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Am J Physiol Renal Physiol 298: F95-F102, 2010. First published November 11, 2009; doi:10.1152/ajprenal.00487.2009.--The kidney is a major site of arginine synthesis where citrulline is converted to arginine via argininosuccinate synthase (ASS) and lyase (ASL). The rate-limiting step in arginine synthesis by the normal kidney is the rate of citrulline delivery and uptake to the renal cortex. We tested whether with chronic kidney disease (CKD) renal arginine synthesis may be compromised. Using the 5/6 renal ablation/infarction (A/I) injury model, we measured renal citrulline delivery and uptake as well as arginine release at early, moderate, and severe stages of CKD vs. healthy controls. The renal plasma flow (RPF) and arterial-renal venous difference was measured at baseline and during citrulline infusion. Citrulline delivery was reduced at all stages of disease due to marked reductions in RPF and despite moderately increased plasma citrulline. Early after 5/6 A/I, the kidney demonstrated a compensatory increase in citrulline uptake while at moderate and severe injury baseline citrulline uptake fell. At all stages of CKD, renal arginine release was markedly reduced. Citrulline infusion increased plasma citrulline in all groups, resulting in increased renal delivery vs. baseline. In healthy kidneys and early injury, citrulline uptake increased with the infusion, but only in the normal kidney did arginine production increase in parallel with the increased citrulline uptake. At moderate and severe injury, there was no increase in citrulline uptake or arginine production. The fall in arginine production in 5/6 A/I was due to an early loss of ASS and ASL conversion of citrulline, which combined with a later reduction in citrulline uptake. argininosuccinate synthase; and nitric oxide

Background/Objectives: Nitric oxide (NO) has been proposed as a mediator of vascular expansion during pregnancy. Inability to increase NO synthesis and/or production of its precursor, arginine, may be a contributor to pregnancy-induced hypertension or preeclampsia. Because maternal weight is associated with blood pressure and risk of preeclampsia during pregnancy, it may also influence arginine and/or NO production. The purpose of this study was to determine the in vivo arginine production and NO synthesis rate in pregnant women with normal (n=10) and low (n=10) body mass indices (BMIS). Subjects/Methods: Arginine flux and NO synthesis rate were measured in the postabsorptive state with constant infusions of [sup.15][N.sub.2]-arginine and [sup.13]C, [sup.2.H.sub.4]-citrulline. Plasma concentrations of arginine and NO metabolites were also measured. Kinetic parameters were correlated to maternal variables, gestational age, birth weight and blood pressure. Results: Endogenous arginine flux was significantly faster in the low-BMI compared with normal-BMI women in the first trimester (63.1 [+ or -] 3.4 vs 50.2 [+ or -] 2.0[[micro]mol/kg per h, P Conclusions: These findings suggest, but do not prove, that maternal BMI may be a factor in the ability to produce NO during pregnancy and may be one way by which BMI influences blood pressure during pregnancy. doi:10.1038/ejcn.2009.24; published online 13 May 2009 Keywords: arginine production; nitric oxide synthesis; maternal weight; maternal body mass index, Introduction A physiological adaptation of pregnancy is expansion of the maternal vascular compartment. It starts with vasodilatation as peripheral resistance falls by 6 weeks and reaches a minimum in mid-pregnancy [...]

The amino acid arginine is the sole precursor for nitric oxide (NO) synthesis. We recently demonstrated that an acute reduction of circulating arginine does not compromise basal or LPS-inducible NO production in mice. In the present study, we investigated the importance of citrulline availability in ornithine transcarbamoylase-deficient [spf.sup.ash] (OTCD) mice on NO production, using stable isotope techniques and C57BL6/J (wild-type) mice controls. Plasma amino acids and tracer-to-tracee ratios were measured by LC-MS. NO production was measured as the in vivo conversion of L-[guanidino-[sup.15][N.sub.2]]arginine to L-[guanidine-[sup.15]N]citrulline; de novo arginine production was measured as conversion of L-[ureido-[sup.13]C-5,5-[sup.2][H.sub.2]]citrulline to L-[guanidino-[sup.13]C-5,5-[sup.2][H.sub.2]]arginine. Protein metabolism was measured using L-[ring-[sup.2][H.sub.5]]phenylalanine and L-[ring-[sup.2][H.sub.2]]tyrosine. OTC deficiency caused a reduction of systemic citrulline concentration and production to 30-50% (P < 0.001), reduced de novo arginine production (P < 0.05), reduced whole-body NO production to 50% (P < 0.005), and increased net protein breakdown by a factor of 2-4 (P < 0.001). NO production was twofold higher in female than in male OTCD mice in agreement with the X-linked location of the OTC gene. In response to LPS treatment (10 mg/kg ip), circulating arginine increased in all groups (P < 0.001), and NO production was no longer affected by the OTC deficiency due to increased net protein breakdown as a source for arginine. Our study shows that reduced citrulline availability is related to reduced basal NO production via reduced de novo arginine production. Under basal conditions this is probably cNOS-mediated NO production. When sufficient arginine is available after LPS stimulated net protein breakdown, NO production is unaffected by OTC deficiency. ornithine transcarbamoylase; sepsis; metabolism; protein breakdown

