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. 1987 Dec 15;248(3):897–901. doi: 10.1042/bj2480897

Specific antibodies and the selective inhibitor ICI 118233 demonstrate that the hormonally stimulated 'dense-vesicle' and peripheral-plasma-membrane cyclic AMP phosphodiesterases display distinct tissue distributions in the rat.

N J Pyne 1, N Anderson 1, B E Lavan 1, G Milligan 1, H G Nimmo 1, M D Houslay 1
PMCID: PMC1148634  PMID: 2829845

Abstract

Polyclonal-antibody preparations DV1 and PM1, raised against purified preparations of rat liver insulin-stimulated 'dense-vesicle' and peripheral-plasma-membrane cyclic AMP phosphodiesterases, were used to analyse rat liver homogenates by Western-blotting techniques. The antibody DV1 identified only the 63 kDa native subunit of the 'dense-vesicle' enzyme, and the antibody PM1 only the 52 kDa subunit of the plasma-membrane enzyme. These antibodies also detected the subunits of these two enzymes in homogenates of kidney, heart and white adipose tissue from rat. Quantitative immunoblotting demonstrated that the amount of these enzymes (by wt.) varied in these different tissues, as did the expression of these two enzymes, relative to each other, by a factor of as much as 7-fold. The ratio of the dense-vesicle enzyme to the peripheral-plasma-membrane enzyme was lowest in liver and kidney and highest in heart and white adipose tissue. ICI 118233 was shown to inhibit selectively the 'dense-vesicle' cyclic AMP phosphodiesterase in liver. It did this in a competitive fashion, with a Ki value of 3.5 microM. Inhibition of tissue-homogenate cyclic AMP phosphodiesterase activity by ICI 118233 was used as an index of the contribution to activity by the 'dense-vesicle' enzyme. By this method, a tissue distribution of the 'dense-vesicle' enzyme was obtained which was similar to that found by using the immunoblotting technique. The differential expression of isoenzymes of cyclic AMP phosphodiesterase activity in various tissues might reflect a functional adaptation, and may provide the basis for the different physiological actions of compounds which act as selective inhibitors.

