Subcellular Localization of Adenosine Diphosphatase in Cultured Pig Arterial Endothelial Cells

Summary

A role for vessel wall adenosine diphosphatase (ADPase) in limiting platelet aggregation has been suggested by a number of studies. The purpose of this study was to determine the properties and subcellular localization of ADPase in endothelial cells.

Homogenates were prepared from subcultured pig aortic endothelial cells and used to determine the kinetics of ADPase activity with a recently described rapid assay employing [²-³²P]ADP. The activity was linear with incubation time and homogenate concentration. The pH optimum was 7.4 and the apparent Km was 43 ¼M. The enzyme displayed an absolute divalent cation requirement and its activity was unaffected by levamisole, a specific alkaline phosphatase inhibitor, or by 2-glycerophosphate, a substrate for non-specific phosphatases.

Post-nuclear supernatants were prepared from cell homogenates and were fractionated by sucrose density centrifugation. The distribution of ADPase activity in the gradient corresponded to 5'nucleotidase, a plasma membrane marker enzyme, and was clearly distinct from the other organelle marker enzymes. When the cells were treated with the cholesterol-binding agent, digitonin, before being homogenized, the activities of both 5'nucleotidase and ADPase shifted to a denser zone in the gradient, confirming their coincident localization to the plasma membrane.

Intact and sonicated cells were incubated with the poorly permeant enzyme inhibitor, diazotized sulphanilic add. With intact cells, ADPase and 5'nucleotidase were inhibited whereas lactate dehydrogenase, an intracellular enzyme, was unaffected; with sonicated cells all three enzymes were inactivated. ADPase is thus an ecto-enzyme on the plasma membrane of cultured pig aortic endothelial cells and is therefore ideally located for a role in limiting platelet aggregation.

Key words

Adenosine diphosphate - Adenosine diphosphatase - Endothelial cells - Platelet aggregation - Subcellular fractionation

