title: Ecto-enzymatic hydrolysis of diadenosine polyphosphates by cultured adrenomedullary vascular endothelial cells.
abstract: We investigated the extracellular degradation of diadenosine polyphosphates (ApnA) by cultured adrenomedullary endothelial cells using fluorogenic analogs of ApnA, the di(1,N6-ethenoadenosine) 5',5"'-P1,Pn-polyphosphates [epsilon-(ApnA)]. Kinetic parameters of epsilon-(ApnA) cleavage and effects of pH, ions, and inhibitors were determined by continuous fluorometric assays, using suspensions of endothelial cells grown on Cytodex-1 microspheres. Ecto-enzyme kinetic parameters for epsilon-(Ap3A), epsilon-(Ap4A), and epsilon-(Ap5A) hydrolysis are as follows: Michaelis-Menten constants of 0.39 +/- 0.07, 0.42 +/- 0.09, and 0.37 +/- 0.05 microM respectively, and maximal velocities of 26.1 +/- 6.8, 74.2 +/- 16.4, and 24.4 +/- 3.4 pmol.min-1.10(6) cells-1, respectively. ApnA and guanosine 5',5"'-P1,P4-tetraphosphate behave as competitor substrates of epsilon-(Ap4A) hydrolysis. The ectoenzyme is activated by Mg2+ and Mn2+ and inhibited by Ca2+, F-, adenosine 5'-tetraphosphate, adenosine 5'-O-(3-thiotriphosphate), and suramin. Optimum pH is around 9.0. High-performance liquid chromatography analysis reveals that the ecto-enzyme hydrolyzes epsilon-(ApnA) to give epsilon-adenosine-5'(n-1)-phosphate and epsilon-AMP, which are then further catabolized up to epsilon-adenosine via the membrane-bound nucleotidase system ecto-ATPase, ecto-ADPase (or apyrase), and ecto-5'-nucleotidase. The endothelial ecto-diadenosine polyphosphate hydrolase studied here exhibits different kinetic parameters and sensitivity to ions with respect to the enzyme from the tissue-related neurochromaffin cells. These different properties may be important in the extracellular signaling by ApnA.
authors.affiliation: Department of Biochemistry, Facultad de Veterinaria, Universidad Complutense, Madrid, Spain.
authors.forename: J
authors.initials: J
authors.lastname: Mateo
authors.forename: M T
authors.initials: MT
authors.lastname: Miras-Portugal
authors.forename: P
authors.initials: P
authors.lastname: Rotllán
mesh_terms: D000313:Adrenal Medulla / Q000098:blood supply*; D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D003911:Dextrans; D015226:Dinucleoside Phosphates / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D005089:Exocytosis; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008863:Microspheres; D008954:Models, Biological
chemical_list: D003911:Dextrans; D015226:Dinucleoside Phosphates; C012275:P(1),P(5)-di(adenosine-5'-)pentaphosphate; C020733:diadenosine tetraphosphate; C011440:diadenosine triphosphate; C052839:Cytodex