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Genotype-specific Influence on Nitric Oxide Synthase Gene Expression, Protein Concentrations, and Enzyme Activity in Cultured Human Endothelial Cells

¹ Department of Laboratory Medicine, Seoul National University College of Medicine, Seoul 110-744, Korea.

² Department of Laboratory Medicine, Cheju National University College of Medicine, Jeju 690-716, Korea.

³ Department of Laboratory Medicine, Korea Cancer Center Hospital, Seoul 139-706, Korea.

⁴ Department of Science Education, Jeju National University of Education, Jeju 690-016, Korea.

^aAddress correspondence to this author at: Department of Laboratory Medicine, Seoul National University College of Medicine, 28 Yongon-dong, Chongno-gu, Seoul 110-799, Korea. Fax 82-2-745-6653; e-mail jqkim{at}plaza.snu.ac.kr.

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^-786C 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 [³H]arginine to [³H]citrulline, respectively.

Results: The T^-786C 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^-786C polymorphism in the promoter region.

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