430 G.E. Morris et al. Figure 4 Depletion of endogenous GRK2 inhibits ETAR desensitization. MSMCs were co-transfected with 0.5 mg eGFP-PH and either negative-control or anti-GRK2 (10 nM) siRNAs. Cells were loaded with Fura-Red and subjected to the standard R1/R2 desensitization protocol (see Methods). (A) Representative images of changes in IP3 and Ca2þ in MSMCs expressing either control siRNA or anti-GRK2 siRNA at both the R1 and R2 stimulation points are shown. Representative traces from single cells transfected with control siRNA (B) or anti-GRK2 siRNA (C ). ETAR desensitization was determined as the relative change in R2 response compared with R1 for both eGFP-PH (black traces) and Fura-Red (broken traces). Cumulative data (D) show means + SEM from 7 – 9 cells from four or more animals. Statistical signiﬁcance is indicated as **P , 0.01; ***P , 0.001 (one-way ANOVA, unpaired t-test). calponin expression, combined with visual evidence of smooth muscle cell contractions elicited by ET-1 (and other contractile agonists) indicated the maintenance of a contractile phenotype in these cultures. In agreement with the previous reports, for example in HEK293 cells,22 the initial increase in [Ca2þ]i stimulated by ET-1 in MSMCs rapidly declined towards basal, even in the continued presence of agonist. Brief (30 s) exposure to ET-1 was sufﬁcient to cause extensive and prolonged loss of ETAR responsiveness to sub- sequent ET-1 re-challenge with respect to both IP3 and Ca2þ signals. As expected, Ca2þ signals showed more rapid recovery than IP3 signals reﬂecting the greater ampliﬁcation of the former signal in the ET-1-stimulated ETAR-PLC signalling pathway. Pre- vious studies in arterial tissue have tended to use prolonged (.60 min) ET-1 exposures leading to marked reductions in arterial contractions on ET-1 re-challenge, indicating profound ETAR desensitization23 and most probably ETAR down-regulation.24 Data from studies in recombinant cell systems suggest that GRKs are able to regulate ETAR signalling.13 Indeed, when expressed in HEK293 cells, ETAR rapidly desensitized, and