L-arginine Transport Research Articles

Insulin resistance in obesity and metabolic syndrome: Is there a connection with platelet l-arginine transport?

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/10 9 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.

P.2.b.023 Rodent magnesium-deficiency models for depression and their relationship to NMDA receptor/Nitric Oxide pathways

Colocalization of demethylating enzymes and NOS and functional effects of methylarginines in rat kidney. NG-monomethylarginine (L-NMA) and asymmetric NG, NG-dimethylarginines (ADMA) are endogenous inhibitors of cellular L-arginine uptake and/or nitric oxide (NO) synthesis that are implicated in renal parenchymal and Dahl salt-sensitive hypertension. Since the L-arginine:(L-NMA+ADMA) ratio determines NO synthase (NOS) activity, we compared the immunohistochemical distribution of NOS with NG, NG-dimethylarginine dimethylaminohydrolase (DDAH), which inactivates dimethylarginines (DMA) and L-NMA by hydrolysis to L-citrulline. Neuronal NOS (nNOS) was expressed predominantly in tubular epithelial cells of macula densa (MD), endothelial NOS (eNOS) in vascular endothelial cells (EC), and inducible NOS (iNOS) quite widely in tubular epithelium, including proximal tubules (PT), thick ascending limbs of Henle (TAL), distal convoluted tubule and intercalated cells (IC) of the collecting duct. Immunostaining for DDAH was present in PT, TAL, MD, and IC, and was also present in the glomerulus, Bowman's capsule, and endothelium of blood vessels. DDAH was detected in small vesicles of TAL and PT by electron microscopic (EM) immunocytochemistry. To study the effects of methylarginines on tubuloglomerular feedback (TGF) response, vehicle or methylarginines (10−3 M) were added to artificial tubular fluid (ATF) perfused orthogradely from the late PT at 40 nl. min−1 while assessing changes in glomerular capillary pressure from proximal stop flow pressure (PSF). Whereas the maximal TGF responses were unchanged by vehicle (ΔTGF 0 ± 0%) or symmetric DMA (SDMA; +1 ± 2%, NS), they were enhanced by L-NMA (+22 ± 4%, P < 0.001) and asymmetric DMA (ADMA; +28 ± 3%, P < 0.001). Since L-arginine transport can regulate renal epithelial NO generation, methylarginines (10−3 M) or vehicle were co-perfused orthogradely with [3H]-L-arginine from the late PT and collected at the early distal tubule to study arginine uptake from the perfused loop of Henle. All methylarginines reduced fractional loop [3H] absorption significantly (P < 0.001; vehicle, 84 ± 6; ADMA, 49 ± 6; SDMA, 56 ± 6; L-NMA, 41 ± 6%). In conclusion, sites of DDAH expression in the vasculature or nephron are all sites of expression of an isoform of NOS. L-NMA, ADMA, and SDMA all inhibit renal tubular L-arginine uptake, whereas L-NMA and ADMA, but not SDMA, enhance TGF responses. Therefore, DDAH may regulate the cellular L-arginine: methylarginine levels in specific renal cells, thereby governing cell-specific L-arginine uptake and NO generation in renal tubular epithelium.

Glycine and l-arginine transport in cultured Müller glial cells (TR-MUL)

We have reported previously that creatine is biosynthesized from glycine and l-arginine in Müller cells, but the mechanism responsible for glycine and l-arginine uptake by Müller cells remains elusive. To explore this issue, [ 14C]glycine and [ 3H] l-arginine uptake by Müller cells was characterized using a conditionally immortalized rat Müller cell line (TR-MUL5 cells). [ 14C]Glycine uptake by TR-MUL5 cells was Na +- and Cl −-dependent, and a saturable process with Michaelis–Menten constants of 48.6 μM and 4.53 mM, and inhibited by glycine transporter 1 (GlyT1) and system A substrates. Under Cl −-free conditions, the high-affinity process was abolished. RT-PCR analysis demonstrated that GlyT1 and system A (encoding ATA1 and ATA2) mRNA are expressed in TR-MUL5 cells. These uptake studies suggest that GlyT1 and system A are involved in [ 14C]glycine uptake for the high- and low-affinity processes, respectively, in TR-MUL5 cells. [ 3H] l-Arginine uptake by TR-MUL5 cells was primarily an Na +-independent and saturable process with Michaelis–Menten constants of 15.0 and 403 μM. This process was inhibited by substrates of cationic amino acid transporter (CAT)s, such as l-arginine, l-lysine, and l-ornithine. The expression of CAT1 protein was detected in TR-MUL5 cells. These results suggest that CAT1 is at least involved in [ 3H] l-arginine uptake by TR-MUL5 cells. In conclusion, GlyT1 and CAT1 most likely mediate glycine and l-arginine uptake, respectively, by Müller cells and are expected to play an important role in supplying precursors for creatine biosynthesis in Müller cells.

