L-arginine transport, metabolism and action
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The "L-arginine paradox" is so termed because L-arginine (ARG) supplementation increases nitric oxide (NO) bioavailability and vascular function in vivo, despite its high endogenous concentrations in plasma (50 – 100 μM) and in endothelial cells (∼ 1 mM) when compared to the Km (∼3 μM) determined for its target enzyme, i.e., purified endothelial nitric oxide synthase (eNOS). To gain a better understanding of the mechanisms involved in ARG-induced NO production, we examined the transport and metabolic fates of exogenously administered ARG using a stable-isotope, i.e., 15 N 4 -ARG, in human endothelial cells. This approach allowed us to examine the transport and metabolism of exogenously administered ARG vs. those of endogenous ARG, and its potential interactions with other ARG related amino acids, particularly those with the endogenous eNOS inhibitor, asymmetric dimethylarginine (ADMA). An LC-MS/ MS assay was first developed for the simultaneous analysis of 15 N 4 -ARG, ARG, L-citrulline (CIT), and dimethylarginines. 15 N 4 -ARG-derived NO formation was determined by analyzing 15 N-nitrite accumulation by an LC-MS/MS assay or by monitoring the formation of 15 N 3 -CIT and 15 N 3 -ARG metabolites. By applying these methods, we found that when EA.hy926 human endothelial cells were exposed to 15 N 4 -ARG, the enhanced nitrite/nitrate accumulation was accompanied by efflux of cellular ADMA by the process of trans-stimulation. The increased cellular ARG concentration showed an inhibitory effect on ADMA metabolism. These findings suggest that other cellular interactions between ARG and ADMA exist beyond competitive eNOS inhibition. Modulation of intracellular ARG concentration by passive transport of ARG-ethyl ester was not associated with enhanced nitrite/nitrate accumulation, whereas attenuation of 15 N 4 -ARG cellular uptake by siRNA transfection of the Cationic Amino Acid Transporter-1 (CAT-1) resulted in reduced eNOS activity. These results indicate that ARG action in NO production is determined by its extracellular transport, the Km of which was determined to be about 50 μM. Our results therefore indicate that the "L-arginine paradox", as it has been formulated in the literature, does not address the pertinent processes or concentrations which critically determine the cellular action of ARG to liberate NO. Based on the rate-limiting step of ARG transport and the extent of increase in ARG plasma concentration during supplementation, the beneficial effects observed with ARG dosing can now be understood. An in vivo preliminary study was conducted to compare the short term ARG vs. CIT supplementation in rats. Our data showed that CIT supplementation was not only more efficient in elevating circulating ARG concentration than ARG supplementation itself, but it was also unaccompanied by the potential negative effect of glucose accumulation exhibited by ARG administration. These results therefore suggest that CIT may be a superior alternative to ARG for increasing in vivo NO bioavailability. This approach may merit further mechanistic and clinical investigations in the future.