Caveolar localization of arginine regeneration enzymes, argininosuccinate synthase, and lyase, with endothelial nitric oxide synthase

Although normal intracellular levels of arginine are well above the K-m, an d should be sufficient to saturate nitric oxide synthase in vascular endoth elial cells, nitric oxide production can, nonetheless, be stimulated by exo genous arginine. This phenomenon, termed the "arginine paradox," has sugges ted the existence of a separate pool of arginine directed to nitric oxide s ynthesis. In this study, we demonstrate that exogenous citrulline was as ef fective as exogenous arginine in stimulating nitric oxide production and th at citrulline in the presence of excess intracellular and extracellular arg inine further enhanced bradykinin stimulated endothelial nitric oxide produ ction. The enhancement of nitric oxide production by exogenous citrulline c ould therefore be attributed to the capacity of vascular endothelial cells to efficiently regenerate arginine from citrulline. However, the regenerati on of arginine did not affect the bulk intracellular arginine levels. This finding not only supports the proposal for a unique pool of arginine, but a lso suggested channeling of substrates that would require a functional asso ciation between nitric oxide production and arginine regeneration. To suppo rt this proposal, we showed that nitric oxide synthase, and the enzymes inv olved in arginine regeneration, argininosuccinate synthase and argininosucc inate lyase, cofractionated with plasmalemmal caveolae, a subcompartment of the plasma membrane. Overall, the results from this study strongly support the proposal for a separate pool of arginine for nitric oxide production t hat is defined by the cellular colocalization of enzymes involved in nitric oxide production and the regeneration of arginine. (C) 2001 Academic Press .