Argininosuccinate Synthase Expression Is Required to Maintain Nitric Oxide Production and Cell Viability in Aortic Endothelial Cells

Abstract

Although cellular levels of arginine greatly exceed the apparent Km for endothelial nitric-oxide synthase, current evidence suggests that the bulk of this arginine may not be available for nitric oxide (NO) production. We propose that arginine regeneration, that is the recycling of citrulline back to arginine, defines the essential source of arginine for NO production. To support this proposal, RNA interference analysis was used to selectively reduce the expression of argininosuccinate synthase (AS), because the only known metabolic role for AS in endothelial cells is in the regeneration of l-arginine from l-citrulline. Western blot analysis demonstrated a significant and dose-dependent reduction of AS protein as a result of AS small interfering RNA treatment with a corresponding diminished capacity to produce basal or stimulated levels of NO, despite saturating levels of arginine in the medium. Unanticipated, however, was the finding that the viability of AS small interfering RNA-treated endothelial cells was significantly decreased when compared with control cells. Trypan blue exclusion analysis suggested that the loss of viability was not because of necrosis. Two indicators, reduced expression of Bcl-2 and an increase in caspase activity, which correlated directly with reduced expression of AS, suggested that the loss of viability was because of apoptosis. The exposure of cells to an NO donor prevented apoptosis associated with reduced AS expression. Overall, these results demonstrate the essential role of AS for endothelial NO production and cell viability. Although cellular levels of arginine greatly exceed the apparent Km for endothelial nitric-oxide synthase, current evidence suggests that the bulk of this arginine may not be available for nitric oxide (NO) production. We propose that arginine regeneration, that is the recycling of citrulline back to arginine, defines the essential source of arginine for NO production. To support this proposal, RNA interference analysis was used to selectively reduce the expression of argininosuccinate synthase (AS), because the only known metabolic role for AS in endothelial cells is in the regeneration of l-arginine from l-citrulline. Western blot analysis demonstrated a significant and dose-dependent reduction of AS protein as a result of AS small interfering RNA treatment with a corresponding diminished capacity to produce basal or stimulated levels of NO, despite saturating levels of arginine in the medium. Unanticipated, however, was the finding that the viability of AS small interfering RNA-treated endothelial cells was significantly decreased when compared with control cells. Trypan blue exclusion analysis suggested that the loss of viability was not because of necrosis. Two indicators, reduced expression of Bcl-2 and an increase in caspase activity, which correlated directly with reduced expression of AS, suggested that the loss of viability was because of apoptosis. The exposure of cells to an NO donor prevented apoptosis associated with reduced AS expression. Overall, these results demonstrate the essential role of AS for endothelial NO production and cell viability. Nitric oxide (NO) 1The abbreviations used are: NO, nitric oxide; AS, argininosuccinate synthase; AL, argininosuccinate lyase; NOS, nitric-oxide synthase; eNOS, endothelial NOS; BAEC, bovine aortic endothelial cells; siRNA, small interfering RNA; LDH, lactate dehydrogenase; AIF, apoptosis-inducing factor; MAP, mitogen-activated protein; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. is an important modulator for a wide range of functions including vasodilation of blood vessels, immune system function, angiogenesis, inhibition of leukocyte adhesion and platelet aggregation, gene regulation, and apoptosis (1Bredt D.S. Snyder S.H. Annu. Rev. Biochem. 