L-arginine Transport Activity Research Articles

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.

Modulation of the Cationic Amino Acid Transport System y+L by Surface Potential, Ouabain and Thrombin in Human Platelets: Effects of Uremia

Background: Nitric oxide (NO), a key endogenous mediator involved in the maintenance of platelet function, is synthesized from the amino acid L-arginine. We have shown that L-arginine transport in platelets is rate-limiting for NO synthesis. A disturbance in the L-arginine-NO pathway in platelets was previously described in chronic renal failure (CRF) patients. Methods: Detailed kinetic studies were performed in platelets from controls (n = 60) and hemodialysis patients (n = 26). Results: The transport of L-arginine in platelets is mediated via system y⁺L, which is competitively inhibited by L-leucine in the presence of Na⁺ and by the irreversible inhibitor pCMB. In platelets, system y⁺L is markedly stimulated by an Na⁺/K⁺-ATPase inhibitor, ouabain, and by changes in surface potential, while it is downregulated by intraplatelet amino acid depletion (zero-trans) and by thrombin. In CRF patients, activation of L-arginine transport was limited to well-nourished patients compared to malnourished patients and controls, where it was reduced and did not differ significantly among the groups under zero-trans conditions. Conclusion: Our results provide the first evidence that system y⁺L in platelets is modulated by zero-trans conditions, surface potential, thrombin and intraplatelet Na⁺ concentration. Our findings suggest that enhanced transport in CRF involves increased L-arginine exchange with intraplatelet neutral amino acids.

Nitric Oxide, Malnutrition and Chronic Renal Failure

The conditionally essential amino acid L-arginine is the substrate for nitric oxide (NO) synthesis, a key second messenger involved in physiological functions including endothelium-dependent vascular relaxation and inhibition of platelet adhesion and aggregation. Extracellular L-arginine transport seems to be essential for the production of NO by the action of NO synthases (NOS), even when the intracellular levels of L-arginine are available in excess (L-arginine paradox). Chronic renal failure (CRF) is a complex clinical condition associated with accelerated atherosclerosis and thrombosis leading to cardiovascular events. Various studies document that markers of malnutrition and inflammation, such as low body mass index (BMI), C-reactive protein (CRP) and interleukin-6 (IL-6), are strong independent predictors of cardiovascular mortality in patients with end-stage renal disease (ESRD). There is considerable literature demonstrating that a disturbance in the nitric oxide control mechanism plays a role in mediating the haemodynamic and haemostatic disorders present in CRF. Endogenous analogues of L-arginine, ADMA and L-NMMA, which can inhibit NO synthesis and L-arginine transport, are increased whilst L-arginine is reduced in plasma from all stages of CRF patients. In this context, the uptake of L-arginine in blood cells is increased in undialysed CRF patients and in patients treated by CAPD and haemodialysis. In platelets obtained from haemodialysis patients, the activation of L-arginine transport and NO production was limited to well-nourished patients. Impairment in nitric oxide bioactivity, coupled with malnutrition and inflammation, may contribute to increased incidence of atherothrombotic events in CRF. This article summarizes the current knowledge of L-arginine-nitric oxide pathway and malnutrition in CRF and briefly describes possible therapeutic interventions.

ACTIVATION OF l‐ARGININE TRANSPORT IN UNDIALYSED CHRONIC RENAL FAILURE AND CONTINUOUS AMBULATORY PERITONEAL DIALYSIS PATIENTS

1. Treatment with haemodialysis and continuous ambulatory peritoneal dialysis (CAPD) presents different pathophysiological profiles and it has been suggested that clinical outcome in chronic renal failure may depend on the mode of dialysis. The transport of L-arginine, a precursor of nitric oxide, into blood cells is increased in uraemic patients on haemodialysis. The present study was designed to investigate L-arginine transport into red blood cells (RBC) in uraemic patients not yet on dialysis and on CAPD therapy. 2. Eleven uraemic patients not yet on dialysis and 17 on CAPD were included in the study. L-Arginine transport into RBC and plasma and RBC amino acid profiles were analysed in these sets of patients. 3. L-Arginine transport via system y(+), but not y(+)L, into RBC, was significantly increased in undialysed uraemic patients (459 +/- 40 micromol/L per cell per h) and CAPD patients (539 +/- 61 micromol/L per cell per h) compared with controls (251 +/- 39 micromol/L per cell per h). High-pressure liquid chromatography measurements demonstrated low levels of plasma L-arginine in uraemic patients both on CAPD (54 +/- 3 micromol/L) and not yet on dialysis (80 +/- 6 micromol/L) compared with control subjects (146 +/- 14 micromol/L). 4. Our findings provide the first evidence that uraemic patients not yet on dialysis and on CAPD present with an activation of L-arginine transport via system y(+) into RBC associated with reduced plasma levels of L-arginine.

