Vol. 53 No. 2/2006, 299–302 Regular paper on-line at: www.actabp.pl

Investigation of the mechanisms of phenotypic realization of allelic polymorphism of the eNOS gene has shown that the level of eNOS mRNA and activity of this enzyme in platelets depends from genotype. We identified a T(-786)-->C polymorphism in the promoter region, a variable number of tandem repeats (4a/4b) in intron 4 and the G(894)-->T polymorphism in exon 7 of the eNOS gene in isolated human platelets. We measured eNOS mRNA in isolated platelets by reverse transcription-PCR and eNOS enzyme activity by fluorimetric detection system FCANOS-1 using diaminofluorescein diacetate (DAF-2A). It was shown that the level of eNOS mRNA is the lowest for the -786C/C promoter genotype. In exon 7 homozygotes (894T/T) the level of RNA is lower than in normal homozygotes (894G/G), but higher than in heterozygotes (894G/T). The eNOS activity in platelets is lower in carriers of the 786C/C promoter genotype than in normal homozygotes (2.1 times; P=0.03), and lower comparing to heterozygotes (2.9 times; P>0.05). The eNOS activity accompanying the 894T/T variant of exon 7 is also lower than in normal homozygotes (P>0.05). Regarding the polymorphism in intron 4 - the enzyme's activity is lower in carriers of the 4a/4a genotype comparing to normal homozygotes (1.7 times; P>0.05) and lower than in heterozygotes (1.9 times; P>0.05). These results allow one to conclude that the T(-786)-->C polymorphism of the eNOS gene promoter most significantly affects the gene expression and eNOS activity.


INTRODUCTION
Allelic polymorphism of endothelial NO-synthase (eNOS) increases the risk of development of cardio-vascular diseases (Hingorani et al., 1999;Yoshimura et al., 2000;Dosenko et al., 2002;2005;Wang et al., 2002;Casas et al., 2004).But it is not clearly elucidated how the gene polymorphism affects the gene expression and enzyme activity in the cells.Studies dealing with this problem using different methodological approaches have not clarified the question (Yoon et al., 2000;Golser et al., 2003, Song et al., 2003).What mechanism of the phenotypic expression of pathologic allelic variants of the eNOS gene is the major cause of NO production decrease is not known.It may be explained either in terms of the NO-synthase functional or quantitative deficiency (abnormalities of transcription, mRNA stability, formation of catalytically defective protein) or by enhanced degradation of the enzyme.We therefore analyzed eNOS gene expression and NO-synthase activity in platelets isolated from individuals with genotyped promoter (Т -786 →С polymorphism), exon 7 (G 894 →T polymorphism) and intron 4 (4a/4b polymorphism) to elucidate these parameters in conditions similar to physiological.
Procedures with human subjects were approved by the regional bioethics committee at the Government of Ukraine.
DNA isolation and PCR.DNA was isolated from whole stabilized blood using a DNA-prep kit (Isogene).Using PCR with following analysis of the length of restriction fragments the Т -786 →С polymorphism of the promoter, the G 894 →T polymorphism of exon 7 and the 4a/4b polymorphism of intron 4 of eNOS gene were determined as described before (Hibi et al., 1998;Wang et al., 2000;Dosenko et al., 2002).PCR was carried out using a GeneAmp System 2700 thermocycler (Applied Biosystems).
RNA isolation and measurement of eNOS mRNA.RNA isolation from platelets was performed using a Trizol RNA-prep kit (Isogen).Reverse transcription was performed using a Re-vertAid ™ H Minus First Strand cDNA Synthesis Kit (Fermentas), using 500 µg of total RNA and a (dT) 18 primer.The single-strand DNA obtained was used for PCR using the following primers: upstream (sense)-5'-TCC CTG AGG AGG GCA GGC-3' and down-stream (antisense)-5'-TGA GGG TCA CAC AGG TTC CT-3'.Amplification mixture contained 5 µl of 5 × PCR-buffer, 2.5 mM of magnesium sulfate, 200 µM mixture of four nucleotide triphosphates, 20 pM of each primer and 0.5 U of Taq polymerase (AmpliSense), the volume was brought up to 25 µl with deionized water.Amplification of the fragment of exon 7 consisted of 35 cycles: denaturation 94°С, 1 min, primer annealing 64°С, 1 min, and elongation 74°С, 1 min.For controlling the quality of RNA isolation and comparison of intensity of the eNOS gene expression, a fragment of β-actin gene was amplified according to (Øvstebø et al., 2003) as an internal control.Amplified fragments were separated in 2.5% agarose gel containing ethidium bromide.Visualization and estimation of the brightness of amplified fragments after horizontal electrophoresis (160 V during 40 min) was performed using transilluminator and Vitran software (Biocom).
eNOS activity.For an assay of eNOS activity we used a fluorimetric detection system FCANOS-1 (Sigma), based on the principle of fluorescence of triazolofluoresceine, which is formed after interaction of NO with 4,5-diaminofluoresceine, which is formed from 4,5-diaminofluoresceine diacetate (DAF-2A) under the action of intracellular esterases.The wavelength of excitation/emission was 492/515 nm.The NOS inhibitor diphenyliodonium chloride (100 µM) inhibited the reaction and this confirms the specificity of the NOS activity assay.Enzyme activity was evaluated in units of fluorescence (UF) per min per 10 6 cells.In mammals there are few cell types, which express only one isoform of NO-synthase.In platelets under normal conditions only endothelial NOS is expressed (Jayachandran & Miller, 2002;Randriamboavonjy et al., 2004), which ensures that only the activity of this particular isoform was assayed.
Statistical analysis.Statistical analysis was performed with Excel 2000 and Оrigin 7.0.All data was expressed as the mean (± S.E.) of replicate experiments performed in each assay.Statistical differences were evaluated using the Student's t-test.P < 0.05 was taken to indicate a statistically significant difference.

