GRK2 regulates endothelin signalling 429 Figure 3 Anti-GRK2 siRNA causes endogenous GRK2 protein depletion. MSMCs were transfected with control or anti-GRK2 siRNAs (10 or 100 nM) using Amaxa nucleofection. After 48 h, cells were lysed and 40 mg of protein loaded for SDS – PAGE separation and immunoblotting. (A) Representative western blots of GRK2 and GRK6 levels are shown: untreated cells (lane 1), cells treated with 10 or 100 nM of anti-GRK2 siRNA (lanes 2 and 3, respectively), 10 or 100 nM negative-control siRNA (lanes 4 and 5, respectively). (B) Cumulative densitometric data show means + SEM from three animals. (C ) Representative images showing depletion of endogenous GRK2 in 4-day-old isolated MSMCs, 48 h after transfection with the same GRK2 siRNA validated in (A and B). On the left are eGFP-transfected cells and the right GRK2 immunoreactivity. (D) Cumulative immunocytochemical data showing that GRK2 siRNA treatment signiﬁcantly decreased GRK2, but not GRK5 or GRK6 expression in MSMCs. Data are means + SEM for 20 – 65 cells for each treatment from three separate animals. Statistical signiﬁcance is indicated as **P , 0.01 vs. untreated cells (one-way ANOVA and Dunnett’s post hoc test). GRK5 translocation to the cytoplasm (Figure 6C). We were unable to detect recruitment of GRK3 (data not shown), and due to its ‘constitutive’ plasma membrane localization GRK611 was not studied here. 3.5 Does protein kinase C have a role in ETAR desensitization ETAR activation can recruit PKC isoenzymes to the plasma mem- brane20 raising the possibility that PKCs may also be involved in ETAR regulation. To assess whether PKC plays a role in ETAR desensitization, we used two complementary approaches: (i) pre- incubation of MSMCs with staurosporine (1 mM) or dimethyl sulphoxide (vehicle control) for 10 min prior to and throughout the standard R1/R2 desensitization protocol and (ii) down- regulation of PKC isoenzymes by a 24 h pre-treatment with phorbol 12,13-dibutyrate (PDBu, 1 mM). Neither acute treatment with staurosporine (see Supplementary material online, Figure S2C, E) nor chronic phorbol ester treatment (see Supplementary material online, Figure S2E) had any effect on ET-1-induced ETAR desensitization. In addition, the magnitude of effect on ETAR desensitization caused by the siRNA GRK2 knockdown strategy was unaltered in MSMCs additionally treated with either staurosporine or chronic PDBu (see Supplementary material online, Figure S2D, E). 4. Discussion ET-1, a potent vasoconstrictor important in the regulation of vas- cular tone, has also been reported to promote vascular hypertro- phy and hyperplasia.1,3,4 In vascular smooth muscle, ETAR has been shown to be the dominant receptor subtype mediating ET-1 smooth muscle contraction.8 In agreement, we ﬁnd that ET-1 acts through the ETAR to activate Gaq/11/PLC signalling and to elevate [Ca2þ]i in our MSMC preparations2,20 (ultimately to modu- late other pathways, including a number of Kþ conductances9 and cause contraction). Despite the clear potential pathophysiological importance of ETAR, relatively little is currently known regarding the regulation of this receptor subtype in native vascular tissue. To address this, we have used simultaneous confocal imaging of IP3 (using eGFP-PH), [Ca2þ]i (using the Ca2þ-sensitive dye Fura-Red), and DAG/PKC (using eGFP-PKCs) to investigate ETAR regulation by ET-1 in smooth muscle cells within days of iso- lation from resistance arteries. MSMC culture conditions were chosen to enable us to examine ETAR regulation in cells maintain- ing as far as possible an in vivo phenotype. High levels of a-actin and