The recruitment of circulating endothelial progenitor cells (EPCs) might have a beneficial effect on the clinical course of several diseases. Endothelial damage and detachment of endothelial cells are known to occur in infection, tissue ischemia, and sepsis. These detrimental effects in EPCs are unknown. Here we elucidated whether human EPCs internalize Bartonella henselae constituting a circulating niche of the pathogen. B. henselae invades EPCs as shown by gentamicin protection assays and transmission electron microscopy (TEM). Dil-Ac-LDL/lectin double immunostaining and fluorescence-activated cell sorting (FACS) analysis of EPCs revealed EPC bioactivity after infection with B. henselae. Nitric oxide (NO) and its precursor L-arginine (L-arg) exert a plethora of beneficial effects on vascular function and modulation of immune response. Therefore, we tested also the hypothesis that L-arg (1-30 mM) would affect the infection of B. henselae or tumor necrosis factor (TNF) in EPCs. Our data provide evidence that L-arg counteracts detrimental effects induced by TNF or Bartonella infections via NO (confirmed by DETA-NO and L-NMMA experiments) and by modulation of p38 kinase phosphorylation. Microarray analysis indicated several genes involved in immune response were differentially expressed in Bartonella-infected EPCs, whereas these genes returned in steady state when cells were exposed to sustained doses of L-arg. This mechanism may have broad therapeutic applications in tissue ischemia, angiogenesis, immune response, and sepsis. immune response | sepsis

While a specific role for nitric oxide (NO) in inducing the hemodynamic alterations of pregnancy is somewhat controversial, it is widely accepted that excess NO is generated during pregnancy. L-Arginine is the sole precursor for NO biosynthesis. Among several transporters that mediate L-arginine uptake, cationic amino acid transporter-1 (CAT-1) acts as the specific arginine transporter for endothelial NO synthase. The present study was designed to test the hypothesis that, during pregnancy, when arginine consumption by the fetus is significantly increased, compensatory changes in maternal arginine uptake affect the endothelium. Uptake of radiolabeled arginine (1.-[[sup.3]H]arginine) by freshly harvested maternal aortic rings from pregnant rats decreased by 65 and 30% in mid- and late pregnancy, respectively, compared with those obtained from virgin animals. This decrease was associated with a significant increase in endothelial protein nitration (the footprint of peroxynitrite generation), as shown by both Western blotting and immunohistochemistry utilizing anti-nitrotyrosine antibodies, reflecting endothelial damage. Northern blot analysis revealed that steady-state aortic CAT-I mRNA levels did not change throughout pregnancy, whereas CAT-1 protein abundance was significantly increased, peaking at mid-pregnancy. Protein content of protein kinase C (PKC)-[alpha], which was previously shown to decrease CAT-1 activity, increased significantly in the pregnant animals and was associated with a significant increase in CAT-1 phosphorylation. Intraperitoneal injection of [alpha]-tocopherol, a PKC-[alpha] inhibitor, prevented the decrease in arginine transport and attenuated protein nitration. In conclusion, aortic arginine uptake is reduced during pregnancy, through posttranslational modulation of CAT-1 protein, presumably via upregulation of PKC-[alpha]. The aforementioned findings are associated with an increase in protein nitration and, therefore, in selected individuals, may lead to the development of certain forms of endothelial dysfunction, like pre-eclampsia. nitric oxide; endothelial function; arginine; pregnancy

L-Arginine (L-arg) is metabolized to nitric oxide (NO) by inducible NO synthase (iNOS) or to urea and L-ornithine (L-orn) by arginase. NO is involved in the inflammatory response, whereas arginase is the first step in polyamine and proline synthesis necessary for tissue repair and wound healing. Mitogen-activated protein kinases (MAPK) mediate LPS-induced iNOS expression, and MAPK phosphatase-1 (MKP-1) plays a crucial role in limiting MAPK signaling in macrophages. We hypothesized that MKP-1, by attenuating iNOS expression, acts as a switch changing L-arg metabolism from NO production to L-ore production after endotoxin administration. To test this hypothesis, we performed studies in RAW264.7 macrophages stably transfected with an MKP-1 expression vector in thioglyollate-elicited peritoneal macrophages harvested from wild-type and [Mkp-1.sup.-/-] mice, as well as in vivo in wild-type and [Mkp-1.sup.-/-] mice. We found that overexpression of MKP-1 resulted in lower iNOS expression and NO production but greater urea production in response to LPS. Although deficiency of MKP-1 resulted in greater iNOS expression and NO production and lower urea production in response to LPS, neither the overexpression nor the deficiency of MKP-1 had any substantial effect on the expression of the arginases. lung injury; macrophage; ornithine; mitogen-activated protein kinases

Previously, we observed an enhanced renal protein synthesis and increased de novo arginine production in the early response to endotoxemia in wild-type Swiss mice (Hallemeesch MM, Soeters PB, and Deutz NE. Am J Physiol Renal Physiol 282: F316-F323, 2002). To establish whether these changes are regulated by nitric oxide (NO) synthesized by NO synthase isoforms NOS2 and NOS3, we studied C57BL6/J wild-type (WT), NOS2-deficient ([NOS2.sup.-/-]), and NOS3-deficient ([NOS3.sup.-/-]) mice under baseline (unstimulated) and LPS-treated conditions. The metabolism of renal protein, amino acid, and arginine was studied at the whole body level and across the kidney by infusing the stable isotopes L-[phenyl-[sup.2][H.sub.5]]phenylalanine, L-[phenyl-[sup.2][H.sub.2]]tyrosine, L-guanidino-[[sup.15][N.sub.2]]arginine, and L-[ureido-[sup.13] C,[sup.2][H.sub.2]]citrulline. Renal blood flow was measured using radioactive PAH extraction. Under baseline conditions, renal blood flow was significantly reduced in [NOS2.sup.-/-] mice (0.29 [+ or -] 0.01 vs. 0.48 [+ or -] 0.07 ml x 10 g body [wt.sup.-1] x [min.sup.-l] in WT) (P < 0.05), and de novo arginine production was lower in [NOS2.sup.-/-] mice. After LPS challenge, renal protein turnover and arginine production increased in all three groups (P < 0.05), even though renal de novo arginine synthesis did not increase. The expected increase in renal citrulline production and disposal after LPS was not observed in [NOS2.sup.-/-] mice (P = 0.06). Collectively, these data show that NOS2 is constitutively expressed in the kidney and remarkably functional as it affects renal blood flow and de novo arginine production under baseline conditions and is important for the increase in renal citrulline turnover during endotoxemia. NOS3, in contrast, appears less important for renal metabolism. The increase in renal protein turnover during endotoxemia does not depend on NOS2 or NOS3 activity. kidney; nitric oxide; nitric oxide synthase; sepsis

