show means + SEM from 8 to 15 cells from MSMCs prepared from more than four separate animals. Statistical signiﬁcance is indicated as *P , 0.01 vs. NC siRNA-treated MSMCs (one-way ANOVA; Dunnett’s post hoc test). G.E. Morris et al. or Ca2+, was similar in the absence or presence of NC, or anti- arrestin2 siRNAs (Figure 6F and H), whereas, in anti-arrestin3 siRNA- transfected MSMCs endothelin-1-induced receptor desensitization was signiﬁcantly attenuated (Figure 6G and H). These data suggest while both P2Y2- and ETA-receptors are desensitized via a GRK2-dependent mechanism in MSMCs, it is likely that different arrestins are recruited to the desensitized receptor, with P2Y2- and ETA-receptors preferentially recruiting arrestin2 and arrestin3, respectively. 4. Discussion The relative importance of different P2 receptor subtypes present in smooth muscle for contraction is complex and delineation of the contributions of speciﬁc subtypes complicated by the lack of highly selective agonists/antagonists. Available data suggest that G protein- coupled P2Y2, P2Y4, and P2Y6 receptors, and ionotropic P2X1 recep- tors contribute to nucleotide-mediated contraction.4,16 Furthermore, evidence from aortic smooth muscle cells suggests that P2Y-receptor mRNA abundance differs between contractile and synthetic pheno- types, with decreases in P2Y4/P2Y6, and increases in P2Y2 mRNA being observed in the synthetic phenotype.17 Our pharmacological and siRNA knockdown data indicate that UTP/PLC responses in isolated MSMCs are primarily mediated by P2Y2 receptors, which concurs with previous ﬁndings that UTP-mediated contractions in mesenteric arteries (.180 mm) are sensitive to blockade by the P2Y2-selective antagonist suramin.16 Furthermore, our UTP- stimulated arterial contractile (EC50 28 mM) and MSMC Ca2+ (EC50 6 mM) responses are comparable to those reported in small (.100 mm) mesenteric arteries.16 Previous studies have described desensitization of P2Y2-receptor responses in several cell backgrounds,8,9,18,19 however, few studies have addressed the regulation of native P2Y2-receptor responsiveness and none to our knowledge have done so in mesenteric arteries. In human promonocytic U937, astrocytoma 132N1 and epithelial HT-29 cells P2Y2-receptor desensitization has been reported to occur rapidly and is enhanced following phorbol ester addition, however in all cases inclusion of PKC inhibitors failed to prevent receptor desensitization.9,18 Indeed, the observed attenuations of sig- nalling may have been the result of PKC-mediated inhibition of PLC activity.20 Interestingly, protein phosphatase inhibitors can attenuate P2Y2-receptor resensitization.18 Collectively, these ﬁndings suggest that P2Y2-receptor phosphorylation, by a kinase(s) other than PKC is required for desensitization.18 Our preliminary experiments demonstrated that a protocol similar to that utilized to study H1 histamine-receptor desensitization allows quantitative assessment of the desensitization of MSMC responses to UTP.15 By employing a pro- tocol where a sub-maximally effective ( EC50) concentration of agonist is added to cells before (R1) and after (R2) a maximal, desen- sitizing agonist exposure (Rmax), attenuations of responsiveness are detected irrespective of P2Y2-receptor reserve. Moreover, we have extended this approach and utilized a similar R1/Rmax/R2 protocol in small, intact mesenteric vessels ( 150 mm diameter) to assess recep- tor desensitization/resensitization at the level of the UTP-evoked con- tractile response. Interestingly, our data indicate that UTP-stimulated