Genotype-specific influence on nitric oxide synthase gene expression, protein concentrations, and enzyme activity in cultured human endothelial cells.
BACKGROUND
The results of studies on the association of ecNOS polymorphisms and vascular diseases are inconsistent. To explore the nature of this interaction in the absence of confounding factors, such as smoking, we measured ecNOS mRNA, protein, and enzyme activity in cultured human umbilical vein endothelial cells (HUVECs) with and without ecNOS polymorphisms.


METHODS
We identified a T(-786)-->C polymorphism in the promoter region, the intron 4 variable number of tandem repeats (VNTR), the E298A polymorphism in exon 7, and the G10-T polymorphism in intron 23 of the ecNOS gene in the DNA from 43 human umbilical cords. We measured ecNOS and GAPDH mRNA from the cultured HUVECs by reverse transcription-PCR and ecNOS protein and enzyme activity by Western blotting (as ratio to positive control band) and by determining the conversion of [(3)H]arginine to [(3)H]citrulline, respectively.


RESULTS
The T(-786)-->C polymorphism showed the same allelic distribution as the intron 4 VNTR. Mean (SD) ecNOS protein from the cultured HUVECs was significantly lower in the 4a/4b genotype [0.84 (1.23); n = 9] of the intron 4 VNTR than in the 4b/4b genotype [2.14 (2.26); n = 34; P = 0.0300]. The enzyme activity was also significantly lower in the 4a/4b genotype [0.84 (0.21) pmol.min(-1).mg protein(-1); n = 9] than in the 4b/4b genotype [1.07 (0.31) pmol.min(-1).mg protein(-1); n = 34; P = 0.0197].


CONCLUSIONS
ecNOS gene expression, protein concentrations, and enzyme activity are genotype-dependent in HUVECs. The intron 4 VNTR has a consistent influence that may be mediated by the T(-786)-->C polymorphism in the promoter region.
Song
Junghan
J
Department of Laboratory Medicine, Seoul National University College of Medicine, Seoul 110-744, Korea.
Yoon
Yeomin
Y
Park
Kyoung Un
KU
Park
Junwan
J
Hong
Young Joon
YJ
Hong
Seung Ho
SH
Kim
Jin Q
JQ
D015153:Blotting, Western; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D005838:Genotype; D006801:Humans; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D011110:Polymorphism, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014471:Umbilical Veins / Q000166:cytology / Q000378:metabolism
D012333:RNA, Messenger; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III