Omithine [alpha]-ketoglutarate (OKG) is a salt formed of 2 molecules of omithine and 1 [alpha]-ketoglutarate. Its administration improves nutritional status in chronically malnourished (e.g., elderly) and acutely malnourished patients (especially burn and trauma patients). There is evidence that OKG activity is not the simple addition of the effects of ornithine (Orn) and [alpha]-ketoglutarate ([alpha]KG), because the presence of both moieties is required to induce the generation of key metabolites such as glutamine, proline, and arginine (Arg), whereas this does not occur when one or the other is given separately. This observation is related to the fact that the main feature of Orn at the whole-body level is to be metabolized through the Orn aminotransferase-dependent pathway, whereas the simultaneous administration of Orn and [alpha]KG saturates this pathway, diverting Orn toward metabolism into Arg. For years, OKG activity has been associated with its ability to induce the secretion of anabolic hormones, such as insulin and growth hormone, and to increase glutamine and polyamine synthesis. Recent studies using chemical inhibitors of nitric oxide synthase (NOS) suggest that nitric oxide derived from Arg could be partly involved in OKG activity. The use of genetically modified animals (i.e., knockout for NOS expression) is required to confirm this hypothesis. Key-words: * ornithine [alpha]-ketoglutarate * arginine * nitric oxide * burn injury

Asymmetric dimethylarginine (ADMA) is a recently recognized endogenous inhibitor of nitric oxide production. Its role in cardiovascular disease is emerging, and ADMA appears to be an important causal factor in dysfunction of the vascular system. Several studies show that ADMA accumulates during renal failure, and ADMA has been identified as causing the cardiovascular complications accompanying renal failure. In addition to the kidney, we recently suggested an important role for the liver as an ADMA-eliminating organ. In a population of critically ill patients, hepatic failure was the most prominent determinant of ADMA concentration, and, notably, high ADMA concentration proved to be a strong and independent risk factor for intensive care unit mortality in these patients. We here summarize the role of both the kidney and the liver in the regulation of ADMA levels. In addition, the potential central role of ADMA as a causative factor in the development of multiple organ failure is discussed. KEY WORDS: * asymmetric dimethylarginine * dimethylarginine dimethylaminohydrolase * nitric oxide * liver * kidney * multiple organ failure

L-Arginine is the biological precursor of nitric oxide (NO), which serves as an important signal and effector molecule in animals. This review summarizes some structure-function aspects of the mammalian nitric oxide synthases, which are enzymes that catalyze the oxidation of L-arginine to NO and L-citrulline. These include aspects related to: 1) the chemical transformations of L-arginine during enzyme catalysis, 2) binding of L-arginine or its structural analogs to the nitric oxide synthases, and 3) how L-arginine levels may affect product formation by the nitric oxide synthases and how this can be modulated by structural analogs of L-arginine. KEY WORDS: * L-arginine, nitric oxide * flavoprotein * reactive oxygen species * hemeprotein

There is abundant evidence that the endothelium plays a crucial role in the maintenance of vascular tone and structure. One of the major endothelium-derived vasoactive mediators is nitric oxide (NO). Asymmetric dimethylarginine (ADMA) is an endogenous competitive inhibitor of NO synthase. ADMA inhibits vascular NO production in concentrations found in pathophysiological conditions; ADMA also causes local vasoconstriction when it is infused intraarterially. Thus, elevated ADMA levels may explain the 'L-arginine paradox,' i.e., the observation that supplementation with exogenous L-arginine improves NO-mediated vascular functions in vivo, although its baseline plasma concentration is about 25-fold higher than the Michaelis-Menten constant [K.sub.m] of the isolated, purified endothelial NO synthase in vitro. The biochemical and physiological pathways related to ADMA are well understood: Dimethylarginines are the result of degradation of methylated proteins; the methyl group is derived from S-adenosylmethionine. Both ADMA and its regioisomer, symmetric dimethylarginine, are eliminated from the body by renal excretion, whereas only ADMA is metabolized via hydrolytic degradation to citrulline and dimethylamine by the enzyme dimethylarginine dimethylaminohydrolase (DDAH). DDAH activity and/or expression may therefore contribute to the pathogenesis of endothelial dysfunction in various diseases. Plasma ADMA levels are increased in humans with hypercholesterolemia, atherosclerosis, hypertension, chronic renal failure, and chronic heart failure. Increased ADMA levels are associated with reduced NO synthesis as assessed by impaired endothelium-dependent vasodilation. In several prospective and cross-sectional studies, ADMA evolved as a marker of cardiovascular risk. With increasing knowledge of the role of ADMA in the pathogenesis of cardiovascular disease, ADMA is becoming a goal for pharmacotherapeutic interventions. Among other potential strategies that are currently being tested, administration of L-arginine has been shown to improve endothelium-dependent vascular functions in subjects with high ADMA levels. Finally, ADMA has gained clinical importance recently because several studies have shown that ADMA is an independent cardiovascular risk factor. KEY WORDS: * endothelium * nitric oxide, atherosclerosis * coronary heart disease * prognosis * oxidative stress

