INVOLVEMENT OF CYCLIC NUCLEOTIDE-DEPENDENT PROTEIN-KINASES IN CYCLIC AMP-MEDIATED VASORELAXATION

1 The involvement of cyclic AMP-dependent protein kinase (PKA) and cyc lic CMP-dependent protein kinase (PKG) in the effects of cyclic AMP-el evating agents on vascular smooth muscle relaxation, cyclic nucleotide dependent-protein kinase activities and ATP-induced calcium signallin g ([Ca2+](i)) was studied in rat aorta. Cyclic AMP-elevating agents us ed were a beta-adrenoceptor agonist (isoprenaline), a phosphodiesteras e 3 (PDE3) inhibitor (SK&F 94120) and a PDE4 inhibitor (rolipram). 2 I n rat intact aorta, the relaxant effect induced by isoprenaline (0.01- 0.3 mu M) was decreased by a specific inhibitor of PKA, H-89, whereas a specific inhibitor of PKG, Rp-8-Br-cyclic GMPS, was without effect. No significant difference in PKA and PKG activity ratios was detected in aortic rings when isoprenaline 10 mu M was used. At the same concen tration, isoprenaline did not modify ATP-induced changes in [Ca2+](i) in smooth muscle cells. Neither H-89 nor Rp-8-Br-cyclic GMPS modified this response. These findings suggest that PKA is only involved in the relaxant effect induced by low concentrations of isoprenaline (0.01-0 .3 mu M), whereas for higher concentrations, other mechanisms independ ent of PKA and PKG are involved. 3 The relaxant effects induced by SK& F 94120 and rolipram were inhibited by Rp-8-Br-cyclic GMPS with no sig nificant effect of H-89. Neither SK&F 94120, nor rolipram at 30 mu M s ignificantly modified the activity ratios of PKA and PKG. Rolipram inh ibited the ATP-induced transient increase in [Ca2+](i). This decrease was abolished by Rp-8-Br-cyclic GMPS whereas H-89 had no significant e ffect. These results suggest that PKG is involved in the vascular effe cts induced by the inhibitors of PDE3 and PDE4. Moreover, since it was previously shown that PDE3 and PDE4 inhibitors only increased cyclic AMP levels with no change in cyclic GMP level, these data also suggest a cross-activation of PKG by cyclic AMP in rat aorta. 4 The combinati on of 5 mu M SK&F 94120 with rolipram markedly potentiated the relaxan t effect of rolipram. This relaxation was decreased by H-89 and not si gnificantly modified by Rp-8-Br-cyclic GMPS. Moreover, the association of the two PDE inhibitors significantly increased the activity ratio of PKA without changing the PKG ratio. The present findings show that PICA rather than PKG is involved in this type of vasorelaxation. The d ifferences in the participation of PKA vs PKG observed when inhibitors of PDE3 and PDE4 were used alone or together could be due to differen ces in the degree of accumulation of cyclic AMP, resulting in the acti vation of PKA or PKG which are differently localized in the cell. 5 Th ese findings support a role for both PKA and PKG in cyclic AMP-mediate d relaxation in rat aorta. Their involvement depends on the cellular p athway used to increase the cyclic AMP level.