Inhibition of L-Arginine Metabolizing Enzymes by L-Arginine-Derived Advanced Glycation End Products

Abstract

Nω-Carboxymethyl-arginine (CMA), Nω-carboxyethyl-arginine (CEA) and Nδ-(5-hydro-5-methyl-4-imidazolon-2-yl)-ornithine (MG-H1) have been identified as L-arginine-derived advanced glycation end products (AGEs) formed by non-enzymatic reactions between reducing sugars such as glucose and amino groups in proteins. These AGEs are structurally analogous to endogenous inhibitors of nitric oxide synthases (NOS) including NG-monomethyl-L-arginine (L-NMMA) and asymmetric NG,NG-dimethyl-L-arginine (ADMA). Increased plasma levels of these NOS inhibitors, and thus impaired generation of NO in vivo has been associated with the pathogenesis of vascular complications such as kidney failure and atherosclerosis. For these reasons we examined whether L-arginine-derived AGEs inhibit the activities of three L-arginine metabolizing enzymes including three isoforms of NOS (endothelium, neuronal and inducible NOS), dimethylarginine dimethylaminohydrolase (DDAH) that catalyzes the hydrolytic degradation of L-NMMA and ADMA to L-citrulline, and arginase that modulates intracellular L-arginine bioavailability. We found that AGEs inhibited the in vitro activities of endothelium type NOS weakly (IC50 values of CMA, CEA and MG-H1 were 830, 3870 and 1280 µM, respectively) and were also potential endogenous inhibitors for arginase (IC50 values of CMA and CML were 1470 and 1060 µM), but were poor inhibitors for DDAH. These results suggest that the tested L-arginine- and L-lysine-derived AGEs appear not to impair NO biosynthesis directly.

Differences Between Tg2576 and Wild Type Mice in the NMDA Receptor–Nitric Oxide Pathway After Prolonged Application of a Diet High in Advanced Glycation End Products

“…In addition, AGE products probably also influenced the homeostasis between nNOS and eNOS or between eNOS and iNOS in the brains of WT mice (Table 3). Our results obtained in WT mice thus support the sensitivity of the cerebral NMDA receptor-NO pathway to AGE products, in good accordance with previous studies [e.g., [20][21][22], and suggest that nNOS activity may be a more sensitive biomarker of alterations mediated by AGE products in vivo than e.g., MDA levels reflecting oxidative injury of lipids (Fig. 1).…”

“…Namely, we found an increase in nNOS activity to 125.6 % ( Table 2) which could be interpreted as a compensatory reaction of a relatively healthy organism (old WT mice used as a model of normal aging), to an AGE-mediated drop in nNOS expression, as reported in many studies [20][21][22]. Nevertheless, some observed trends to alterations in particular components of the NMDA receptor-NO pathway [e.g., the drop in NR1 expression to 96.8 %, the increase in NR2A expression to 104.8 % and the increase in NR2B expression to 104.9 % (Table 1) and the increases in eNOS and iNOS activities to 107.9 and 127.6 %, respectively (Table 2)] should not be neglected e.g., with respect to the results of the correlation analysis.…”

“…Since MDA levels were significantly increased in both groups of Tg2576 mice compared to the corresponding WT mice (to 138.5 % in animals kept on a regular AGE diet and to 150.2 % in these kept on a high AGE diet, Fig. 1), the pronounced negative correlation between iNOS activity and MDA levels exclusively in Tg2576 mice kept on a high AGE diet could be interpreted as a trend toward an AGE-mediated drop in iNOS activity [20][21][22] under conditions of overexpressed Ab (see also the non-significant decrease in iNOS activity to 82.9 % in Tg2576 mice kept on a regular AGE diet and to 85.4 % in Tg2576 mice kept on a high AGE diet when compared to the corresponding WT mice, Table 2). This result could also support an increasing role of oxidative lipid injury in alterations in iNOS activity in Tg2576 mice.…”

“…One would expect alterations in eNOS activity with regard to the significant changes reported in AD patients [10], diabetic people [18,19] and Tg2576 mice [34,35] and to the high sensitivity of eNOS protein to AGE products in vitro experiments [20][21][22]. We suggest that our results of the relatively intact eNOS activity in WT as well as Tg2576 mice (Table 2) could be based on antagonistic trends evoked either by a high AGE product diet in WT mice (eNOS activity could be mildly increased with regard to the significant shift from a negative to a positive correlation with increasing nNOS activity and to the significant negative correlation with moderately decreased iNOS activity, Table 3) either by overexpressed Ab in Tg2576 (eNOS activity could be decreased, on the contrary [34,35], see also the non-significant decrease to 82.9 % in Tg2576 mice kept on a regular AGE diet and to 85.4 % in Tg2576 mice kept on a high AGE diet when compared to the corresponding WT mice, Table 2).…”

“…Very pronounced effects of AGE products, especially on the NO mediator system, may be mediated either indirectly via RAGE mechanisms or directly through L-argininederived AGE products that are structurally analogous to endogenous inhibitors of NOS isoforms. Various in vitro experiments have demonstrated that AGE products inhibit the activities of nNOS/eNOS/iNOS and the expression of nNOS/eNOS [20][21][22], and that AGE-modified proteins reduce the release of NO and quench it [21]. In sinoaortic denervated rats, treatment with soluble RAGE daily for 3 weeks restored aortic NO formation through the upregulation of eNOS [23].…”