NO control: nitric oxide directly regulates substrate delivery to NOS. Focus on “Nitric oxide can acutely modulate its biosynthesis through a negative feedback mechanism on l-arginine transport in cardiac myocytes”

although the amino acid arginine is commonly associated with nitric oxide (NO) production via NO synthase (NOS), it also participates in the synthesis of urea, creatine, creatinine, agmatine, polyamines, as well as overall protein synthesis. Furthermore, it also influences hormone release (insulin,

High fat diets modulate nitric oxide biosynthesis and antioxidant defence in red blood cells from C57BL/6 mice

The consumption of a high fat (HF) diet is considered a risk factor for the development of obesity. On the other hand, a monounsaturated HF diet has beneficial cardiometabolic effects. Since nitric oxide (NO) modulates vascular homeostasis, we investigate whether HF diets that vary in fatty acid composition have a different effect on the l-arginine-NO pathway and oxidative stress in C57BL/6 mice red blood cells (RBC). The olive oil diet induced an activation of l-arginine transport compared to other diets. NO synthase (NOS) activity was increased in all unsaturated HF diets (olive, sunflower and canola oils). Moreover, the expression of endothelial NOS (eNOS) and inducible NOS (iNOS) was increased in the olive oil group. In contrast, NOS activity from the lard group was decreased associated with diminished l-arginine transport. Olive oil also induced superoxide dismutase activation. Inhibition of the l-arginine-NO pathway in the lard group could contribute to cardiovascular diseases, while unsaturated HF diets may have a protector effect via enhanced NO bioavailability.

Functional relationship between cationic amino acid transporters and β-defensins: Implications for dry skin diseases and the dry eye

The ocular surface, constantly exposed to environmental pathogens, is particularly vulnerable to infection. Hence an advanced immune defence system is essential to protect the eye from microbial attack. Antimicrobial peptides, such as β-defensins, are essential components of the innate immune system and are the first line of defence against invaders of the eye. High concentrations of l-arginine and l-lysine are necessary for the expression of β-defensins. These are supplied by epithelial cells in inflammatory processes. The limiting factor for initiation of β-defensin production is the transport of l-arginine and l-lysine into the cell. This transport is performed to 80% by only one transporter system in the human, the y +-transporter. This group of proteins exclusively transports the cationic amino acids l-arginine, l-lysine and l-ornithine and is also known under the term cationic amino acid transporter proteins (CAT-proteins). Various infections associated with l-arginine deficiency (for example psoriasis, keratoconjuctivitis sicca) are also associated with an increase in β-defensin production. For the first time, preliminary work has shown the expression of human CATs in ocular surface epithelia and tissues of the lacrimal apparatus indicating their relevance for diseases of the ocular surface. In this review, we summarize current knowledge on the human CATs that appear to be integrated in causal regulation cascades of β-defensins, thereby offering novel concepts for therapeutic perspectives.

Platelet aggregation in arterial hypertension: Is there a nitric oxide–urea connection?

