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Insulin regulates the l -arginine/nitric oxide (NO) pathway in human umbilical vein endothelial cells (HUVECs), increasing the plasma membrane expression of the l -arginine transporter hCAT-1 and inducing vasodilation in umbilical and placental veins. Placental vascular relaxation induced by insulin is dependent of large conductance calcium-activated potassium channels (BKCa), but the role of KCa channels on l -arginine transport and NO synthesis is still unknown. The aim of this study was to determine the contribution of KCa channels in both insulin-induced l -arginine transport and NO synthesis, and its relationship with placental vascular relaxation. HUVECs, human placental vein endothelial cells (HPVECs) and placental veins were freshly isolated from umbilical cords and placenta from normal pregnancies. Cells or tissue were incubated in absence or presence of insulin and/or tetraethylammonium, 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole, iberiotoxin or N G-nitro- l -arginine methyl ester. l -Arginine uptake, plasma membrane polarity, NO levels, hCAT-1 expression and placenta vascular reactivity were analyzed. The inhibition of intermediate-conductance KCa (IKCa) and BKCa increases l -arginine uptake, which was related with protein abundance of hCAT-1 in HUVECs. IKCa and BKCa activities contribute to NO-synthesis induced by insulin but are not directly involved in insulin-stimulated l -arginine uptake. Long term incubation (8 h) with insulin increases the plasma membrane hyperpolarization and hCAT-1 expression in HUVECs and HPVECs. Insulin-induced relaxation in placental vasculature was reversed by KCa inhibition. The results show that the activity of IKCa and BKCa channels are relevant for both physiological regulations of NO synthesis and vascular tone regulation in the human placenta, acting as a part of negative feedback mechanism for autoregulation of l -arginine transport in HUVECs. Image 1 • Inhibition of KCa channels increases the l -arginine transport and NO levels in HUVECs. • Insulin-induced NO synthesis is partially dependent of KCa channels. • Insulin induces hyperpolarization in plasma membrane of HUVECs. • Insulin-induced placental vascular relaxation is blocked by KCa inhibitors. [ABSTRACT FROM AUTHOR]

L-arginine is a semi-essential amino acid that serves as precursor for the production of urea, nitric oxide (NO), polyamines, and other biologically important metabolites. Hence, a fast and reliable assessment of its intracellular concentration changes is highly desirable. Here, we report on a genetically encoded Förster resonance energy transfer (FRET)-based arginine nanosensor that employs the arginine repressor/activator ahrC gene from Bacillus subtilis. This new nanosensor was expressed in HEK293T cells, and experiments with cell lysate showed that it binds L-arginine with high specificity and with a K of ∼177 μM. Live imaging experiments showed that the nanosensor was expressed throughout the cytoplasm and displayed a half maximal FRET increase at an extracellular L-arginine concentration of ∼22 μM. By expressing the nanosensor together with SLC7A1, SLC7A2B, or SLC7A3 cationic amino acid transporters (CAT1-3), it was shown that L-arginine was imported at a similar rate via SLC7A1 and SLC7A2B and slower via SLC7A3. In contrast, upon withdrawal of extracellular L-arginine, intracellular levels decreased as fast in SLC7A3-expressing cells compared with SLC7A1, but the efflux was slower via SLC7A2B. SLC7A4 (CAT4) could not be convincingly shown to transport L-arginine. We also demonstrated the impact of membrane potential on L-arginine transport and showed that physiological concentrations of symmetrical and asymmetrical dimethylarginine do not significantly interfere with L-arginine transport through SLC7A1. Our results demonstrate that the FRET nanosensor can be used to assess L-arginine transport through plasma membrane in real time. [ABSTRACT FROM AUTHOR]

