426 2.5 Single cell confocal imaging MSMCs were transfected (24 – 48 h after isolation) with the pleckstrin- homology domain of PLCd1 tagged with enhanced green ﬂuorescent protein (eGFP-PH, 0.5 mg), a previously extensively characterized IP3 biosensor.18 Cells were maintained at 378C by a Peltier unit and con- tinually perfused with a modiﬁed Krebs-Henseleit buffer (in mM: NaCl 134, KCl 6, MgCl2 1, glucose 10, HEPES 10, CaCl2 1.3, pH 7.4). Real- time images were taken using an Olympus FV500 laser scanning con- focal IX70 inverted microscope (oil immersion objective 60). Cells transfected with eGFP-PH and loaded with Fura-Red were excited (Ex) at 488 nm and eGFP-PH and Fura-Red emissions (Em) were col- lected at 505 – 560 and .660 nm, respectively. ET-1 (50 nM) was applied via the perfusion line as indicated. Changes in cytosolic eGFP-PH ﬂuorescence are represented as the ﬂuorescence emission (F )/initial basal ﬂuorescence (F0) (F/F0) and increases in intracellular Ca2þ ([Ca2þ]i) are reported by Fura-Red as decreases in F/F0 ﬂuor- escence. Additional Ca2þ experiments in the absence of eGFP-PH were conducted using Fluo-4-AM (3 mM, 60 min) and imaged as for eGFP above. To assess GRK2 effects on ET-1-stimulated DAG/PKC signalling, MSMCs were transfected with eGFP-PKCa20 or eGFP-PKC120 (0.5 mg), and anti-GRK2 siRNA as above. After 48 h, cells were loaded with Fura-Red (3 mM) for 50 min before commencing imaging experiments. 2.6 Data and statistical analysis Data presented are from cells obtained from at least three separate preparations and are expressed as means + SEM. Data were analysed using one-way ANOVA as indicated, with appropriate post hoc testing (GraphPad Prism, San Diego, CA, USA). 3. Results 3.1 ETAR desensitization and re-sensitization ET-1 activation of PLC signalling was assessed in MSMCs trans- fected with the eGFP-PH biosensor and loaded with the Ca2þ- sensitive dye Fura-Red to allow simultaneous measurement of changes in IP3 and [Ca2þ]i.18 Continual ET-1 (50 nM) challenge produced transient [Ca2þ]i increases, which rapidly returned to basal within 100 s (Figure 1A). Short applications (30 s) of ET-1 stimulated transient concentration-dependent translocations of eGFP-PH from the plasma membrane to the cytoplasm and increases in [Ca2þ]i, with maximal effects at concentrations of ET-1 of 50 nM (data not shown). The ETAR antagonist BQ123 (1 mM) blocked ET-1-stimulated signals, suggesting that in our MSMC preparations ETARs are primarily responsible for PLC sig- nalling (data not shown). To assess the time courses of ETAR desensitization and re-sensitization MSMCs were challenged with a short desensitizing pulse of ET-1 (50 nM, for 30 s, termed R1) followed by a wash period (of 10, 20, 30, or 60 min) and a second ET-1 challenge (50 nM, 30 s, termed R2). Initial ET-1 chal- lenge (R1) increased eGFP-PH translocation and [Ca2þ]i with responses returning to basal within 5 min of agonist washout (Figure 1B). A second ET-1 challenge applied 10 min after R1 caused less translocation of eGFP-PH and a reduced [Ca2þ]i response (Figure 1B). The reduction in the R2/R1 ratio can be inter- preted as an indicator of ETAR desensitization.14,19 Increasing the G.E. Morris et al. washout period provided evidence of a slow and incomplete re-sensitization, with R2 for the IP3 response still 70% less than R1 after 60 min (Figure 1C, D). 3.2 GRK expression in MSMCs Immnuoblotting data showed that GRKs 2, 5, 6 are expressed in rat MSMCs, however, in agreement with the previous ﬁndings16 we were unable to detect GRK3 expression (see Supplementary material online, Figure S1). Immunocytochemical evidence high- lights an anticipated cytoplasmic distribution of GRK2 (Figure 6A), whereas GRK5 expression appears to be primarily nuclear (Figure 6B and C ). 3.3 Effects of inhibiting endogenous GRK activities on ETAR signalling To determine which endogenous GRKs modulate ETAR signalling, we inhibited individual GRK isoenzymes by over-expressing cataly- tically inactive, dominant-negative GRK mutants. This approach has been successfully used in a number of studies to bring about highly selective GRK isoenzymic inhibition.14,15 MSMCs were co-transfected with eGFP-PH (0.5 mg) and pcDNA3 (control), D110A,K220RGRK2,19 D110A,K220RGRK3, K215RGRK5 or K215RGRK6 (0.5 mg).21 MSMCs transfected with dominant-negative GRKs were subjected to the standard desensitization protocol (R1/R2) with 10 min washout between ET-1 additions. Control exper- iments, co-transfecting monomeric red ﬂuorescent protein and eGFP-tagged dominant-negative GRKs indicated . 90% co-transfection of cells (data not shown). In MSMCs transfected with pcDNA3 (Figure 2A, B, G), D110A,K220RGRK3 (Figure 2D, G), K215RGRK5 (Figure 2E, G), or K215RGRK6 (Figure 2F, G), the reduction in R2 compared with R1 was consistently 80% for eGFP-PH and 60% for [Ca2þ]i signals, indicating similar reductions in ETAR responsiveness to those observed in untrans- fected cells. However, in the presence of D110A,K220RGRK2, the R2 IP3 response was decreased by 40% relative to R1, whereas the reduction in R2 for the ET-1-stimulated Ca2þ signal was almost completely ablated (Figure 2A, C, G). These data indicate that inhibition of endogenous GRK2 activity results in a highly sig- niﬁcant attenuation of ET-1-induced ETAR desensitization. To conﬁrm and extend our ﬁndings, MSMCs were transfected with siRNAs designed to target GRK2. Optimal depletion of endogenous GRK2 was achieved 48 h after siRNA transfection at concentrations of siRNA of 10 nM (Figure 3A, B). This effect was shown to be GRK2-speciﬁc as the anti-GRK2-siRNA did not affect GRK6 expression (Figure 3A, B). A negative-control siRNA had no effect on either GRK2 or GRK6 expression (Figure 3B). In addition, we co-transfected MSMCs with eGFP (0.5 mg) and either negative-control or anti-GRK2 siRNAs (10 or 100 nM) and 48 h later GRK2 expression was determined immunocyto- chemically. Our previous work indicates that co-transfection rates are .90%; therefore, we have assumed that all eGFP-expressing cells were also transfected with siRNA con- structs. When compared with non-eGFP-expressing cells, the presence of eGFP and negative-control siRNA did not affect GRK2 expression (data not shown), however, transfection with anti-GRK2 siRNA caused a marked reduction ( 75%) in GRK2