Figure 5.

β₂AR stimulates the GRK5-dependent phosphorylation and inhibition of the cardiac MR. (A,B) Immunoblotting for phosphoserine content of the MR in response to 15-min-long treatments with 100 nM Aldo, 10 µM isoproterenol (Iso), 1 µM dobutamine (Dob), or 10 µM salbutamol (Salb) in native ARVM lysates. Representative blots are shown in (A), and the relative densitometric quantitation of five independent experiments done in duplicate is shown in (B). * p < 0.05, vs. any other treatment; n = 5. (C,D) Immunoblotting for phosphoserine content of the MR in response to 10 µM salbutamol (Salb) for 15 min or vehicle (DMSO) in ARVMs transfected with a GRK5 kinase-dead dominant negative mutant (GRK5 KD) lentivirus or control, mock (empty vector, EV) lentivirus. Representative blots are shown in (C), and the relative densitometric quantitation of six independent experiments done in duplicate is shown in (D). * p < 0.05, vs. EV/−Salb; ^#, p < 0.05, vs. EV/+Salb; n = 6. IP: immunoprecipitation; IB: Immunoblotting. (E) mRNA levels of plasminogen activator inhibitor (PAI)-1 in response to a 2-hr-long treatment of 100 nM Aldo alone or in the presence of 10 µM salbutamol (Salb + Aldo) in ARVMs. Lenti-GRK5 KD: Cells transfected with a GRK5 kinase-dead mutant lentivirus. 18S rRNA levels were used for normalization of the results. NS: Not significant at p = 0.05; one-way ANOVA with Bonferroni test; n = 6 independent experiments done in duplicate samples/condition.