Reduced nitric oxide (NO) bioavailability plays a central role in the pathogenesis of myocardial ischemia-reperfusion injury (I-R), and reduced l -arginine transport via cationic amino acid transporter-1 is a key contributor to the reduced NO levels. Insulin can increase NO levels by increasing the transport of its substrate l -arginine but insulin alone exerts minimal cardiac protection in I-R. We hypothesized that combined insulin and l -arginine may provide cardioprotective effects in the setting of myocardial I-R. The effect of supplemental insulin, l -arginine and the combination was examined in cardiomyocytes exposed to hypoxia/reoxygenation and in isolated perfused mouse hearts undergoing ischemia/reperfusion. When compared to controls, cardiomyocytes treated upon reoxygenation with 1 nM insulin+1 mM l -arginine exhibited significant (all P <0.05) improvements in NO generation and mitochondrial membrane potential, with a concomitant fall in reactive oxygen species production and LDH release. Insulin also increased l -arginine uptake following hypoxia-reoxygenation ( P <0.05; n =4–6). In langendorff perfused isolated mouse hearts, combined l -arginine-insulin treatment upon reperfusion significantly (all P <0.05; n =9–11) improved recovery of left ventricular developed pressure, rate pressure product and end diastolic pressure following ischemia, independent of any changes in post-ischemic coronary flow, together with significantly lower LDH release. The observed improvements were greater than l -arginine or insulin treatment alone. In isolated cardiomyocytes ( n =3–5), 1 nM insulin caused cationic amino acid transporter-1 to redistribute to the cellular membrane from the cytosol and the effects of insulin on l -arginine uptake were partially dependent on the PI3K/Akt pathway. l -arginine-insulin treatment may be a novel strategy to ameliorate I-R injury. [ABSTRACT FROM AUTHOR]

Observational studies indicate that psychological stress may contribute to the pathogenesis of hypertension and this may be further accentuated by factors such as endothelial dysfunction. On this basis, we aimed to determine whether oxidative stress enhances pressor responses to stressful stimuli and whether augmenting endothelial function by increasing the transport of l-arginine can counter the effects of oxidative stress. Telemetry probes were used to measure mean arterial pressure ( MAP) in wild-type ( WT; n = 6) and endothelial cationic amino acid transporter-1 ( CAT-1)-overexpressing ( CAT+) mice ( n = 6) before and during an aversive (restraint) and non-aversive (almond feeding) stressor. The superoxide dismutase inhibitor diethyldithiocarbamic acid ( DETCA; 30 mg/kg per day; 14 days) was then administered via a minipump to induce oxidative stress. Stress responses to feeding and restraint were repeated during Days 11-12 of DETCA infusion. In WT mice, pressor responses to restraint and feeding were augmented during infusion of DETCA (35 ± 1 and 28 ± 1 mmHg, respectively) compared with respective pretreatment responses (28 ± 2 and 24 ± 1 mmHg, respectively; P ≤ 0.01). In CAT+ mice, pressor responses to feeding were blunted during DETCA (20 ± 1 mmHg) compared with the control response (23 ± 1 mmHg; P = 0.03). In these mice, pressor responses to restraint were similar before (28 ± 1 mmHg) and during (26 ± 1 mmHg) DETCA infusion ( P = 0.26). We conclude that endothelial CAT-1 overexpression can counter the ability of oxidative stress to augment pressor responses to behavioural stress. [ABSTRACT FROM AUTHOR]

Aim Hypertension is a major clinical complication of obesity. Our previous studies show that abnormal uptake of the nitric oxide precursor L-arginine, via the cationic amino acid transporter-1 ( CAT1), contributes to endothelial dysfunction in cardiovascular disease. In this study, we tested the hypothesis that abnormal L-arginine transport may be a key mediator of obesity-induced hypertension. Methods Mean arterial pressure ( MAP) was monitored by telemetry in conscious wild-type ( WT; n = 13) mice, and transgenic mice with endothelial-specific overexpression of CAT1 ( CAT+; n = 14) fed a normal or a high fat diet for 20 weeks. Renal angiotensin II ( Ang II), CAT1 m RNA and plasma nitrate/nitrite levels were then quantified. In conjunction, plasma nitrate/nitrite levels were assessed in obese normotensive ( n = 15) and obese hypertensive subjects ( n = 15). Results Both genotypes of mice developed obesity when fed a high fat diet ( P ≤ 0.002). Fat fed WT mice had 13% greater MAP and 78% greater renal Ang II content, 42% lesser renal CAT1 m RNA levels and 42% lesser plasma nitrate/nitrite levels, than WT mice fed a normal fat diet ( P ≤ 0.02). In contrast, none of these variables were significantly altered by high fat feeding in CAT+ mice ( P ≥ 0.36). Plasma nitrate/nitrite levels were 17% less in obese hypertensives compared with obese normotensives ( P = 0.02). Conclusion Collectively, these data indicate that obesity-induced down-regulation of CAT1 expression and subsequent reduced bioavailability of nitric oxide may contribute to the development of obesity-induced hypertension. [ABSTRACT FROM AUTHOR]