1994; 63: 175-195Google Scholar, 2Vallance P. Chan N. Heart. 2001; 85: 342-350Google Scholar, 3Gow A.J. Ischiropoulos H. J. Cell. Physiol. 2001; 187: 277-282Google Scholar). Moreover, NO has a dual role in cell viability depending on the tissue type and concentration. Either very high or very low concentrations of NO may induce cell death, whereas basal concentrations may inhibit apoptosis (4Melino G. Catani M.V. Corazzari M. Guerrieri P. Bernassola F. Cell. Mol. Life Sci. 2000; 57: 612-622Google Scholar, 5Dimmeler S. Zeiher A.M. Nitric Oxide. 1997; 1: 275-281Google Scholar, 6Haendeler J. Zeiher A.M. Dimmeler S. Vitam. Horm. 1999; 57: 49-77Google Scholar, 7Shen Y.H. Wang X.L. Wilcken D.E. FEBS Lett. 1998; 433: 125-131Google Scholar). Previous work has shown that NO protects against serum starvation- (8Kwon Y.G. Min J.K. Kim K.M. Lee D.J. Billiar T.R. Kim Y.M. J. Biol. Chem. 2001; 276: 10627-10633Google Scholar), H2O2- (9Hermann C. Zeiher A.M. Dimmeler S. Arterioscler. Thromb. Vasc. Biol. 1997; 17: 3588-3592Google Scholar), TNF-α- (10Estrada C. Gomez C. Martin C. Moncada S. Gonzalez C. Biochem. Biophys. Res. Commun. 1992; 186: 475-482Google Scholar), and oxidized low density lipoprotein-induced apoptosis (11Kotamraju S. Hogg N. Joseph J. Keefer L.K. Kalyanaraman B. J. Biol. Chem. 2001; 276: 17316-17323Google Scholar, 12Dimmeler S. Hermann C. Galle J. Zeiher A.M. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 656-664Google Scholar) in endothelial cells. Argininosuccinate synthase (AS), the rate-limiting step (13Xie L. Gross S.S. J. Biol. Chem. 1997; 272: 16624-16630Google Scholar) in the regeneration of arginine from citrulline, catalyzes the synthesis of argininosuccinate, AMP, and inorganic pyrophosphate from citrulline, ATP, and aspartate. Argininosuccinate is then cleaved by argininosuccinate lyase (AL) to produce l-arginine and fumarate. In the liver, AS and AL function together as components of the urea cycle, ultimately to form arginine from citrulline. Although the expression of AS and AL in the liver is high, both enzymes are found in most mammalian tissues, although at significantly lower levels. The discovery of arginine-derived NO, catalyzed by nitric-oxide synthases (NOSs), revealed a second role for AS and AL (1Bredt D.S. Snyder S.H. Annu. Rev. Biochem. 1994; 63: 175-195Google Scholar, 2Vallance P. Chan N. Heart. 2001; 85: 342-350Google Scholar, 3Gow A.J. Ischiropoulos H. J. Cell. Physiol. 2001; 187: 277-282Google Scholar). Together with NOS, they function as part of a citrulline-NO cycle where AS and AL convert citrulline to arginine, which is then oxidized to form NO and citrulline by NOS. Although both extracellular and intracellular concentrations of arginine are much higher than the reported Km of arginine for endothelial nitric-oxide synthase (eNOS), NO production still appears to be limited by the availability of arginine (13Xie L. Gross S.S. J. Biol. Chem. 1997; 272: 16624-16630Google Scholar, 14Xie L. Hattori Y. Tume N. Gross S.S. Semin. Perinatol. 2000; 24: 42-45Google Scholar, 15Sessa W.C. Hecker M. Mitchell J.A. Vane J.R. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8607-8611Google Scholar, 16Hattori Y. Campbell E.B. Gross S.S. J. Biol. Chem. 1994; 269: 9405-9408Google Scholar, 17Su Y. Block E.R. Am. J. Physiol. 1995; 269: L581-L587Google Scholar, 18Flam B.R. Hartmann P.J. Harrell-Booth M. Solomonson L.P. Eichler D.C. Nitric Oxide. 2001; 5: 187-197Google Scholar, 19Shuttleworth C.W. Burns A.J. Ward S.M. O'Brien W.E. Sanders K.M. Neuroscience. 