196 concentration (Rmax ¼ 100 mM, 5 min). Arteries were washed for 5 min between R1 and Rmax, and for 2 – 15 min between Rmax and R2 agonist challenges. Reduced R2/R1 ratios are interpreted as an index of receptor/tissue desensitization.14,15 R2 contractile responses com- pared with R1 were dramatically reduced (by 80%) following Rmax exposure when the inter-Rmax/R2 delay was 2 min (Figure 1E); extend- ing the inter-Rmax/R2 wash period revealed a time-dependent resensi- tization of UTP-induced contractions with the R1/R2 ratio returning to 1 after 12 – 15 min (Figure 1C – E). 3.2 Characterization of UTP-mediated Ca21 signalling in isolated MSMC Vascular smooth muscle cells are known to express several UTP-responsive (types 2/4/6) P2Y-receptors,1 although it has not been conclusively determined which receptors contribute to UTP- stimulated Ca2+ signals, particularly in mesenteric smooth muscle. Therefore, various P2Y2-, P2Y4- and P2Y6-selective agonists were utilized to characterize the P2Y-receptor subtypes present in our preparation. The P2Y2-selective agonist 2-thio-UTP was more potent [pEC50 (M) ¼ 6.56], but less efﬁcacious than UTP [pEC50 (M) ¼ 5.23] in causing concentration-dependent [Ca2+]i increases in MSMCs. The P2Y4-selective agonists, inosine 5′-triphosphate, and UTPgS produced almost identical responses, again showing a slightly reduced efﬁcacy relative to UTP (Supplementary material online, Figure S1B). The P2Y6-selective agonist PSB0474 failed to cause any Ca2+ signals, while the P2Y6-selective antagonist MRS2578 (10 mM) had no signiﬁcant effect on the UTP-mediated [Ca2+]i response (Supplementary material online, Figure S1C). Since our data suggested that MSMCs express a mixed P2Y2/ P2Y4-receptor population, we applied an siRNA approach speciﬁ- cally to deplete the expression of these P2Y-receptor subtypes indi- vidually. The effectiveness of siRNA constructs to suppress P2Y2- and P2Y4-receptor mRNAs was determined using real-time reverse transcription-polymerase chain reactions (RT-PCR) (see Section 2.6) in rat aortic smooth muscle cells 48 h after nucleofec- tion with siRNA constructs targeting P2Y2- or P2Y4-receptor mRNAs or a NC (non-targeting) siRNA. Data show that nucleofec- tion with NC siRNA had no effect on P2Y2 or P2Y4 signals (data not shown), while in the presence of anti-P2Y2 siRNA, mean P2Y2 cycle threshold values were suppressed (DCT ¼ 22.0, relative to NC siRNA-transfected samples), with no effect on the P2Y4 signal (Figure 2A). Conversely, anti-P2Y4 siRNA suppressed mean cycle threshold values for P2Y4 (DCT ¼ 23.4), but was without effect on P2Y2 signals. Neither P2Y-targeting siRNA altered b-actin mRNA levels signiﬁcantly (data not shown). Collectively, these data suggest that ≥75% and ≥ 90% P2Y mRNA transcripts are selectively depleted following transfection of rat vascular smooth muscle with anti-P2Y2 or anti-P2Y4 siRNAs, respectively. The contri- butions P2Y2- and P2Y4-receptors play in UTP-mediated PLC signal- ling were determined in MSMCs co-transfected with eGFP-PH and NC, anti-P2Y2 or anti-P2Y4 siRNAs. NC siRNA nucleofection had no signiﬁcant effect on the magnitude and proﬁle of UTP-stimulated responses in MSMCs (data not shown). UTP-induced eGFP-PH translocation was virtually ablated in MSMCs treated with anti-P2Y2 siRNA, whereas anti-P2Y4 siRNA treatment was without effect (Figure 2C – E). Furthermore, responses to endothelin-1 were also unaltered following P2Y2-depletion (Figure 2C). Collectively, these data strongly suggest that in cultured MSMCs, UTP-mediated G.E. Morris et al. signalling via PLC is mediated primarily and perhaps exclusively by P2Y2-receptors. 