Aim Oxidative stress may play an important role in the pathogenesis of hypertension. The aim of our study is to examine whether increased expression of the predominant endothelial l-arginine transporter, cationic amino acid transporter-1 ( CAT1), can prevent oxidative stress-induced hypertension. Methods Wild-type mice ( WT; n = 9) and endothelial CAT1 overexpressing ( CAT+) mice ( n = 6) had telemetry probes implanted for the measurement of mean arterial pressure ( MAP), heart rate ( HR) and locomotor activity. Minipumps were implanted for infusion of the superoxide dismutase inhibitor diethyldithiocarbamic acid ( DETCA; 30 mg kg−1 day−1; 14 days) or its saline vehicle. Baseline levels of MAP, HR and locomotor activity were determined before and during chronic DETCA administration. Mice were then killed, and their plasma and kidneys collected for analysis of F2-isoprostane levels. Results Basal MAP was less in CAT+ (92 ± 2 mmHg; n = 6) than in WT (98 ± 2 mmHg; n = 9; P < 0.001). During DETCA infusion, MAP was increased in WT (by 4.2 ± 0.5%; P < 0.001) but not in CAT+, when compared to appropriate controls ( PDETCA*genotype = 0.006). DETCA infusion increased total plasma F2-isoprostane levels (by 67 ± 11%; P = 0.05) in WT but not in CAT+. Total renal F2-isoprostane levels were greater during DETCA infusion in WT (by 72%; P < 0.001), but not in CAT+, compared to appropriate controls. Conclusion Augmented endothelial l-arginine transport attenuated the prohypertensive effects of systemic and renal oxidative stress, suggesting that manipulation of endothelial CAT1 may provide a new therapeutic approach for the treatment of cardiovascular disease associated with oxidative stress. [ABSTRACT FROM AUTHOR]

Supplementation with L-arginine or fresh food with high content of this amino acid is associated with favorable effects in the metabolic control of diabetes. We aimed to determine whether supplementation with apples enriched with L-arginine offer additional benefits compared to L-arginine by itself in a preclinical study of diabetes. This study combines food-engineer technologies with in vivo and in vitro analysis. In vitro experiments show that cells derived from non-diabetic animals and exposed to high glucose (25 mM, 12 H) and cells isolated from alloxan-induced diabetic animals exhibited a reduction (∼50%) in the L-arginine uptake. This effect was reverted by L-arginine pretreatment (12 H) in both the normal and diabetes-derived cells. In preclinical studies, normoglycemic ( n = 25) and diabetic groups ( n = 50) were divided into subgroups that received either L-arginine (375 mg/kg per 10 days) or apple enriched with L-arginine or vehicle (control). In a preliminary analysis, supplementation with L-arginine by itself (50%) or apple enriched with L-arginine (100%) improve survival rate in the diabetic group compared to control (0%) at the end of the follow up (17 days). This phenomenon was associated with a partial but sustained high plasma level of L-arginine, as well as plasma concentration of nitrites and insulin in the L-arginine or apple + L-arginine groups after supplementation. Apple + L-arginine supplementation in diabetic animals induced the highest and longest effects in the level of these three markers among the studied groups. Therefore, apple enriched by L-arginine offers more benefits than L-arginine by itself in this preclinical study. © 2013 BioFactors 39(5):564-574, 2013 [ABSTRACT FROM AUTHOR]

