PKA‐mediated phosphorylation and activation of cAMP‐specific phosphodiestearses in pulmonary endothelial cells

We have recently presented functional expressions of the cAMP‐specific type 4 phosphodiesterase in pulmonary microvascular endothelial cells (PMVECs), and both type 4 and type 7 phosphodiesterases, PDE4 and PDE7, in pulmonary arterial endothelial cells (PAECs). While both enzymes regulate intracellular cAMP concentration ([cAMP]i), they have markedly different kinetic parameters, leading to the possibility that these enzymes have distinct subcellular distributions. To elucidate the roles of PDE4 and PDE7 in regulating cAMP signals, we monitored changes in phosphorylation status of PDE4 and PDE7 as well as vasodilator‐stimulated phosphoprotein (VASP) in response to agents that increase [cAMP]i using phospho‐site specific antibodies. Antibodies were specific for consensus PKA sites of VASP, PDE4A, 4D, and PDE7A/B. Results showed that PKA‐mediated VASP phosphorylations were increased after forskolin stimulation and indicated differently between PMVECs and PAECs in membrane and cytosol based fractions. Forskolin also stimulated cAMP‐PDE activities, which were both PDE4 and PDE7 related and blunted by PKA inhibitor H89. Interestingly, the time course of forskolin‐stimulated PDE4 activity correlated with the time course of PDE4A phosphorylation, but not with PDE4D. PDE7A/B phosphorylations were detected at high basal levels and appeared in less degree of responses to forskolin and H89 as compared with PDE4A. Although additional experiments are required to confirm antibody specificity in immunofluorescence studies, preliminary data indicate distinct spatial distributions of phosphorylated PDE4 and PDE7A/B. Therefore, our results suggest that PDE4A, 4D, and 7 regulate different cAMP signals in distinct subcellular microdomains, and that their subcellular localizations are different in PAECs and PMVECs. (R01HL094455)