1995; 68: 1295-1304Google Scholar, 20Solomonson L.P. Flam B.R. Pendleton L.C. Goodwin B.L. Eichler D.C. J. Exp. Biol. 2003; 206: 2083-2087Google Scholar). Recently, a DNA microarray analysis of shear stress-induced NO production demonstrated that up-regulation of AS was coordinated with an increase in NO production (21McCormick S.M. Eskin S.G. McIntire L.V. Teng C.L. Lu C.M. Russell C.G. Chittur K.K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8955-8960Google Scholar), supporting an important role for AS in endothelial NO production. If AS is essential for endothelial NO production then even in the presence of excess arginine, depletion of AS should decrease the capacity of endothelial cells to produce NO. To test this hypothesis, RNA interference analysis was carried out using AS-specific RNA duplexes in bovine aortic endothelial cells (BAEC). In this report, we demonstrate a significant and dose-dependent reduction of AS protein following siRNA transfection. We also show a concomitant decrease in enzyme activity corresponding to a decrease in stimulated and unstimulated NO production in endothelial cells with reduced AS expression, despite excess arginine in the media. In addition, we demonstrate that the viability of endothelial cells grown in excess arginine and treated with AS siRNA was significantly diminished compared with control cells. Cell Culture—Bovine aortic endothelial cells in with bovine serum and at and to in a at of siRNA was carried out with as by the siRNA was with of in and to cells at a transfection. RNA siRNA was used to the RNA following the by and S.M. J. A. J. 2001; Scholar). S. J. and The siRNA to AS to to the of the control siRNA, was by the of the AS to a to out to known Cell and and then in and in in by by The was on for and the protein was by of protein on and Western was as L.C. Goodwin B.L. Flam B.R. Solomonson L.P. Eichler D.C. J. Biol. Chem. Scholar). for in in and with Bcl-2 and in for with a in for The was by using and then to using RNA and RNA was from using a available to the RNA was treated with and to which was as L.C. Goodwin B.L. Flam B.R. Solomonson L.P. Eichler D.C. J. Biol. Chem. Scholar). was using AS-specific and L.C. Goodwin B.L. Flam B.R. Solomonson L.P. Eichler D.C. J. Biol. Chem. Scholar). to Argininosuccinate AS activity in with AS and control siRNA as for cells of arginine by in citrulline and for in this and then stimulated with and Flam B.R. Eichler D.C. Solomonson L.P. Arterioscler. Thromb. Vasc. Biol. 2000; Scholar) for Nitric oxide production was as by blue exclusion following and as cells. In a in AS activity was by of to in cell as by O'Brien W.E. Scholar) with the that was cell by the cells in citrulline, and by of in a of protein for AS siRNA and control siRNA to which citrulline and in a of for at the of the of was and the to for to with and to in of was to the in the of was by in Cell cells and by blue of the cell was with of blue and cell using a Cell was by the cells in by the (10Estrada C. Gomez C. Martin C. Moncada S. Gonzalez C. Biochem. Biophys. Res. Commun. 1992; 186: 475-482Google Scholar), by and for cell to was also using a that reduction as A. M. Scholar). siRNA carried out in AS and control with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium for The was the and was used to the The was on a at cell was using a available activity was in cell from AS and control cells using a with and as the of the which was by of cells. activity was using in with AS and control siRNA as and activity was by of the on a with and analysis of the an NO donor on AS with siRNA as the with and for to caspase Nitric with siRNA, stimulated with and Flam B.R. Eichler D.C. Solomonson L.P. Arterioscler. Thromb. Vasc. Biol. 2000; Scholar). was in the as an of cellular NO using a Biochem. Scholar). on a at and at cells by blue and are as cells. are as the was at of AS by siRNA is known to the rate-limiting step (13Xie L. Gross S.S. J. Biol. Chem. 1997; 272: 16624-16630Google Scholar) in arginine synthesis from citrulline and aspartate. Argininosuccinate lyase (AL) is also for this M. W.C. Vane J.R. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: Scholar). To the function of AS in endothelial with to role in NO we the of siRNA to selectively reduce AS expression. RNA to the AS was as by and S.M. J. A. 2001; Scholar) S. J. and and compared with known using to to The AS siRNA was in using the siRNA control siRNA was the as the AS siRNA in a with AS or control siRNA where in the for AS protein levels by and as shown in AS siRNA treatment reduced cellular AS protein levels to than of control levels. To reduction of AS protein correlated to the reduction of AS RNA was and AS was by shown in AS siRNA treatment in a reduction of AS that correlated with the reduction of AS Argininosuccinate in AS we demonstrated that citrulline was to NO production in the of arginine, on the presence of the recycling enzymes AS and To that the activity of AS was decreased in corresponding to the decrease in protein expression, cells in citrulline for and then stimulated with and to NO production Flam B.R. Eichler D.C. Solomonson L.P. Arterioscler. Thromb. Vasc. Biol. 2000; Scholar). Nitric oxide production was as using the Biochem. Scholar). In the endothelial cells with reduced AS expression, nitric oxide was decreased by than a significant reduction in activity of the enzyme a of in AS activity, of to in from cells was W.E. Scholar). in control cells was whereas activity in AS cells was only that expression and activity of AS protein was reduced as a result of AS siRNA of AS siRNA on endothelial NO AS in a of AS siRNA on AS AS in a of NO in the production of arginine from citrulline, catalyzed by the enzymes AS and AL, an essential source of arginine for NO production in endothelial a reduction of AS protein expression should result in a decrease in the of NO (13Xie L. Gross S.S. J. Biol. Chem. 1997; 272: 16624-16630Google Scholar, 14Xie L. Hattori Y. Tume N. Gross S.S. Semin. Perinatol. 2000; 24: 42-45Google Scholar, 15Sessa W.C. Hecker M. Mitchell J.A. Vane J.R. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8607-8611Google Scholar, 16Hattori Y. Campbell E.B. Gross S.S. J. Biol. Chem. 1994; 269: 9405-9408Google Scholar, 17Su Y. Block E.R. Am. J. Physiol. 1995; 269: L581-L587Google Scholar) despite saturating levels of extracellular To test AS is essential for endothelial NO AS stimulated with and in the presence of excess extracellular arginine and NO was shown in reduction in AS expression in a decrease in stimulated NO as compared with levels to the of cells by blue the decrease in NO be to a decrease in production for cell as to on cell viability. results demonstrate that levels of AS be to NO production and that arginine regeneration an essential role in stimulated NO production in endothelial cells even in the presence of excess AS in a of NO significant of AS on stimulated NO production in endothelial cells to basal levels of NO production in also reduced by AS siRNA with AS and control the with excess arginine and and for Cell was by blue exclusion and results are as cells. shown in a significant reduction in basal production of NO was in BAEC, whereas AS expression decreased by siRNA transfection. AS expression is also to unstimulated levels of NO by endothelial cells even in the presence of excess levels of extracellular in Cell in AS siRNA treatment of endothelial cells in a and decrease in cell viability control cells. To this AS cells for viability by blue shown in a decrease in cell was compared with To this cellular was as a of viability using the AS siRNA transfection. also a decrease in cell in endothelial cells reduction of AS protein the levels in this was not because of cell associated with concentrations of the AS in cells the of the media. used