Low renal nitric oxide (NO) bioavailability contributes to the development and maintenance of chronic hypertension. We investigated whether impaired L-arginine transport contributes to low renal NO bioavailability in hypertension. Responses of renal medullary perfusion and NO concentration to renal arterial infusions of the L-arginine transport inhibitor L-lysine (10 μmol.kg-1.min-1; 30 min) and subsequent superimposition of L-arginine (100 μmol.kg-1.min-1; 30 min), the NO synthase inhibitor NG-nitro-L-arginine (2.4 mg/kg; iv bolus), and the NO donor sodium nitroprusside (0.24 μg.kg-1.min-1) were examined in Sprague-Dawley rats (SD) and spontaneously hypertensive rats (SHR). Renal medullary perfusion and NO concentration were measured by laser-Doppler flowmetry and polarographically, respectively, 5.5 mm below the kidney surface. Renal medullary NO concentration was less in SHR (53 ± 3 nM) compared with SD rats (108 ± 12 nM; P = 0.004). L-Lysine tended to reduce medullary perfusion (-15 ± 7%; P = 0.07) and reduced medullary NO concentration (-9 ± 3%; P = 0.03) while subsequent superimposition of L-arginine reversed these effects of L-lysine in SD rats. In SHR, L-lysine and subsequent superimposition of L-arginine did not significantly alter medullary perfusion or NO concentration. Collectively, these data suggest that renal L-arginine transport is impaired in SHR. Renal L-[3H]arginine transport was less in SHR compared with SD rats (P = 0.01). Accordingly, we conclude that impaired arginine transport contributes to low renal NO bioavailability observed in the SHR kidney. [ABSTRACT FROM AUTHOR]

Abstract: We examined the relative contributory roles of extracellular vs. intracellular l-arginine (ARG) toward cellular activation of endothelial nitric oxide synthase (eNOS) in human endothelial cells. EA.hy926 human endothelial cells were incubated with different concentrations of -ARG, ARG, or l-arginine ethyl ester (ARG-EE) for 2h. To modulate ARG transport, siRNA for ARG transporter (CAT-1) vs. sham siRNA were transfected into cells. ARG transport activity was assessed by cellular fluxes of ARG, -ARG, dimethylarginines, and l-citrulline by an LC–MS/MS assay. eNOS activity was determined by nitrite/nitrate accumulation, either via a fluorometric assay or by15N-nitrite or estimated -citrulline concentrations when -ARG was used to challenge the cells. We found that ARG-EE incubation increased cellular ARG concentration but no increase in nitrite/nitrate was observed, while ARG incubation increased both cellular ARG concentration and nitrite accumulation. Cellular nitrite/nitrate production did not correlate with cellular total ARG concentration. Reduced -ARG cellular uptake in CAT-1 siRNA transfected cells vs. control was accompanied by reduced eNOS activity, as determined by 15N-nitrite, total nitrite and -citrulline formation. Our data suggest that extracellular ARG, not intracellular ARG, is the major determinant of NO production in endothelial cells. It is likely that once transported inside the cell, ARG can no longer gain access to the membrane-bound eNOS. These observations indicate that the “l-arginine paradox” should not consider intracellular ARG concentration as a reference point. [Copyright &y& Elsevier]

Abstract: Objective: Nitric oxide (NO) is a short-lived gaseous messenger with multiple physiological functions including regulation of blood flow, platelet adhesion and aggregation inhibition. NO synthases (NOS) catalyze the conversion of cationic amino acid l-arginine in l-citrulline and NO. Despite an increasing prevalence of obesity and metabolic syndrome (MetS) in the last decades, the exact mechanisms involved in the pathogenesis and cardiovascular complications are not fully understood. We have examined the effects of obesity and MetS on the l-arginine–NO–cGMP pathway in platelets from a population of adolescents. Materials: A total of twenty six adolescent patients (13 with obesity and 13 with MetS) and healthy volunteers (n=14) participated in this study. Transport of l-arginine, NO synthase (NOS) activity and cGMP content in platelets were analyzed. Moreover, platelet function, plasma levels of l-arginine, metabolic and clinical markers were investigated in these patients and controls. Results: l-arginine transport (pmol/109 cells/min) in platelets via system y+L was diminished in obese subjects (20.8±4.7, n=10) and MetS patients (18.4±3.8, n=10) compared to controls (52.3±14.8, n=10). The y+L transport system correlated negatively to insulin levels and Homeostasis Model Assessment of Insulin Resistance (HOMA IR) index. No differences in NOS activity and cGMP content were found among the groups. Moreover, plasma levels of l-arginine were not affected by obesity or MetS. Discussion: Our study provides the first evidence that obesity and MetS lead to a dysfunction of l-arginine influx, which negatively correlates to insulin resistance. These findings could be a premature marker of future cardiovascular complications during adulthood. [ABSTRACT FROM AUTHOR]