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Selected References

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

  1. Appleman M. M., Thompson W. J., Russell T. R. Cyclic nucleotide phosphodiesterases. Adv Cyclic Nucleotide Res. 1973;3:65–98. [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. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  4. Bristol J. A., Evans D. B. Agents for the treatment of heart failure. Med Res Rev. 1983 Jul-Sep;3(3):259–287. doi: 10.1002/med.2610030304. [DOI] [PubMed] [Google Scholar]
  5. Farah A. E., Alousi A. A., Schwarz R. P., Jr Positive inotropic agents. Annu Rev Pharmacol Toxicol. 1984;24:275–328. doi: 10.1146/annurev.pa.24.040184.001423. [DOI] [PubMed] [Google Scholar]
  6. Francis S. H., Kono T. Hormone-sensitive cAMP phosphodiesterase in liver and fat cells. Mol Cell Biochem. 1982 Feb 5;42(2):109–116. doi: 10.1007/BF00222697. [DOI] [PubMed] [Google Scholar]
  7. Harrison S. A., Reifsnyder D. H., Gallis B., Cadd G. G., Beavo J. A. Isolation and characterization of bovine cardiac muscle cGMP-inhibited phosphodiesterase: a receptor for new cardiotonic drugs. Mol Pharmacol. 1986 May;29(5):506–514. [PubMed] [Google Scholar]
  8. Heyworth C. M., Wallace A. V., Houslay M. D. Insulin and glucagon regulate the activation of two distinct membrane-bound cyclic AMP phosphodiesterases in hepatocytes. Biochem J. 1983 Jul 15;214(1):99–110. doi: 10.1042/bj2140099. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Heyworth C. M., Wallace A. V., Wilson S. R., Houslay M. D. An assessment of the ability of insulin-stimulated cyclic AMP phosphodiesterase to decrease hepatocyte intracellular cyclic AMP concentrations. Biochem J. 1984 Aug 15;222(1):183–187. doi: 10.1042/bj2220183. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Houslay M. D. Insulin, glucagon and the receptor-mediated control of cyclic AMP concentrations in liver. Twenty-second Colworth medal lecture. Biochem Soc Trans. 1986 Apr;14(2):183–193. doi: 10.1042/bst0140183. [DOI] [PubMed] [Google Scholar]
  11. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  12. Londos C., Honnor R. C., Dhillon G. S. cAMP-dependent protein kinase and lipolysis in rat adipocytes. III. Multiple modes of insulin regulation of lipolysis and regulation of insulin responses by adenylate cyclase regulators. J Biol Chem. 1985 Dec 5;260(28):15139–15145. [PubMed] [Google Scholar]
  13. Loten E. G., Francis S. H., Corbin J. D. Proteolytic solubilization and modification of hormone-sensitive cyclic nucleotide phosphodiesterase. J Biol Chem. 1980 Aug 25;255(16):7838–7844. [PubMed] [Google Scholar]
  14. Marchmont R. J., Ayad S. R., Houslay M. D. Purification and properties of the insulin-stimulated cyclic AMP phosphodiesterase from rat liver plasma membranes. Biochem J. 1981 Jun 1;195(3):645–652. doi: 10.1042/bj1950645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Marchmont R. J., Houslay M. D. A peripheral and an intrinsic enzyme constitute the cyclic AMP phosphodiesterase activity of rat liver plasma membranes. Biochem J. 1980 May 1;187(2):381–392. doi: 10.1042/bj1870381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Marchmont R. J., Houslay M. D. Characterization of the phosphorylated form of the insulin-stimulated cyclic AMP phosphodiesterase from rat liver plasma membranes. Biochem J. 1981 Jun 1;195(3):653–660. doi: 10.1042/bj1950653. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Marchmont R. J., Houslay M. D. Insulin trigger, cyclic AMP-dependent activation and phosphorylation of a plasma membrane cyclic AMP phosphodiesterase. Nature. 1980 Aug 28;286(5776):904–906. doi: 10.1038/286904a0. [DOI] [PubMed] [Google Scholar]
  18. Milligan G., Gierschik P., Spiegel A. M., Klee W. A. The GTP-binding regulatory proteins of neuroblastoma x glioma, NG108-15, and glioma, C6, cells. Immunochemical evidence of a pertussis toxin substrate that is neither Ni nor No. FEBS Lett. 1986 Jan 20;195(1-2):225–230. doi: 10.1016/0014-5793(86)80165-x. [DOI] [PubMed] [Google Scholar]
  19. Mumby M. C., Martins T. J., Chang M. L., Beavo J. A. Identification of cGMP-stimulated cyclic nucleotide phosphodiesterase in lung tissue with monoclonal antibodies. J Biol Chem. 1982 Nov 25;257(22):13283–13290. [PubMed] [Google Scholar]
  20. 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]
  21. Pyne N. J., Cooper M. E., Houslay M. D. The insulin- and glucagon-stimulated 'dense-vesicle' high-affinity cyclic AMP phosphodiesterase from rat liver. Purification, characterization and inhibitor sensitivity. Biochem J. 1987 Feb 15;242(1):33–42. doi: 10.1042/bj2420033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Saltiel A. R., Steigerwalt R. W. Purification of putative insulin-sensitive cAMP phosphodiesterase or its catalytic domain from rat adipocytes. Diabetes. 1986 Jun;35(6):698–704. doi: 10.2337/diab.35.6.698. [DOI] [PubMed] [Google Scholar]
  23. Sharma R. K., Wang T. H., Wirch E., Wang J. H. Purification and properties of bovine brain calmodulin-dependent cyclic nucleotide phosphodiesterase. J Biol Chem. 1980 Jun 25;255(12):5916–5923. [PubMed] [Google Scholar]
  24. Takemoto D. J., Hansen J., Takemoto L. J., Houslay M. D. Peptide mapping of multiple forms of cyclic nucleotide phosphodiesterase. J Biol Chem. 1982 Dec 25;257(24):14597–14599. [PubMed] [Google Scholar]
  25. Thompson W. J., Appleman M. M. Multiple cyclic nucleotide phosphodiesterase activities from rat brain. Biochemistry. 1971 Jan 19;10(2):311–316. [PubMed] [Google Scholar]
  26. 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]
  27. Wells J. N., Hardman J. G. Cyclic nucleotide phosphodiesterases. Adv Cyclic Nucleotide Res. 1977;8:119–143. [PubMed] [Google Scholar]
  28. Wilson S. R., Wallace A. V., Houslay M. D. Insulin activates the plasma-membrane and dense-vesicle cyclic AMP phosphodiesterase in hepatocytes by distinct routes. Biochem J. 1983 Oct 15;216(1):245–248. doi: 10.1042/bj2160245. [DOI] [PMC free article] [PubMed] [Google Scholar]

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