ECTO-ENZYMATIC HYDROLYSIS OF DIADENOSINE POLYPHOSPHATES BY CULTURED ADENOMEDULLARY VASCULAR ENDOTHELIAL-CELLS

We investigated the extracellular degradation of diadenosine polyphosp hates (Ap(n)A) by cultured adrenomedullary endothelial cells using flu orogenic analogs of Ap(n)A, the di(1,N-6-ethenoadenosine) 5',5'''-P-1, P-n-polyphosphates [epsilon-(Ap(n)A)]. Kinetic parameters of epsilon-( Ap(n)A) cleavage and effects of pH, inns, and inhibitors were determin ed by continuous fluorometric assays, using suspensions of endothelial cells grown on Cytodex-1 microspheres. Ecto-enzyme kinetic parameters far epsilon-(AP(3)A), epsilon-(AP(4)A), and epsilon-(Ap(5),A) hydroly sis are as follows: Michaelis-Menten constants of 0.39 +/- 0.07, 0.42 +/- 0.09, and 0.37 +/- 0.05 mu M respectively, and maximal velocities of 26.1 +/- 6.8, 74.2 +/- 16.4, and 24.4 +/- 3.4 pmol.min(-1).10(6) ce lls(-1), respectively. Ap(n)A and guanosine 5',5'''-P-1,P-4-tetraphosp hate behave as competitor substrates of epsilon-(AP(4)A) hydrolysis. T he 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 chromatogr aphy analysis reveals that the ecto-enzyme hydrolyzes epsilon-(Ap(n)A) to give epsilon-adenosine-5'(n-1)-phosphate and epsilon-AMP, which ar e then further catabolized up to epsilon-adenosine via the membrane-bo und nucleotidase system ecto-ATPase, ecto-ADPase (or apyrase), and ect o-5'-nucleotidase. The endothelial ecto-diadenosine polyphosphate hydr olase studied here exhibits different kinetic parameters and sensitivi ty to ions with respect to the enzyme from the tissue-related neurochr omaffin cells. These different properties may be important in the extr acellular signaling by Ap(n)A.