Obesity related hypertension is a major risk factor for resistant hypertension. We do not completely understand the mechanism(s) underlying the development of obesity related hypertension which hinders the development of novel treatment strategies for this condition. Data from experimental studies and small clinical trials indicate that transport of L-arginine, the substrate for nitric oxide (NO), and subsequent NO production are reduced in obesity induced hypertension. Reduced NO bioavailability can induce hypertension via multiple mechanisms. Mirmiran et al. recently analyzed data from a large population study and found that the association between dietary L-arginine and serum nitrate and nitrite was weakened in obese hypertensive subjects compared to obese normotensives. These data suggest that L-arginine dependent NO production is impaired in the former group compared to the latter which may represent a novel mechanism contributing to hypertension in the setting of obesity. [ABSTRACT FROM AUTHOR]

Nitric oxide (NO) inhibits platelet function and plays a key role in the regulation of cardiovascular homeostasis. Essential hypertension is characterized by an increased risk of thrombus formation, and by an inhibition of intraplatelet NO bioactivity. We have previously shown that membrane transport ofl-arginine is a rate-limiting step for platelet-derived NO synthesis. This study examined the effects of exercise on the plateletl-arginine–NO pathway and aggregation and systemic inflammation markers in 13 sedentary hypertensive patients subjected to 60 min of training activity (exercise group), predominantly aerobic, three times a week for a period of 12 weeks. Six sedentary hypertensive patients participated in the control group. After 12 weeks,l-arginine transport was significantly increased and associated with increased platelet NO synthase activity and cGMP levels and reduced platelet aggregation. Moreover, exercise training reduced plasma concentrations of fibrinogen and C-reactive protein and blood pressure. The control group did not change their previous intraplateletl-arginine–NO results and systemic inflammatory markers levels. Thus, exercise training reduces inflammatory responses, restores NO synthesis in platelets and thereby contributes to the beneficial effects of exercise in hypertension. The present study adds exercise as a new tool to reduce morbidity and mortality associated with platelet activation in hypertension. [ABSTRACT FROM AUTHOR]

Background. L-arginine or its metabolites may be important pathogenetic factors in ischemic acute renal failure (iARF) in rats. It was found that the L-arginine-nitric oxide synthase-nitric oxide system plays an important role in the renal hemodynamic alterations in the early stages of diabetes. The iARF in diabetic rats is much more severe than the normal rats exposed to a same ischemia time. The purpose of the present study was to evaluated L-arginine uptake and its transporters and nitric oxide synthase isoform expression in tubuli and glomeruli of STZ-induced diabetic rats with iARF. Methods. iARF was induced by right nephrectomy and left renal artery clamping for 60 min followed by a 60 min reflow period. iARF was induced in STZ diabetes rats two weeks after intraperitoneal streptozotocin (60 mg/kg body weight) and in normal control rats. L-arginine uptake, L-arginine transporters (CAT1 and CAT2) and nitric oxide synthases (iNOS, eNOS, and bNOS) were determined by RT-PCR) in both glomeruli and tubuli preparations. Results. The STZ diabetic rats compared with the non diabetic normal rats have a higher glomerular L-arginine uptake, higher iNOS mRNA, lower eNOS mRNA, and lower tubular CAT1 mRNA, eNOS mRNA, and bNOS mRNA. The diabetic iARF after one hour of reperfusion had lower glomerular L-arginine uptake, lower CAT1 mRNA, lower eNOS mRNA, lower bNOS, and higher tubular iNOS mRNA compared with iARF in normal rats. Conclusions. Our findings suggest a prolonged and more severe post-glomerular vasoconstriction very early after the reflow in the iARF of STZ diabetic rats compared with the iARF in the normal control rats. That may be a plausible explanation to the very significant decline in GFR and tubular necrosis that characterize the iARF in diabetic rats. [ABSTRACT FROM AUTHOR]