Arginine is a dibasic, cationic, semiessential amino acid with numerous roles in cellular metabolism. It serves as an intermediate in the urea cycle and as a precursor for protein, polyamine, creatine and nitric oxide (NO) biosynthesis. Arginine is conditionally essential since it becomes necessary under periods of growth and after recovery after injury. Arginine also promotes wound healing and functions as a secretagogue stimulating the release of growth hormone, insulin-like growth factor 1, insulin, and prolactin. Furthermore, arginine has several immunomodulatory effects such as stimulating T- and natural killer cell activity and influencing pro-inflammatory cytokine levels. The discover that L-arginine is the sole precursor for the multifunctional messenger molecule nitric oxide (NO) led to investigation into the role of arginine in numerous physiologic and pathophysiologic phenomena including cancer. Although NO was first identified in endothelial cells, it is now recognized to be generated by a variety of cell types, including several tumor cell lines and solid human tumors. Unfortunately, the precise role of NO in cancer is poorly understood but it may influence tumor initiation, promotion, and progression, tumor-cell adhesion, apoptosis angiogenesis, differentiation, chemosensitivity, radiosensitivity, and tumor-induced immunosuppression. The biological effects of NO are complex and dependent upon numerous regulatory factors. Further research is necessary to enhance our understanding of the complex mechanisms that regulate NO's role in tumor biology. A better understanding of the role of arginine-derived NO in cancer may lead to novel antineoplastic and chemopreventative strategies. KEY WORDS: * arginine * nitric oxide * apoptosis * angiogenesis * cancer

Nitric oxide (NO) is synthesized from arginine by NO synthase (NOS), and the availability of arginine is one of the rate-limiting factors in cellular NO production. Citrulline that is formed as a by-product of the NOS reaction can be recycled to arginine by successive actions of argininosuccinate synthetase (AS) and argininosuccinate lyase (AL), forming the citrulline-NO cycle. AS and sometimes AL have been shown to be coinduced with inducible NOS (iNOS) in various ceil types including activated macrophages, microglia, vascular smooth muscle cells, glial cells, neuronal PC12 cells, retinal pigment epithelial cells, and pancreatic [beta]-cells. Coinduction of endothelial NOS (eNOS), AS, and AL are observed in human umbilical vein endothelial cells. In contrast, arginase can downregulate NO production by decreasing intracellular arginine concentrations, iNOS and arginase activities are regulated reciprocally in macrophages by cytokines, and this may guarantee the efficient production of NO. In contrast, iNOS and arginase isoforms (type I and/or II) are coinduced in immunostimulated macrophages, but not in PC12 cells and glial cells. These results indicate that NO production is modulated by the recycling and degradation of arginine. Arginase also plays an important role in regulation of polyamine and proline synthesis. KEY WORDS: * arginine * arginase * argininosuccinate synthetase * nitric oxide * nitric oxide synthase

Urea cycle disorders (UCD) are human conditions caused by the dysregulation of nitrogen transfer from ammonia nitrogen into urea. The biochemistry and the genetics of these disorders were well elucidated. Earlier diagnosis and improved treatments led to an emerging, longer-lived cohort of patients. The natural history of some of these disorders began to point to pathophysiological processes that may be unrelated to the primary cause of acute morbidity and mortality, i.e., hyperammonemia. Carbamyl phosphate synthetase I single nucleotide polymorphisms may be associated with altered vascular resistance that becomes clinically relevant when specific environmental stressors are present. Patients with argininosuccinic aciduria due to a deficiency of argininosuccinic acid lyase are uniquely prone to chronic hepatitis, potentially leading to cirrhosis. Moreover, our recent observations suggest that there may be an increased prevalence of essential hypertension. In contrast, hyperargininemia found in patients with arginase 1 deficiency is associated with pyramidal tract findings and spasticity, without significant hyperammonemia. An intriguing potential pathophysiological link is the dysregulation of intracellular arginine availability and its potential effect on nitric oxide (NO) metabolism. By combining detailed natural history studies with the development of tissue-specific null mouse models for urea cycle enzymes and measurement of nitrogen flux through the cycle to urea and NO in UCD patients, we may begin to dissect the contribution of different sources of arginine to NO production and the consequences on both rare genetic and common multifactorial diseases. J. Nutr. 134: 2775S-2782S, 2004. KEY WORDS: * urea cycle * arginine * nitric oxide

In mammals, L-arginine is classified as a semiessential or conditionally essential amino acid, depending on the developmental stage and health status of the individual. It can be derived from proline or glutamate, with the ultimate synthetic step catalyzed by argininosuccinate lyase. L-arginine is catabolized by arginases, nitric oxide synthases, arginine:glycine amidinotransferase, and possibly also by arginine decarboxylase, resulting ultimately in the production of urea, proline, glutamate, polyamines, nitric oxide, creatine, or agmatine. There is considerable diversity in tissue-specific and stimulus-dependent regulation of expression within this group of enzymes, and the expression of several of them can be regulated at transcriptional and translational levels by changes in the concentration of L-arginine itself. Consequently, the interplay among these enzymes in the regulation of specific aspects of arginine metabolism can be quite complex. For example, nitric oxide production can be affected by the interplay between nitric oxide synthases, arginases, and argininosuccinate synthetase. This metabolic complexity can pose challenges for analyses of arginine metabolism not only because L-arginine is a substrate for several different enzymes but also because ornithine and citrulline, key products of arginine metabolism, can each be produced by multiple enzymes. This overview highlights key features of the arginine metabolic enzymes and their interactions. KEY WORDS: * arginine * ornithine * citrulline * nitric oxide * polyamines

