Isolation and Regulation of the cGMP-lnhibited cAMP Phosphodiesterase in Human Erythroleukemia Cells

Summary

The predominant cAMP phosphodiesterase in human platelets is the low K_m cGMP-inhibited phosphodiesterase (PDE 3A). We have isolated native PDE3A from platelets and human erythroleukemia (HEL) cells and studied its kinetics. The platelet and HEL cell enzymes hydrolyze cAMP with K_m = 0.5 μM. Incubation of cell supernatant with cAMP dependent protein kinase resulted in a rapid increase in activity within minutes, which resulted from a 2-fold decrease in K_m with no increase in V_max. HEL cells grown for 24 h in the presence of 50 (iM forskolin, an adenylate cyclase activator, demonstrate further increase in PDE3A of 274% of control (p = 0.03). Cells incubated with forskolin and cycloheximide or actinomycin D demonstrated no increase suggesting that cAMP stimulates PDE3A synthesis by transcriptional regulation. The results indicate that cAMP affects both the short and long-term regulation of PDE3A. The latter effect may play a role in the developing hematopoietic cell and the cardiovascular system to regulate cAMP levels.

• References

• 1 Hidaka H, Endo T. Human blood platelet 3’5’-cyclic nucleotide phosphodiesterase. Isolation of low-km and high-km phosphodiesterase. Biochim Biophys Acta 1976; 429: 485-497
• 2 Sheth SB, Colman RW. Regulatory and catalytic domains of platelet cAMP phosphodiesterases: targets for drug design. [Review]. Semin Heme 1996; 110-119
• 3 Grant PG, Colman RW. Purification and characterization of a human platelet cyclic nucleotide phosphodiesterase. Biochem 1984; 23: 1801-1807
• 4 Honerjager P. Pharmacology of positive inotropic phosphodiesterase III inhibitors. Eur Heart J 1989; 10 Suppl C 25-31
• 5 Weishaar RE, Kobylarz-Singer DC, Kaplan HR. Subclasses of cyclic AMP phosphodiesterase in cardiac muscle. J Mol Cell Card 1987; 19: 1025-1036
• 6 Weishaar RE, Kobylarz-Singer DC, Quade MM, Steffen RP, Kaplan HR. Multiple molecular forms of phosphodiesterase and the regulation of cardiac muscle contractility. J Cyc Nuc Prot Phos Res 1986; 11: 513-527
• 7 Weishaar RE, Burrows SD, Kobylarz DC, Quade MM, Evans DB. Multiple molecular forms of cyclic nucleotide phosphodiesterase in cardiac and smooth muscle and in platelets. Isolation, characterization, and effects of various reference phosphodiesterase inhibitors and cardiotonic agents. Biochem Pharm 1986; 35: 787-800
• 8 Suttorp N, Weber U, Welsch T, Schudt C. Role of phosphodiesterases in the regulation of endothelial permeability in vitro. J Clin Invest 1993; 91: 1421-1428
• 9 Degerman E, Moos jr M, Rascón A, Vasta V, Meacci E, Smith CJ, Andersson KE, lingersn S, Belfrage P, Manganiello V. Single-step affinity p urification partial structure and properties of human platelet cGMP inhibited cAMP phosphodiesterase. Biochim Biophys Acta Protein Struct Mol Enzymol 1994; 1205: 189-198
• 10 Charbonneau H. Structure-function relationships among cyclic nucleotide phophodiesterases. In: Cyclic Nucleotide Phosphodiesterases: Structure, Regulation and Drug Action. Beavo J, Houslay MD. (eds) New York: John Wiley and Sons; 1990. pp 267-296
• 11 Tabilio A, Rosa JP, Testa U, Kieffer N, Nurden AT, Del Canizo MC, Breton-Gorius J, Vainchender W. Expression of platelet membrane glycoproteins and α-granule proteins by a human erythroleukemia cell line (HEL). EMBOJ 1984; 03: 453-459
• 12 Ashby B, Almonor GO, Wernick E, Selak MA. Prostaglandin-concentration-dependent desensitization of adenylate cyclase in human erythroleu-kaemia (HEL)cells is abolished by pertussis toxin and enhanced by induction by dimethyl sulphoxide. Biochem J 1991; 280: 801-804
• 13 Macphee CH, Reifsnyder DH, Moore TA, Beavo JA. Intact cell and cell-free phosphorylation and concomitant activation of a low Km cAMP phosphodiesterase found in human platelets. J Cyc Nuc Prot Phos Res 1986; 11: 487-496