The aim of the present study was to investigate the effect of hydrogen sulfide (H2S) signaling by nitric oxide (NO) in isolated rat aortas and cultured human umbilical vein endothelial cells (HUVECs). Both administration of H2S and NariS, as well as endogenous H2S, reduced NO formation, endothelial nitric oxide synthase (eNOS) activity, eNOS transcript abundance, and L-arginine (L-Arg) transport (all P < 0.01). The kinetics analysis of eNOS activity and L-Arg transport showed that H2S reduced Vmax values (all P < 0.01) without modifying Km parameters. Use of selective NOS inhibitors verified that eNOS [vs. inducible NOS (iNOS) and neuronal NOS (nNOS)] was the specific target of H2S regulation. H2S treatment (100 μmol/1) reduced Akt phosphorylation and decreased eNOS phosphorylation at Ser1177. H2S reduced L-Arg uptake by inhibition of a system y+ transporter and decreased the CAT-1 transcript. H2S treatment reduced protein expression of eNOS but not of nNOS and iNOS. Pinacidil (KATP channel opener) exhibited the similar inhibitory effects on the L-Arg/NOS/NO pathway. Glibenclamide (KATP channel inhibitor) partly blocked the inhibitory effect of H2S and pinacidil. An in vivo experiment revealed that H2S downregulated the vascular L-Arg/eNOS/NO pathway after intraperitoneal injection of NariS (14 μmol/kg) in rats. Taken together, our findings suggest that H2S downregulates the vascular L-Arg/NOS/NO pathway in vitro and in vivo, and the KATP channel could be involved in the regulatory mechanism of H2S. [ABSTRACT FROM AUTHOR]

Background. It has become evident that increased nitric oxide (NO) generation may be associated with production of reactive oxygen species, such as peroxynitrite (ONOO−). Peroxynitrite has been postulated to be responsible for several of the cytotoxic effects previously ascribed to NO. Since cellular arginine uptake has been shown to modulate nitric oxide synthase activity, we were intrigued to study the effect of ONOO− on arginine traffic in renal mesangial cells. [ABSTRACT FROM PUBLISHER]

There is clear evidence that chronic inhibition of nitric oxide synthase (NOS) in animals causes hypertension and also leads to progressive kidney damage. There is also evidence that nitric oxide (NO) deficiency occurs in man with chronic kidney disease (CRD) and this may contribute to further progression of CRD, to hypertension, and to other cardiovascular complications. There are multiple ways in which NO deficiency develops in CRD. At end stage there are uremic factors in plasma that inhibit L-arginine transport into cells and this may cause a “net” substrate deficiency. Also, increases occur in endogenous NOS inhibitors, in particular asymmetric dimethylarginine (ADMA). The increased oxidative stress of CRD is likely to be a primary cause of the increased plasma ADMA since the catabolic enzyme, dimethylarginine dimethylaminohydrolase (DDAH) is extremely sensitive to inhibition by oxidants. Animal studies demonstrate a decrease in abundance of the neuronal NOS within the injured kidney that correlates with extent of injury. Overall, there is substantial clinical, “in vitro,” and animal data to suggest that systemic, endothelial, and renal NO deficiency is a common feature of CRD irrespective of the primary genesis of the disease. This NO deficiency, which is multifactorial, contributes to the progressive nature of the CRD and the endothelial dysfunction and associated risk for cardiovascular events. Strategies that reverse NOS inhibition and/or can boost the ability of the damaged kidney to produce NO might help preserve residual renal function and/or slow down the rate of progression to end stage. [ABSTRACT FROM AUTHOR]

