Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1987 Jan 15;241(2):535–541. doi: 10.1042/bj2410535

The identification of a new cyclic nucleotide phosphodiesterase activity in human and guinea-pig cardiac ventricle. Implications for the mechanism of action of selective phosphodiesterase inhibitors.

M L Reeves, B K Leigh, P J England
PMCID: PMC1147593  PMID: 3036066

Abstract

Four cyclic nucleotide phosphodiesterase (PDE) activities were separated from low-speed supernatants of homogenates of human cardiac ventricle by DEAE-Sepharose chromatography, and designated PDE I-PDE IV in order of elution with an increasing salt gradient. PDE I was a Ca2+/calmodulin-stimulated activity, and PDE II was an activity with a high Km for cyclic AMP which was stimulated by low concentrations of cyclic GMP. Human ventricle PDE III had Km values of 0.14 microM (cyclic AMP) and 4 microM (cyclic GMP), and showed simple Michaelis-Menten kinetics with both substrates. PDE IV is a previously unrecognized activity in cardiac muscle, the human enzyme having Km values of 2 microM (cyclic AMP) and 50 microM (cyclic GMP). PDE III and PDE IV were not activated by cyclic nucleotides or calmodulin. Four PDE activities were also isolated from guinea-pig ventricle, and had very similar kinetic properties. By gel filtration, the Mr of PDE III was 60,000, and that of PDE IV 45,000. The drug SK&F 94120 selectively and competitively inhibited PDE III with a Ki value of 0.8 microM (human), showing simple hyperbolic inhibition kinetics. Rolipram (Schering ZK 62711) and Ro 20-1724 (Roche), which have previously been reported to inhibit PDE III-like activities strongly, were shown to be weak inhibitors of human and guinea-pig PDE III enzymes (Ki values greater than 25 microM), but potent inhibitors of PDE IV [Ki values 2.4 microM (Rolipram) and 3.1 microM (Ro 20-1724) with human PDE IV]. The inhibition in all cases demonstrated simple hyperbolic competition. These observations suggest that the previously reported complex inhibition of PDE III-type activities from cardiac muscle was caused by incomplete separation of the PDE III from other enzymes, particularly PDE IV.