The amino acid L-arginine, the precursor of nitric oxide (NO) synthesis, induces vasodilation in vivo, but the mechanism behind this effect is unclear. There is, however, some evidence to assume that the L-arginine membrane transport capacity is dependent on insulin plasma levels. We hypothesized that vasodilator effects of L-arginine may be dependent on insulin plasma levels. Accordingly, we performed two randomized, double-blind crossover studies in healthy male subjects. In protocol 1 (n = 15), subjects received an infusion of insulin (6 mU*[kg.sup.-1]*[min.sup.-1] for 120 min) or placebo and, during the last 30 min, L-arginine or Darginine (1 g/min for 30 min). In protocol 2 (n = 8), subjects received L-arginine in stepwise increasing doses in the presence (1.5 mU*[kg.sup.-1]*[min.sup.-1]) or absence of insulin. Renal plasma flow and glomerular filtration rate were assessed by the para-aminohippurate and inulin plasma clearance methods, respectively. Pulsatile choroidal blood flow was assessed with laser interferometric measurement of fundus pulsation, and mean flow velocity in the ophthalmic artery was measured with Doppler sonography. L-arginine, but not D-arginine, significantly increased renal and ocular hemodynamic parameters. Coinfusion of L-arginine with insulin caused a dose-dependent leftward shift of the vasodilator effect of L-arginine. This stereospecific renal and ocular vasodilator potency of L-arginine is enhanced by insulin, which may result from facilitated L-arginine membrane transport, enhanced intracellular NO formation, or increased NO bioavailability. renal plasma flow; ocular blood flow; nitric oxide

The decrease in glomerular filtration rate (GFR) that is characteristic of sepsis has been shown to result from inhibition of glomerular endothelial nitric oxide synthase (eNOS) by nitric oxide (NO) generated from the inducible isoform of NOS (iNOS). Although L-arginine is the sole precursor for NO biosynthesis, its intracellular availability in glomeruli from septic animals has never been investigated. Arginine uptake was measured in freshly harvested glomeruli from the following experimental groups: 1) untreated rats; 2) rats pretreated with LPS (4 mg/kg body wt, 4 h before experiments); 3) rats treated with LPS as above with either L-[N.sup.6]-(1-iminoethyl)lysine hydrochloride (L-NIL), a selective iNOS antagonist, or 7-nitroindazole, a selective neuronal NOS antagonist; and 4) rats treated with L-NIL only. Both glomeular and mesangial arginine transport characteristics were found compatible with a [y.sup.+] system. Arginine uptake was augmented in glomeruli from LPS-treated rats. Treatment with L-NIL completely abolished this effect whereas L-NIL alone had no effect. Similar results were obtained when primary cultures of rat mesangial cells were preincubated with LPS (10 [micro]g/ml for 24 h) with or without L-NIL. Using RT-PCR, we found that in vivo administration of LPS resulted in a significant increase in glomerular cationic amino acid transporter-2 (CAT-2) mRNA expression whereas CAT-1 mRNA was undetected. Northern blotting further confirmed a significant increase in glomerular CAT-2 by LPS. In mesangial cells, the expression of both CAT-1 and CAT-2 mRNA was augmented after incubation with LPS. In conclusion, in vivo administration of LPS augments glomerular arginine transport through upregulation of steady-state CAT-2 mRNA while downregulating CAT-1 mRNA. These results may correspond to the changes in glomerular iNOS and eNOS activity in sepsis. cationic amino acid transporter-2; sepsis; nitric oxide; arginine transport

Atherogenesis is enhanced in arterial segments exposed to disturbed blood flow, indicating the active participation of the hemodynamic environment in lesion formation. Turbulent shear stress selectively regulates responsive genes in the endothelium and increases the damage induced by free radicals. The purpose of the present study was to evaluate the effects of intervention with antioxidants and L-arginine on endothelial NO synthase (eNOS) and oxidation-sensitive gene perturbation induced by disturbed flow in vitro and in vivo. Both human endothelial cells exposed to shear stress and high atherosclerosis-prone areas of hypercholesterolemic low-density lipoprotein receptor knockout (LDL[R.sup.-/-]) mice showed increased activities of redox-transcription factors (ELK-1, p-Jun, and p-CREB) and decreased expression of eNOS. Intervention with antioxidants and L-arginine reduced the activation of redox-transcription factors and increased eNOS expression in cells and in vivo. These results demonstrate that atherogenic effects induced by turbulent shear stress can be prevented by cotreatment with antioxidants and L-arginine. The therapeutic possibility to modulate shear stress-response genes may have important implications for the prevention of atherosclerosis and its clinical manifestations. eNOS | ELK-1 | p-CREB | p-Jun | vascular disease

We examined the hypothesis that changes in heart rate at rest influence bioactivity of nitric oxide (NO) in humans by examining forearm blood flow responses during cardiac pacing in six subjects. Peak forearm and mean forearm blood flows across the cardiac cycle were continuously recorded at baseline and during pacing, with the use of high-resolution brachial artery ultrasound and Doppler flow velocity measurement. The brachial artery was cannulated to allow continuous infusion of saline or [N.sup.G]monomethyl-L-arginine (L-NMMA). As heart rate increased, no changes in pulse pressure and mean or peak blood flow were evident. L-NMMA had no effect on brachial artery diameter, velocity, or flows compared with saline infusion. These results contrast with our recent findings that exercise involving the lower body, associated with increases in heart rate and pulse pressure, also increased forearm blood flow, the latter response being diminished by L-NMMA. These data suggest that changes in blood pressure, rather than pulse frequency, may be the stimulus for shear stress-mediated NO release in vivo. blood flow; high-resolution ultrasound; Doppler velocity; shear stress