Background. Malnutrition is a common feature in chronic renal failure and adversely affects patient morbidity and mortality. We here investigate the effects of nutritional status on the l-arginine–nitric oxide signaling pathway and platelet function in chronic renal failure patients on regular hemodialysis. Methods. Platelet aggregation was correlated with plasma amino acid profiles, l-arginine transport, and nitric oxide synthase (NOS) activity determined by conversion of l-[3H]-arginine to l-[3H]-citrulline and accumulation of intracellular cyclic guanosine monophospate (cGMP) in platelets from malnourished and well-nourished chronic renal failure patients on regular hemodialysis ( N= 78). Results. Transport of l-arginine (pmol/109cells/min) via y+ L system was increased in well-nourished (104 ± 15) compared to controls (57 ± 11) or malnourished chronic renal failure patients (55 ± 13). Basal NOS activity (pmol/108cells) was enhanced in well-nourished chronic renal failure patients (0.51 ± 0.01) compared to controls (0.18 ± 0.01) or malnourished chronic renal failure patients (0.08 ± 0.03). In addition, basal cGMP levels are elevated in platelets from well-nourished chronic renal failure compared to malnourished uremic patients. Platelet aggregation induced by collagen is impaired in well-nourished chronic renal failure patients compared to malnourished patients and controls. Plasma l-arginine levels are reduced in chronic renal failure patients and even lower in malnourished patients. Conclusion. Our findings provide the first evidence that l-arginine transport via the high affinity system y+ L and nitric oxide synthesis are only stimulated in platelets from well-nourished chronic renal failure patients, leading to impaired platelet aggregation. The absence of this adaptive response in the l-arginine–nitric oxide pathway in platelets from malnourished chronic renal failure patients may account for the enhanced occurrence of thrombotic events in these patients. [ABSTRACT FROM AUTHOR]

Abstract: Expression of inducible nitric oxide synthase (iNOS) is generally accompanied by a parallel upregulation in l-arginine transport which is dependent, at least in part, on the synthesis of new carrier proteins. It is not clear however whether the induction of iNOS and its subsequent utilisation of l-arginine for NO synthesis contribute to the enhancement in l-arginine transport rates observed following induction of cells with pro-inflammatory mediators. To address this issue, we have transfected an iNOS construct in a pEGFP-N1 vector into HEK-293 cells and investigated the effects this has on l-arginine transport. The expression of iNOS through transfection resulted in the production of significant quantities of NO as detected by the standard Griess assay. Under these conditions, the transport of l-arginine was found to be unaltered, with rate of uptake being comparable in both transfected and non-transfected cells. Characterisation of the transporter(s) involved with uptake of l-arginine revealed features characteristic of the classical cationic amino acid transport system y+. Further analysis of the expression profile of the cationic amino acid transporter (CAT) involved revealed the presence of transcripts for CAT-1 and CAT-2B. These data demonstrate that iNOS activity does not drive or enhance l-arginine transport despite the fact that HEK-293 cells transport l-arginine via the CATs, including CAT-2B which is thought to be critical for supply of substrate to iNOS. [Copyright &y& Elsevier]

Abstract Background Recent evidence indicates that hyperhomocysteinaemia is an independent risk factor for atherosclerosis, thrombosis and other cardiovascular diseases. This may be secondary to impaired fibrinolysis or increased platelet reactivity. Nitric oxide (NO), a product from l-arginine by NOS and potent antiaggregating agent, plays an important role in the regulation of platelet function. Design The present study aimed to define the effect of homocysteine on the l-arginine/NO pathway in human platelets. l-arginine uptake, NO formation and Ca2+ levels were measured. Moreover the homocysteine effect on platelet activation induced by thrombin was tested. Results Homocysteine causes a concentration-dependent inhibition of l-arginine transport. Results show that homocysteine does not modify the Km parameter, but it significantly decreases the Vmax value. The nitrite and nitrate formation, strictly correlated with the l-arginine transport, also significantly decreased. In contrast, cNOS activity remained unchanged upon homocysteine treatment. In addition homocysteine in a dose dependent manner increased the intracellular Ca2+ concentration and platelet response to thrombin. Conclusions Results indicate that the l-arginine/NO pathway is one of the various targets of homocysteine in human platelets. The increased Ca2+ levels associated with reduced NO formation may generate hyperactivation and may contribute to the thrombogenic processes. [ABSTRACT FROM AUTHOR]