• References

• 1 Heyns Adu P, Van den Berg DJ, Potgieter GM, Retief FP. The inhibition of platelet aggregation by an aorta intima extract. Thrombos Diathes Haemorrh 1974; 32: 417-431
• 2 Lieberman GE, Lewis GP, Peters TJ. A membrane-bound enzyme in rabbit aorta capable of inhibiting adenosine-diphosphate-induced platelet aggregation. Lancet 1977; 2: 330-332
• 3 Cooper DR, Lewis GP, Lieberman GE, Webb H, Westwick J. ADP metabolism in vascular tissue, a possible thrombo-regulatory mechanism. Thromb Res 1979; 14: 901-914
• 4 Marchesi VT, Barrnett RJ. The demonstration of enzymatic activity in pinocytic vesicles of blood capillaries with the electron microscope. J Cell Biol 1963; 17: 547-556
• 5 Crutchley DJ, Eling TE, Anderson MW. ADPase activity of isolated perfused rat lung. Life Sci 1978; 22: 1413-1420
• 6 Odegaard AE, Skalhegg BA, Hellem AJ. Investigation on adenosine diphosphate (ADP) induced platelet adhesiveness in vitro. Part III. The inactivation of ADP in plasma. Thrombos Diathes Haemorrh 1964; 11: 317-326
• 7 Haslam RJ, Mills DCB. The adenylate kinase of human plasma, erythrocytes and platelets in relation to the degradation of adenosine diphosphate in plasma. Biochem J 1967; 103: 773-784
• 8 Holmsen I, Holmsen H. Partial purification and characterization of an ADP phosphohydrolase from human plasma. Thrombos Diathes Haemorrh 1971; 26: 177-191
• 9 Richardson DJ, Smith GP, Meade TW, Langley P, Peters TJ. Assay, kinetics and properties of plasma adenosine diphosphatase. The relationship to add and alkaline phosphatase and variations in disease. Clin Chim Acta 1982; 121: 87-94
• 10 Smith GP, Peters TJ. Subcellular localization and properties of adenosine diphosphatase activity in human polymorphonuclear leukocytes. Biochim Biophys Acta 1981; 673: 234-242
• 11 Smith GP, Shah T, Webster ADB, Peters TJ. Studies on the kinetic properties and subcellular localization of adenosine diphosphatase activity in human peripheral blood lymphocytes. Clin Exp Immunol 1981; 46: 321-326
• 12 Dosne AM, Legrand C, Bauvois B, Bodevin E, Caen JP. Comparative degradation of adenylnucleotides by cultured endothelial cells and fibroblasts. Biochem Biophys Res Commun 1978; 85: 183-189
• 13 Glasgow JG, Schade R, Pitlick FA. Evidence that ADP hydrolysis by human cells is related to thrombogenic potential. Thromb Res 1978; 13: 255-266
• 14 Pearson JD, Carleton JS, Gordon JL. Metabolism of adenine nucleotides by ectoenzymes of vascular endothelial and smooth-muscle cells in culture. Biochem J 1980; 190: 421-429
• 15 Wilson PD, Lieberman GE, Peters TJ. Electron microscope localisation of ADPase in aortic endothelial cells. Histochem J. 1982 (in press)
• 16 Born GVR. Aggregation of blood platelets by adenosine diphosphate and its reversal. Nature 1962; 194: 927-929
• 17 Aursnes I, Gjesdal K, Abildgaard U. Platelet aggregation induced by ADP from unsheared erythrocytes at physiological Ca²⁺-concentration. Br J Haematol 1981; 47: 149-152
• 18 DePierre JW, Kamovsky ML. Plasma membranes of mammalian cells. A review of methods for their characterization and isolation. J Cell Biol 1973; 56: 275-303
• 19 DePierre JW, Kamovsky ML. Ecto-enzymes of the guinea pig polymorphonuclear leucocyte. I. Evidence for an ecto-adenosine monophosphatase, adenosine triphosphatase and p nitrophenyl phosphatase. J Biol Chem 1974; 249: 7111-7120
• 20 Lieberman GE, Lewis GP. Properties of vascular ADP-ase. Artery 1980; 8: 374-380
• 21 Lieberman GE, Leake DS, Peters TJ. Subcellular localisation of ADP-ase in cultured, porcine, endothelial cells. Thromb Haemostas 1981; 46: 36 (Abstr.)
• 22 Leake DS, Bowyer DE. Quantitative studies of pinocytosis by arterial endothelial and smooth muscle cells in culture. Exp Mol Pathol 1981; 35: 84-97
• 23 Leake DS, Peters TJ. Proteolytic degradation of low density lipoproteins by arterial smooth muscle cells. The role of individual cathepsins. Biochim Biophys Acta 1981; 664: 108-116
• 24 Peters TJ, Müller M, de Duve C. Lysosomes of the arterial wall. I. Isolation and subcellular fractionation of cells from normal rabbit aortas. J Exp Med 1972; 136: 1117-1139
• 25 Peters TJ. Analytical subcellular fractionation of jejunal biopsy specimens: methodology and characterization of the organelles in normal tissue. Clin Sci Mol Med 1976; 51: 557-574
• 26 Seymour CA, Peters TJ. Enzyme activities in human liver biopsies: assay methods and activities of some lysosomal and membrane-bound enzymes in control tissue and serum. Clin Sci Mol Med 1977; 52: 229-239
• 27 Smith G, Smith GD, Peters TJ. A direct, rapid radioassay for adenosine diphosphatase. Clin Chim Acta 1980; 101: 287-291
• 28 Mann FG, Saunders BC. Practical Organic Chemistry. 169-170 272 Longmans, Green and Company; London: 1952
• 29 Eisenthal R, Cornish-Bowden A. The direct linear plot. A new graphical prodecure for estimating enzyme kinetic parameters. Biochem J 1974; 139: 715-720
• 30 Leake DS, Heald B, Peters TJ. A large proportion of the add phosphatase activity in arterial smooth-muscle cells is assodated with the plasma membrane. Biochem Soc Trans 1982; 10: 35-36
• 31 Smolen JE, Weissmann G. Mg²⁺-ATPase as a membrane ecto-enzyme of human granulocytes. Inhibitors, activators and response to phagocytosis. Biochim Biophys Acta 1978; 512: 525-538
• 32 Hayes LW, Goguen CA, Stevens AL, Magargal WW, Slakey LI. Enzyme activities in endothelial cells and smooth muscle cells from swine aortas. Proc Natl Acad Sci USA 1979; 76: 2532-2535