1. Systemic arterial hypertension (SAH) is a major independent risk factor for cardiovascular disease. The physiopathology of SAH is multifactorial, complex and remains to be elucidated. Nitric oxide (NO) is an important regulator of vascular and haemostatic functions. The cationic amino acid l-arginine serves as the substrate for NO synthases (NOS) and arginase, an enzyme of the urea cycle. We have previously reported inhibition of l-arginine transport in erythrocytes and platelets in hypertension. 2. The aim of the present study was to investigate the l-arginine-NO pathway and urea cycle in platelets and their role in platelet function and systemic inflammatory responses in SAH patients. The expression and activity of NOS and arginase in platelets, platelet aggregation and plasma levels of C-reactive protein (CRP) were evaluated in 20 SAH patients and 18 age-matched healthy volunteers. 3. There was a reduction of NOS activity in hypertensive patients that was associated with activation of platelet aggregability induced by collagen, but not by ADP. Platelets from SAH patients exhibited compensatory overexpression of inducible NOS, but not endothelial NOS. Intraplatelet arginase activity in SAH patients was not affected, but systemic concentrations of CRP were increased compared with controls. 4. It is likely that diminished NO bioavailability in SAH contributes to cardiovascular complications. Our findings may provide the basis for developing new therapeutic approaches for the treatment of hypertension.

Oxidative stress, l-arginine–nitric oxide and arginase pathways in platelets from adolescents with anorexia nervosa

Anorexia nervosa (AN) is associated with high cardiovascular mortality. Nitric oxide (NO) inhibits platelet function and regulates the cardiovascular homeostasis. The aim of this study was to investigate the l-arginine–NO–GMPc and arginase pathways and oxidative stress in platelets from patients with AN. Intraplatelet l-arginine transport, NOS expression and activity, cGMP levels, platelet aggregation, arginase expression and oxidative stress were measured in adolescent patients with AN ( n = 11) and healthy controls ( n = 12). Plasma l-arginine levels were significantly reduced in AN. l-arginine transport, NOS activity and cGMP basal levels were reduced in platelets associated with unchanged platelet aggregability. The expression of NOS isoforms was not affected. TBARS production was diminished, while the activity of superoxide dismutase was elevated in AN patients. There was an overexpression of arginase II in AN. Alterations of l-arginine–NO–GMPc and arginase pathways in platelets can be early predictors of the incidence of cardiovascular disease into adult life in AN.

The Activity of a Low-Affinity L-Arginine Transporter Quenches Peroxynitrite-Induced Fluorescence in Ventricular Cardiomyocytes

We discovered a low-affinity, high-capacity L-arginine (L-Arg) transport process in rat cardiomyocytes consistent with the activity of the CAT-2A member of the y(+) family of cationic amino acid transporters (Peluffo, J Physiol, 580:925-936, 2007), set to function in parallel with the previously described high-affinity, low-capacity CAT-1 (Lu et al., Biosci Rep, 29:271-281, 2009). In assessing the role of a low-affinity transporter in this setting, we propose that CAT-2A protects cardiac muscle cells by ensuring the availability of proper L-Arg levels for the synthesis of nitric oxide (NO) via NO synthase (NOS). To test this hypothesis, acutely-isolated cardiomyocytes were loaded with the dye coelenterazine that greatly increases its fluorescence quantum yield in the presence of peroxynitrite (ONOO-) and superoxide radicals. Cells were then exposed to 20 or 100 μM ONOO- and changes in fluorescence were followed with a spectrofluorometer. Addition of extracellular L-Arg reduced ONOO-induced fluorescence in a concentration-dependent manner, an effect that was not mimicked by D-arginine or L-lysine and was fully blocked by the NOS inhibitor L-NAME. L-Arg reduced fluorescence with Ki values of 0.84 ± 0.12 and 1.26 ± 0.16 mM at 20 and 100 μM ONOO-, respectively. L-Arg “zero effect” on ONOO-induced fluorescence was also dependent on ONOO- concentration, with values of 145 and 363 μM for 20 and 100 μM ONOO-, respectively. Below these values, decreasing concentrations of L-Arg progressively increased ONOO-induced fluorescence, an effect that was also blocked by L-NAME. All these effects can be explained by NOS-mediated NO synthesis, which may turn to ONOO- production at limiting L-Arg. Since ONOO- has detrimental effects on cardiac contractility, these results suggest a cardioprotective role for the low-affinity L-Arg transporter, ensuring proper supply of NOS substrate under a variety of physiological and pathological conditions.

