1 Rat cultured aortic vascular smooth muscle cells (VSMC) express both cyclic GMP-inhibited cyclic AMP phosphodiesterase (PDE3) and Ro 20-1724-inhibited cyclic AMP phosphodiesterase (PDE4) activities. By utilizing either cilostamide, a PDE3-selective inhibitor, or Ro 20-1724, a PDE4-selective inhibitor, PDE3 and PDE4 activities were shown to account for 15% and 55% of total VSMC cyclic AMP phosphodiesterase (PDE) activity. 2 Treatment of VSMC with either forskolin or 8-bromo-cyclic AMP caused signi®cant concentrationand time-dependent increases in total cellular cyclic AMP PDE activity. Using cilostamide or Ro 20-1724, we demonstrated that both PDE3 and PDE4 activities were increased following forskolin or 8-bromo-cyclic AMP treatment, with a relatively larger e ect observed on PDE3 activity. The increase in cyclic AMP PDE activity induced by forskolin or 8-bromo-cyclic AMP was inhibited by actinomycin D or cycloheximide, demonstrating that new mRNA synthesis and protein synthesis were required. An analogue of forskolin which does not activate adenylyl cyclase (1,9-dideoxyforskolin) or an analogue of cyclic GMP (8-bromo-cyclic GMP) did not a ect total cyclic AMP PDE activity. 3 Incubation of VSMC with 8-bromo-cyclic AMP for 16 h caused a marked rightward shift in the concentration-response curves for both isoprenaline-and forskolin-mediated activation of adenylyl cyclase. A role for up-regulated cyclic AMP PDE activity in this reduced potency is supported by our observation that cyclic AMP PDE inhibitors (IBMX, cilostamide or Ro 20-1724) partially normalized the e ects of isoprenaline or forskolin in treated cells to those in untreated cells. 4 We conclude that VSMC cyclic AMP PDE activity is increased following long-term elevation of cyclic AMP and that increases in PDE3 and PDE4 activities account for more than 70% of this e ect. Furthermore, we conclude that increases in cyclic AMP PDE activity contribute to the reduced potency of isoprenaline or forskolin in treated VSMC. These results have implications for long-term use of cyclic AMP PDE inhibitors as therapeutic agents.

Longitudinal smooth muscle contraction induced by trypsin involves capsaicin-sensitive neurons and subsequent activation of NK-2, which is identical to the pathway involved in acid-induced LSM contraction and oesophageal shortening. This suggests that acid-induced LSM contraction may involve mast cell-derived mediators that activate capsaicin-sensitive neurons via PAR-2.

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