Influence of G894T polymorphism of endothelial nitric oxide synthase gene on endothelium-depedent vasodilation

Author(s)

Rodrigo Gonçalves Dias

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
• Defense date: 2008-08-28

Examining board members

Marta Helena Krieger; Dora Maria Grassi Kassisse; Denise Vaz de Macedo; Maria Helena Catelli de Carvalho; Francisco Rafael Martins Laurindo; Helena Coutinho Franco de Oliveira

• Advisor: Marta Helena Krieger

Abstract

Nitric oxide (NO) is a free radical synthesized in vessels mainly by endothelial nitric oxide synthase (eNOS) enzyme. In the cardiovascular system NO is involved in the modulation of regional blood flow, mainly in the skeletal muscle. The G894T polymorphism of the eNOS gene has been extensively investigated, and the 894T allele that results in glutamate to aspartate exchange at the position 298 of the eNOS enzyme, has been associated with cardiovascular diseases. However, the effects of 894T allele on muscle vasodilatation are unknown. We hypothesized that the reflex forearm blood flow responses during handgrip isometric exercise would be attenuated in individuals carrying the 894T allele. In addition, these responses would be mediated by impairment in nitric oxide availability and/or an increase in vasoconstrictor sympathetic nerve activity to skeletal muscle. To test the previous hypothesis, 287 volunteers were previously genotyped, and 33 healthy individuals selected to represent three genotypes: GG (n=15, age=43±3 years, BMI=22.9±0.3 kg/m2), GT (n=9, age=39±3 years, BMI=24.6±1.2 kg/m2) and TT (n=9, age=40±4 years, BMI=23.5±0.9 kg/m2). Heart rate (HR; ECG), mean blood pressure (MBP; oscillometric cuff) and forearm blood flow (FBF; venous occlusion plethysmography) were recorded for 3 minutes at baseline and 3 minutes during isometric handgrip exercise performed at 30% of maximal voluntary contraction. Baseline HR, MBP, FBF and forearm vascular conductance (FVC) were similar among GG (69±2 bpm; 91±3 mmHg; 1.81±0.09 ml.min-1.100 ml-1; 2.03±0.12 units, respectively), GT (69±4 bpm; 94±2 mmHg; 1.80±0.23 ml.min-1.100 ml-1; 1.90±0.24 units) and TT (63±3 bpm; 95±3 mmHg; 1.79±0.17 ml.min-1.100 ml-1; 1.89±0.17 units). FVC responses to exercise were significantly lower in TT ('delta'=0.07±0.14 units, P=.002) when compared with GG ('delta'=0.57±0.09 units) and GT ('delta'=0.64±0.20 units). To investigate the mechanisms underlying the lowered vasodilatation in TT genotype, we studied FBF during brachial intra-arterial infusion of L-NMMA (4 mg.min-1) and L- NMMA + phentolamine (4 mg.min-1 and 100µg.min-1, respectively) in a subset of 8 GG and 7 TT individuals. In addition, we directly measured muscle sympathetic nerve activity (MSNA) by means of microneurography. L-NMMA did not significantly change FVC responses to exercise in TT when compared with saline control infusion ('delta'=- 0.05±0.20 vs. -0.09±0.18 units). In contrast, L-NMMA significantly reduced FVC in GG xviii genotype ('delta'=0.79±0.14 vs. 0.14±0.09 units), and the difference between genotypes were no longer observed (P=.62). L-NMMA + phentolamine increased similarly FVC responses to exercise in GG and TT individuals ('delta'=1.16±0.26 vs. 0.56±0.24 units, respectively, P=.43). MBP and MSNA increased significant and similarly throughout experimental protocols in GG and TT genotypes. We concluded that G894T gene eNOS polymorphism is functionally associated with impaired reflex muscle vasodilatation to exercise. In addition, the attenuated vasodilatation responsiveness to exercise in individuals who are homozygous for the 894T allele of the eNOS gene seems to be primarily mediated by a reduced nitric oxide bioavailability, but not increase in MSNA (AU)