Reduced Mitochondrial l-Arginine Transport Contributes to the Pathogenesis of Heart Failure

Reduced Mitochondrial l-Arginine Transport Contributes to the Pathogenesis of Heart Failure

Exercise training in doxorubicin-induced heart failure: effects on the L-arginine–NO pathway and vascular reactivity

Heart failure (HF) is the end-stage of cardiovascular disease and is associated with a high incidence of thrombotic events. Nitric oxide (NO) mediates vasodilation and prevents platelet activation, providing an important antithrombotic effect. The aim of this study was to investigate the effects of aerobic training on survival, platelet L-arginine–NO pathway, and vasodilator properties in doxorubicin (DOX)-induced HF. Sprague Dawley rats were randomly assigned to saline/sedentary (SAL/SED), saline/exercise (SAL/EX), DOX/sedentary (DOX/SED), and DOX/exercise (DOX/EX) groups. Four weeks after intraperitoneal DOX injection (1 mg/kg -1/d -1; 10 days), shortening fraction in DOX/SED and DOX/EX was significantly reduced. Treadmill exercise was performed during 6 weeks, 5 days/week -1, 30 minutes/day -1, 50% to 60% of maximum velocity. Survival was higher in DOX/EX (67%) than DOX/SED (33%). No differences were observed in intraplatelet L-arginine transport assessed by incubation with L- [ 3H]-arginine, nor in NOS activity measured by the conversion of L- [ 3H]-arginine into L- [ 3H]-citrulline among the groups. Vasodilation response to acetylcholine was impaired in DOX/SED and DOX/EX; in nitroglycerine, it was limited to DOX/SED. Aerobic training reduced mortality in DOX-induced HF animals and restored vascular smooth muscle relaxation properties. However, it did not ameliorate intraplatelet NO bioavailability and endothelial function during the period studied.

Dysregulation of Corticosterone Secretion in Streptozotocin-Diabetic Rats: Modulatory Role of the Adrenocortical Nitrergic System

An increased activity of the hypothalamo-pituitary-adrenal axis resulting in exaggerated glucocorticoid secretion has been repeatedly described in patients with diabetes mellitus and in animal models of this disease. However, it has been pointed out that experimental diabetes is accompanied by a decreased glucocorticoid response to ACTH stimulation. Because previous studies from our laboratory demonstrate the involvement of nitric oxide (NO) in the modulation of corticosterone production, present investigations were designed to evaluate 1) the impact of streptozotocin (STZ)-induced diabetes on the adrenocortical nitrergic system and 2) the role of NO in the modulation of adrenal steroidogenesis in STZ-diabetic rats. Four weeks after STZ injection, increased activity and expression levels of proteins involved in L-arginine transport and in NO synthesis were detected, and increased levels of thiobarbituric acid reactive species, carbonyl adducts, and nitrotyrosine-modified proteins were measured in the adrenocortical tissue of hyperglycemic rats. An impaired corticosterone response to ACTH was evident both in vivo and in adrenocortical cells isolated from STZ-treated animals. Inhibition of NO synthase activity resulted in higher corticosterone generation in adrenal tissue from STZ-treated rats. Moreover, a stronger inhibition of steroid output from adrenal cells by a NO donor was observed in adrenocortical Y1 cells previously subjected to high glucose (30 mM) treatment. In summary, results presented herein indicate an inhibitory effect of endogenously generated NO on steroid production, probably potentiated by hyperglycemia-induced oxidative stress, in the adrenal cortex of STZ-treated rats.