• 14 Grant PG, Mannarino AF, Colman RW. cAMP-mediated phosphorylation of the low-Km cAMP phosphodiesterase markedly stimulates its catalytic activity. Proc Natl Acad Sci USA 1988; 85: 9071-9075
• 15 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680-685
• 16 Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 1979; 76: 4350-4354
• 17 Torphy TJ, Zhou HL, Foley JJ, Sarau HM, Manning CD, Barnette MS. Salbutamol up-regulates PDE4 activity and induces a heterologous desensitization of U937 cells to prostaglandin E₂ . Implications for the therapeutic use of beta-adrenoceptor agonists. J Biol Chem 1996; 270: 23598-23604
• 18 Sinha AK, Shattil SJ, Colman RW. Cyclic AMP metabolism in cholesterol-rich platelets. J Biol Chem 1977; 252: 3310-3314
• 19 Torphy TJ, Cieslinski LB. Characterization and selective inhibition of cyclic nucleotide phosphodiesterase isozymes in canine tracheal smooth muscle. Mol Pharmacol 1990; 37: 206-214
• 20 Thompson WJ, Terasaki WL, Epstein PM, Strada SJ. Assay of cyclic nucleotide phosphodiesterase and resolution of multiple molecular forms of the enzyme [Review]. Adv Cyc Nuc Res 1979; 10: 69-92
• 21 Macphee CH, Reifsnyder DH, Moore TA, Lerea KM, Beavo JA. Phosphorylation results in activation of a cAMP phosphodiesterase in human platelets. J Biol Chem 1988; 263: 10353-10358
• 22 Macphee CH, Harrison SA, Beavo JA. Immunological identification of the major platelet low-Km cAMP phosphodiesterase: probable target for antithrombotic agents. Proc Natl Acad Sci USA 1986; 83: 6660-6663
• 23 Rascon A, Degerman E, Taira M, Meacci E, Smith CJ, Manganiello V, Belfrage P, Tornqvist H. Identification of the phosphorylation site in vitro for cAMP-dependent protein kinase on the rat adipocyte cGMP-inhibited cAMP phosphodiesterase. J Biol Chem 1994; 269: 11962-11966
• 24 Rahn T, Ronnstrand L, Leroy MJ, Wemsted C, Tornqvist H, Manganiello VC, Belfrage P, Degerman E. Identification of the site in the cGMP-inhibited phosphodiesterase phosphorylated in adipocytes in response to insulin and isoproterenol. J Biol Chem 1996; 271: 11575-11580
• 25 Sette C, Vicini E, Conti M. The ratPDE3/IVd phosphodiesterase gene codes for multiple proteins differentially activated by cAMP-dependent protein kinase [published erratum appears in J Biol Chem 1994 Aug 12; 269(32): 20806]. J Biol Chem 1994; 269: 18271-18274
• 26 Gettys TW, Blackmore PF, Redmon JB, Beebe SJ, Corbin JD. Short-term feedback regulation of cAMP by accelerated degradation in rat tissues. J Biol Chem 1987; 262: 333-339
• 27 Cheung P, Xu H, McLaughlin MM, Ghazeleh FA, Livi GP, Colman RW. Human platelet cGI-PDE: Expression in yeast and localization of the catalytic domain by deletion mutagenesis. Blood 1996; 88: 1321-1329
• 28 Meacci E, Taira M, Moos jr M, Smith CJ, Movsesian MA, Degerman E, Belfrage P, Manganiello V. Molecular cloning and expression of human myocardial cGMP-inhibited cAMP phosphodiesterase. Proc Natl Acad Sci USA 1992; 89: 3721-3725
• 29 Taira M, Hockman SC, Calvo JC, Belfrage P, Manganiello VC. Molecular cloning of the rat adipocyte hormone-sensitive cyclic GMP-inhibited cyclic nucleotide phosphodiesterase. J Biol Chem 1993; 268: 18573-18579
• 30 Mochly-Rosen D. Localization of protein kinases by anchoring proteins: a theme in signal transduction. [Review]. Science 1995; 268: 247-251
• 31 Richardson MD, Goka TJ, Barber R, Butcher RW. Growth of S49 wild type cells in 3 nM epinephrine increases cyclic AMP phosphodiesterase activity. Life Sciences 1994; 54: 863-875
• 32 Torphy TJ, Zhou HL, Cieslinski LB. Stimulation of beta adrenoceptors in a human monocyte cell line (U937) up-regulates cyclic AMP-specific phosphodiesterase activity. J Pharmacol Exp Ther 1992; 263: 1195-1205
• 33 Mazur EM, Rosmarin AG, Sohl PA, Newton JL, Narendran A. Analysis of the mechanism of anagrelide-induced thrombocytopenia in humans. Blood 1992; 79: 1931-1937