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. 2021 Nov;100(5):513–525. doi: 10.1124/molpharm.121.000363

Fig. 3.

Fig. 3.

Cmpd-6 augments cellular activity of carvedilol at the β2AR. (A) HEK293 cells stably expressing GloSensor were pretreated with either vehicle (DMSO) alone or cmpd-6 (C6) (30 µM) for 15–20 minutes. The extent of cAMP generation by endogenously expressed β2AR was subsequently measured after stimulation of the cells with either epinephrine (EPI) or carvedilol (CARV) for 5–10 minutes in a dose-dependent manner. Values were normalized to the maximal level of EPI-induced activity in the vehicle (0.3% DMSO) control, expressed as a percentage, and represent means ± S.D. (B, C) Cells were pretreated with either vehicle DMSO alone (B) or cmpd-6 (C) for 15–20 minutes, and inhibition of 1 µM EPI-stimulated cAMP generation was monitored in the presence of a dose of either carvedilol or metoprolol. Values were normalized to the uninhibited EPI signal in the DMSO control. Dose-dependent curve fits were generated with data points obtained from three or four independent experiments done in duplicate. (D) HEK293 cells stably expressing the β2AR were serum-starved overnight and subsequently pretreated with either vehicle (DMSO) alone or cmpd-6 at 5 µM for 15–20 minutes. The cells were either non-stimulated (NS) or stimulated with carvedilol for 5 minutes in a dose-dependent manner. ERK phosphorylation (p-ERK) and total ERK expression (t-ERK) in each sample were visualized by immunoblotting as described. (E) Each of the p-ERK and t-ERK bands in the immunoblot was quantified as described, and the extent of ERK phosphorylation was determined through dividing the p-ERK signal by the t-ERK. Each data point was expressed as percent of the maximal response in the vehicle-treated control cells and represents the mean ± S.D. from five independent experiments. Dose-response curves and EC50 values between vehicle (DMSO)– and cmpd-6–treated samples were obtained by using GraphPad Prism. Statistical significance for the difference in LogKi values (carvedilol versus metoprolol) between vehicle (DMSO)– and cmpd-6–treated curve fits (P < 0.001) was determined by two-way ANOVA. ***P < 0.001.