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Figure 3 In Vitro Effects of B2AR Stimulation on VSMC Biology
Panel A shows the proliferative response in subconfluent vascular smooth muscle cells (VSMCs). Adenoviral beta2-adrenergic receptor–wild type (Ad B2AR-WT) (open circles) but not the Ad B2AR-Ile164 mutation (in which isoleucine is located at position 164) (open triangles) produced a proliferative response (*p < 0.05 vs. control [CTR]; analysis of variance [ANOVA], with Bonferroni post-hoc test). Isoproterenol (ISO) (10–7 mol/l) caused an increase of cell numbers over time (solid symbols). Panel B shows deoxyribonucleic acid (DNA) synthesis by [3H]-thymidine incorporation. The activation of B2AR by ISO (10–7 mol/l) caused increased DNA synthesis. The same effect was obtained in Ad B2AR-WT but not in Ad B2AR-Ile164. The maximal response was observed in ISO-stimulated Ad B2AR-WT cells (*p < 0.05 vs. no ISO; #p < 0.05 vs. control; ANOVA, with Bonferroni post-hoc test; n = 3 in duplicate). Panels C and D show the progression in cell cycle assessed by retinoblastoma protein (Rb) phosphorylation. This protein regulates cell-cycle progression through the restriction point within the G1 phase. By Western blot at 24 h of stimulation, ISO (10–7 mol/l), caused Rb phosphorylation (P-Rb). Equal amount of proteins were confirmed via blotting for actin. Densitometric analysis (bar graph) shows that B2AR stimulation caused Rb activation, and Ad B2AR-WT treatment induced Rb activation. The ISO (10–7 mol/l) response was enhanced. These amounts were measured in arbitrary densitometry units (ADU); *p < 0.05 versus control; #p < 0.05 versus the ThrIle164 polymorphism (threonine is replaced by isoleucine at position 164) ANOVA, with Bonferroni post-hoc test (n = 3 in duplicate). Panels E and F show extracellular signal-related kinase/mitogen-activated protein kinase (ERK/MAPK) activation. Western blot of activated phosphorylated ERK (pERK) after ISO treatment. Equal amount of proteins were confirmed via blotting for total ERK. Representative blots are presented in the inset. Densitometric analysis (bar graph) shows that B2AR stimulation caused ERK/MAPK activation, and that Ad B2AR-WT treatment induced ERK activation. The ISO (10–7mol/l) response was enhanced (*p < 0.05 vs. no ISO; #p < 0.05 vs. basal ANOVA, with Bonferroni post-hoc test; n = 3 in duplicate).