Recent evidence indicates that chronic hyperhomocysteinemia is an independent risk factor for the development of atherosclerosis, thrombosis, and other cardiovascular diseases. This may be secondary to impaired fibrinolysis, or increased platelet reactivity. l-Arginine/nitric oxide synthase/nitric oxide (NO) plays an important role in the regulation of platelet function. The present study was undertaken to determine the effect of homocysteine (HCY) on the l-arginine/NO pathway of human platelets. Washed human platelets were incubated in the presence or absence of HCY for 2 h at 37°C followed by a measurement of indices of the l-arginine/NO pathway. HCY caused a concentration-dependent reduction in the platelet up-take of l-[3H]arginine. HCY also caused a concentration-dependent decrease in nitrite production concurrent with a decrease in cyclic guanosine monophosphate, whereas NO synthase activity of the platelets, measured as conversion of l-[3H]arginine to l-[3H]citrulline, remained unchanged on incubation with HCY. These observations indicate that the l-arginine/NO pathway is involved in the mechanism responsible for the effects of HCY on platelets by diminishing NO production through decreased uptake of l-arginine. [ABSTRACT FROM AUTHOR]

Background: Chronic renal disease (CRD) is associated with hypertension and reduced synthesis of nitric oxide (NO). Here, we investigated whether there is a circulating endothelial NO synthase (eNOS) inhibitory factor(s) in some patients with CRD that might directly influence endothelial NOS.Methods: Human dermal microvascular endothelial cells (HDMECs) were incubated for six hours with 20% plasma from subjects with normal renal function (PCr = 0.8 +/- 0.2 mg%), and patients with moderate renal insufficiency of various causes (PCr = 4.0 +/- 1.5 mg%) and impact on NOS activity, transport of L-arginine, and abundance of eNOS protein were measured. Plasma concentrations of asymmetric and symmetric dimethyl L-arginine (ADMA and SDMA) were also measured.Results: There was no effect of any human plasma on L-arginine transport. The NOS activity was variable in CRD patients and fell into two subgroups: CRD I, individual values similar to control, and CRD II, individual values lower than control mean. The effect of CRD plasma on NOS activity in cultured cells was not related to the primary disease, but was predicted by plasma ADMA levels since plasma ADMA was elevated in CRD II versus both control and CRD I. Blood urea nitrogen and creatinine levels were uniformly elevated in CRD plasma. The abundance of eNOS protein was unaffected by plasma.Conclusion: High plasma levels of ADMA in CRD patients are independent of reduced renal clearance, suggesting an alteration in ADMA synthesis and/or degradation. High ADMA is a marker and is partly responsible for the inhibition of eNOS activity in cultured cells and may also result in reduced eNOS activity in vivo, with consequent hypertension. [ABSTRACT FROM AUTHOR]

Although the numbers of patients affected by cardiorenal syndrome keeps increasing, we lack a complete understanding of the molecular pathways involved in its development and progression. Nitric oxide synthase (NOS) may play a role in cardiorenal syndrome, particularly cardiorenal syndrome type 2 (CRS2). However, complexities and paradoxical clinical findings have limited translation. In the current Clinical Science, Giam et al. (Clinical Science (2020) 134, 2755-2769) highlight the role of a key NOS substrate transporter, the cationic amino acid transporter-1, in preserving renal function in CRS2. In this commentary, we introduce the cardiorenal syndrome and the putative role that nitric oxide (NO) may play in the development of this disease and discuss the exciting findings of Giam et al. (Clinical Science (2020) 134, 2755-2769) and their tantalizing translational implications., (© 2021 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

The uptake of L-arginine into purified rat brain synaptosomes was investigated with respect to time and various concentrations of L-[H] arginine. Specific uptake was found to be linear with time for up to 5 min of incubation at 37°C. Electrolytes, including sodium chloride, potassium chloride, magnesium chloride and calcium chloride, inhibited uptake of 3 μM L-arginine, and the inhibitory effect increased with increased electrolyte concentration under constant osmolarity. It was found that L-arginine was transported into synaptosomes by two uptake components - a high affinity component (3.5 μM) and a low affinity component (100 μM). These two components were similar to the Ly system because of their extreme sensitivity to inhibition by L-lysine and L-ornithine but were distinguishable from each other by kinetic analysis of the uptake data and by their relative sensitivity to inhibition by several amino acids. [ABSTRACT FROM AUTHOR]