PIG AORTIC ENDOTHELIAL-CELL CYCLIC-NUCLEOTIDE PHOSPHODIESTERASES - USE OF PHOSPHODIESTERASE INHIBITORS TO EVALUATE THEIR ROLES IN REGULATING CYCLIC-NUCLEOTIDE LEVELS IN INTACT-CELLS

Two cyclic nucleotide phosphodiesterase (PDE) activities were identified in pig aortic endothelial cells, a cyclic GMP-stimulated PDE and a cyclic AMP PDE. Cyclic GMP-stimulated PDE had K(m) values of 367 μM for cyclic AMP and 24 μM for cyclic GMP, and low concentrations (1 μM) of cyclic GMP increased the affinity of the enzyme for cyclic AMP (K(m) = 13 μM) without changing the V(max). This isoenzyme was inhibited by trequinsin [IC50 (concn. giving 50% inhibition of substrate hydrolysis) = 0.6 μM for cyclic AMP hydrolysis in the presence of cyclic GMP; IC50 = 0.6 μM for cyclic GMP hydrolysis] and dipyridamole (IC50 = 5 μM for cyclic AMP hydrolysis in the presence of cyclic GMP; IC50 = 3 μM for cyclic GMP hydrolysis). Cyclic AMP PDE exhibited a K(m) of 2 μM for cyclic AMP and did not hydrolyse cyclic GMP. This activity was inhibited by trequinsin (IC50 = 0.2 μM), dipyridamole (IC50 = 6 μM) and, selectively, by rolipram (IC50 = 3 μM). Inhibitors of cyclic GMP PDE (M and B 22948) and of low K(m) (Type III) cyclic AMP PDE (SK and F 94120) only weakly inhibited the two endothelial PDEs. Incubation of intact cells with trequinsin and dipyridamole induced large increases in cyclic GMP, which were completely blocked by LY-83583. Rolipram, SK and F 94120 and M and B 22948 did not significantly influence cyclic GMP accumulation. Dipyramidamole enhanced the increase in cyclic GMP induced by sodium nitroprusside. Cyclic AMP accumulation, was stimulated by dipyridamole and trequinsin with and without forskolin. Rolipram, although without effect alone, increased cyclic AMP in the presence of forskolin, whereas M and B 22948 and SK and F 94120 had no effects on resting or forskolin-stimulated levels. These results suggest that cyclic GMP-stimulated PDE regulates cyclic GMP levels and that both endothelial PDE isoenzymes contribute to the control of cyclic AMP.