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Effects of homocysteine on endothelial nitric oxide production
1Departments of Medicine and Physiology, State University of New York, Stony Brook, New York 11794-8152, USA.
Insights
High homocysteine (Hcy) levels impair endothelial nitric oxide (NO) production by increasing oxidative stress, not by affecting eNOS expression. Folate supplementation and antioxidants can restore NO function in hyperhomocysteinemia.
Area of Science:
- Cardiovascular Science
- Renal Medicine
- Biochemistry
Background:
- Hyperhomocysteinemia (HHCy) is a significant cardiovascular risk factor, particularly prevalent in chronic renal failure patients.
- The precise impact of HHCy on nitric oxide (NO) production remains debated, with conflicting reports on its stimulatory or suppressive effects.
Purpose of the Study:
- To investigate the effect of homocysteine (Hcy) on endothelial nitric oxide (NO) production using electrochemical detection.
- To elucidate the mechanisms underlying Hcy-induced alterations in NO synthesis and endothelial cell function.
Main Methods:
- Amperometric electrochemical NO detection with porphyrinic microelectrodes to measure NO release from endothelial cells.
- Incubation of endothelial cells with varying concentrations of Hcy (10-50 microM) followed by stimulation with bradykinin, calcium ionophore, or L-arginine.
- Assessment of endothelial nitric oxide synthase (eNOS) expression, superoxide anion formation, and tyrosine nitration to detect peroxynitrite.
Main Results:
- Hcy preincubation (≥20 microM) significantly suppressed endothelial cell responsiveness to NO-stimulating agents.
- Hcy increased superoxide anion and peroxynitrite formation but did not alter eNOS expression.
- 5-methyltetrahydrofolate, peroxynitrite scavengers, and superoxide dismutase mimetics restored NO production, indicating a role for cellular redox state.
Conclusions:
- Hcy at concentrations above 20 microM indirectly suppresses eNOS activity in endothelial cells, primarily through oxidative stress mechanisms.
- Cellular redox state modulation, influenced by factors like folate availability and reactive oxygen/nitrogen species, is critical in Hcy-associated NO dysfunction.
- These findings highlight potential therapeutic strategies targeting oxidative stress to manage cardiovascular risks in HHCy.
Abstract:
Hyperhomocysteinemia (HHCy) is an independent and graded cardiovascular risk factor. HHCy is prevalent in patients with chronic renal failure, contributing to the increased mortality rate. Controversy exists as to the effects of HHCy on nitric oxide (NO) production: it has been shown that HHCy both increases and suppresses it. We addressed this problem by using amperometric electrochemical NO detection with a porphyrinic microelectrode to study responses of endothelial cells incubated with homocysteine (Hcy) to the stimulation with bradykinin, calcium ionophore, or L-arginine. Twenty-four-hour preincubation with Hcy (10, 20, and 50 microM) resulted in a gradual decline in responsiveness of endothelial cells to the above stimuli. Hcy did not affect the expression of endothelial nitric oxide synthase (eNOS), but it stimulated formation of superoxide anions, as judged by fluorescence of dichlorofluorescein, and peroxynitrite, as detected by using immunoprecipitation and immunoblotting of proteins modified by tyrosine nitration. Hcy did not directly affect the ability of recombinant eNOS to generate NO, but oxidation of sulfhydryl groups in eNOS reduced its NO-generating activity. Addition of 5-methyltetrahydrofolate restored NO responses to all agonists tested but affected neither the expression of the enzyme nor formation of nitrotyrosine-modified proteins. In addition, a scavenger of peroxynitrite or a cell-permeant superoxide dismutase mimetic reversed the Hcy-induced suppression of NO production by endothelial cells. In conclusion, electrochemical detection of NO release from cultured endothelial cells demonstrated that concentrations of Hcy >20 microM produce a significant indirect suppression of eNOS activity without any discernible effects on its expression. Folates, superoxide ions, and peroxynitrite scavengers restore the NO-generating activity to eNOS, collectively suggesting that cellular redox state plays an important role in HCy-suppressed NO-generating function of this enzyme.