Evidence for the Pathophysiological Role of Endogenous Methylarginines in Regulation of Endothelial NO Production and Vascular Function

In endothelium, NO is derived from endothelial NO synthase (eNOS)-mediated l-arginine oxidation. Endogenous guanidinomethylated arginines (MAs), including asymmetric dimethylarginine (ADMA) and NG-methyl-l-arginine (l-NMMA), are released in cells upon protein degradation and are competitive inhibitors of eNOS. However, it is unknown whether intracellular MA concentrations reach levels sufficient to regulate endothelial NO production. Therefore, the dose-dependent effects of ADMA and l-NMMA on eNOS function were determined. Kinetic studies demonstrated that the Km for l-arginine is 3.14 μm with a Vmax of 0.14 μmol mg–1 min–1, whereas Ki values of 0.9 μm and 1.1 μm were determined for ADMA and l-NMMA, respectively. EPR studies of NO production from purified eNOS demonstrated that, with a physiological 100 μm level of l-arginine, MA levels of >10μm were required for significant eNOS inhibition. Dose-dependent inhibition of NO formation in endothelial cells was observed with extracellular MA concentrations as low 5 μm. Similar effects were observed in isolated vessels where 5 μm ADMA inhibited vascular relaxation to acetylcholine. MA uptake studies demonstrated that ADMA and l-NMMA accumulate in endothelial cells with intracellular levels greatly exceeding extracellular concentrations. l-Arginine/MA ratios were correlated with cellular NO production. Although normal physiological levels of MAs do not significantly inhibit NOS, a 3- to 9-fold increase, as reported under disease conditions, would exert prominent inhibition. Using a balloon model of vascular injury, ∼4-fold increases in cellular MAs were observed, and these caused prominent impairment of vascular relaxation. Thus, MAs are critical mediators of vascular dysfunction following vascular injury. In endothelium, NO is derived from endothelial NO synthase (eNOS)-mediated l-arginine oxidation. Endogenous guanidinomethylated arginines (MAs), including asymmetric dimethylarginine (ADMA) and NG-methyl-l-arginine (l-NMMA), are released in cells upon protein degradation and are competitive inhibitors of eNOS. However, it is unknown whether intracellular MA concentrations reach levels sufficient to regulate endothelial NO production. Therefore, the dose-dependent effects of ADMA and l-NMMA on eNOS function were determined. Kinetic studies demonstrated that the Km for l-arginine is 3.14 μm with a Vmax of 0.14 μmol mg–1 min–1, whereas Ki values of 0.9 μm and 1.1 μm were determined for ADMA and l-NMMA, respectively. EPR studies of NO production from purified eNOS demonstrated that, with a physiological 100 μm level of l-arginine, MA levels of >10μm were required for significant eNOS inhibition. Dose-dependent inhibition of NO formation in endothelial cells was observed with extracellular MA concentrations as low 5 μm. Similar effects were observed in isolated vessels where 5 μm ADMA inhibited vascular relaxation to acetylcholine. MA uptake studies demonstrated that ADMA and l-NMMA accumulate in endothelial cells with intracellular levels greatly exceeding extracellular concentrations. l-Arginine/MA ratios were correlated with cellular NO production. Although normal physiological levels of MAs do not significantly inhibit NOS, a 3- to 9-fold increase, as reported under disease conditions, would exert prominent inhibition. Using a balloon model of vascular injury, ∼4-fold increases in cellular MAs were observed, and these caused prominent impairment of vascular relaxation. Thus, MAs are critical mediators of vascular dysfunction following vascular injury. The biological significance of guanidino-methylated arginine derivatives has been known since the inhibitory actions of NG-methyl-l-arginine (l-NMMA) 3The abbreviations used are: l-NMMA, NG-methyl-l-arginine; NOS, nitric oxide synthase; eNOS, endothelial NOS; ADMA, asymmetric dimethylarginine; MGD, N-methyl-d-glucamine dithiocarbamate; BH4, tetrahydrobiopterin; BAEC, bovine aortic endothelial cell; PBS, phosphate-buffered saline; HPLC, high-performance liquid chromatography. on macrophage induced cytotoxicity were first demonstrated. It was subsequently realized that these effects were mediated through inhibition of NO release (1Hibbs Jr., J.B. Vavrin Z. Taintor R.R. J. Immunol. 