Arginine vasopressin (AVP) plays a central role in water balance. In principal cells of the collecting duct system, AVP controls the expression of several genes, including aquaporin-2. Because nitric oxide (NO) participates in the regulation of water reabsorption by the collecting duct system, we analyzed the effect of AVP on the expression of NO synthase (NOS) isoforms in the kidney. Rats were either water restricted or water loaded to modify the circulating AVP levels, and expressions of NOS isoforms were assessed by Western blot analysis. In water-restricted rats, endothelial NOS (eNOS) expression increased in the outer medulla, and neuronal NOS (nNOS) expression rose in both the outer medulla and the papilla. Conversely, water loading induced a decrease in expression of nNOS in the outer medulla and papilla but did not alter eNOS expression. Oral administration of the specific V2-receptor antagonist SR-121463B decreased nNOS expression in the outer medulla and papilla but did not alter eNOS expression levels. Finally, the very low nNOS expression levels observed in AVP-deficient Brattleboro rats was restored by AVP infusion for I wk. Thus AVP specifically increases nNOS expression levels in the renal outer medulla and papilla. Because nNOS is specifically expressed in principal cells of the collecting duct system, the stimulation of nNOS expression by AVP may participate in the control of water reabsorption. principal cells; aquaporin-2; collecting duct; nitric oxide synthase

Research has been conducted on the endothelial nitric oxide synthase heme domain. The results of the electron paramagnetic resonance and electron nuclear double resonance studies of the activated-oxygen reaction intermediates formed in this domain are presented.

Research has been conducted on the guanidines related to L-arginine. The oxidation of these compounds carried out via the recombinant NO-synthase II has produced a non-alpha-amino acid guanidine NO-synthase II substrate which acts as NO precursor.

We examined whether endogenous inhibitors of nitric oxide (NO) synthesis are involved in the impaired cavernosal relaxation with ischemia in rabbits. Two weeks after cavernosal ischemia caused by partial vessel occlusion, endothelium-dependent and electrical field stimulation (EFS)-induced neurogenic NO-mediated relaxations, but not sodium nitroprusside (SNP)-induced relaxation, were significantly impaired in the isolated corpus cavernosum. The [Ca.sup.2+]-dependent NO synthase (NOS) activity and the basal and stimulated cGMP productions with carbachol or EFS were significantly decreased after ischemia. Supplementation of excess L-arginine partially recovered both of the impaired relaxations. The contents of [N.sup.G]-monomethyl-L-arginine (L-NMMA) and asymmetric [N.sup.G],[N.sup.G]-dimethyl-L-arginine (ADMA) but not L-arginine and symmetric [N.sup.G],[N.sup.'G]-dimethyl-L-arginine (SDMA) were increased in the cavernosal tissues after ischemia. Authentic L-NMMA and ADMA but not SDMA concentration dependently inhibited both relaxations without affecting the relaxation produced by SNP in the control. Excess L-arginine abolished the inhibition with L-NMMA and ADMA. These results suggest that the impaired NO-mediated cavernosal relaxations after ischemia are closely related to the decreased NOS activity and the increased accumulation of L-NMMA and ADMA. asymmetric [N.sup.G],[N.sup.G]-dimethyl-L-arginine; L-arginine; [N.sup.G]-monomethyl-L-arginine; corpus cavernosum

Research has been conducted on the ferric endothelial nitric oxide synthase holoenzyme. The low-frequency resonance Raman spectra of this holoenzyme have been investigated in the presence of the L-arginine substrate and the results are reported.

The authors review the publications on physiological activity of nitric oxide synthases. The topics of interest include the enzymology of nitric oxide derived from nitric oxide synthase, superoxide generation and secondary free-radical formation by nitric oxide synthase.

Protective effects of L-arginine were evaluated in a human ventricular heart cell model of low-volume anoxia and reoxygenation independent of alternate cell types. Cell cultures were subjected to 90 min of low-volume anoxia and 30 min of reoxygenation. L-Arginine (0-5.0 mM) was administered during the preanoxic period or the reoxygenation phase. Nitric oxide (NO) production, NO synthase (NOS) activity, cGMP levels, and cellular injury were assessed. To evaluate the effects of the L-arginine on cell signaling, the effects of the NOS antagonist [N.sup.G]-nitro-L-arginine methyl ester, NO donor S-nitroso-N-acetyl-penicillamine, guanylate cyclase inhibitor methylene blue, cGMP analog 8-bromo-cGMP, and ATP-sensitive [K.sup.+] channel antagonist glibenclamide were examined. Our data indicate that low-volume anoxia and reoxygenation increased NOS activity and facilitated the conversion of L-arginine to NO, which provided protection against cellular injury in a dose-dependent fashion. In addition, L-arginine cardioprotection was achieved by the activation of guanylate cyclase, leading to increased cGMP levels in human heart cells. This action involves a glibenclamide-sensitive, NO-cGMP-dependent pathway. ventricular myocytes; cardiac surgery; nitric oxide

Two-way arginine transport in human endothelial cells: TNF-[alpha] stimulation is restricted to system [y.sup.+]. Am J Physiol Cell Physiol 282: C134-C143, 2002.--Human umbilical vein endothelial cells transport arginine through two [Na.sup.+]-independent systems. System [y.sup.+]L is insensitive to N-ethylmaleimide (NEM), inhibited by L-leucine in the presence of [Na.sup.+], and referable to the expression of SLC7A6/[y.sup.+]LAT2, SLC7A7/[y.sup.+]LAT1, and SLC3A2/4F2hc. System [y.sup.+] is referable to the expression of SLC7A1/CAT1 and SLC7A2/CAT2B. Tumor necrosis factor-[alpha] (TNF-[alpha]) and bacterial lipopolysaccharide induce a transient stimulation of arginine influx and efflux through system [y.sup.+]. Increased expression of SLC7A2/CAT2B is detectable from 3 h of treatment, while SLC7A1 expression is inhibited at later times of incubation. System [y.sup.+]L activity and expression remain unaltered. Nitric oxide synthase type 2 mRNA is not detected in the absence or presence of TNF-[alpha], while the latter condition lowers nitric oxide synthase type 3 expression at the mRNA and the protein level. Nitrite accumulation is comparable in cytokine-treated and control cells up to 48 h of treatment. It is concluded that modulation of endothelial arginine transport by TNF-[alpha] or lipopolysaccharide occurs exclusively through changes in CAT2B and CAT1 expression and is dissociated from stimulation of nitric oxide production. system [y.sup.+]L; cationic amino acid transporters; SLC7A genes; lipopolysaccharide; nitric oxide