Pertussis toxin activates L-arginine uptake in pulmonary endothelial cells through downregulation of PKC-alpha activity.

Pertussis toxin (PTX) induces activation of l-arginine transport in pulmonary artery endothelial cells (PAEC). The effects of PTX on l-arginine transport appeared after 6 h of treatment and reached maximal values after treatment for 12 h. PTX-induced changes in l-arginine transport were not accompanied by changes in expression of cationic amino acid transporter (CAT)-1 protein, the main l-arginine transporter in PAEC. Unlike holotoxin, the beta-oligomer-binding subunit of PTX did not affect l-arginine transport in PAEC, suggesting that Galpha(i) ribosylation is an important step in the activation of l-arginine transport by PTX. An activator of adenylate cyclase, forskolin, and an activator of protein kinase A (PKA), Sp-cAMPS, did not affect l-arginine transport in PAEC. In addition, inhibitors of PKA or adenylate cyclase did not change the activating effect of PTX on l-arginine uptake. Long-term treatment with PTX (18 h) induced a 40% decrease in protein kinase C (PKC)-alpha but did not affect the activities of PKC-epsilon and PKC-zeta in PAEC. An activator of PKC-alpha, phorbol 12-myristate 13-acetate, abrogated the activation of l-arginine transport in PAEC treated with PTX. Incubation of PTX-treated PAEC with phorbol 12-myristate 13-acetate in combination with an inhibitor of PKC-alpha (Go 6976) restored the activating effects of PTX on l-arginine uptake, suggesting PTX-induced activation of l-arginine transport is mediated through downregulation of PKC-alpha. Measurements of nitric oxide (NO) production by PAEC revealed that long-term treatment with PTX induced twofold increases in the amount of NO in PAEC. PTX also increased l-[(3)H]citrulline production from extracellular l-[(3)H]arginine without affecting endothelial NO synthase activity. These results demonstrate that PTX increased NO production through activation of l-arginine transport in PAEC.

Activation of Intestinal Arginine Transport by Protein Kinase C Is Mediated by Mitogen-Activated Protein Kinases

L-Arginine uptake by the small intestine can play a pivotal role in regulating nitric oxide synthesis and immune functions in catabolic states. We previously showed that protein kinase C (PKC) activation stimulates intestinal brush-border membrane arginine transport. However, the signaling pathways implicated in this activation have not been studied. The purpose of this study was to investigate the intracellular signal transduction pathways involved in the protein kinase C stimulation of arginine transport across the apical membrane of intestinal epithelial Caco-2 cells. [ 3H]- l-arginine transport activity, Northern blot analysis of mRNA levels of the intestinal arginine transporter CAT1, and Western blot analysis of the mitogen-activated protein (MAP) kinases phospho-p44/42 activity and phospho-MEK1/2 were measured in cultured Caco-2 cells treated with phorbol ester (phorbol 12-myristate 13-acetate, TPA; 0 to 0.5 μmol/L), and the MEK1 inhibitor PD 98059 (0 to 50 μmol/L). Phorbol ester stimulated intestinal arginine transport activity. Arginine transporter gene CAT1 mRNA, phospho-p44/42, and phospho-MEK1/2 levels were stimulated in phorbol ester–treated cells, compared with the control group. Phorbol ester stimulation of arginine transport activity and transporter CAT1 mRNA levels was blocked by PD 98059. These data suggest that phorbol ester stimulates arginine transport in Caco-2 cells via signaling pathways that lead to increased transcription and/or stabilization of CAT1 mRNA. Protein kinase C and MAP kinases MEK1/2 and p44/42 are key intracellular regulators involved in this signal transduction cascade. ( J Gastrointest Surg 2002;6:876–882.)