1987; 138: 550-565PubMed Google Scholar). NO has been demonstrated as a critical effector molecule in the maintenance of vascular function (2Arnold W.P. Mittal C.K. Katsuki S. Murad F. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 3203-3207Crossref PubMed Scopus (1206) Google Scholar, 3Gruetter D.Y. Gruetter C.A. Barry B.K. Baricos W.H. Hyman A.L. Kadowitz P.J. Ignarro L.J. Biochem. Pharmacol. 1980; 29: 2943-2950Crossref PubMed Scopus (50) Google Scholar, 4Martin W. Villani G.M. Jothianandan D. Furchgott R.F. J. Pharmacol. Exp. Ther. 1985; 232: 708-716PubMed Google Scholar). In the vasculature, NO is derived from the oxidation of l-Arg, catalyzed by the constitutively expressed enzyme, eNOS (5Hecker M. Walsh D.T. Vane J.R. FEBS Lett. 1991; 294: 221-224Crossref PubMed Scopus (70) Google Scholar, 6Janssens S.P. Shimouchi A. Quertermous T. Bloch D.B. Bloch K.D. J. Biol. Chem. 1992; 267: 14519-14522Abstract Full Text PDF PubMed Google Scholar, 7Sessa W.C. Harrison J.K. Barber C.M. Zeng D. Durieux M.E. D'Angelo D.D. Lynch K.R. Peach M.J. J. Biol. Chem. 1992; 267: 15274-15276Abstract Full Text PDF PubMed Google Scholar). This endothelial-derived NO diffuses from the vascular endothelium into the smooth muscle cell layer where it activates soluble guanylate cyclase leading to smooth muscle relaxation (2Arnold W.P. Mittal C.K. Katsuki S. Murad F. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 3203-3207Crossref PubMed Scopus (1206) Google Scholar, 3Gruetter D.Y. Gruetter C.A. Barry B.K. Baricos W.H. Hyman A.L. Kadowitz P.J. Ignarro L.J. Biochem. Pharmacol. 1980; 29: 2943-2950Crossref PubMed Scopus (50) Google Scholar, 4Martin W. Villani G.M. Jothianandan D. Furchgott R.F. J. Pharmacol. Exp. Ther. 1985; 232: 708-716PubMed Google Scholar). In addition to its role in the maintenance of vascular tone, NO helps to maintain the anti-atherogenic character of the normal vascular wall. NO, in concert with various cell signaling molecules, has been demonstrated to maintain smooth muscle cell quiescence and as such, counteracts pro-proliferative agents, specifically those involved in the propagation of athero-proliferative disorders (8Cooke J.P. Oka R.K. Curr. Atheroscler. Rep. 2001; 3: 252-259Crossref PubMed Scopus (52) Google Scholar, 9Holm A.M. Andersen C.B. Haunso S. Hansen P.R. Scand. Cardiovasc. J. 2000; 34: 28-32Crossref PubMed Scopus (20) Google Scholar, 10Janero D.R. Ewing J.F. Free Radic. Biol. Med. 2000; 29: 1199-1221Crossref PubMed Scopus (48) Google Scholar, 11Jeremy J.Y. Yim A.P. Wan S. Angelini G.D. J. Cardiovasc. Surg. 2002; 17: 324-327Google Scholar, 12Le Tourneau T. Van Belle E. Corseaux D. Vallet B. Lebuffe G. Dupuis B. Lablanche J.M. McFadden E. Bauters C. Bertrand M.E. J. Am. Coll. Cardiol. 1999; 33: 876-882Crossref PubMed Scopus (56) Google Scholar, 13Sarkar R. Webb R.C. J. Vasc. Res. 1998; 35: 135-142Crossref PubMed Scopus (76) Google Scholar, 14Sarkar R. Gordon D. Stanley J.C. Webb R.C. Am. J. Physiol. 1997; 272: H1810-H1818Crossref PubMed Google Scholar). As such, eNOS dysfunction is an early symptom of vascular disease and is manifested through insufficient NO bioavailability. Several potential causes of NO deficiency in disease settings have been proposed. Among these, high circulating levels of the endogenous methylarginine NOS inhibitor asymmetric dimethylarginine (ADMA) has been hypothesized to be of particular importance (15Boger R.H. Sydow K. Borlak J. Thum T. Lenzen H. Schubert B. Tsikas D. Bode-Boger S.M. Circ. Res. 2000; 87: 99-105Crossref PubMed Scopus (472) Google Scholar, 16Boger R.H. Vallance P. Cooke J.P. Atheroscler. Suppl. 2003; 4: 1-3Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 17Dayoub H. Achan V. Adimoolam S. Jacobi J. M. Vallance P. Cooke J.P. 2003; PubMed Scopus Google Scholar, Bode-Boger S.M. J.C. H. R.H. Suppl. 2001; PubMed Google Scholar, M.J. Cardiol. 2003; PubMed Scopus Google Scholar, H. H. Cooke J.P. M. S. S. T. 1999; PubMed Scopus Google Scholar, T. M. Am. J. Physiol. PubMed Scopus (50) Google Scholar). In and the it has been that the arginine l-NMMA is the levels of methylarginine have not been in studies vascular dysfunction J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). ADMA and l-NMMA are endogenous inhibitors of NOS and are derived from the of arginine on various The is by a of to as J. Vallance P. Cardiovasc. Res. 1999; PubMed Scopus Google Scholar). of methylarginine to the release of methylarginine into the and sufficient levels are NO production from NOS would be inhibited Vallance P. J. Pharmacol. PubMed Scopus Google Scholar, P. A. A. J. S. 1992; PubMed Scopus Google Scholar). of the have been with various levels of and J. Vallance P. Cardiovasc. Res. 1999; PubMed Scopus Google Scholar, J. S. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). are subsequently by the dimethylarginine the of ADMA l-NMMA to and V. F. J.M. Vallance P. Circ. Res. 2002; PubMed Scopus Google Scholar, A. Adimoolam S. M. T. Cooke J.P. 1999; PubMed Scopus Google Scholar, A. V. M. K. M. T. Vallance P. 1997; Full Text PDF PubMed Scopus Google Scholar). In of the role of ADMA and l-NMMA as endogenous inhibitors of NOS studies have demonstrated that endothelial function through NO W. S. M. R.R. P.J. 1997; PubMed Scopus Google Scholar, R.H. Bode-Boger S.M. M. R. A. J.C. 1997; PubMed Scopus Google Scholar, Cooke J.P. Circ. Res. PubMed Scopus Google Scholar, Z. D. Z. J. Med. J. Google Scholar). This be to However, into that the intracellular levels of are the Km of the enzyme, NO would not be with J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). This of NO with has been the to is that the endogenous inhibitory actions of cellular (8Cooke J.P. Oka R.K. Curr. Atheroscler. Rep. 2001; 3: 252-259Crossref PubMed Scopus (52) Google Scholar, J. Vallance P. Cardiovasc. Res. 1999; PubMed Scopus Google Scholar, R.H. J. PubMed Google Scholar, P. J. Vasc. Biol. PubMed Scopus Google Scholar, D. R.H. J. Bode-Boger S.M. J.C. FEBS Lett. 2000; PubMed Scopus Google Scholar). However, as to whether normal physiological levels of these endogenous NOS the levels in significantly inhibit NOS vascular Therefore, studies were to endothelial methylarginine levels and the dose-dependent effects of cellular on NO formation and vascular eNOS eNOS was expressed in a and purified as M. V. A.L. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). eNOS were in and eNOS expressed in has a low was by of purified protein with was by through a of were by and under liquid aortic endothelial cells were from and in a EPR and of NO were a with as the J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, Free Radic. Biol. Med. 2000; 29: PubMed Scopus Google Scholar). the of purified eNOS in μm μm and 100 μm The were into a and EPR were the in a The and was used to of NO by cells were as and were on the of cells in cells were to and with NO The from cells to the of was and the cells were in and in of and of the NO was and the cells were with of the effects of on NO production were following a from cellular were the following and 100 of NO production was by the formation of J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). This is on the of NO with to and with an in were on and a were in 100 of of μm μm BH4, μm μm and μm of purified eNOS and was by the addition of of for the were a The in NO formation as by the for the first Km and Vmax values were derived the and Ki values were determined inhibitor concentrations with as a function of inhibitor and the for competitive inhibition J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). were from and in by a were with and on a and and were and an with was to the as an to for the of The of and with a of The following was with and with a from to J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, M. R. A. G. Biochem. 2003; PubMed Scopus Google Scholar). levels of and were determined from values derived from of the intracellular of M. W.C. Vane J.R. Proc. Natl. Acad. Sci. U. S. A. 87: PubMed Scopus Google Scholar). ADMA and l-NMMA uptake of ADMA was determined with of cellular were to in to uptake the cells were in and for in ADMA l-NMMA the the was and the cells were in the the cells were in 5 of and by 5 The was in of and a as l-Arg, ADMA, and l-NMMA levels were determined and relaxation of isolated were in an and with were into to and on a was a with for a the were to a of The of the aortic was determined by the by under The dose-dependent effects of ADMA on vascular relaxation were in the and of were with μm The vascular relaxation was determined concentrations of to were and The was in with and were with in The was and a balloon was an The was to the to a of and in a and The was and the was the vessels were and endothelial function was by vascular The the of Kinetic of eNOS studies were to the Ki for of the methylarginine NO formation was the as under Km and Vmax values were derived the and values of 3.14 μm and 0.14 The Ki values were determined inhibitor concentrations with as a function of inhibitor and the for competitive inhibition and The Ki for ADMA was determined to be 0.9 μm and for l-NMMA 1.1 μm. of ADMA and of NO studies were to the dose-dependent inhibition of ADMA and l-NMMA upon NO formation from eNOS. eNOS was in 100 μm as with ADMA l-NMMA and NO production was a from these studies demonstrated that ADMA inhibited NO formation with inhibition observed μm ADMA and a of inhibition μm ADMA Similar inhibitory effects were observed with l-NMMA, inhibition μm l-NMMA and μm methylarginine levels in endothelial cells and were to normal physiological ADMA and l-NMMA concentrations. demonstrated the levels of l-Arg, ADMA, and l-NMMA in to be μm μm and μm respectively. from in demonstrated l-Arg, ADMA, and l-NMMA levels to be μm μm and μm respectively. studies that, under normal physiological conditions, endogenous would exert effects on endothelial cell NO production. However, methylarginine levels have been to be 3- to 9-fold under various conditions, the effects of cellular ADMA and l-NMMA levels on endothelial NO production R.H. R. F. E. Chem. Med. PubMed Scopus Google Scholar). of ADMA and on NO studies were with the NO were in and 5 with The dose-dependent effects of ADMA and l-NMMA were demonstrated that ADMA inhibited NO with 5 μm ADMA inhibition and 100 μm ADMA, inhibition in the of inhibitory effects were the studies were in the of in the of levels ADMA a dose-dependent inhibition of NO production with inhibition observed 5 μm ADMA and inhibition 100 μm ADMA Similar were with l-NMMA, inhibition 5 μm l-NMMA and 100 μm l-NMMA, in the of In the of physiological levels of 5 μm l-NMMA inhibited NO production by whereas 100 μm levels in inhibition The inhibition observed with low concentrations of ADMA and l-NMMA in the of that the cells are to Therefore, methylarginine uptake studies were on and intracellular levels were of ADMA and by studies demonstrated that, cells were with ADMA l-NMMA in low methylarginine levels significantly inhibited NO would that are to and accumulate ADMA and l-NMMA the Therefore, studies were to intracellular ADMA and were to in that μm the dose-dependent effects of ADMA on the intracellular ADMA ADMA was to with and and the cells were to for demonstrated that, under these conditions, levels in the cell were μm and ADMA was μm. In the of μm ADMA, the intracellular of inhibitor to μm effects were dose-dependent and demonstrated a significant in intracellular ADMA μm μm was to the was the were in the of the addition of μm ADMA to the to an intracellular ADMA of μm. that ADMA is by endothelial cells and be an in NOS under where are that uptake studies were with were to those observed with ADMA and demonstrated that in the of extracellular l-Arg, were to l-NMMA with intracellular levels values the cell l-NMMA uptake was inhibited by that these effects are on the not l-NMMA uptake was inhibited by was to the that has a for l-NMMA Although the studies in the of do not physiological conditions, to and the intracellular of ADMA l-NMMA for extracellular Thus, by intracellular methylarginine and l-arginine levels following addition of were to NO production as a function of intracellular and that the for NO production from were the a to the inhibition from the of the isolated was observed and The was from the for competitive inhibition for the where is the that the of cellular NOS inhibition be on the the cellular ratios are of ADMA on from the isolated and cellular studies demonstrated dose-dependent eNOS to whether the effects of on eNOS physiological as vascular Therefore, vascular studies were and the dose-dependent effects of ADMA were were in the of ADMA and to for the the vessels were with and the relaxation to was The relaxation to μm was the and studies were in of extracellular that the intracellular ADMA as demonstrated that ADMA inhibited relaxation with a 5 μm The ADMA effects were μm a in vascular relaxation The studies were in the of physiological levels of demonstrated that ADMA inhibited relaxation with a μm ADMA and μm of on NO that methylarginine levels would be to inhibit endothelial NO it is whether the intracellular levels of ADMA and reach levels sufficient to significantly inhibit NOS conditions, 3- to 9-fold increases in ADMA levels have been reported R.H. R. F. E. Chem. Med. PubMed Scopus Google the to levels in the is Therefore, studies were a balloon model of vascular to the of vascular on the levels of the critical ADMA, and injury, from vascular demonstrated a in cellular MA with in ADMA and l-NMMA In addition to in MA levels a of in levels was in MA prominent impairment in vascular relaxation was with a of The NOS inhibitory of in ADMA and levels is through the of l-arginine to the relaxation to In the of l-Arg, the vessels in the of the relaxation to This was not observed in the and the of inhibition to that from the cellular inhibition these for the first that cellular MA are following vascular injury, and endothelial NO production with of and methylarginine levels and inhibitory effects on eNOS in a is a of ADMA as a in endothelial dysfunction and that ADMA is involved in the of a of including and (8Cooke J.P. Oka R.K. Curr. Atheroscler. Rep. 2001; 3: 252-259Crossref PubMed Scopus (52) Google Scholar, Bode-Boger S.M. J.C. H. R.H. Suppl. 2001; PubMed Google Scholar, J. Vallance P. Cardiovasc. Res. 1999; PubMed Scopus Google Scholar, R.H. Bode-Boger S.M. A. J.R. Cooke J.P. 1998; PubMed Scopus Google Scholar, H. Tsikas D. R.H. J. Pharmacol. Scopus Google Scholar). are with methylarginine levels with values as high as μm reported R.H. R. F. E. Chem. Med. PubMed Scopus Google Scholar). these methylarginine values levels sufficient to significantly NO production and vascular function was Therefore, to be to the of ADMA l-NMMA level on NO production from eNOS, the of the and the effects of the methylarginine NOS inhibitors were determined. Although the Vmax and Km of eNOS have been determined by the Ki values for the methylarginine NOS Although is that are competitive NOS is the Ki values D. R.H. J. Bode-Boger S.M. J.C. FEBS Lett. 2000; PubMed Scopus Google Scholar, Biochem. Res. 1991; PubMed Scopus Google Scholar, Biochem. PubMed Scopus Google Scholar). Therefore, of the of NO formation from eNOS as a function of and ADMA l-NMMA concentrations. The Km determined for was 3.14 μm with a Vmax of 0.14 Ki values of 0.9 μm and 1.1 μm were determined for the inhibitors ADMA and l-NMMA, respectively. are with of eNOS, have reported Km values from to μm and a Vmax of to P. J. L.J. W.C. Biochem. Res. PubMed Scopus Google Scholar, W.C. Barber C.M. Lynch K.R. Circ. Res. PubMed Scopus Google Scholar). The observed reported Ki values for l-NMMA and those be to the in the used for the studies Biochem. PubMed Scopus Google Scholar). EPR were to the dose-dependent inhibitory effects of ADMA and l-NMMA on NO production from eNOS. Although on the Ki values it would be that would be inhibition of in of the physiological effects of these endogenous NOS it was to whether is NOS is by inhibition to NO to the and be by it is to of NO formation ADMA and l-NMMA demonstrated dose-dependent inhibition of eNOS in the of physiological levels of l-Arg, and inhibition was ADMA and l-NMMA inhibition μm with inhibition μm. from these studies would that the normal cellular levels of ADMA and l-NMMA, to be μm and would have on endothelial NO production. It has been that in endothelial cells the of the l-Arg, in the of NO from eNOS S. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar, A. A. U. M. Circ. Res. 2003; PubMed Scopus (90) Google Scholar, J.R. Biochem. PubMed Scopus (56) Google Scholar). the NO from eNOS and its inhibition by studies were in endothelial cells to the critical cellular methylarginine levels to NOS inhibition. were in demonstrated that the extracellular concentrations of ADMA and l-NMMA were to 5 NOS was inhibited by were in the of physiological levels of and methylarginine effects on NO production were The NOS inhibitory effects of ADMA and l-NMMA on derived NO were in the of extracellular ADMA a dose-dependent inhibition of NO with inhibition observed with μm extracellular ADMA and inhibition with 100 μm extracellular ADMA In the of physiological levels of l-Arg, μm l-NMMA inhibited NO production by whereas 100 μm levels in inhibition. The of inhibition observed in the cellular studies was that on studies and that the cells are to In Vallance P. Am. J. Physiol. PubMed Google demonstrated that are through the As a of extracellular methylarginine levels would be to cellular levels of these NOS inhibitors and a for NOS inhibition. Therefore, studies of methylarginine uptake by were ADMA and demonstrated that were to with the intracellular methylarginine levels values the studies were in the of physiological methylarginine uptake was inhibited by in the of that protein has a for ADMA and This have physiological the methylarginine levels in disease J. Vallance P. Cardiovasc. Res. 1999; PubMed Scopus Google Scholar, R.H. Bode-Boger S.M. A. J.R. Cooke J.P. 1998; PubMed Scopus Google Scholar, H. Tsikas D. R.H. J. Pharmacol. Scopus Google Scholar, R. S. E. C. F. D. C. F. T. Circ. Res. PubMed Google Scholar). on methylarginine levels would in uptake and cellular levels of these endogenous NOS that the dimethylarginine is the for methylarginine and as such, of of would be to in significantly cellular methylarginine levels H. Achan V. Adimoolam S. Jacobi J. M. Vallance P. Cooke J.P. 2003; PubMed Scopus Google Scholar, V. F. J.M. Vallance P. Circ. Res. 2002; PubMed Scopus Google Scholar, J. Sydow K. G. H. B. M. Cooke J.P. PubMed Scopus Google Scholar, M. Sydow K. F. Jacobi J. R.C. Cooke J.P. PubMed Scopus Google Scholar). In intracellular of ADMA has been as a in the of a of through the impairment of the normal physiological of NO P. A. A. J. S. 1992; PubMed Scopus Google Scholar, P. J. Vasc. Biol. PubMed Scopus Google Scholar, R.H. R. F. E. Chem. Med. PubMed Scopus Google Scholar, S. R. M. G. PubMed Scopus Google Scholar). However, the levels of endogenous required for NOS inhibition. on have not been on cellular and that have been by S. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar, A. A. U. M. Circ. Res. 2003; PubMed Scopus (90) Google Scholar, J.R. Biochem. PubMed Scopus (56) Google Scholar). Therefore, in an to these NO production from endothelial cells and these values as a function of the intracellular The intracellular was by the addition of extracellular ADMA these were the for competitive a was observed that cellular inhibition would be from the on the Ki values for ADMA and l-NMMA, that under normal physiological endogenous methylarginine levels would inhibit NO production by This studies ADMA in the arginine The arginine is a in with vascular function the that endothelial concentrations are to the However, as the endogenous NOS the addition of would be to and NO bioavailability. the importance of ADMA and l-NMMA in NOS and a role for in the arginine In addition to these methylarginine levels are known to in a of are studies that under methylarginine levels are by 3- to 9-fold P. J. Vasc. Biol. PubMed Scopus Google Scholar, R.H. Bode-Boger S.M. A. J.R. Cooke J.P. 1998; PubMed Scopus Google Scholar, H. Tsikas D. R.H. J. Pharmacol. Scopus Google Scholar, R. S. E. C. F. D. C. F. T. Circ. Res. PubMed Google Scholar, M. Sydow K. F. Jacobi J. R.C. Cooke J.P. PubMed Scopus Google Scholar, S. R. M. G. PubMed Scopus Google Scholar, R.H. Bode-Boger S.M. Sydow K. D.D. Vasc. Biol. 2000; PubMed Scopus Google Scholar, A. T. Adimoolam S. M. H. G.M. Cooke J.P. 2002; PubMed Scopus Google on would be to inhibit NO from eNOS by The for inhibition be to studies cellular methylarginine levels in disease and of the of NOS inhibition. from the isolated and cellular studies demonstrated dose-dependent eNOS inhibition. The to was whether the through effects on NOS, vascular studies were and the dose-dependent effects of ADMA were demonstrated that, in the of extracellular l-Arg, ADMA inhibited relaxation with inhibition 5 μm ADMA and inhibition μm In the of the ADMA effects were inhibition μm and μm these studies that NOS inhibition the relaxation to effects were dose-dependent and that with levels of 100 μm intracellular methylarginine levels μm to a significant on physiological Although these methylarginine levels physiological studies that levels sufficient to NOS be under where ADMA levels are P. A. A. J. S. 1992; PubMed Scopus Google Scholar, P. J. Vasc. Biol. PubMed Scopus Google Scholar, R.H. R. F. E. Chem. Med. PubMed Scopus Google Scholar). As ADMA levels have been reported to to 9-fold in a of However, these studies do not the intracellular levels of these endogenous endothelial cells dimethylarginine and are to it is whether intracellular levels sufficient to inhibit eNOS are Therefore, studies were to intracellular ADMA and levels following vascular injury. In a model of balloon injury, studies were to whether intracellular methylarginine levels accumulate to values sufficient to inhibit endothelial NOS and observed that was a significant impairment in vascular relaxation with a in ADMA and levels with a in The NOS inhibitory of in methylarginine levels was by the of l-arginine to the relaxation to acetylcholine. The of to relaxation is to NOS of NOS inhibition in to the injury. for the first that intracellular are under and reach levels sufficient to inhibit NOS function and In that the ADMA and l-NMMA, inhibit NO production from isolated and cellular The from these studies to the vasculature, demonstrated impairment of vascular relaxation. of intracellular methylarginine levels that normal physiological concentrations would have a on NOS However, under as vascular and where endogenous are observed NOS inhibition with endothelial This is the first that cellular are under and reach concentrations sufficient to inhibit NO production. these a in of the and role of in