to this is the of the C. J. Scholar). treatment with AS and control siRNA, cell for activity in the media. Although treatment with AS siRNA in a loss of cell was significant in activity in the in AS cells as compared with the control results suggested that the loss of cell viability from AS depletion was not necrosis. in Bcl-2 in with AS blue exclusion as as the of suggested that was not the cell in cells with reduced AS expression, the of apoptosis was Bcl-2 is known to be an important protein in cell including endothelial that protects against apoptosis C. Mol. 2003; Scholar). To apoptosis for the loss of viability in AS from cells with AS and control and Bcl-2 expression was by Western shown in a significant decrease in Bcl-2 protein was in cells as compared with control with the that the loss of cell viability results from the of apoptosis. by of a in endothelial apoptosis-inducing was as an important of apoptosis that the for in the of apoptosis M. Biochem. Biophys. Res. Commun. 2003; Scholar). To levels of result from AS siRNA cell from AS and control cells to Western blot analysis to in protein expression. shown in was significant on levels from AS siRNA treatment that for the loss of cell viability by an in in of of the of Bcl-2 protein and the of on the of AS on caspase was is a that on of the is cleaved from an form to an AS was an increase in activity compared with control cells finding the that the reduction of AS expression results in of apoptosis. of AS with an NO NO production in endothelial cells has suggested to the of apoptosis S. J. M. Zeiher A.M. J. Exp. 1997; Scholar) by the of L. J. Hermann C. P. C. Zeiher A.M. Dimmeler S. J. Biol. Chem. 2000; Scholar) and by directly of L. B. J. Zeiher A.M. A. Dimmeler S. J. Biol. Chem. 1999; Scholar) To a reduction in basal NO production by AS correlated with the increase in treated with an NO donor with a of was to the following the with AS caspase activity was to the of the shown in AS apoptosis in treated endothelial cells was decreased by the of NO donor suggests that of apoptosis may be in to the reduction in basal NO production that results from AS although a of AS protein on apoptosis be of AS apoptosis with an NO with AS siRNA in following concentrations of the NO was to the media. activity was as an of NO production appears to be limited by the availability of arginine, despite extracellular and intracellular concentrations that are much higher than the reported Km for (13Xie L. Gross S.S. J. Biol. Chem. 1997; 272: 16624-16630Google Scholar, 14Xie L. Hattori Y. Tume N. Gross S.S. Semin. Perinatol. 2000; 24: 42-45Google Scholar, 15Sessa W.C. Hecker M. Mitchell J.A. Vane J.R. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8607-8611Google Scholar, 16Hattori Y. Campbell E.B. Gross S.S. J. Biol. Chem. 1994; 269: 9405-9408Google Scholar, 17Su Y. Block E.R. Am. J. Physiol. 1995; 269: L581-L587Google Scholar, 18Flam B.R. Hartmann P.J. Harrell-Booth M. Solomonson L.P. Eichler D.C. Nitric Oxide. 2001; 5: 187-197Google Scholar, 19Shuttleworth C.W. Burns A.J. Ward S.M. O'Brien W.E. Sanders K.M. Neuroscience. 1995; 68: 1295-1304Google the source of arginine to NO production in endothelial cells has and of this to be with the finding that the the of arginine for endothelial with in K.K. S. Block E.R. J. Biol. Chem. 1997; 272: Scholar). was suggested that endothelial NO production was extracellular of arginine K.K. S. Block E.R. J. Biol. Chem. 1997; 272: Scholar). was a because serum arginine levels from to the Km for evidence that endothelial NO production was limited by the capacity to arginine from citrulline (13Xie L. Gross S.S. J. Biol. Chem. 1997; 272: 16624-16630Google Scholar, 14Xie L. Hattori Y. Tume N. Gross S.S. Semin. Perinatol. 2000; 24: 42-45Google Scholar, 15Sessa W.C. Hecker M. Mitchell J.A. Vane J.R. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8607-8611Google Scholar, 16Hattori Y. Campbell E.B. Gross S.S. J. Biol. Chem. 1994; 269: 9405-9408Google Scholar, 17Su Y. Block E.R. Am. J. Physiol. 1995; 269: L581-L587Google Scholar, 18Flam B.R. Hartmann P.J. Harrell-Booth M. Solomonson L.P. Eichler D.C. Nitric Oxide. 2001; 5: 187-197Google Scholar, 19Shuttleworth C.W. Burns A.J. Ward S.M. O'Brien W.E. Sanders K.M. Neuroscience. 1995; 68: 1295-1304Google Scholar). has shown B.R. Hartmann P.J. Harrell-Booth M. Solomonson L.P. Eichler D.C. Nitric Oxide. 2001; 5: 187-197Google Scholar) that extracellular citrulline was as as arginine in NO even in saturating levels of arginine extracellular citrulline levels on intracellular arginine these results suggested that citrulline of NO production was the regeneration of arginine to NO production and be despite saturating levels of extracellular G. 2000; Scholar) that the synthesis of arginine from citrulline was stimulated by of citrulline. In this the that the function of citrulline recycling was to the to l-arginine to NO of evidence that the of arginine regeneration for NO production the of AS in cells (13Xie L. Gross S.S. J. Biol. Chem. 1997; 272: 16624-16630Google Scholar). a result of AS was a in the of the cells to produce NO that of despite saturating levels of extracellular (13Xie L. Gross S.S. J. Biol. Chem. 1997; 272: 16624-16630Google Scholar, 14Xie L. Hattori Y. Tume N. Gross S.S. Semin. Perinatol. 2000; 24: 42-45Google Scholar) that the capacity to citrulline back to arginine is to NO production. Y. Block E.R. Am. J. Physiol. 1995; 269: L581-L587Google Scholar) at a that in endothelial cells of AS by a the production of NO, of extracellular arginine was significantly Although on the role of recycling for endothelial NO production by eNOS, demonstrated the of recycling for NO production by both and Billiar T.R. S.M. J. Biol. Chem. 1994; 269: Scholar, A. M. Y. M. H. M. J. Biol. Chem. Scholar, S. H. M. Exp. Res. 2003; Scholar, S. M. A. Y. S. H. M. Res. Mol. Res. 2001; Scholar, Wang A.M. M. 2000; Scholar, J. Scholar, S. D.S. Snyder S.H. J. Biol. Chem. 1992; Scholar, H. J. 1995; Scholar, A.M. S. A. C. L. J. 1995; Scholar, J. Billiar T.R. Kim Y.M. Biochem. Biophys. Res. Commun. 1997; Scholar, S. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: Scholar). AS and are in Billiar T.R. S.M. J. Biol. Chem. 1994; 269: Scholar, A. M. Y. M. H. M. J. Biol. Chem. Scholar) as as in stimulated cells where the citrulline-NO cycle is shown to be S. H. M. Exp. Res. 2003; Scholar). In addition, of and AS in cells that both arginine by and recycling are important in the production of of NO in cells S. M. A. Y. S. H. M. Res. Mol. Res. 2001; Scholar). In of NOS, AS, and AL was in the evidence of a citrulline-NO cycle Wang A.M. M. 2000; Scholar). in the evidence of recycling is by of AS, AL, and J. Scholar). expression using DNA microarray demonstrated that a significant and up-regulation of AS gene expression in to shear of NO production by endothelial cells (21McCormick S.M. Eskin S.G. McIntire L.V. Teng C.L. Lu C.M. Russell C.G. Chittur K.K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8955-8960Google Scholar). extracellular arginine was not in these the that a for shear stress-induced NO in the of synthesis of eNOS, was an increase in arginine regeneration AS expression (21McCormick S.M. Eskin S.G. McIntire L.V. Teng C.L. Lu C.M. Russell C.G. Chittur K.K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8955-8960Google Scholar). In expression of AS in the capacity to the to NO production associated with the shear results evidence supporting the for the regeneration of arginine for NO production. AS expression was demonstrated to be and to both stimulated and levels of NO synthesis in endothelial cells. the finding that reduction of AS expression in endothelial cells in a decrease in cell viability was we that cell may be a of which result when is of arginine S. D.S. Snyder S.H. J. Biol. Chem. 1992; Scholar). production was by AS depletion not Moreover, because blue exclusion and not that was the of cell death, we the that these endothelial cells to apoptosis. NO has a role in cell or inhibit The of NO and the type of cell the NO has on cell viability S. Zeiher A.M. Nitric Oxide. 