The therapeutic benefits of L-arginine (ARG) supplementation in humans, often clearly observed in short-term studies, are not evident after long-term use. The mechanisms for the development of ARG tolerance are not known and cannot be readily examined in humans. We have developed a sensitive in vitro model using a low glucose/low arginine culture medium to study the mechanisms of ARG action and tolerance using two different human endothelial cells, i.e., Ea.hy926 and human umbilical venous endothelial cells (HUVEC). Cultured cells were incubated with different concentrations of ARG and other agents to monitor their effects on eNOS expression and function, as well as glucose and superoxide (O2•−) accumulation. Short-term (2 h) exposure to at least 50 μM ARG moderately increased eNOS activity and intracellular glucose (p<0.05), with no change in eNOS mRNA or protein expression. In contrast, 7-day continuous ARG exposure suppressed eNOS expression and activity. This was accompanied by increase in glucose and O2•− accumulation. Co-incubation with 100 μM ascorbic acid, 300 U/ml PEG-superoxide dismutase (PEG-SOD), 100 μM L-lysine or 30 μM 5-chloro-2-(N-2,5-dichlorobenenesulfonamido))-benzoxazole (a fructose-1,6-bisphosphatase inhibitor) prevented the occurrence of cellular ARG tolerance. Short-term co-incubation of ARG with PEG-SOD improved cellular nitrite accumulation without altering cellular ARG uptake. These studies suggest that ARG-induced oxidative stress may be a primary causative factor for the development of cellular ARG tolerance.
Membrane preparations containing only the four acetylcholine receptor polypeptide subunits (40, 50, 60, and 65 x 10(3) daltons) were purified from Torpedo californica electroplax. The receptor protein was extracted from these membranes with 2% aqueous sodium cholate, and complete dissolution into discrete molecular species was confirmed by sedimentation analysis. The solubilized preparation reassociated with exogenous phospholipids when the detergent was removed by dialysis and formed spherically sealed vesicles 400--600 A in diameter. The reconstituted receptor preparations had nearly 90% of their alpha-bungarotoxin binding sites exposed on the exterior surface of the vesicles. In a reproducible manner, the reconstituted acetylcholine receptor responded to carbamoylcholine by exhibiting a rapid efflux of 22Na+ from within the vesicles. Such preparations were successfully reconstituted only from acetylcholine receptor stabilized by asolectin above a minimal level upon dissolution of the original membrane preparation by detergent. In addition to the response to carbamoylcholine, the reconstituted preparations also exhibited pharmacological characteristics that resemble those observed for the original electroplax membranes. In terms of the carbamoylcholine-induced signal, a significantly large fraction of the total receptor was functionally reconstituted. The results also confirm the notion that only the four polypeptides considered to constitute the receptor are essential for acetylcholine-mediated cation translocation and rule out possible roles for other polypeptide species.
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