FIGURE 10.
β2AR coupling to Gαi is impaired in PDE4DKO MEFs. A and B, after a 16-h pretreatment with 100 ng/ml PTX, wild type (A) and PDE4DKO (B) MEFs were stimulated with 10 μm ISO for the indicated times. Incubations were then terminated, and intracellular cAMP was determined by RIA. All data represent the means ± S.E. of three independent experiments. Statistical significance was determined with two-way ANOVA. C and D, similar amounts of detergent extracts from wild type and PDE4DKO MEFs were separated on SDS-PAGE. The expression level of Gαs and Gαi proteins was determined by Western blotting, and the signal intensities were subsequently quantified (D). E, after a 16-h pretreatment with 100 ng/ml PTX, wild type MEFs were stimulated with 10 μm ISO for 5 min. At the end of the incubation, cells were lysed and PDE activity was measured in the presence or absence of 10 μm rolipram. The change in PDE4 activity, which is defined as the PDE activity inhibited by rolipram, is reported. Data shown in D and E represent the means ± S.E. of three independent experiments. Statistical analysis was performed using Student's t test. n.s., not significant.