Cationic amino acid transporter 1‐mediated l‐arginine transport at the inner blood–retinal barrier

The purpose of this study was to identify the transporter mediating l-arginine transport at the inner blood-retinal barrier (BRB). The apparent uptake clearance of [(3)H]L-arginine into the rat retina was found to be 118 microL/(min.g retina), supporting a carrier-mediated influx transport of L-arginine at the BRB. [(3)H]L-arginine uptake by a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2 cells), used as an in vitro model of the inner BRB, was primarily an Na(+)-independent and saturable process with Michaelis-Menten constants of 11.2 microM and 530 microM. This process was inhibited by rat cationic amino acid transporter (CAT) 1-specific small interfering RNA as well as substrates of CATs, L-arginine, L-lysine, and L-ornithine. The expression of cationic amino acid transporter (CAT) 1 mRNA was 25.9- and 796-fold greater than that of CAT3 in TR-iBRB2 and magnetically isolated rat retinal vascular endothelial cells, respectively. The expression of CAT1 protein was detected in TR-iBRB2 cells and immunostaining of CAT1 was observed along the rat retinal capillaries. In conclusion, CAT1 is localized in retinal capillary endothelial cells and at least in part mediates L-arginine transport at the inner BRB. This process seems to be closely involved in visual functions by supplying precursors of biologically important molecules like nitric oxide in the neural retina.

A Role for Insulin on L-Arginine Transport in Fetal Endothelial Dysfunction in Hyperglycaemia

Endothelial cells are key in the regulation of vascular tone through the release of vasoactive molecules, including nitric oxide (NO). NO is a gas synthesized from the cationic amino acid L-arginine via the endothelial NO synthase (eNOS). The semi-essential amino acid L-arginine is a taken up by endothelial cells via systems y(+) and y(+)L in primary cultures of human umbilical vein endothelial cells (HUVEC). System y(+) is a family of membrane transporters including at least five transport systems for cationic amino acids (CAT) of which HUVEC express human CAT-1 (hCAT-1) and hCAT-2B. Exposure of HUVEC to high extracellular concentrations of D-glucose increases L-arginine transport, hCAT-1 mRNA expression and eNOS activity. These phenomena are also related with increased production of reactive oxygen species (ROS), thus supporting the possibility that changes in L-arginine/NO signalling pathway result from elevated ROS. It has been shown that insulin blocks D-glucose-increased L-arginine transport and cGMP accumulation in HUVEC, whereas in this cell type insulin also modulates high D-glucose effects by activating the transcriptional factors Sp1 and NFkappaB. These transcription factors have response elements in SLC7A1 (for hCAT-1) gene promoter region, thus representing 2 possible targets for regulation of the expression of this transporter by D-glucose and/or insulin in this cell type. Recent evidences suggest that insulin blocks the stimulatory effect of D-glucose on L-arginine transport by reducing the transcriptional activity of SLC7A1 via Sp1-, NFkappaB- and ROS-dependent mechanisms. Thus, a role for these transcription factors in response to insulin is proposed in fetal endothelial cells exposed to hyperglycaemia.

Sorting of Leishmania-bearing dendritic cells reveals subtle parasite-induced modulation of host-cell gene expression

Once in the mouse skin, Leishmania (L) amazonensis amastigotes are hosted by professional mononuclear phagocytes such as dendritic cells (DCs). When monitored after parasite inoculation, the frequency of amastigote-hosting DCs is very low (<1%) in both the skin and skin-draining lymph nodes. Therefore, we designed and validated an efficient procedure to purify live amastigotes-hosting DCs with the objective to facilitate quantitative and qualitative analysis of such rare cells. To this end, a L. amazonensis transgenic parasite expressing DsRed2 fluorescent protein was generated and added to mouse bone marrow-derived DC cultures. Then, a high speed sorting procedure, performed in BSL-2 containment, was setup to pick out only DCs hosting live amastigotes. This study reveals, for the first time, a unique transcript pattern from sorted live amastigotes-hosting DCs that would have been undetectable in unsorted samples. It was indeed possible to highlight a significant and coordinated up-regulation of l-arginine transporter and arginase2 transcripts in Leishmania-hosting DCs compared to un-parasitized DCs. These results indicate that arginine catabolism for polyamine generation is dominating over L-arginine catabolism for NO generation. In conclusion, this approach provides a powerful method for further characterisation, of amastigote-hosting DCs in the skin and the skin-draining lymph nodes.

