Studies of L-arginine transport in bovine aortic endothelial cells.

We have previously demonstrated that p(1),p(4)-diadenosine 5'-tetraphosphate induces the release of NO and modulates the uptake of L-arginine by bovine aortic endothelial cells (BAEC) [Hilderman, R. H., and Christensen, E. F. (1998) FEBS Lett. 407, 320-324; Hilderman, R. H., Casey, T. E., and Pojoga, L. H. (2000) Arch. Biochem. Biophys. 375, 124-130]. In this communication we characterize the uptake of L-Arg by BAEC. L-Arg is transported into BAEC by at least two different transporter systems. One transporter system is protein synthesis dependent, and L-Arg transported by this system is incorporated into proteins. The second transporter system involved in L-Arg uptake is protein synthesis independent, and uptake occurs by facilitated diffusion. The L-Arg transported by facilitated diffusion is metabolized into L-argininosuccinate. Homologous and heterologous competition uptake studies were performed using a fixed concentration of radiolabeled L-Arg, L-lysine, and L-leucine with varying concentrations of competing nonradiolabeled amino acids. The results of these competition uptake studies are consistent with the protein-synthesis-dependent uptake of L-Arg taking place through a transporter system that is highly specific for L-Arg and with the facilitated diffusion uptake taking place through a transporter that is specific for L-Arg and L-Leu.

Modification of the cadmium reduction assay for detection of nitrite production using fluorescence indicator 2,3-diaminonaphthalene.

The measurement of nitric oxide (NO) is important for characterizing the regulatory roles of NO in various biological systems. In this communication we report that cadmium (Cd) reduction of nitrate (NO(-)(3)) to nitrite (NO(-)(2)) can be quantitated by using the fluorescence indicator, 2,3-diaminonaphthalene (DAN) to detect the sum of NO(-)(3) and NO(-)(2) (NO(-)(x)) from endothelial cells. This assay is at least 10-fold more sensitive than when Cd reduction is coupled with the spectrophotometric Greiss reaction and can be used to quantitate the small amounts of NO(-)(x) generated from the constitutive form of endothelial nitric oxide synthase (eNOS). In addition various P(2) purinoceptor agonists and antagonists do not interfere the Cd reduction/DAN assay. Thus the Cd reduction/DAN assay can be used not only to characterize P(2) purinoceptor release of NO(-)(x) from cultured endothelial cells but also to quantitate NO(-)(x) levels in serum.

P(1),P(4)-Diadenosine 5'-tetraphosphate modulates l-arginine and l-citrulline uptake by bovine aortic endothelial cells.

We have previously demonstrated that P(1),P(4)-diadenosine 5'-tetraphosphate (Ap(4)A) interacts with high-affinity and low-affinity binding sites on the bovine aortic endothelial cell (BAEC) surface. In this report we demonstrate that Ap(4)A interaction with the lower affinity site modulates l-arginine (l-Arg) and l-citrulline (l-Cit) uptake by BAEC. Competition uptake studies demonstrate that l-Arg and l-Cit uptake occurs through a common transporter system that is sensitive to Ap(4)A. Evidence is also presented that is consistent with Ap(4)A modulating l-Arg uptake by increasing the affinity of l-Arg for the transporter.