eNOS polymorphism frequency
In the case of the Т -786 →С promoter polymorphism it turned out that 11 of 30 individuals had the T/T genotype, 13 had the T/C genotype and 6 had the C/C genotype.The G 894 →T exon 7 polymorphism showed the following allelic distribution: 11 G/G, 14 G/Т and 5 Т/Т.Intron 4 VNTR had the following distribution: 18 of 30 individuals had the 4b/4b genotype, 9 had the 4b/4а genotype and 3 had the 4а/4а genotype.The observed ratio of the frequencies of the allelic variants in general corresponds to such a ratio in the Ukrainian population (Dosenko et al., 2005).

eNOS mRNA quantification by RT-PCR
We observed a dependence of eNOS mRNA quantity on the genotype (Figs. 1, 2).The eNOS mRNA level was the lowest in persons with the C/ C promoter genotype.The pathologic homozygotes (T/T) of exon 7 had a lower eNOS mRNA level than normal homozygotes (G/G), but higher than the het-erozygotes (G/T).eNOS mRNA concentration was higher in subjects with the 4a/4a genotype than in normal homozygotes and heterozygotes.

eNOS enzyme activity
The eNOS activity in platelets according to genotype is summarized in Fig. 3.The C/C promoter genotype gave a 2.1 times lower NO-producing activity than in normal homozygotes (P = 0.03) and 2.9 lower than in heterozygotes (P > 0.05).So, the data about gene expression entirely corresponds to the level of eNOS activity in relation to the promoter polymorphism.The pathologic genotype of exon 7 exhibits a lower eNOS activity than normal homozygotes, but the difference is not significant.The subjects with the 4a/4a genotype of intron 4 VNTR have a 1.7 times lower NO-producing activity comparing to normal homozygotes (P > 0.05) and 1.9 times lower comparing to heterozygotes (P > 0.05).There was no positive correlation between the eNOS gene expression and eNOS activity for intron 4 polymorphism.On the contrary, while eNOS expression was high in the 4a/4a genotype, the enzyme activity was low.

DISCUSSION
Different approaches used by investigators have permitted the establishing of main rules of expression, translation and catalytic activity of NOS depending on the NOS gene polymorphism.However, evident controversies remain: in the work of Golser et al. (2003) it was shown that the Glu/Asp polymorphism does not affect the protein's catalytic activity.The same conclusion was made by Song et al. (2003).But in both those studies investigation of pathologic Asp/Asp variant was absent, while it is this allelic variant, according to Casas et al. (2004), that is the most significant predictor of cardio-vascular diseases.According to our data, NOS gene expression is lower in subjects with the Glu/Asp and Asp/Asp genotype compared to the Glu/Glu one, while the enzyme's activity is not significantly changed.In any case, for elucidating the role of this polymorphism additional studies are needed.Nakayama et al. (1999) were the first to report about the loss of promoter activity upon a T→C substitution in position -786.But the results of population studies were ambiguous (Nakayama et al., 1999;Yoshimura et al., 2000;Casas et al., 2004).Using isolated platelets we managed to show that eNOS gene expression with the C/C genotype is significantly lower than in the case of the T/T and T/C allelic variants and the enzyme activity is significantly decreased, too.The obtained data explain to a certain extent the pathogenic meaning of eNOS gene promoter polymorphism.In case of disturbances of coronary blood flow in individuals with the pathologic variant of the promoter, eNOS gene expression is activated to lower extent, insufficient number of eNOS molecules are produced, thus resulting in an inability to produce additional NO.As a consequence, the vessels are dilated to a lesser extent, platelet adhesion and aggregation is not prevented and other mechanisms of the angio-and cardioprotective action of NO are inoperative as well.

Figure 1 .
Figure 1.Results of electrophoresis of amplified fragments of eNOS gene exon 7 (A) and β-actin gene (B).The data show dependence of eNOS gene expression on the allelic variant of the T -786 →C promoter polymorphism.M, marker of molecular size (500, 400 etc. base pairs); T/T, normal homozygotes; T/C, heterozygotes; C/C, pathologic homozygotes.

Figure 2 .
Figure 2. level of eNOS mRNA in isolated platelets at different allelic variants of the promoter (A), exon 7 (B) and intron 4 (C) of eNOS gene.Results are expressed as means (± S.E.; bars).Values of eNOS mRNA are given as the ratio of the intensity of the eNOS band to the β-actin band.*P < 0.05 comparing to eNOS gene expression at the T/T promoter variant, obtained by the Student's t-test.

Figure 3 .
Figure 3. eNOS activity in isolated platelets at different allelic variants of the promoter (A), exon 7 (B) and intron 4 (C) of eNOS gene.Results are expressed as means (±S.E.; bars).*P < 0.05 comparing to eNOS activity at T/T promoter variant, obtained by the Student's t-test.