Li, Yi-Fan, William G. Mayhan, and Kaushik P. Patel. NMDA-mediated increase in renal sympathetic nerve discharge within the PVN: role of nitric oxide. Am J Physiol Heart Circ Physiol 281: H2328-H2336, 2001.--The paraventricular nucleus (PVN) of the hypothalamus is an important site of integration in the central nervous system for sympathetic outflow. Both glutamate and nitric oxide (NO) play an important role in the regulation of sympathetic nerve activity. The purpose of the present study was to examine the interaction of NO and glutamate within the PVN in the regulation of renal sympathetic nerve activity in rats. Renal sympathetic nerve discharge (RSND), arterial blood pressure (BP), and heart rate (HR) were measured in response to administration of N-methyl-D-aspartic acid (NMDA) and [N.sup.G]-monomethyl-L-arginine (L-NMMA) into the PVN. We found that microinjection of NMDA (25, 50, and 100 pmol) into the PVN increased RSND, BP, and HR in a dose-dependent manner, reaching 53 [+ or -] 9%, 19 [+ or -] 3 mmHg, and 32 [+ or -] 12 beats/min, respectively, at the highest dose. These responses were significantly enhanced by prior microinjection of L-NMMA. On the other hand, inhibition of NO within the PVN by microinjection of L-NMMA also induced increases in RSND, BP, and HR in a dose-dependent manner, reaching 48 [+ or -] 6.5%, 11 [+ or -] 4 mmHg, and 55 [+ or -] 16 beats/min, respectively, at the highest dose. This sympathoexcitatory response was eliminated by prior microinjection of DL-2-amino-5-phosphonovaleric acid, an antagonist of the NMDA receptor. Furthermore, with the use of the push-pull technique, perfusion of glutamate (0.5 [micro]mol) or NMDA (0.1 nmol) into the PVN induced an increase in NO release. In conclusion, our data indicate that NMDA receptors within the PVN mediate an excitatory effect on renal sympathetic nerve activity, arterial BP, and HR. NO in the PVN, which is released by activation of the NMDA receptor, also inhibits NMDA-mediated increases in sympathetic nerve activity. This negative feedback of NO on the glutamate system within the PVN may play an important role in maintaining the overall balance and tone of sympathetic outflow in normal and pathophysiological conditions known to have increased sympathetic tone. sympathetic nerve activity; N-methyl-L-arginine receptor; paraventricular nucleus Received 8 June 2001; accepted in final form 5 September 2001

Low-temperature optical absorption spectra indicate that there may be a tetrahydrobiopterin (BH4) redox role in both steps of nitric oxide synthase catalysis. The spectral changes that follow addition of oxygen to the reduced oxygenase domain of endothelial nitric oxide synthase (NOS) in the presence of various pteridines at -30 degrees Celsius were analyzed. Incomplete and slow formation of high-spin ferric heme with 4-amino-BH4 was seen and suggests a structural basis for inhibition of NOS activity by this pteridine.

A study was conducted to test the hypothesis that endogenously produced nitric oxide (NO) directly reduces NaCl transport by the thick ascending limb of the loop of Henle (TALH). Results indicated a significant role for locally produced NO, which may act through an autocrine mechanism to directly affect TALH sodium chloride transport. Hence, TALH NO synthesis and inhibition of chloride transport may contribute to the diuretic and natriuretic effects of NO seen in vivo.

A study was conducted to investigate the role and regulation of arginase and inducible nitric oxide synthase (iNOS) in the macrophage cells of a mouse. Results show that increases in arginase mRNAs increase arginase activity. Moreover, complex patterns of iNOS and arginase expressions suggest that arginase isoforms may play distinct but partially overlapping functional roles in macrophage arginine metabolism.

The mechanisms by which N(supra G)-hydroxy-L-arginine (NOHA) and nitric oxide (NO) suppress the proliferation of a cultured human colon carcinoma cell line (Caco-2) was examined. Results showed that both NOHA and NO were able to inhibit Caco-2 in a concentration-dependent manner. It was observed that NOHA inhibits the Caco-2 cell arginase activity both in cell-free extracts and in intact cells. On the other hand, NO inhibits Caco-2 by inhibiting the ornithine decarboxylase present in Caco-2 cells.

A study was conducted on the effects of 'long-term' oral intake of L-arginine (L-Arg), the endogenous source for nitric oxide synthesis, on postprandial lower esophageal sphincter pressure (LESP), esophageal motility, gastroesophageal reflux and gallbladder motility. The findings indicate that prolonged oral L-Arg suppresses late postprandial LESP increase, extends transient LES relaxations and increases fasting and residual gallbladder volumes.

Research was conducted to test whether nitric oxide (NO) production in lipopolysaccharide-activated macrophages is influenced by arginase and to examine whether arginase-regulated NO production is modulated by the extracellular level of L-arginine. NO synthase was assayed for cultured cells while cell lysates were utilized for enzyme assays in the presence and absence of L-norvaline. Results indicated that inducible NO synthase activity was not influenced by L-norvaline.

