Effect of adenine nucleotides on cyclooxygenase and lipoxygenase enzyme products of arachidonic acid in human platelets.
Nucleotides are known to enhance cyclooxygenase product formation in several tissues and, in addition, are believed to function as cofactors for mammalian 5-lipoxygenases. Since nucleotides are released by stimulated platelets and by damaged tissue, we examined the hypothesis that nucleotides can affect the metabolism of arachidonic acid (AA) in washed human platelets. The various nucleotides were given 15 sec prior to the addition of 3 microM arachidonic acid and 1 muCi [3H]AA. We found that the phosphorylated adenine derivatives (ATP, ADP, and AMP) increased the formation of 12-hydroxyeicosatetraenoic acid (12-HETE) by 2-fold without altering the formation of cyclooxygenase products. Adenosine was without effect on 12-HETE formation. ATP also stimulated 12-HETE formation in lysed platelets. This suggests that the 12-lipoxygenase enzyme of platelets can be regulated by adenine nucleotides. We next determined the portion of the nucleotide molecule responsible for the enhanced 12-lipoxygenase activity of platelets. Alteration of the nucleotide base led to a decrease in stimulation, with GTP less active than ATP, and UTP even less active than GTP. Studies with adenine nucleotides showed that the length of the phosphate chain was not important. We also found that the stable methylene isosters of ATP (alpha, beta-methylene ATP and beta, gamma-methylene ATP) increased 12-HETE formation, suggesting that the conformation and hydrolysis of the phosphate chain are not responsible for the stimulatory activity. Cyclic 3',5'AMP and 3'AMP were inactive, implying the necessity for a free phosphate at the 5' position for nucleotide stimulation of 12-HETE synthesis. In conclusion, platelet 12-lipoxygenase was stimulated by ATP, as is true for several mammalian 5-lipoxygenases. However, cyclooxygenase product formation by platelets was not altered by nucleotide addition. These studies suggest that following in vivo injury or platelet aggregation, when local concentrations of nucleotides are high, platelet lipoxygenase activity may be stimulated.
Dragan
Y P
YP
Department of Pharmacology and Toxicology, Medical College of Virginia, Richmond 23298.
Ellis
E F
EF
D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; D000227:Adenine Nucleotides / Q000494:pharmacology*; D000244:Adenosine Diphosphate / Q000494:pharmacology; D000249:Adenosine Monophosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000328:Adult; D001092:Arachidonate 12-Lipoxygenase / Q000097:blood*; D001091:Arachidonate Lipoxygenases / Q000097:blood*; D016718:Arachidonic Acid; D001095:Arachidonic Acids / Q000097:blood*; D001792:Blood Platelets / Q000187:drug effects / Q000201:enzymology*; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006801:Humans; D006893:Hydroxyeicosatetraenoic Acids / Q000097:blood; D011451:Prostaglandin-Endoperoxide Synthases / Q000097:blood*; D014544:Uridine Triphosphate / Q000494:pharmacology
D000227:Adenine Nucleotides; D001095:Arachidonic Acids; D006893:Hydroxyeicosatetraenoic Acids; D016718:Arachidonic Acid; D000249:Adenosine Monophosphate; D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; D000244:Adenosine Diphosphate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D001091:Arachidonate Lipoxygenases; D001092:Arachidonate 12-Lipoxygenase; D011451:Prostaglandin-Endoperoxide Synthases; D014544:Uridine Triphosphate