Evidence for the Pathophysiological Role of Endogenous Methylarginines in Regulation of Endothelial NO Production and Vascular Function

Abstract

In endothelium, NO is derived from endothelial NO synthase (eNOS)-mediated L-arginine oxidation. Endogenous guanidinomethylated arginines (MAs), including asymmetric dimethylarginine (ADMA) and N G -methyl-L-arginine (L-NMMA), are released in cells upon protein degradation and are competitive inhibitors of eNOS. However, it is unknown whether intracellular MA concentrations reach levels sufficient to regulate endothelial NO production. Therefore, the dose-dependent effects of ADMA and L-NMMA on eNOS function were determined. Kinetic studies demonstrated that the K m for L-arginine is 3.14 M with a V max of 0.14 mol mg The biological significance of guanidino-methylated arginine derivatives has been known since the inhibitory actions of N G -methyl-L-arginine (L-NMMA) 3 on macrophage induced cytotoxicity were first demonstrated. It was subsequently realized that these effects were mediated through inhibition of NO release (1). NO has been demonstrated as a critical effector molecule in the maintenance of vascular function (2-4). In the vasculature, NO is derived from the oxidation of L-Arg, catalyzed by the constitutively expressed enzyme, eNOS (5-7). This endothelial-derived NO diffuses from the vascular endothelium into the smooth muscle cell layer where it activates soluble guanylate cyclase leading to smooth muscle relaxation (2-4). In addition to its role in the maintenance of vascular tone, NO helps to maintain the anti-atherogenic character of the normal vascular wall. NO, in concert with various cell signaling molecules, has been demonstrated to maintain smooth muscle cell quiescence and as such, counteracts pro-proliferative agents, specifically those involved in the propagation of athero-proliferative disorders (8 -14). As such, eNOS dysfunction is an early symptom of vascular disease and is manifested through insufficient NO bioavailability. Several potential causes of NO deficiency in disease settings have been proposed. Among these, high circulating levels of the endogenous methylarginine NOS inhibitor asymmetric dimethylarginine (ADMA) has been hypothesized to be of particular importance (15)(16)(17)(18)(19)(20)(21). In neurons and the brain, it has been shown that the methyl arginine L-NMMA is also present, however, the levels of this methylarginine have not been previously considered in studies evaluating vascular dysfunction (22).ADMA and L-NMMA are endogenous inhibitors of NOS and are derived from the proteolysis of methylated arginine residues on various proteins. The methylation is carried out by a group of enzymes referred to as protein-arginine methyltransferase (23). Subsequent proteolysis of proteins containing methylarginine groups leads to the release of free methylarginine into the cytoplasm, and if sufficient levels are reached NO production from NOS would be inhibited (24, 25). To date, six different isoforms of the enzyme have been identified with each

Role of Dimethylarginine Dimethylaminohydrolases in the Regulation of Endothelial Nitric Oxide Production

“…In the presence of physiologically relevant L-Arg levels (100 M), ADMA treatment resulted in a dose-dependent inhibition of endothelial NO with Ͻ20% inhibition seen at ADMA concentrations of 5 M. Overall, these results demonstrate that L-Arg supplementation can only partially restore the loss in NO production occurring after ADMA administration. Although the addition of exogenous L-Arg would be expected to fully restore ADMA-mediated eNOS inhibition, these results are consistent with previous studies demonstrating partial restoration of endothelial NO with L-Arg following exposure to exogenous ADMA (18) (Fig. 9).…”

“…In support of this hypothesis, we and others have demonstrated that endothelial ADMA levels increase 3-4-fold in restenotic lesions and in the ischemia reperfused myocardium (18,40). Based on the kinetics of cellular inhibition, these concentrations of ADMA would be expected to elicit a 30 -40% inhibition in NOS activity (18). These studies, however, involve lesionspecific increases in ADMA and are not associated with increased plasma levels of ADMA and would not be expected to contribute to systemic cardiovascular pathology.…”

“…It is more likely that elevated plasma ADMA levels reflect increased endothelial concentrations of ADMA. In support of this hypothesis, we and others have demonstrated that endothelial ADMA levels increase 3-4-fold in restenotic lesions and in the ischemia reperfused myocardium (18,40). Based on the kinetics of cellular inhibition, these concentrations of ADMA would be expected to elicit a 30 -40% inhibition in NOS activity (18).…”

“…Given that normal intracellular methylarginines are in the low micromolar range, it would not be expected that physiological levels of these competitive NOS inhibitors would elicit pathological eNOS inhibition. Therefore, additional studies were performed in the presence of exogenously added ADMA to assess whether DDAH overexpression can overcome ADMA accumulation at levels observed with cardiovascular disease states (18). Results demonstrated that exogenously added ADMA (10 M) resulted in 40% inhibition of endothelial cell NO production from BAECs and that overexpression of either DDAH-1 or DDAH-2 was able to restore ϳ50% of the loss in endothelial NO production (Fig.…”

Competitive Interaction Between Fibroblast Growth Factor 23 And Asymmetric Dimethylarginine in Patients With CKD

“…ADMA is the most abundant endogenous inhibitor of NO system, 33 and it dose-dependently inhibits NO synthase at concentration in the range of values actually attained in patients with CKD. 34 FGF-23 and the associated co-receptor Klotho impinge on the same system. Indeed, Klotho knockout mice 35,36 (i.e., animal models characterized by high plasma levels of FGF-23 37 ) do not express NO synthase in the vascular system and exhibit almost abolished response to acetylcholine, the strongest stimulant of NO activity.…”