Peroxynitrite Stimulates l-Arginine Transport Systemy+ in Glial Cells: A POTENTIAL MECHANISM FOR REPLENISHING NEURONALl-ARGININE

We have reported previously that peroxynitrite stimulates L-arginine release from astrocytes, but the mechanism responsible for such an effect remains elusive. To explore this issue, we studied the regulation of L-[(3)H]arginine transport by either exogenous or endogenous peroxynitrite in glial cells. A 2-fold peroxynitrite-mediated stimulation of l-arginine release in C6 cells was found to be Na(+)-independent, was prevented by 5 mm L-arginine and, although only in the presence of Na(+), was blocked by 5 mm L-alanine or L-leucine. Peroxynitrite-mediated stimulation of L-arginine uptake was trans-stimulated by 10 mm L-arginine and was inhibited in a dose-dependent fashion (k(i) of approximately 40 microm) by the system y(+) inhibitor N-ethylmaleimide in C6 cells. Endogenous production of peroxynitrite in lipopolysaccharide-treated astrocytes triggered an increased L-arginine transport activity without affecting Cat1 l-arginine transporter mRNA levels. However, Western blot analyses of peroxynitrite-treated astrocytes and C6 glial cells revealed a 3-nitrotyrosinated anti-Cat1-immunopositive band, strongly suggesting peroxynitrite-mediated Cat1 nitration. Furthermore, peroxynitrite stimulation of L-arginine release was abolished in fibroblast cells homozygous for a targeted inactivation of the Cat1 gene. Finally, peroxynitrite-triggered L-arginine released from astrocytes was efficiently taken up by neurons in an insert-based co-culture system. These results strongly suggest that peroxynitrite-mediated activation of the Cat1 transporter in glial cells may serve as a mechanism focused to replenish L-arginine in the neighboring neurons.

Specific Reversible Stimulation of System y +l-Arginine Transport Activity in Human Intestinal Cells

l-Arginine, which is intimately involved in cellular immune functions and nitric oxide biology, is transported by intestinal cells largely via transport System y +. The gut epithelium is exposed to various luminal amino acids at any given time, and therefore the purpose of this study was to study the regulation of luminal arginine transport by other amino acids. System y + l-arginine transport activity was measured in Caco-2 monolayers exposed to various amino acids. l-arginine and/or other System y + substrates specifically upregulated System y + transport activity twofold after 1 hour, with a response noted as early as 5 minutes. Non–System y + substrates did not affect l-arginine absorption. Kinetic analysis indicated that l-arginine exposure increased both System y + K m and V max. Neither cycloheximide nor actinomycin affected this stimulation, indicating that the regulation did not involve transcription or translation. The System y + substrate activation effect was reversible. l-arginine transport activity returned to baseline within 3 hours when cells were reincubated in amino acid–free media. These data indicate that System y + arginine transport activity is rapidly and reversibly activated by System y + substrates via a mechanism consistent with transmembrane stimulation. These findings identify a mechanism by which luminal nutrients regulate arginine uptake by the gut. ( J Gastrointest Surg 2002;6:379–386.)

Association of L-arginine transporters with fodrin: implications for hypoxic inhibition of arginine uptake.

In this study, we investigated the possible interaction between the cationic amino acid transporter (CAT)-1 arginine transporter and ankyrin or fodrin. Because ankyrin and fodrin are substrates for calpain and because hypoxia increases calpain expression and activity in pulmonary artery endothelial cells (PAEC), we also studied the effect of hypoxia on ankyrin, fodrin, and CAT-1 contents in PAEC. Exposure to long-term hypoxia (24 h) inhibited L-arginine uptake by PAEC, and this inhibition was prevented by calpain inhibitor 1. The effects of hypoxia and calpain inhibitor 1 were not associated with changes in CAT-1 transporter content in PAEC plasma membranes. However, hypoxia stimulated the hydrolysis of ankyrin and fodrin in PAEC, and this could be prevented by calpain inhibitor 1. Incubation of solubilized plasma membrane proteins with anti-fodrin antibodies resulted in a 70% depletion of CAT-1 immunoreactivity and in a 60% decrease in L-arginine transport activity in reconstituted proteoliposomes (3,291 +/- 117 vs. 8,101 +/- 481 pmol. mg protein(-1). 3 min(-1) in control). Incubation with anti-ankyrin antibodies had no effect on CAT-1 content or L-arginine transport in reconstituted proteoliposomes. These results demonstrate that CAT-1 arginine transporters in PAEC are associated with fodrin, but not with ankyrin, and that long-term hypoxia decreases L-arginine transport by a calpain-mediated mechanism that may involve fodrin proteolysis.

Activation of A2-purinoceptors by adenosine stimulates L-arginine transport (system y+) and nitric oxide synthesis in human fetal endothelial cells.

