Inhibition of extracellular ATP degradation in endothelial cells.
The plasma membrane ATPase on the human umbilical vein endothelial cell line (ECV304) was demonstrated to be an ecto-enzyme. Hydrolysis of ATP was measured by monitoring the appearance of inorganic phosphorus. Hydrolysis of extracellular ATP was insensitive to oligomycin, vanadate, ouabain and N-ethylmaleimide, compounds that inhibit the intracellular ion pumping ATPases. Beta-Glycerophosphate (1-10 mM) or p-nitrophenyl phosphate (1-10 mM) did not inhibit hydrolysis of ATP, ruling out the involvement of non-specific phosphatases. Enzyme activity in buffer that had previously been incubated with cells was < 7%, showing that the enzyme activity measured did not result from release of intracellular enzymes. Consistent with this, the cell preparations used were estimated to be > 95% intact as judged by release of cytosolic enzyme lactate dehydrogenase. The enzyme activity was Ca2-/Mg2- dependent. Gramicidin S (20 microM), suramin (100-300 microM), chlorpromazine (250 microM), trifluoperazine (50-250 microM), and thioridazine (100 microM) inhibited the hydrolysis of ATP (3 mM) by 45-80%. The percentage inhibition produced by these substances was not altered in the presence of a concentration of alpha, beta-methylene ADP (10 microM) which inhibited hydrolysis of AMP (3 mM) by 90%, suggesting that these compounds inhibit ecto-ATPase and/or ecto-ADPase. Measurements of absolute amounts of ATP released from various tissues, including the heart, have been hindered because ATP is rapidly and sequentially hydrolysed to adenosine. Identification of compounds that inhibit ATP degradation would prove to be useful to overcome this problem and would lead to the development of invaluable pharmacological tools in many other aspects of purine research.
Meghji
P
P
Department of Anatomy and Developmental Biology, University College London, United Kingdom.
Burnstock
G
G
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors; D000255:Adenosine Triphosphate / Q000378:metabolism*; D002118:Calcium / Q000494:pharmacology; D002460:Cell Line; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D005110:Extracellular Space / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D008274:Magnesium / Q000494:pharmacology
D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D008274:Magnesium; D002118:Calcium