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.</jats:p