Identification of constitutive and inducible forms of nitric oxide synthase in human platelets.
A number of cell types possess an L-arginine-nitric oxide (NO) pathway. We studied the presence of constitutive and inducible forms of NO synthase in human platelets. N omega-nitro-L-arginine, an inhibitor of NO synthase, potentiated thrombin-induced aggregation of washed human platelets, whereas L-arginine inhibited it. The direct evidence for the presence of constitutive form of NO synthase came from the observation of conversion of tritium-labeled L-arginine to tritium-labeled L-citrulline by washed platelets suspended in Ca(++)-rich but not in Ca(++)-free buffer. Incubation of washed platelets in Ca(++)-free buffer with cytokines (tumor necrosis factor-alpha and interferon-gamma) or cytokines plus lipopolysaccharide caused a marked increase in the conversion of [3H]L-arginine to [3H]L-citrulline, suggesting the presence of inducible form of NO synthase. Gel electrophoresis identified an approximately 130 kd protein band with NO synthase in the platelet cytosol, which on isolation converted [3H]L-arginine to [3H]L-citrulline. This 130 kd protein required the presence of Ca++, reduced nicotinamide adenine dinucleotide phosphate tetrahydro-L-biopterin, and flavin adenine dinucleotide for expression of NO synthase activity. Platelet sonicates demonstrated presence of nitrite, and its concentrations were lowered by preincubation of platelets with NG-nitro-L-arginine methyl ester and enhanced in cytokine-treated platelets. Reverse-transcription polymerase chain reaction demonstrated messenger RNA expression of the constitutive endothelial (but not brain) and inducible isoforms of NO synthase in platelets. These observations indicate that human platelet cytosol possesses both constitutive and inducible forms of NO synthase.
Mehta
J L
JL
Department of Medicine, University of Florida College of Medicine, Gainesville 32610-0277.
Chen
L Y
LY
Kone
B C
BC
Mehta
P
P
Turner
P
P
D000594:Amino Acid Oxidoreductases / Q000097:blood* / Q000235:genetics; D001120:Arginine / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D002956:Citrulline / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004790:Enzyme Induction; D006801:Humans; D019001:Nitric Oxide Synthase; D009573:Nitrites / Q000097:blood; D019335:Nitroarginine; D010974:Platelet Aggregation; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism; D014158:Transcription, Genetic
D009573:Nitrites; D012333:RNA, Messenger; D019335:Nitroarginine; D002956:Citrulline; D001120:Arginine; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases