The effects of long-term smoking on endothelial nitric oxide synthase mRNA expression in human platelets as detected with real-time quantitative RT-PCR.
Endothelium-derived nitric oxide (NO) plays an important role in the prevention of platelet aggregation and adhesion to the vascular wall. Endothelial nitric oxide synthase (eNOS) and L-arginine/NO pathway are both present in human platelets. Platelet-derived NO inhibits excessive activation and aggregation of platelets. However, the expression level of the eNOS gene in human platelets has yet to be elucidated. The current study investigates the individual expression level of platelet eNOS mRNA using the real-time reverse transcriptase-polymerase chain reaction (RT-PCR) detection method. eNOS mRNA expression was examined in platelets isolated from 50 subjects: 11 male smokers, 15 male nonsmokers, and 24 female non-smokers. After extraction of platelet total RNA, eNOS (target) and GAPDH (internal control) mRNA expression levels were quantitated using real-time RT-PCR. The expression levels of eNOS mRNA (relative copy numbers) were significantly lower in male smokers (59+/-17) than in male nonsmokers (195+/-71, P < .03), and higher in female nonsmokers (285+/-60) than in the male nonsmokers (195+/-71, P < .03). By multiple linear regression analysis, cigarette smoking (P = .008) and diabetes mellitus (P = .047) were found to be significantly negative predictors, and antioxidant (vitamin E) treatment (P = .01) was a significantly positive predictor of platelet eNOS mRNA expression. Age, other medications, and other risk factors for coronary artery disease were not significant. Using this method, eNOS mRNA abundance in human platelets was detected and quantitated in real-time. The intraplatelet eNOS mRNA expression levels were significantly decreased in cigarette smokers. Low platelet NO synthesis in smokers may result in the augmentation of platelet aggregation and thrombus formation, developing into acute coronary syndromes.
Shimasaki
Yukio
Y
Department of Cardiovascular Medicine, Kumamoto University Graduate School of Medical Sciences, Kumamoto, Japan. yuki7nao@mocha.ocn.ne.jp
Saito
Yoshihiko
Y
Yoshimura
Michihiro
M
Kamitani
Shigeki
S
Miyamoto
Yoshihiro
Y
Masuda
Izuru
I
Nakayama
Masafumi
M
Mizuno
Yuji
Y
Ogawa
Hisao
H
Yasue
Hirofumi
H
Nakao
Kazuwa
K
D000328:Adult; D000368:Aged; D000975:Antioxidants / Q000494:pharmacology / Q000627:therapeutic use; D001792:Blood Platelets / Q000201:enzymology / Q000378:metabolism*; D016022:Case-Control Studies; D003324:Coronary Artery Disease / Q000209:etiology; D003920:Diabetes Mellitus; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D052250:Nitric Oxide Synthase Type III / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012307:Risk Factors; D012907:Smoking / Q000009:adverse effects*; D013997:Time Factors
D000975:Antioxidants; D012333:RNA, Messenger; D052250:Nitric Oxide Synthase Type III