Effects of homocysteine on endothelial nitric oxide production

Hyperhomocysteinemia (HHCy) is an independent and graded cardiovascular ris k factor. HHCy is prevalent in patients with chronic renal failure, contrib uting 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 amperometri c electrochemical NO detection with a porphyrinic microelectrode to study r esponses of endothelial cells incubated with homocysteine (Hcy) to the stim ulation with bradykinin, calcium ionophore, or L-arginine. Twenty-four-hour preincubation with Hcy (10, 20, and 50 mu M) resulted in a gradual decline in responsiveness of endothelial cells to the above stimuli. Hcy did not a ffect the expression of endothelial nitric oxide synthase (eNOS), but it st imulated formation of superoxide anions, as judged by fluorescence of dichl orofluorescein, 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 oxidat ion of sulfhydryl groups in eNOS reduced its NO-generating activity. Additi on of 5-methyltetrahydrofolate restored NO responses to all agonists tested but affected neither the expression of the enzyme nor formation of nitroty rosine-modified proteins. In addition, a scavenger of peroxynitrite or a ce ll-permeant superoxide dismutase mimetic reversed the Hcy-induced suppressi on of NO production by endothelial cells. In conclusion, electrochemical de tection of NO release from cultured endothelial cells demonstrated that con centrations of Hcy >20 mu M produce a significant indirect suppression of e NOS activity without any discernible effects on its expression. Folates, su peroxide ions, and peroxynitrite scavengers restore the NO-generating activ ity to eNOS, collectively suggesting that cellular redox state plays an imp ortant role in HCy-suppressed NO-generating function of this enzyme.