1997; 1: 275-281Google Scholar, 6Haendeler J. Zeiher A.M. Dimmeler S. Vitam. Horm. 1999; 57: 49-77Google Scholar). concentrations of NO shown to induce cell apoptosis. In a NO apoptosis to (4Melino G. Catani M.V. Corazzari M. Guerrieri P. Bernassola F. Cell. Mol. Life Sci. 2000; 57: 612-622Google Scholar, 5Dimmeler S. Zeiher A.M. Nitric Oxide. 1997; 1: 275-281Google Scholar, 6Haendeler J. Zeiher A.M. Dimmeler S. Vitam. Horm. 1999; 57: 49-77Google Scholar, 7Shen Y.H. Wang X.L. Wilcken D.E. FEBS Lett. 1998; 433: 125-131Google Scholar). In lower concentrations of NO shown to cells as endothelial cells S. J. M. Zeiher A.M. J. Exp. 1997; Scholar), H. J. 1995; Scholar), and A.M. S. A. C. L. J. 1995; Scholar) from apoptosis. In endothelial of NO by protects endothelial cells from apoptosis (8Kwon Y.G. Min J.K. Kim K.M. Lee D.J. Billiar T.R. Kim Y.M. J. Biol. Chem. 2001; 276: 10627-10633Google Scholar). are that in the of NO is by of the enzyme by NO L. B. J. Zeiher A.M. A. Dimmeler S. J. Biol. Chem. 1999; Scholar, J. Billiar T.R. Kim Y.M. Biochem. Biophys. Res. Commun. 1997; Scholar). In addition, Bcl-2 is by NO as as the of S. Zeiher A.M. Nitric Oxide. 1997; 1: 275-281Google Scholar, A.M. S. A. C. L. J. 1995; Scholar). RNA is an and the cell from the L. J. Hermann C. P. C. Zeiher A.M. Dimmeler S. J. Biol. Chem. 2000; Scholar). NO induce apoptosis by directly a decrease in the of the and of S. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: Scholar). In AS apoptosis was by the of an NO finding suggests that basal levels of NO in endothelial by the recycling of citrulline back to arginine, may against apoptosis. We that AS may be function in the cell that is apoptosis. The of AS as a protein S. J.R. A. S. J. Biol. Chem. 2001; 276: Scholar), the of AS by C. P. T.R. Proc. Natl. Acad. Sci. U. S. A. 2000; Scholar), and the of AS expression by the U. B. Proc. Natl. Acad. Sci. U. S. A. 2003; Scholar) an role of the enzyme in cell viability. The of endothelial cell viability basal NO production has important In are by reduced shear which with reduced NO synthesis S. J. M. Zeiher A.M. FEBS Lett. Scholar, M. M. S. M. M. S. G. A. Res. 1995; Scholar). endothelial cell because of of also these 17: Scholar). NO production by endothelial cells may a role in where endothelial cell apoptosis is by endothelial NO production S. Hermann C. Zeiher A.M. 1998; Scholar). of these however, demonstrate a from the of not of viability as we demonstrate in the current levels of NO may of viability by the in cell cycle cell in viability. decrease in endothelial NO production associated with endothelial is a in as and with arginine in has In arginine results in function by for and NO production S.M. J. 1992; Scholar, S.M. H. A. Scholar, P. A.J. J. A. D.S. J. Scholar). not an in and may S.M. G. P.J. J. Am. 2001; Scholar, Wang Arterioscler. Thromb. Vasc. Biol. Scholar, C.M. Arterioscler. Thromb. Vasc. Biol. 2000; Scholar, J. Arterioscler. Thromb. Vasc. Biol. 2003; Scholar). We demonstrate in this that a citrulline-NO cycle is essential for endothelial NO an increase in AS activity in endothelial cells may a for endothelial In NO production by endothelial cells an important role in the function of the and cell as Bcl-2 expression and caspase that endothelial NO synthesis is on the availability of a of arginine the of citrulline to arginine by the enzymes AS and