Effects of homocysteine on intracellular nitric oxide and superoxide levels in the renal arterial endothelium

Abstract

The present study was designed to test the hypothesis that homocysteine (Hcys) reduces intracellular nitric oxide (NO) concentrations ([NO](i)) and stimulates superoxide (O.) production in the renal arterial endothelium, thereby resulting in endothelial dysfunction. With the use of fluorescence microscopic imaging analysis, a calcium ionophore, A-23187 (2 microM), and bradykinin (2 microM) were found to increase endothelial [NO](i) in freshly dissected lumen-opened small renal arteries loaded with 4,5-diaminofluorescein diacetate (DAF-2DA; 10 microM). Preincubation of the arteries with L-Hcys (20-40 microM) significantly attenuated the increase in endothelial [NO](i). However, L-Hcys had no effect on NO synthase activity in the renal arteries, as measured by the conversion rate of [(3)H]arginine to [(3)H]citrulline, but it concentration dependently decreased DAF-2DA-sensitive fluorescence induced by PAPA-NONOate in the solution, suggesting that L-Hcys reduces endothelial [NO](i) by its scavenging action. Because other thiol compounds such as L-cysteine and glutathione were also found to reduce [NO](i), it seems that decreased NO is not the only mechanism resulting in endothelial dysfunction or arteriosclerosis in hyperhomocysteinemia (hHcys). By analysis of intracellular O. levels using dihydroethidium trapping, we found that only L-Hcys among the thiol compounds studied markedly increased O. levels in the renal endothelium. These results indicate that L-Hcys inhibits the agonist-induced NO increase but stimulates O. production within endothelial cells. These effects of L-Hcys on [NO](i) and [O.] may contribute to endothelial injury associated with hHcys.

Performance of diamino fluorophores for the localization of sources and targets of nitric oxide

“…Prior investigations have called attention to the quenching of DAF-2 nitrosation by naturally occurring reducing agents such as Asc and GSH [7,46,67]. Consistent with these findings, our …”

“…As we struggled with this seemingly straight-forward proposition, it became apparent that, while the development of diamino fluorophores represent a great analytical advance in NO bioimaging, there are a number of unresolved questions regarding the performance of these probes in biological tissues that impact their utility for the localization of sources and targets of NO. One important issue is that their sensitivity may be significantly limited in biological milieus by reducing agents such as ascorbate (Asc) and reduced glutathione (GSH), since these are known to scavenge nitrosating products such as N 2 O 3 , or their precursor NO 2. Several studies [6,46,66] have demonstrated this effect in buffer solutions, in which substantial drops or even a total abolishment of formation of the fluorescent triazole form DAF-2 T were observed. Yet, only modest inhibition of DAF-2 activation is observed within cultured cells containing comparable levels of reducing agents [66], and all other cell-culture or tissue studies published to date document its successful activation.…”

Chronic diet-induced hyperhomocysteinemia impairs eNOS regulation in mouse mesenteric arteries

“…E: relative eNOS mRNA (means Ϯ SE) normalized to relative smooth muscle alpha actin mRNA (n ϭ 7 in each group). mechanism for endothelium dysfunction in HHcy (2,8,21,28). This study reveals decreased eNOS expression and decreased ability to phosphorylate eNOS at S1179 with HHcy.…”

“…While the mechanistic link between HHcy and vascular disease is not entirely understood, one consistent finding is that HHcy reduces endotheliumdependent vasodilation (13,19,20,29,31), which is likely to be an underlying mechanism for its contribution to the vascular disease process. Evidence indicates that HHcy increases superoxide levels, thereby reducing the bioavailability of nitric oxide and the ability of arteries to dilate (2,8,21,28). It is unknown, however, whether chronic HHcy decreases vascular expression and activation of endothelial nitric oxide synthase (eNOS), the enzyme that produces nitric oxide.…”

Role of homocysteinylation of ACE in endothelial dysfunction of arteries

“…Not only does Hcy itself have prooxidant properties, but also accumulating evidence from clinical and basic science studies indicate that the primary pathogenesis of diseases associated with HHcy is a concomitant generation of superoxide anion, which inactivates NO (7,27,28,51,56,58). To date, several studies have suggested the existence of an interaction between Hcy and angiotensin II (ANG II) signaling as a pathway that contributes significantly to the potential oxidative stress in the vasculature (2,31).…”