1. Human umbilical vein endothelial cells were challenged acutely with adenosine and its analogues to examine whether adenosine modulates L-arginine transport (system y+) and synthesis of nitric oxide (NO) and prostacyclin (PGI2). 2. L-Arginine transport was stimulated by adenosine (10 microM, 2 min) and the A2-receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS-21680; 100 nM), but not by the A1-receptor agonist N6-cyclopentyladenosine (CPA). 3. Activation of L-arginine transport was inhibited by the A2-receptor antagonists ZM-241385 and 3,7-dimethyl-1-propargylxanthine (DMPX), but unaffected by the A1-receptor antagonists 8-cyclopentyl-1,3-dipropylxanthine and 8-phenyltheophylline or the adenosine transport inhibitor nitrobenzylthioinosine. 4. Adenosine and CGS-21680 evoked a rapid membrane hyperpolarization. 5. Adenosine and CGS-21680 induced increases in intracellular cGMP levels, whereas release of PGI2 was unaffected. NG-nitro-L-arginine methyl ester (an NO synthase inhibitor) and the A2-receptor antagonists ZM-241385 and DMPX prevented increases in cGMP accumulation. 6. Our findings provide the first evidence that activation of human fetal endothelial cell A2-purinoceptors, but not A1-purinoceptors, leads to a membrane hyperpolarization and stimulation of basal rates of L-arginine transport and NO biosynthesis.

Protein kinase C-dependent regulation of l-arginine transport activity in Caco-2 intestinal cells

The regulation of plasma membrane l-arginine transport activity was investigated in differentiated and undifferentiated states of the human intestinal cell line, Caco-2. The sodium-independent, leucine-insensitive uptake of l-arginine measured in this study has been assigned by us previously to system y + in Caco-2 cells. Treatment of cells with serum-free media containing epidermal growth factor (EGF), transforming growth factor α (TGFα), or the protein kinase C (PKC) activator 12- O-tetradecanoylphorbol 13-acetate (TPA), stimulated system y + arginine transport activity in Caco-2 cells. Transport upregulation by these growth factors or by TPA was blocked by cycloheximide or the PKC inhibitor chelerythrine. Arginine uptake was diminished during the course of differentiation, attributable to a reduction in the transport system y + capacity ( V max) with no change in apparent affinity ( K m). TPA stimulated arginine uptake required at least 3 h of continual exposure, and increased the membrane's transport capacity ( V max) in both undifferentiated and differentiated cells. TPA elevated the diminished transport V max of differentiated cells TPA to the elevated V max value associated with undifferentiated cells. We conclude that upregulation of arginine transport is part of a pleiotropic response to EGF/TGFα, and that this involves PKC and de novo synthesis of polypeptides associated with system y + transport activity.

Cytokines regulate endotoxin stimulation of endothelial cell arginine transport

Endotoxin (lipopolysaccharide) stimulates transmembrane L-arginine transport in pulmonary artery endothelial cells (PAECs). The proinflammatory cytokines tumor necrosis factor (TNF) and interleukin-1 (IL-1) mediate many of the pathophysiologic effects of endotoxemia and sepsis. Endothelial cells secrete TNF and IL-1 in response to endotoxin. We hypothesize that lipopolysaccharide stimulation of plasma membrane L-arginine transport is mediated via an autocrine cytokine loop involving TNF and IL-1. Confluent porcine PAECs were incubated with various concentrations of lipopolysaccharide, TNF, or IL-1, and arginine uptake was determined by assaying the uptake of 3H-L-arginine in the presence or absence of Na+ at different time points. PAECs were then incubated with lipopolysaccharide or saline solution after pretreatment with either anti-TNF antibody or IL-1-receptor antagonist, and transport was measured 12 hours later. Lipopolysaccharide, IL-1, and TNF all increased both Na+-dependent and Na+-independent carrier-mediated L-arginine transport in a fashion that was both time and dose dependent. Maximal increases in stimulated arginine uptake occurred 8 hours after exposure to the cytokines and 12 hours after exposure to lipopolysaccharide. Pretreatment of endothelial cells with anti-TNF antibody blocked lipopolysaccharide stimulation of both Na+-independent and Na+-dependent transport by 100% and 90%, respectively. In addition, IL-1-receptor antagonist inhibited lipopolysaccharide stimulation of both Na+-independent and Na+-dependent transport by 65% and 85%, respectively. The marked increase in carrier-mediated L-arginine transport activity produced by lipopolysaccharide, IL-1, and TNF may represent an adaptive response by the pulmonary endothelium to support arginine-dependent biosynthetic pathways during sepsis. Furthermore, lipopolysaccharide stimulation of arginine transport is mediated in part through an autocrine mechanism involving IL-1 and TNF.