Figure 5

β-AR stimulation activates the MAPK pathway after treatment with selective PKA inhibitors. After a 30 min preincubation with 100 nM of the PKA inhibitors H-89 (panel A) or 10 μM of Rp-cAMPS (panel B), UROtsa cells were stimulated with 100 nM isoproterenol for the indicated times and immunoblotted with anti-pERK or anti-ERK2. Peak levels of pERK were observed within 5 min after the addition of isoproterenol with no changes in the total cell lysate levels of ERK2. Semi-quantitave analysis of the immunoblots revealed a time-dependent increase in ERK phosphorylation (panel C). Five minutes after treatment, isoproterenol significantly induced a 1.9 ± 0.4 and 2.6 ± 0.7 fold increase in pERK levels over basal after H-89 or Rp-cAMPS pretreatment, respectively. These peak values are not significantly different than the fold increase over basal for pERK measured in the absence of PKA inhibitor (2.6 ± 0.9). Values are presented as the mean ± S.E. and the autoradiographs are representative immunoblots of n = 3–4 independent UROtsa cell treatments.