Effect of peroxynitrite on endothelial L-arginine transport and metabolism

Under conditions of oxidative stress it is well known that the bioavailability of nitric oxide (NO) is known to be significantly reduced. This process is in part due to the combination of NO with superoxide radicals to form peroxynitrite (ONOO −). While this process inactivates NO per se, it is not certain to which extent this process may also further impair ongoing NO production. Given the pivotal role of arginine availability for NO synthesis we determined the impact of ONOO − on endothelial arginine transport and intracellular arginine metabolism. Peroxynitrite reduced endothelial [ 3H]- l-arginine transport and increased the rate of arginine efflux in a concentration-dependent manner (both p < 0.05). In conjunction, exposure to ONOO − significantly reduced the intracellular concentration of l-arginine, N G-hydroxy- l-arginine (an intermediate of NO biosynthesis) and citrulline by 46%, 45% and 60% respectively (all p < 0.05), while asymmetric dimethyl arginine (ADMA) levels rose by 180% ( p < 0.05). ONOO − exposure did not alter the cellular distribution of the principal l-arginine transporter, CAT1, rather the effect on CAT1 activity appeared to be mediated by protein nitrosation. Conclusion Peroxynitrite negatively influences NO production by combined effects on arginine uptake and efflux, most likely due to a nitrosative action of ONOO − on CAT-1.

Reduced L‐arginine transport contributes to the pathogenesis of myocardial ischemia‐reperfusion injury

Myocardial injury due to ischemia-reperfusion (I-R) damage remains a major clinical challenge. Its pathogenesis is complex including endothelial dysfunction and heightened oxidative stress although the key driving mechanism remains uncertain. In this study we tested the hypothesis that the I-R process induces a state of insufficient L-arginine availability for NO biosynthesis, and that this is pivotal in the development of myocardial I-R damage. In neonatal rat ventricular cardiomyocytes (NVCM), hypoxia-reoxygenation significantly decreased L-arginine uptake and NO production (42 +/- 2% and 71 +/- 4%, respectively, both P < 0.01), maximal after 2 h reoxygenation. In parallel, mitochondrial membrane potential significantly decreased and ROS production increased (both P < 0.01). NVCMs infected with adenovirus expressing the L-arginine transporter, CAT1, and NVCMs supplemented with L-arginine both exhibited significant (all P < 0.05) improvements in NO generation and mitochondrial membrane potentials, with a concomitant significant fall in ROS production and lactate dehydrogenase release during hypoxia-reoxygenation. In contrast, L-arginine deprived NVCM had significantly worsened responses to hypoxia-reoxygenation. In isolated perfused mouse hearts, L-arginine infusion during reperfusion significantly improved left ventricular function after I-R. These improved contractile responses were not dependent on coronary flow but were associated with a significant decrease in nitrotyrosine formation and increases in phosphorylation of both Akt and troponin I. Collectively, these data strongly implicate reduced L-arginine availability as a key factor in the pathogenesis of I-R injury. Increasing L-arginine availability via increased CAT1 expression or by supplementation improves myocardial responses to I-R. Restoration of L-arginine availability may therefore be a valuable strategy to ameliorate I-R injury.

Reduced l-Arginine Transport and Nitric Oxide Synthesis in Human Umbilical Vein Endothelial Cells from Intrauterine Growth Restriction Pregnancies is Not Further Altered by Hypoxia

Intrauterine growth restriction (IUGR) is associated with chronic fetal hypoxia, altered placental vasodilatation and reduced endothelial nitric oxide synthase (eNOS) activity. In human umbilical vein endothelial cells (HUVEC) from pregnancies complicated with IUGR (IUGR cells) and in HUVEC from normal pregnancies (normal cells) cultured under hypoxia l-arginine transport is reduced; however, the mechanisms leading to this dysfunction are unknown. We studied hypoxia effect on l-arginine transport and human cationic amino acid transporters 1 (hCAT-1) expression, and the potential NO and protein kinase C α (PKCα) involvement. Normal or IUGR HUVEC monolayers were exposed (0–24 h) to 5% O 2 (normoxia), and 1 or 2% O 2 (hypoxia). l-Arginine transport and hCAT-1 expression, phosphorylated and total PKCα or eNOS protein and mRNA expression were quantified. eNOS involvement was tested using a siRNA against eNOS (eNOS-siRNA) adenovirus. IUGR cells in normoxia or hypoxia, and normal cells in hypoxia exhibited reduced l-arginine transport, hCAT-1 expression, NO synthesis and eNOS phosphorylation at Serine 1177, effects reversed by calphostin C (PKC inhibitor) and S-nitroso- N-acetyl- l, d-penicillamine (SNAP, NO donor). However, N G-nitro- l-arginine methyl ester ( l-NAME, NOS inhibitor) reduced hCAT-1 expression only in normal cells in normoxia. Increased Thr 638-phosphorylated PKCα was exhibited by IUGR cells in normoxia or hypoxia and normal cells in hypoxia. The effects of hypoxia in normal cells were mimicked in eNOS-siRNA transduced cells; however, IUGR phenotype was unaltered by eNOS knockdown. Thus, IUGR- and hypoxia-reduced l-arginine transport could result from increased PKCα, but reduced eNOS activity leading to a lower hCAT-1 expression in HUVEC. In addition, IUGR endothelial cells are either not responsive or maximally affected by hypoxia. These mechanisms could be responsible for placental dysfunction in diseases where fetal endothelium is chronically exposed to hypoxia, such as IUGR.

ROLE OF l‐ARGININE IN NITRIC OXIDE PRODUCTION IN HEALTH AND HYPERTENSION

1. l-Arginine is the substrate for vascular nitric oxide (NO) formation. Under normal physiological conditions, intracellular l-arginine levels far exceed the K(m) of NO synthase for l-arginine. However, endogenous NO formation is dependent on extracellular l-arginine concentrations, giving rise to the concept of the 'l-arginine paradox'. 2. Nitric oxide production in epithelial and endothelial cells is closely coupled to cellular l-arginine uptake, indicating that l-arginine transport mechanisms play a major role in the regulation of NO-dependent function. 3. Consistent with the data in endothelial and epithelial cells are functional data indicating that exogenous l-arginine can increase renal vascular and tubular NO bioavailability and thereby influence kidney perfusion, function and arterial pressure. The integrated effect of increased cellular l-arginine transport is to lower arterial pressure. Therefore, the use of l-arginine in the treatment of hypertension warrants investigation. 4. Low NO bioavailability is central to the development and maintenance of hypertension and to related endothelial dysfunction and target organ damage. We propose that l-arginine can interrupt the vicious cycle that initiates and maintains low NO in hypertension by increasing the formation of NO.

Norfloxacin treatment for clinically significant portal hypertension: results of a randomised double‐blind placebo‐controlled crossover trial

While selective intestinal decontamination (SID) can alter the hyperdynamic circulatory state of cirrhosis, the impact of SID on portal pressure remains unclear especially in the setting of clinically significant portal hypertension. To examine the impact of SID with norfloxacin on portal pressure in subjects with clinically significant portal hypertension and explore the potential mechanisms by which norfloxacin exerts its haemodynamic effects. Randomised, double blind, placebo-controlled, crossover trial of norfloxacin 400 mg twice daily for 4 weeks. The portal pressure was assessed by hepatic venous pressure gradient (HVPG). Endotoxaemia was assessed by the Limulus amebocyte lysate (LAL) assay. l-arginine (l-Arg) transporter function was assessed in peripheral blood mononuclear cells (PBMCs). Plasma levels of urotensin II (UII) and tumour necrosis factor were measured before and after therapy. Sixteen subjects with clinically significant portal hypertension (16.5+/-1.1 mmHg) completed the study. Norfloxacin therapy was not superior to placebo in reducing HVPG (13.8+/-1.0 mmHg vs 13.6+/-1.2 mmHg, P=0.3). Furthermore, no alteration in l-Arg transport was detected after 4 weeks of norfloxacin therapy. Plasma UII levels correlated positively with HVPG (P=0.01) and the Child-Pugh score (P<0.05). However, UII levels following therapy did not parallel HVPG changes. Norfloxacin is not superior to placebo in reducing HVPG in subjects with clinically significant portal hypertension. Furthermore, norfloxacin does not appear to modulate the l-Arg transporter mechanism in this patient population. Although plasma UII correlates positively with HVPG, UII does not appear to have a direct role in modulating HVPG.