CYCLIC AMP-MEDIATED REGULATION OF PDE3 AND PDE4 EXPRESSION IN VASCULAR SMOOTH MUSCLE: RELATIONSHIP TO cGMP MODULATION OF cAMP BREAKDOWN

Abstract

We have described importpnt role for cGMP inhibition of a cAMP phosphodic?tcraw (PDE3) in mediating the relaxant effects of cAMP in rat aoxta. More recently, we have utilized cultured rat aortic vascular smooth muscle cells (VSMC) to further delineate the roles of PDE3 and a non cGMP-inhibited cAMP phosphodiesta;lse (PDE4) in regulating CAMP-mediated events in the vssculature. Thus, incubation of prelabelled ([rnhypoxanthinc, Anal. Biochem. 215:llO-117, 1993) VSMC for 1 min with citha isoprcarlinc (1 nM), nitroprusside (1 uM), cilostamide (1 uM) or Ro 20-1724 (10 uM) increased basal cAMP by 99 -+ 5196, 98 f 5846, 25 f 14% or 56 & 1496, respectively (Mean L S.D., n 2 3). Incubation of VSMC with both nitroprusside and cilostamide did not nsult in a greata increase in CAMP than was achieved using either agent alone, suggesting that cilostamide and nitroprusside operate via a similar mechanism (i.e. inhibition of PDE3). In contrast, concurrent ddition of cilostamide and Ro 20-1724 did result in a sup-additive inc-in cAMP levels. induced by this submaximal concentration of isoprenaline (1 nM) was further increased by the simultaneous addition of either nitroplusside (1 11 -+ 15%), cilostamide (141 & 69%) or Ro 20-1724 (139 L 26%). To assess the relative importance of PDE3 and PDE4 in the regulated hydrolysis of CAMP in VSMC, we have ddermined the specific activities of these two types of p h o s p h o d i e s w in rat aorta and in cultured VSMC.Although the specific activity in rat aorta was 3-fold higher than in cultured VSMC (166 pmollmio/mg of protein vs 47 pmol/min/mg of protein at 1 uM CAMP as subshate), the ratio of PDE3 to PDE4 activities in both prcp.ntions was similar. These two activities accounted for approximately 80% of the total cAMP phosphodiestcmse activity, as measured by cilostamide and Ro 20-1724 inhibition and accounted for approximately 39% (PDE3) and 65% (PDE4) of the total activity. To study CAMP-mediated regulation of expression of these two CAMP phosphodiestcrases, VSMC wen incubated for 4, 8 or 16 h with 10 uM forskolm. A tim6-dependent increase in both PDE3 and PDE4 activities was observed following forskolin treatment and the ratio of the PDE3 to PDE4 activities in these cells was maintained.Also, the inc-in cAMP