The effects of endotoxin on the activity of argininosuccinate synthetase (AS) were analyzed in cultured pulmonary artery endothelial cells (PAEC) under hypoxia. Incubation of porcine PAEC with endotoxin elevated AC activity and messenger RNA levels. However, exposure of porcine PAEC to hypoxia reduced AC activity and messenger RNA stability. The inhibitory effects of hypoxia were also mediated by reductions in intracellular L-arginine.

The role of nitric oxide synthase (NOS) on the effects of exogenously administered agmantine on glomerular filtration rate was analyzed in isolated rat kidneys. Infusions of BHT-933 did not increase nephron filtration rate compared to the stimulatory effects of agmantine. Furthermore, agmantine exerted an opposite effect on the constitutive enzyme system involving endothelial NOS in the rat kidneys.

The effects of NG-monomethyl-L-arginine (L-NMMA) nitric oxide synthase (NOS)inhibitor and L-arginine on NOS inhibition of renal hemodynamics were analyzed in human volunteers. Analysis of L-NMMA and L-arginine renal activity in human subjects indicated the presence of reduced renal perfusions without alterations in blood pressure and pulse rate. The effects of L-arginine on NOS inhibition of renal hemodynamics indicated the susceptibility of human renal vasculature to endogenous NO.

Resident mouse peritoneal exudate cells (MPEC) were analyzed to the mechanisms that regulate arginine metabolism and its role in wound healing. The presence of interferon-gamma caused reciprocal changes in the activities of nitric oxide (NO) synthase and arginase in mouse peritoneal macrophages. The factors which regulated arginine metabolism also affected the function of cells at the site of injury. Furthermore, cells in the site of the wound create a cytoxic environment by generating arginine to NO synthase.

The rates of whole body nitric oxide (NO) synthesis, plasma arginine flux, and de novo arginine synthesis and their relationships to urea production, were examined in a total of seven healthy adults receiving an L-amino acid diet for 6 days. NO synthesis was estimated by the rate of conversion of the [15N]guanidino nitrogen of arginine to plasma [15N]ureido citrulline and compared with that based on urinary nitrite [Mathematical Expression Omitted]/nitrate [Mathematical Expression Omitted] excretion. Six subjects received on dietary day 7, a 24-hr (12-hr fed/12-hr fasted) primed, constant, intravenous infusion of L-[guanidino-15[N.sub.2]]arginine and [13C]urea. A similar investigation was repeated with three of these subjects, plus an additional subject, in which they received L-[ureido-13C]citrulline, to determine plasma citrulline fluxes. The estimated rates (mean [+ or -] SD) of NO synthesis over a period of 24 hr averaged 0.96 [+ or -] 0.1 [micro]mol[multiplied by][kg.sup.-1][multiplied by][hr.sup.-1] and 0.95 [+ or -] 0.1 [micro]mol[multiplied by][kg.sup.-1][multiplied by][hr.sup.-1], for the [15N]citrulline and the nitrite/nitrate methods, respectively. About 15% of the plasma arginine turnover was associated with urea formation and 1.2% with NO formation. De novo arginine synthesis averaged 9.2 [+ or -] 1.4 [micro]mol[multiplied by][kg.sup.-1][multiplied by][hr.sup.-1], indicating that [approximately equal to]11% of the plasma arginine flux originates via conversion of plasma citrulline to arginine. Thus, the fraction of the plasma arginine flux associated with NO and also urea synthesis in healthy humans is small, although the plasma arginine compartment serves as a significant precursor pool (54%) for whole body NO formation. This tracer model should be useful for exploring these metabolic relationships in vivo, under specific pathophysiologic states where the L-arginine-NO pathway might be altered.

The baroreflex control of heart rate (HR) and renal sympathetic nerve activity (RSNA) increases in the absence of nitric oxide (NO) synthesis in conscious rabbits. The use of N(super G)-nitro-L-arginine (L-NNA) helps in the blockade of NO. The effects of NO on baroreflex control also involve both arms of the automatic innervation of the sinoatrial node and central nervous system. The control of RSNA, however, remains almost unaffected.

The presence of nitric oxide (NO) in the initial stage of mechanically induced osteogenesis influences the osteogenic response to mechanical stimulation. A study using a model of mechanically-induced osteogenesis shows that the introduction of N(super G)-monomethyl-L-arginine (L-NMMA), an inhibitor of NO, before mechanical stimulation suppresses bone formation. However, coadministration of L-arginine can prevent the oppression, implying that NO has a major role in the transduction of mechanical stimuli into subsequent bone formation.

The physiological effects of myoglobin on renal function, wherein myoglobin acts as a source of nitric oxide (NO), were investigated in the rabbit. Rabbits with myoglobin-induced renal failure had reduced renal blood flow, increased renal vascular resistance and reduced creatinine clearance, and decreased synthesis of cGMP. Co-administration of L-arginine reversed the effects of renal failure. It is suggested that renal vasoconstriction leads to renal dysfunction which is reversed by the addition of L-arginine.

Isolated perfused rat livers in a recirculation model were used in an investigation of the source of arginine for hepatic nitric oxide (NO) synthesis by inducible NO synthase (iNOS). Expression of iNOS was induced by injecting killed Corynebacterium parvum or Escherichia coli endotoxin. The results indicate the existence of endogenous and exogenous sources of arginine for NO synthesis. The source of endogenous arginine cannot be identified, but the results indicate that it does not come from the urea cycle.

A study of the influence of NO on regional blood circulation and diaspirin cross-linked hemoglobin (DCLHb) reveals that L-arginin, a precursor of NO inhibits the systemic hemodynamic activities of DCLHb. DCLHb increases blood pressure, total peripheral resistance cardiac output and stroke volume. L-NAME substantially increases regional vascular resistance thereby reducing the blood flow to all organs except the heart. Infusion of DCLHb to L-NAME pretreated rats accentuates the decreased blood flow to the gastrointestinal system.