Full text

PDF
535

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. BUTCHER R. W., SUTHERLAND E. W. Adenosine 3',5'-phosphate in biological materials. I. Purification and properties of cyclic 3',5'-nucleotide phosphodiesterase and use of this enzyme to characterize adenosine 3',5'-phosphate in human urine. J Biol Chem. 1962 Apr;237:1244–1250. [PubMed] [Google Scholar]
  2. Beavo J. A., Hansen R. S., Harrison S. A., Hurwitz R. L., Martins T. J., Mumby M. C. Identification and properties of cyclic nucleotide phosphodiesterases. Mol Cell Endocrinol. 1982 Nov-Dec;28(3):387–410. doi: 10.1016/0303-7207(82)90135-6. [DOI] [PubMed] [Google Scholar]
  3. Davis C. W., Daly J. W. A simple direct assay of 3',5'-cyclic nucleotide phosphodiesterase activity based on the use of polyacrylamide-bononate affinity gel chromatography. J Cyclic Nucleotide Res. 1979;5(1):65–74. [PubMed] [Google Scholar]
  4. Elks M. L., Manganiello V. C. Selective effects of phosphodiesterase inhibitors on different phosphodiesterases, adenosine 3',5'-monophosphate metabolism, and lipolysis in 3T3-L1 adipocytes. Endocrinology. 1984 Oct;115(4):1262–1268. doi: 10.1210/endo-115-4-1262. [DOI] [PubMed] [Google Scholar]
  5. England P. J. Studies on the phosphorylation of the inhibitory subunit of troponin during modification of contraction in perfused rat heart. Biochem J. 1976 Nov 15;160(2):295–304. doi: 10.1042/bj1600295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Grant P. G., Colman R. W. Purification and characterization of a human platelet cyclic nucleotide phosphodiesterase. Biochemistry. 1984 Apr 10;23(8):1801–1807. doi: 10.1021/bi00303a034. [DOI] [PubMed] [Google Scholar]
  7. Hidaka H., Endo T. Selective inhibitors of three forms of cyclic nucleotide phosphodiesterase--basic and potential clinical applications. Adv Cyclic Nucleotide Protein Phosphorylation Res. 1984;16:245–259. [PubMed] [Google Scholar]
  8. Hidaka H., Yamaki T., Ochiai Y., Asano T., Yamabe H. Cyclic 3':5'-nucleotide phosphodiesterase determined in various human tissues by DEAE-cellulose chromatography. Biochim Biophys Acta. 1977 Oct 13;484(2):398–407. doi: 10.1016/0005-2744(77)90095-x. [DOI] [PubMed] [Google Scholar]
  9. Ho H. C., Wirch E., Stevens F. C., Wang J. H. Purification of a Ca2+-activatable cyclic nucleotide phosphodiesterase from bovine heart by specific interaction with its Ca2+-dependent modulator protein. J Biol Chem. 1977 Jan 10;252(1):43–50. [PubMed] [Google Scholar]
  10. Kariya T., Wille L. J., Dage R. C. Biochemical studies on the mechanism of cardiotonic activity of MDL 17,043. J Cardiovasc Pharmacol. 1982 May-Jun;4(3):509–514. doi: 10.1097/00005344-198205000-00024. [DOI] [PubMed] [Google Scholar]
  11. Kincaid R. L., Manganiello V. C., Odya C. E., Osborne J. C., Jr, Stith-Coleman I. E., Danello M. A., Vaughan M. Purification and properties of calmodulin-stimulated phosphodiesterase from mammalian brain. J Biol Chem. 1984 Apr 25;259(8):5158–5166. [PubMed] [Google Scholar]
  12. Krebs E. G., Beavo J. A. Phosphorylation-dephosphorylation of enzymes. Annu Rev Biochem. 1979;48:923–959. doi: 10.1146/annurev.bi.48.070179.004423. [DOI] [PubMed] [Google Scholar]
  13. Kukovetz W. R., Holzmann S., Wurm A., Pöch G. Evidence for cyclic GMP-mediated relaxant effects of nitro-compounds in coronary smooth muscle. Naunyn Schmiedebergs Arch Pharmacol. 1979 Dec;310(2):129–138. doi: 10.1007/BF00500277. [DOI] [PubMed] [Google Scholar]
  14. LaPorte D. C., Toscano W. A., Jr, Storm D. R. Cross-linking of iodine-125-labeled, calcium-dependent regulatory protein to the Ca2+-sensitive phosphodiesterase purified from bovine heart. Biochemistry. 1979 Jun 26;18(13):2820–2825. doi: 10.1021/bi00580a021. [DOI] [PubMed] [Google Scholar]
  15. Martins T. J., Mumby M. C., Beavo J. A. Purification and characterization of a cyclic GMP-stimulated cyclic nucleotide phosphodiesterase from bovine tissues. J Biol Chem. 1982 Feb 25;257(4):1973–1979. [PubMed] [Google Scholar]
  16. Purvis K., Olsen A., Hansson V. Calmodulin-dependent cyclic nucleotide phosphodiesterases in the immature rat testis. J Biol Chem. 1981 Nov 25;256(22):11434–11441. [PubMed] [Google Scholar]
  17. Puurunen J., Lücke C., Schwabe U. Effect of the phosphodiesterase inhibitor 4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidone (ZK 62711) on gastric secretion and gastric mucosal cyclic AMP. Naunyn Schmiedebergs Arch Pharmacol. 1978 Aug;304(1):69–75. doi: 10.1007/BF00501379. [DOI] [PubMed] [Google Scholar]
  18. Pyne N. J., Cooper M. E., Houslay M. D. Identification and characterization of both the cytosolic and particulate forms of cyclic GMP-stimulated cyclic AMP phosphodiesterase from rat liver. Biochem J. 1986 Mar 1;234(2):325–334. doi: 10.1042/bj2340325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Russell T. R., Thompson W. J., Schneider F. W., Appleman M. M. 3':5'-cyclic adenosine monophosphate phosphodiesterase: negative cooperativity. Proc Natl Acad Sci U S A. 1972 Jul;69(7):1791–1795. doi: 10.1073/pnas.69.7.1791. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Tucker M. M., Robinson J. B., Jr, Stellwagen E. The effect of proteolysis on the calmodulin activation of cyclic nucleotide phosphodiesterase. J Biol Chem. 1981 Sep 10;256(17):9051–9058. [PubMed] [Google Scholar]
  21. Weishaar R. E., Burrows S. D., Kobylarz D. C., Quade M. M., Evans D. B. 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 Pharmacol. 1986 Mar 1;35(5):787–800. doi: 10.1016/0006-2952(86)90247-9. [DOI] [PubMed] [Google Scholar]
  22. Weishaar R. E., Cain M. H., Bristol J. A. A new generation of phosphodiesterase inhibitors: multiple molecular forms of phosphodiesterase and the potential for drug selectivity. J Med Chem. 1985 May;28(5):537–545. doi: 10.1021/jm50001a001. [DOI] [PubMed] [Google Scholar]
  23. Weishaar R. E., Quade M., Schenden J. A., Boyd D. K., Evans D. B. Studies aimed at elucidating the mechanism of action of CI-914, a new cardiotonic agent. Eur J Pharmacol. 1985 Dec 17;119(3):205–215. doi: 10.1016/0014-2999(85)90297-3. [DOI] [PubMed] [Google Scholar]
  24. Wells J. N., Hardman J. G. Cyclic nucleotide phosphodiesterases. Adv Cyclic Nucleotide Res. 1977;8:119–143. [PubMed] [Google Scholar]
  25. Yamamoto T., Lieberman F., Osborne J. C., Jr, Manganiello V. C., Vaughan M., Hidaka H. Selective inhibition of two soluble adenosine cyclic 3',5'-phosphate phosphodiesterases partially purified from calf liver. Biochemistry. 1984 Feb 14;23(4):670–675. doi: 10.1021/bi00299a013. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES