A Tale of Two Molecules: Nitric Oxide and Asymmetric Dimethylarginine Ken ¥Lin'? and Shik-Chung Lin! Despite monumental progres in bath molecular and interventions cardiology, atherosclerosis remains the leading cause of motlity and morbidity inthe US, claiming more lve than te next 7 causes of morality combined, Not only is this chronic disease the beginning of many faiardownstram clinical manifestation suchas acute myocardial infarction, bt esearch ha demonstrated tha the pathogenesis of atherosclerosis sina inked to many other cardiovascular abnormalities, from hypethomoeysteinemia to diabetes melius Inflammation and cuidatve sven, fr intane, ere commen. medisir for tess deans. The inferceltedoes bas elicited noch interes in fading a molecular marker with both prognostic and diagnostic valucs.Asymmevic dimethylarginine (ADMA) is one such molecule, recently gaining wider acceptance as a novel cardiovascular risk factor. As an endogenous competitive inhibitor of ntric oxide synthase (NOS), ADMA is levate in the plain of patients with typerbotesteolemi, hypertension, hypertomocyaeinenia,hypenglycemia, an ion ceitnce, Not ony i plasma ADMA concentration predictive of extdiovascular evens, ADMA reduees the production of the 2nt-inflammatory and an-atherogeic molecule nitric oxide. As such, ADMA is both disease maker and a ‘metstoe, whotepethophysilogy merits tention, Thistle reviews te cutent ko vdedge of ADMA wii the larger context of the NOS pathway. Key Words: The endothelium ‘The endothelium is a monolayer of cell that invests the Iumen of all blood vessels. The normal endothelium acts like a Teflon coat, preventing circulating blood ele- ments from adhering to the vessel wall. Strategically stationed between the smooth muscle cells and the circu- lating blood, endothelial cells play a seminal role in the intricate signal pathways that network our circulation to local tissues. To maintain these functions, endothelial cells synthesize and respond to a plethora of molecules. ‘Most famous among them in the past decade has been, Received: February 16, 2004 Accepted: June 4, 2004 'Division of Cardiovascular Medicine, Sunford University School of “Medicine, Stanford, CA, USA. ’MD-PhD Program, Harvard Medical School, Boston, MA, USA. ‘Lin Heart Cline, Tainan, Taiwan, ‘Address correspondence and reprint requests to: Shil-Chung Lin, MD, FAC, FCCM, President, Lin Heart Clini 403, Soe. 2, Min ‘Sheng Road, Tainan, 703, Taiwan, Tel: 886-6-228.0808, E-mail: ken_lin@stadent hms hervard.edu 201 Nitric oxide + Asymetric dimethyl-arginine # Endothelium © Nitric oxide synthesis (NOS) nitric oxide (NO), Nitric oxide ‘The pioneering work that led to the identification of NO as the endothelium-derived relaxing factor (EDRF) ‘was no less celebrated than the molecule itself. As a parasympathetic molecule, acetylcholine was known to relax vascular smooth muscles, causing vasodilation. In- triguingly, when a vessel ring was removed for ex vivo studies in which acetylcholine was added, @ contradic- tory vasoconstriction was observed in the vessel section, Moreover, vascular constriction was observed if acetyl- choline was added directly to vascular smooth muscle cells. Robert Furchgott and colleagues explained these observations with a hypothetical EDRF, which was re- leased from endothelial cells upon their stimulation by acetylcholine. Subsequent chemical characterization of EDRE by the laboratories of Louis Ignarro and Ferid Murrad ted to the striking conclusion that NO, a com- Acta Cardio Sin 2004:20:201-11Ken ¥ Linet al mon air pollutant, is indeed the EDRF. For their contributions, Furchgott, Ignarro, and Murrad were awarded the Nobel Prize in Physiology and Medicine in 1998, Because its nitrogen has an unpaired electron, NO is a free radical. As such, its in vivo half-life is on the order of microseconds, the molecule being rapidly oxidized to nitrate (NO;"). This short half-life gives NO just enough time to diffuse from the endothelium, where the gas is produced, to the neighboring smooth muscle cells, where the molecule induces its vasodilatory effect. Reduced NO bioavailability occurs through 2 mechanisms: decreased production and/or inereased degradation. In the former, nitric oxide synthase (NOS) makes less NO because of re- duced substrate, cofactors, the enzyme itself, and/or inereased antagonists. In the latter, oxidative stress, char~ acterized by an increase in superoxide anion (07), quenches NO, turning it into peroxynitrite (ONOO’), which is then oxidized to the inactive nitrate. Decreased NO bioavailability is the defining feature of endothelial dysfunction. In patients with atherosclero- sis, endothelial dysfunction, indicated by reduced forearm blood flow following acetylcholine infusion, ccan be observed before any morphological changes typi- ‘eal of atherosclerosis. NO inhibits monocyte adhesion,! platelet aggregation,”* and vascular smooth muscle pro- liferation, all of which are key pathological processes in GTP ————+ cGMP —> Ge Adauatesustate evel | Arginine or oO, - Reduced substrate level atherosclerosis. As such, deficiency of NO increases vascular resistance and promotes atherogenesis; it also plays a key role in the progression of a constellation of cardiovascular abnormalities, including diabetes mellitus,°* hypercholesteralemia,’ and hypethomocysteinemia,"” Nitric oxide synthesis ‘NO is converted from L-arginine by the enzyme NOS. ‘The reaction involves the transfer of electrons from NADPH, via the flavins FAD and FMN from the reductase region (C-terminal) of NOS to its oxidase region (N-termi nal), where L-arginine is converted to L-citrulline and NO." Other cofactors in this enzymatic reaction include calmodulin, zinc, and tetrahydrobiopterin. In conditions where the substrate and/or cofactors become limited, the electrons obtained from oxidizing NADPH would be di rected to oxygen, instead of L-arginine. Subsequently, superoxide anion, rather than NO, is produced, Under this condition, NOS is said to be “decoupled,” in the sense that the enzyme’s reductase is short-circuited from the oxidase region by molecular oxygen. In other words, depending on substrate availability, NOS would produce either NO or superoxide anion (Figure 1). Upon its release from NOS, NO diffuses across the en dothelial cell membrane and travels to the neighboring smooth muscle cells, where the gas activates soluble ‘guanylate cyclase (SGC). The cytosolic sGC converts gua- ‘Smooth muscle relaxation NO oe "} onoo-+ No, (inactive) ADMA —————> Ctrutine + bimetnytamine Figure 1. The nitric ovide synthase pathway: NOS generates either NO or superoxide anon, depending onthe substrate aval. NO stimulates the enzyme soluble guanylate eyelase (sGC), which generates COMP, leading to vascular relaxation, Superoxide anion quenches NO and inactivates itto irae. ADMA san endogenous inhibitor of NOS and site degraded DAH and thereby elevates the level of ADMA and reduces NO production. eta Cardiol Sin 2004;20:201-11 202 bythe enzyme DAH, Superoide anon lower the enzymatic activity ofNitric Oxide and Asymetric Dimethyl-Arginine nine triphosphate (GTP) to cyclic guanine monophosphate (CGMP), which triggers @ phosphorylation eascade that leads to the relaxation ofthe smooth muscle cells, ‘There are three isoforms of NOS; namely, neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial ‘NOS (eNOS). Both nNOS and eNOS are constitutively expressed, with the latter predominantly found in the cardiovascular system.'™" It must be noted that the ex- pression of eNOS is nonetheless subject to change, A variety of agents can augment eNOS expression. These include cyclosporin, glucose at high concentration, low-density lipoprotein at low concentration, shear stress, and estrogen, In fact, the promoter region of the eNOS gene contains an estrogen response element, an observation that could in part explain estrogen’s cardioprotective effect Increase in endothelial intracellular calcium concen- tration following agonist stimulation (such as acetylcholine) activates eNOS because the enzyme requires the cal- cium-bound calmodulin as a cofactor. Indeed, the calcium-dependency of €NOS was confirmed by studies in which NO production and eNOS activity were both abrogated after calcium removal.''* iNOS, on the other hhand, is ealeium-independent because it binds tightly to calmodulin, and the binding interaction is irreversible; its regulation henceforth depends almost exclusively on the transcriptional level, for which the enzyme receives the term “inducible.” The arginine paradox NOS is an intracellular enzyme, Half-saturating arginine concentration (K,.) was reported as 1.4 uM for nNOS, 2.83 uM for iNOS, and 2.9 uM for eNOS." Freshly isolated endothelial cells have been found to,con- tain up to 2 uM of arginine.”” Given that the intracellular arginine concentration in endothelial cells is hundred-fold higher than the enzyme's Ke, eNOS should always be sat- urated. In other words, an increase in the substrate arginine's concentration would not increase the produc- tion of NO. Intriguingly, numerous studies have indicated that intravenous or oral supplementation of arginine in humans augments endothelial NO production (a list of| these studies can be found in the study by Flam et al)."* Furthermore, in hypercholesterolemic humans and animals, in whom NO-mediated endothelium-dependent vasodilation is impaired, arginine supplementation was 203 found to improve endothelial function by restoring NO production." This phenomenon is generally known as the arginine paradox, So far, no conclusive studies have provided a satisfactory explanation. Recently, intracellular localization has been hypothesized to provide an insight into the paradox. NOS localization The.cell biology of eNOS is fascinating, and the field is still growing, eNOS is an N-myristoylated pro- tein, N-myristoylation is a post-translational process whereby a fatty acid chain is covalently attached to a protein, making it membrane-bound. This modification, is important for subcellular targeting of proteins to dis- crete microdomains of cells. Confocal imaging revealed that eNOS is distributed on the cytoplasmic face of Golgi and caveolae, Caveolae are specialized 50-100 nm flask-shaped invaginations of the plasma membrane. Given that eNOS is localized, the aforementioned intracellular arginine concentration, which is an average ‘over the entire cell, might not necessarily reflect the lo- cal arginine concentration in the enzyme’s vicinity. Emerging evidence, derived from our accumulating un- derstanding of caveolae, suggests that this is a valid speculation, ‘The phospholipids that typically decorate the plasma membrane are virtually absent in caveolae, which are enriched in certain lipid cholesterols such as glycosphingolipids and structural proteins such as caveolin, The fatty acid chains of these lipids tend to be extended and unsaturated, rendering the lipids more tightly packed. Their tightness gives caveolae a higher ordered state in a typically fluid membrane. Accumulat- ing evidence suggests that caveolae are sites where many signal transduction pathways converge, because many signaling molecules, including eNOS, are found in this specialized membrane region. Of note, colocalized with eNOS in caveolae is the y" transporter, a protein that transports cationic amino acid, including arginine, into the cell.” Also clustered around eNOS are argininosuccinate synthase and argininosuecinate lyase, which are enzymes that convert L-citrulline, a by-prod- uct of NO production, back to L-arginine, the substrate for NOS.” In fact, a recent study showed that infusion of L-citrulline could also enhance NO production, mim= icking the effect of arginine infusion. Colocalization of Acta Cardiol Sin 2004;20:201-11Ken ¥ Lin etal these proteins with NOS suggests that the local arginine level around NOS could be highly regulated. Accord- ingly, the total intracellular arginine concentration should be cautiously interpreted. Nevertheless, the hypothesis that eNOS localization answers the arginine paradox raises one critical question. Membrane-bound eNOS is inactive. In caveolae, eNOS is, associated with the protein caveolin-1, which prevents calmodulin from binding to eNOS when intracellular cal- cium concentration is low. The activation of eNOS requires, in addition to a rise in calcium level, phosphorylation of the enzyme by a serine kinase, and the enzyme’s depalmytoylation. This process releases eNOS from the membrane, That eNOS is inactive when localized and that the enzyme makes NO only when it leaves caveolae raise a question regarding the extent to which eNOS lo- alization contributes to the arginine paradox. Further experiments are needed to answer this question, Here is ‘an example using cell culture: Dr. William Sessa’s labo- ratory at Yale University has developed eNOS mutants that are kinetically indistinguishable from the wildtype.” However, the mutants cannot be myristoylated; hence, they remain cytosolic. After transfecting these mutants, to endothelial cells, we could expose the cells to patho- logical stimuli that are known to reduce NO production (stimulus such as oxidized LDL). We could then apply arginine to the medium to determine whether increasing, the substrate would lead to greater amount of NO. If arginine fails to restore NO production in the transfected. cells, we can conclude that eNOS localization does not, contribute to the arginine paradox. ‘ADMA in cardiovascular diseases While the precise role of eNOS localization in the NO pathway still remains unsettled, another mechanism for endothelial dysfunction in general, and arginine para- ox in specifi, focuses on asymmetric dimethylarginine (ADMA). As an endogenous, intracellular competitive antagonist ofall isoforms of NOS,' ADMA ean reduce NO synthesis if its concentration increases. In other words, the level of ADMA could serve as a biochemical marker for NO availability. Given that NO deficiency is the defining feature of endothelial dysfunction — the crit- ial step in the onset of many cardiovascular diseases ~ ADMA appears a promising candidate for cardiovascular risk evaluation, Acta Cardio Si 2004;20:201-11 208 ‘Accumulating evidence from both basic and clinical investigations supports this concept. Vallance and col- leagues were among the first to document ADMA’s clinical significance, They noted that in patients with end-stage renal disease, plasma ADMA accumulate due to reduced renal clearance.”* Reduced clearance of ADMA in renal failure is associated with endothelial vasodilator dysfunction, reversible by administration of L-arginine or by dialysis, which removes plasma ADMA.* In subsequent years, several laboratories independ- ently demonstrated and confirmed the correlation between elevated plasma ADMA and nearly all major cardiova cular risk factors such as aging,”* hypertension,””"* diabetes,” insulin resistance," hypercholesterolemia, hypertriglyceridemia,”” and hyperhomocysteinemia."” In hypercholesterolemic humans, elevated plasma ADMA inversely correlated with endothelial-dependent vasodilation in the forearm vasculature. Consistent withthe idea of a competitive antagonism between ADMA and L-arginine, intravenous infusion of L-arginine restored endothelial function and increased urinary nitrate excretion, which served as a surrogate parameter of NO production. Plasma ADMA levels are also associated with other cardiovascular complications such as stroke,** conges- tive heart failure,” and peripheral arterial disease (PAD)." The level of ADMA was found to correlate with the severity of atherosclerotic disease in patients with PAD, Intravenous infusion of L-arginine both re- duced pain and improved walking distance. In addi ADMA levels have been shown to correlate with carotid intima-media thickness in patients with end-stage renal disease,” and more strikingly, in a group of 116 appar- ently healthy individuals.” Most recently, plasma concentrations of ADMA wwere reported a strong predictor of coronary events in non-smoking male subjects with a history of coronary heart disease.” Of note, patients in the upper quartile of ADMA plasma levels had a 3.9-fold enhanced risk of an Acute coronary event. Furthermore, Zoccali and colleagues recently pub- lished a large, prospective clinical study of 225 patients with end-stage renal disease undergoing hemodialysis (mean duration of hemodialysis 42 months).”” In this study cohort, both fatal and non-fatal cardiovascular events were recorded during a mean follow-up period of 33.4 months. Strikingly, plasma ADMA levels emerged ion,Nitric Oxide and Asymetric Dimethyl-Arginine as the second strongest predictor ofall causes of mortal- ity after age, outweighing conventional risk factors such as hypertension, diabetes, hypercholesterolemia, and smoking Commensurate with these human studies, animal and cell-culture works not only confirm the association between elevated ADMA and reduced NO, but also es- tablish the causal relation between the 2 molecules. Exogenously added ADMA in concentrations between 1 and 10 mol/L dose-dependently reduces endothe- lium-dependent NO-mediated vasodilation in isolated rat mesenteric vessels” and cerebral vessels,™ inhibits NO production by cultured macrophages, and increases en- dothelial adhesiveness to monocytes." ‘ADMA is arguably the newest addition to an already sizable list of cardiovascular risk markers such as fibrinogen, LDL, triglyceride, interleukin-6, von Willebrand factor, plasminogen activator inhibitor-1 and C-reactive protein, However, unlike some of these markers, ADMA is more than a marker; it plays a pivotal role in cardio- vascular physiology by regulating the amount of NO synthesized. Hence, understanding the regulation of plasma ADMA elevation could lead to novel therapeutic approaches and treatment modalities. Presently, the ‘mechanism of increased ADMA level is not fully charac~ terized, Plasma ADMA level reflects a dynamic balance between the molecule’s generation and its degradation. [As such, inereased ADMA is a consequence of increased ‘generation and/or decreased clearance, ADMA generation In endothelial cells, ADMA is generated from the proteolysis of ubiquitous proteins containing methyl- ated arginine residue. In the nucleus, transcriptional regulators are often methylated by enzymes called methy! transferases. In particular, protein arginine methyl transferase (PRMT) transfers methyl group to one of the guanidino nitrogens on an arginine residue Either one or two methyl groups can be added to arginine, subsequently resulting in monomethylarginine (NMMA) and dimethylarginine., respectively. In the case of dimethylarginine, both methyl groups can oc- cupy the same guanidino nitrogen, giving rise to asymmetric dimethylarginine (ADMA). Alternatively, the 2 methyl groups can be distributed symmetrically such that each guanidino nitrogen is only methylated NH NH: 4 AR, ~ a NH cu, NO ONA ome Y oe an Figure 2. Structure of arginine and endogenous methylarginines. Arginine is the substrate for nitric oxide synthase. Asymmetric dimethylarginine (ADMA) and Monomethylarginine (NMMA) are competitive inhibitors of NOS, while symmetric dimethylarginine (SDMA) ie inactive toward the enzyme. once. This configuration gives rise to symmetric dimethylarginine (SDMA).” Both NMMA and ADMA. block the activity of NOS; SDMA, however, is inactive to NOS, presumably because its guanidino group is too large to fit into the active site of NOS (Figure 2) ADMA clearance Renal excretion is one common way by which ADMA, NMA, and SDMA are removed."! This observa- tion might in part explain the rise in plasma ADMA in patients with renal failure. In that particular context, the kkidney acts as a sink for ADMA. Once the sink has mal- functioned, ADMA accumulates in the source - the endo- thelial cells - where it reduces the production of nitric oxide, The degradation of ADMA and NMA, but not SDMA, occurs via an additional pathway. The enzyme dimethylarginine dimethylaminohydrolase (DDAH) me- tabolizes ADMA to dimethylamine and citrulline. Currently, two DDAH isoforms have been identified. DDAH-I is found mostly in neural tissue, while DDAH-II predominates in peripheral and vascular tis sues, DDAH is an intracellular protein. Its distribution does not seem to be localized within any intracellular ‘compartments, nor is the enzyme membrane-bound. In- terestingly, red blood cells also contain DDAH, suggesting a possible role of erythrocytes in regulating, 20s Acta Cardio Sin 2004;20:201-11Ken ¥ Lin etal plasma level of ADMA. ‘The role of DDAH in a variety of disease states has been intensely investigated *"" Given the enzyme’s role in removing ADMA, reduction of DDAH enzymatic ex- pression or activity would elevate ADMA and subsequently reduce NO — the hallmark of endothelial dysfunction Indeed, John Cooke’s laboratory has shown that an impair- ment of DDAH activity almost always accompanies a rise in plasma ADMA. In fact, a 30-50% reduction in DDAH en- zyme activity was observed in hyperhomocysteinemia, hypercholesterolemia, and hyperglycemia, both in vitro and in vivo. In several papers, Cooke's colleagues'have also shown a strong negative correlation between DDAH, activity and plasma ADMA level.* Taken together, this line of evidence suggests that DDAH plays a key role in regulating the concentration of ADMA. As such, it be- comes imperative to identify how DDAH activity is lowered. DAN is an oxidant-sensitive enzyme. At its active site, a cysteine residue initiates the first chemical reac~ tion that leads to formation of the enzymatic reaction intermediate, This cysteine residue 249 is vulnerable to oxidative attack from many intracellular oxidants, in- cluding homocysteine and superoxide anion (0;)) An imbalanced cellular oxidative state might oxidatively ‘modify the cysteine residue of DAH. In the case of homocysteine, a disulfide bond actually forms between the oxidant and the cysteine residue of DDAH."® This and other oxidative interaction with the cysteine residue drastically lowers the activity of DDAH. Administration of antioxidants such as pyrollidine dithiocarbamate and polyethylene glycol conjugated superoxide dismutase have been shown to restore the enzymatic activity of DDAH in endothelial cell culture.*"” There is already a sizable list of pathways activated by oxidative stress that render cells in a pro-inflamma- tory state, The vulnerability of DDAH to oxidative stress presents another mechanism whereby oxidative stress aggravates endothelial dysfunction. Pathological condi- tions that elevate oxidative stress lower the activity of DDAK, leading to higher cellular and plasma levels of ADMA, which in turn reduces the production of NO. ADMA assay in biological samples Literature on ADMA has been on a rapid rise over the last few years. Concurrent with our increasing appre- Acta Cardiol Sin 2004;20.201-11 206 ciation of this molecule’s clinical importance rests the question of how to quantify ADMA. There are two chal- lenges that any ADMA assay must overcome. First, the detection must be sufficiently sensitive to distinguish ADMA from its endogenous isomer, SDMA. Second, the assay must possess sub-micromolar sensitivity, because plasma level of ADMA is usually in the range of 0.5 to 3 uM. Virtually all of the laboratories around the world currently investigating ADMA measure the molecule us- ing high pressure liquid chromatography (HPLC).*“* Briefly, the method involves pre-concentration of ADMA using solid-phase extraction, with a strong cat jon exchange column (available from Varian Inc., Palo Alto, USA). Solid-phase extraction removes most of the chemical components from the sample (which could be plasma, urine, ot culture medium), leaving only moder- ately basic compounds, which include ADMA, SDMA, and arginine.* The concentrated sample is labeled with the fluorochrome, o-phthaldialdehyde or naphthalene-2, 3-dicarboxaldehyde.”” After the labeling (or the “derivatization”), components within the sample are separated using reverse-phase HPLC with @ nucleosil phenyl column, A fluorescence detector then quantifies each sample component as it exits the column. HPLC separates compounds based on their structural dissimilarity, and the time a compound takes to exit the ‘column is in part determined by its relative hydrophobicity compared to other compounds present in the sample, Be- ‘cause ADMA is among the most hydrophobic chemicals in the concentrated sample, it is among the last com- pound to exit the column. The literature reports an elution time between 20 to 50 minutes for ADMA. The total time of the assay also includes the time spent on solid phase extraction and on cleaning the column after each sample run. In sum, one sample takes at least 40 ‘minutes to process. As such, HPLC is very time- and la- bor-intensive, and is prone to human and mechanical errors HPLC's intrinsic variability notwithstanding, there is no unified and consistent protocol of measuring ADMA using HPLC. Different mobile phases, column types, and derivatization times are usually employed in various laboratories. As such, there is often a significant difference between the control groups from 2 differentNitric Oxide and Asymetric Dimethyl-Arginine laboratories A study, initiated by Dr. Boger from Germany, is, currently under its way to collect data from the major ADMA laboratories around the world and to compare their differences, Results from this study would certainly stimulate more communication and collaboration among laboratories investigating ADMA. Other methods to quantify ADMA have also been published and proposed. A French group has reported successful separation and quantification of ADMA and SDMA using laser-induced fluorescence capillary elec- trophoresis (LIF-CE).“* This technique separates compounds based on difference in their charge-to-mass ratio. However, ADMA and SDMA have the same mo- lecular mass, and their charge difference under any pH is so subtle that separation would be difficult under any buffer system. In fact, 2 groups from Stanford University have failed to measurg ADMA using the reported LIF-CE protocol, but are currently working on a modi- fied protocol that introduces organic additive into the buffer system to enhance the electrochemical difference among the chemicals (unpublished data). Several private firms have also started developing immunoassay for de- tecting ADMA. Such an approach relies on an ADMA-specific antibody to identify and quantify the molecule. If realized, this technique would enable ADMA detection using enzyme-linked immuno-sorbent assay (ELISA), which offers both high sensitivity (at the level of nanomole) and high throughput (96 samples in a few seconds). The field of ADMA is a wonderful example of how interdisciplinary approach could accelerate and catalyze progress in today’s medicine, The number of analytical chemistry laboratories looking at ways to improve ADMA measurement is growing as rapidly as the num- ber of clinical and basic laboratories investigating the pathophysiology of ADMA. These trends only under- score ADMA’s diagnostic and prognostic values. ADMA: a critical appraisal NO plays a critical role in maintaining vascular tone and integrity. When NO becomes deficient, the endothe- lium becomes adhesive, attracting monocytes and other blood elements to adhere and infiltrate through the endo- thelium, In the presence of oxidized LDL in the sub-enthdothelial subspace, the infiltrated monocytes be- 207 come foam cells, setting the stage ready for the development of fatty streak and early lesions of athero- sclerosis, ADMA is among most potent and abundant endoge- nous inhibitors of eNOS. Today some doubts remain regarding the relevance of ADMA to the NO pathway. Most of the eritics of ADMA argue that ADMA’s plasma level is too low, and the extent of its variation between normal and pathological condition is insignificant, to ef fectively compete with arginine, which has a ten-fold higher concentration in the plasma. The same ADMA- to-arginine ratio also holds true within the endothelial cells. However, as we have pointed out earlier, caution must be taken when interpreting intracellular concentra- tions, because the numbers reflect only the average, but not the local concentration of ADMA and arginine, which, given the localization of eNOS, are biochemically more relevant. Cellular compartmentalization theory addresses the crit- ies’ concems; however, it does so by simply citing the “unknown ~ the cell biology of ¢NOS, ADMA, and arginine. Another more direct piece of evidence supporting the role of ADMA comes from a standard practice in a pharmacology laboratory. Monomethylarginine (L-NMMA) and L-ni- ‘ro-arginine methylester (L-NAME) are 2 commonly used agents to induce systemic vasoconstriction in humans and laboratory animals. They are usually administered with a low dose that would elevate their plasma concentrations 3- to 4-fold, which are still below 5 uM. The systemic effects of these eNOS inhibitors have been well documented for many years. Given that they share similar pharmacokinetic profile with ADMA, ADMA should likewise affect eNOS at micromolar quantity. More importantly, this line of logic suggests that eNOS” access to arginine might not be as straightforward as vascular biologists once thought. Many factors could upset arginine’s availability to €NOS, from a decrease in arginine itself (possibly caused by increased arginine tumover by arginase) to an increase in eNOS inhib- itor such as ADMA, which could be caused by oxidative stress and DDAH dysfunction, If the level of ADMA is clinically important, the fac~ tors that influence DDAH enzymatic activity are also critical, for the latter directly regulates the former. Ac- cordingly, a clinical study has been launched in Europe to investigate the relation between DDAH single nucleo- tide polymorphisms and a variety of cardiovascular Acta Cardio Sin 2004;20:201-11Ken ¥ Lin etal diseases. In the preliminary data, DDAH actually merged a stronger predictor for the onset of Type 2 di betes than markers such as triglyceride and cholesterol Meanwhile, the laboratory of John Cooke at Stanford University has also developed transgenic mice over-ex- pressing human DDALH I gene. Recently, we have shown that acute peritoneal injection of ADMA instantly at- tenuates insulin sensitivity in mice.*” Meanwhile, the insulin sensitivity of transgenic mice over-expressing DDAH is not significantly different from that of mice re- ceiving the control treatment, Presumably, this is due to the transgenic mice’s enhanced ability to degrade the ex- ogenously added ADMA. Taken together, this line of initial evidence suggests that DDAH dysfunetion in par- ticular, and very possibly endothelial dysfunction in general, could lead to insulin resistance and Type 2 dia- betes. A recent clinical study corroborated this hypothesis by showing that eNOS polymorphism is associated with ‘Type 2 diabetes and insulin resistance syndrome."* While this concept still requires more rigorous proof, these studies embody the now decades-old belief that metabolic abnormality is also a vascular disease The etiology of atherosclerosis is certainly multifae- ted, ADMA and DDAH might also have yet-unknown interactions with other components of the NOS and in- flammatory pathways, such as interleukin-8, protein kinase C and phosphoinositol-3-kinase. Treatments that aim to restore I pathway might bestow the benefits at the expense of another pathway. Hence, future studies should be directed to untangling these intricate interac- tions and based on these mechanistic understanding, to design better clinical guidelines and therapeutic inter- ventions. ACKNOWLEDGEMENTS The authors of this paper would like to thank Dr. John P. Cooke, who directs Stanford University Medical School’s Division in Cardiovascular Medicine and who hhas mentored and inspired Ken Lin during his undergrad- uate years. Ken Lin is an American Heart Association Westcm Affiliates Undergraduate Research Fellow, and a recipient of the Howard Hughes Medical Institute's Un- dergraduate Grant, These 2 institutions have in part supported this study. Ken Lin is currently a first-year Acta Cardiol Sin 2004;20:201—11 208 MD-PhD student at Harvard Medical School; he receives a fellowship from the Medical Scientist Training Program of the National Institution of Health. REFERENCES 1. Boger RH, Bode-Boger SM, Tsao PS, et al. An endogenous Intbitor of nitric oxide synthase regulates endothelial adhesiveness for monocytes. JA Coll Cardio! 2000;36:2287-95 ‘Tsao PS, Theilmeior G, Singer AH, et al. L-Arginine attenuates platelet reactivity in hypercholesterolemic rabbits, Arterioscler Thromb 1994;14:1829-33, Shukla SD, Paul A, Klachko DM. 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