GRK2 and arrestin2 regulate UTP-mediated arterial contraction 197 Figure 2 Dissecting the P2Y2/P2Y4-receptor dependency of UTP signalling. (A) Real-time PCR data showing changes in cycle threshold (DCT) values for P2Y2 or P2Y4 transcripts, relative to negative-control (NC) and untransfected cells, following transfection with 100 nM of anti-P2Y2 or anti-P2Y4 receptor siRNAs. Data shown are means + SEM for n ¼ 6 experiments (in triplicate) undertaken in cell preparations from six separate animals. Repre- sentative traces (B – D) and cumulative data (E) showing affects of NC (B), anti-P2Y2 (C ), or anti-P2Y4 (D) siRNAs on UTP-stimulated (10 or 100 mM) IP3 signals. Data expressed as means + SEM for n ¼ 8 – 10 cells from cell preparations produced from four or more separate animals. Statistical sig- niﬁcance is indicated as asterisk **P , 0.01 vs. NC siRNA (one-way ANOVA, Dunnett’s post hoc test). arrestin depletion (.70% for arrestin2 and arrestin3) was observed at this time-point, and both arrestin2- and arrestin3-targeted siRNAs appeared to be highly selective for their respective targets (Figure 6A and B). To determine the effects of arrestin depletion on the desensitization of UTP-stimulated PLC signalling, MSMCs were co-transfected with eGFP-PH (0.5 mg) and anti-arrestin2 or anti-arrestin3, before being subjected to the standard R1/Rmax/R2 desensitization protocol. In the absence or presence of NC siRNA reductions in R2 relative to R1 responses of ≥80% for eGFP-PH and ≥70% for [Ca2+]i signals were observed (Figure 6C and E). Selective depletion of arrestin2 markedly reversed UTP-induced decreases in R2 relative to R1 IP3/Ca2+ responses (Figure 6E). Con- trastingly, while arrestin3 depletion had a small effect on R2/R1 ratios (Figure 6E), this did not achieve statistical signiﬁcance, suggesting that attenuation of the UTP signal is primarily effected by the arrestin2 isoform. Previously we showed that GRK2 regulates endothelin (ETA) receptor desensitization,14 suggesting that ETARs are also likely sub- strates for arrestin recruitment in MSMCs. Consequently, the poten- tial involvement of arrestin proteins in the regulation of ETA receptor signalling was assessed in MSMCs co-transfected with eGFP-PH and either anti-arrestin2 or anti-arrestin3 siRNAs. Here, ETA receptor desensitization was assessed by exposing cells to a short desensitizing pulse of endothelin-1 (50 nM, 30 s, termed R1) followed by a 10 min wash period and second endothelin-1 challenge (50 nM, 30 s, termed R2). The extent of receptor desensitization, assessed at the level of IP3