3.3 Desensitization and resensitization of UTP-signalling in isolated MSMC Time-courses of desensitization/resensitization of receptor – PLC sig- nalling in response to UTP were assessed using similar protocols as those described above for myography experiments, however shorter agonist applications at lower concentrations (R1/R2 10 mM UTP, 30 s; Rmax 100 mM UTP, 30 s) were sufﬁcient to induce signiﬁcant desensitization (Figure 3). Comparison of UTP-induced changes in IP3 (eGFP-PH translocation) and [Ca2+]i (Fluo4), before (R1) and after (R2) the desensitizing UTP (Rmax) challenge resulted in a 64% decrease for [Ca2+]i and 84% decrease for IP3 in R2 vs. R1 measured 2 and 3 min after Rmax challenge, respectively (Figure 3A, C, and E). Prolonging the inter-Rmax/R2 washout period revealed a time-dependent resensitization of UTP-stimulated IP3 and [Ca2+]i responses (Figure 3B, D, and E), with essentially complete recovery of UTP-stimulated IP3 and [Ca2+]i responses after 15 and 10 min, respectively (Figure 3E). 3.4 GRK isoenzymic dependency of P2Y-receptor desensitization in MSMCs To determine whether endogenous GRKs regulate UTP signalling in MSMCs, individual GRK isoenzymes were inhibited using catalytically inactive, dominant-negative mutant GRKs. MSMCs were co-transfected with eGFP-PH (0.5 mg) together with pcDNA3 (control), D110A,K220R GRK2, D110A,K220RGRK3, K215RGRK5, or K215RGRK6 (0.5 mg).14 When subjected to the previously described desensitization protocol (with 5 min washes between R1/Rmax/R2 agonist additions), MSMCs transfected with pcDNA3, D110A,K220RGRK3, K215RGRK5, or K215RGRK6 displayed similar reductions in R2 responses ( 70%) compared with R1, compar- able to responses observed in non-transfected cells (Figure 4). However, the presence of D110A,K220RGRK2 signiﬁcantly attenuated reductions in the R2 response when compared with R1 (Figure 4B and F), suggesting that endogenous GRK2 activity plays a signiﬁcant role in regulating UTP-stimulated P2Y-receptor-mediated PLC/Ca2+ signalling. To corroborate these ﬁndings, we applied a previously validated siRNA that speciﬁcally depletes endogenous GRK2 (by ≥75%) without altering the expression of non-targeted GRKs.14 MSMCs were co-transfected with eGFP-PH (0.5 mg) and either 10 nM anti-GRK2 or NC siRNAs and 48 h later subjected to the standard R1/Rmax/R2 desensitization protocol (Figure 5A and B). In agreement with our data from D110A,K220RGRK2 co-transfections, siRNA-induced depletion of GRK2 attenuated the decrease in R2 (relative to R ) 1 caused by Rmax UTP-addition (Figure 5B and C). This was most striking for the eGFP-PH/IP3 response (R2/R1 (%), negative control siRNA, 32 + 8; anti-GRK2 siRNA, 71 + 7; P , 0.05; data are means + SEM). Taken together, these ﬁndings strongly suggest that GRK2 is a key mediator of UTP-induced P2Y2 receptor desensitization. 3.5 Arrestin dependency of P2Y2-receptor desensitization in MSMCs To examine the potential role that arrestin proteins play in regulat- ing P2Y2-receptor signalling, we utilized an siRNA approach to selec- tively deplete endogenous arrestin2/3 expression. MSMCs were transfected with anti-arrestin2, anti-arrestin3, or NC siRNAs (100 nM) 48 h prior to cell lysis and immunoblotting. Substantial