Role of phosphodiesterase isoenzymes in regulating intracellular cyclic AMP in adenosine-stimulated smooth muscle cells

Abstract

Three phosphodiesterase (PDE) isoenzymes were separated by Mono Q h.p.l.c. column chromatography from the soluble fraction of a homogenate of pig aortic smooth muscle cells. The first peak of PDE activity was stimulated by calmodulin in the presence of calcium. The second broad peak contained at least two activities, which were sensitive to inhibition by CI-930 or rolipram respectively. The distribution of total cellular enzyme activity in different subcellular fractions was also determined. The majority (78%) of the total activity was present in the cytosolic fraction, 18% of activity was in a membrane-bound form and 4% of activity was associated with the cytoskeleton. Rolipram-sensitive PDE was present predominantly in the cytosolic fraction, whereas cyclic GMP-inhibited, CI-930-sensitive PDE was evenly distributed between the cytosolic and particulate fractions. All of the calmodulin-dependent PDE activity was found in the soluble fraction. CI-930 and rolipram enhanced, by 2-fold and 3-4-fold respectively, the adenosine-stimulated rise in cellular cyclic AMP level. The increase in cyclic AMP levels due to CI-930 or rolipram was dose-dependent. Removal of adenosine once cyclic AMP had risen resulted in a rapid fall in cyclic AMP levels even in the presence of rolipram and CI-930. M&B 22,948, the calmodulin-dependent PDE inhibitor, caused less than a 25% increase of the adenosine-stimulated cyclic AMP levels by itself, but it contributed substantially to controlling the cyclic AMP levels after the removal of adenosine when used together with CI-930 and rolipram. These phenomena suggested that all three PDE isoenzymes participated in modulating cellular cyclic AMP levels after adenosine stimulation, and that differential importance of the individual isoenzymes depends on cellular cyclic AMP levels.

Inhibition of thrombosis by a novel platelet selective S-nitrosothiol compound without hemodynamic side effects

“…Samples were then mixed for 10 min, sonicated for 10 s and centrifuged at 1600 Â g for 15 min at 4 jC. Platelet subfraccionation was performed as described [31]. Total protein concentration in platelet extracts was measured using the Pierce BCA (bicinchoninic acid) protein assay method (Pierce, Rockford, IL, USA) [32].…”

Cyclic Nucleotide Phosphodiesterases and Vascular Smooth Muscle

“…Takahashi et a1 (76) showed that cilostaml produced concentration-dependent attenuation of proliferation assessed by [3H]-thymidine incorporation into DNA of rat aortic SMCs stimulated with fetal calf serum, PDGF, insulin, or IGF-I. In addition, CI-930 was found to enhance the adenosineinduced elevation of CAMP in pig aortic SMCs (73). Souness et al (72) found that the PDE3 inhibitor SK&F 94836 caused a weak concentration-dependent attenuation of [3H]-thymidine incorporation into the DNA of pig aortic SMCs and that it also enhanced the antiproliferative and CAMP-elevating effects of forskolin.…”

“…Shirotani et al (77) showed that the PDE4 inhibitor Ro 20-1724 alone produced little inhibition of DNA synthesis in bovine aortic SMCs but that it enhanced the attenuation of DNA synthesis and CAMP elevations induced by the adenylyl cyclase stimulator U-61,43 lF, a stable prostacyclin analogue. In cultured porcine aortic SMCs, rolipram and Ro 20-1724 have been shown to enhance the antiproliferative and CAMP-elevating effects of forskolin (72), and rolipram has been shown to enhance the CAMP-elevating effects of adenosine (73). In cultured porcine aortic SMCs, rolipram and Ro 20-1724 have been shown to enhance the antiproliferative and CAMP-elevating effects of forskolin (72), and rolipram has been shown to enhance the CAMP-elevating effects of adenosine (73).…”

Effects of a Novel Platelet Nitric Oxide Donor (LA816), Aspirin, Clopidogrel, and Combined Therapy in Inhibiting Flow- and Lesion-Dependent Thrombosis in the Porcine Ex Vivo Model

“…Platelet subfractionation was performed as described. 23 The protein concentration was measured using the bicinchoninic acid (BCA) method (Pierce Biotechnology). Equal amounts of protein (25 g) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes.…”