The effects of oral arginine on its metabolic pathways in Sprague-Dawley rats.

Oral arginine supplements are popular mainly for their presumed vasodilatory benefit. Arginine is a substrate for at least four enzymes including nitric oxide synthase (NOS) and arginase, but the impact of oral supplements on its different metabolic pathways is not clear. Deficiencies of arginine-metabolising enzymes are associated with conditions such as hyperammonaemia, endothelial dysfunction, central nervous system and muscle dysfunction, which complicate the use of oral arginine supplements. We examined the effect of l-arginine (l-Arg) and d-arginine (d-Arg), each at 500 mg/kg per d in drinking water administered for 4 weeks to separate groups of 9-week-old male Sprague-Dawley rats. We quantified the expression of enzymes and plasma, urine and organ levels of various metabolites of arginine. l-Arg significantly decreased cationic transporter-1 expression in the liver and the ileum and increased endothelial NOS expression in the aorta and the kidney and plasma nitrite levels, but did not affect the mean arterial pressure. l-Arg also decreased the expression of arginase II in the ileum, arginine:glycine amidinotransferase in the liver and the kidney and glyoxalase I in the liver, ileum and brain, but increased the expression of arginine decarboxylase and polyamines levels in the liver. d-Arg, the supposedly inert isomer, also unexpectedly affected the expression of some enzymes and metabolites. In conclusion, both l- and d-Arg significantly affected enzymes and metabolites in several pathways that use arginine as a substrate and further studies with different doses and treatment durations are planned to establish their safety or adverse effects to guide their use as oral supplements.

Dimethyl sulfoxide attenuates nitric oxide generation via modulation of cationic amino acid transporter-1 in human umbilical vein endothelial cells.

Dimethyl sulfoxide (DMSO) is a solvent that is commonly used in medicine. Conflicting data exist as to its effects on endothelial function. Endothelial cell dysfunction (ECD) is characterized by decreased endothelial nitric oxide synthase (eNOS) activity. Cationic amino acid transporter-1 (CAT-1), the specific arginine transporter for eNOS, has been shown to modulate eNOS activity. We hypothesize that DMSO inhibits eNOS activity through modulation of its selective arginine supplier CAT-1. We studied the effect of DMSO on arginine transport, NO2/NO3 generation as an index of NO production, as well as CAT-1 and Protein Kinase C alpha (PKC-α) (CAT-1 inhibitor) protein expression in human umbilical vein endothelial cell cultures (HUVECs). DMSO 2.5% and 3.5% (v/v) significantly attenuated arginine transport, a phenomenon which was prevented by co-incubation with l-arginine (1 mM). The aforementioned findings were accompanied by a decrease in NO2/NO3 generation. DMSO significantly increased the abundance of phosphorylated CAT-1 (the inactive form) and phosphorylated PKC-α protein, an effect that was attenuated by l-arginine. GO 6976 (PKC-α antagonist) prevented the decrease in arginine transport caused by DMSO. DMSO also induced profound transient morphological changes in HUVECs' structure but these were not related to its effect on arginine transport. In conclusion, DMSO inhibits NO generation by endothelial cells through modulation of CAT-1 activity.

Cyclosporine attenuates arginine transport, in human endothelial cells, through modulation of cationic amino acid transporter-1.

BACKGROUND: The spectrum of cardiovascular toxicity by cyclosporine (CsA) includes hypertension, accelerated atherosclerosis, and thrombotic microangiopathy, all of which are the result of endothelial cell dysfunction. Endothelial cell dysfunction is characterized by decreased endothelial nitric oxide synthase (eNOS) activity. Cationic amino acid transporter-1 (CAT-1) is the specific arginine transporter for eNOS. CsA has been shown to attenuate nitric oxide (NO) generation. However, the mechanism remains elusive. We hypothesize that CsA inhibits eNOS activity through modulation of its selective arginine supplier CAT-1. METHODS: We studied the effect of CsA on arginine uptake, NO2/NO3 generation, and CAT-1, protein kinase Cα (PKCα), and phosphorylated PKCα protein expression in human umbilical vein endothelial cell cultures (HUVEC) in the absence and presence of L-arginine. RESULTS: CsA (0.5-2 µg/ml) significantly attenuated arginine transport in a dose- and time-dependent manner, a phenomenon which was prevented by co-incubation with L-arginine (1 mM). The aforementioned findings were accompanied by increased protein nitration, a measure for peroxynitrite accumulation. In contrast, no changes were observed in NO2/NO3 generation. CsA significantly decreased the abundance of CAT-1 protein, an effect that was attenuated by L-arginine. PKCα and phosphorylated PKCα (CAT-1 inhibitors) protein contents were not affected by CsA. CONCLUSION: CsA inhibits arginine transport and induces protein nitration in HUVEC through modulation of CAT-1.

y+ cationic amino acid transport of arginine in packed red blood cells.

BACKGROUND: Transfusion of packed red blood cells (PRBCs) is associated with morbidity and mortality. The mechanisms are not fully understood. Packed red blood cells deplete extracellular arginine and possess transporters for arginine, an amino acid essential for normal immunity. We hypothesize that the membrane y+ amino acid transporter contributes to arginine depletion in PRBCs. MATERIALS AND METHODS: We titrated PRBCs to a 10% hematocrit with phosphate-buffered saline, blocked PRBC y+ transporters using n-ethylmaleimide (0.2 mM), and measured arginine and ornithine levels using liquid chromatography-mass spectroscopy. We added radiolabeled L-arginine [4,5-(3)H] (10 µmol/L) added to similar culture conditions and measured arginine uptake in counts per minute (CPM). We examined storage periods of 6-9 d, 1-4 wk, and 6 wk, and correlated donor demographics with arginine uptake. RESULTS: n-Ethylmaleimide blockade of y+ transporters impaired PRBC arginine depletion from culture media (117.6 ± 8.6 µM versus 76.9 ± 5.8 µM; P < 0.001) and reduced intracellular L-arginine (7,574 ± 955 CPM versus 18,192 ± 1,376 CPM; P < 0.01). Arginine depletion increased with storage duration (1 wk versus 6 wk; P < 0.002). With n-ethylmaleimide treatment, 6-wk-old PRBCs preserved more culture arginine (P < 0.008) than at shorter durations. Nine-day storage duration increased L-arginine uptake compared with 6- to 8-day storage (n = 77, R = 0.225, P < 0.05). Extracellular arginine depletion and extracellular ornithine synthesis varied among donors and correlated inversely (R = -0.5, P = 0.01). CONCLUSIONS: Membrane y+ transporters are responsible for arginine depletion by PRBCs. Membrane y+ activity increases with storage duration. Arginine uptake varies among donors. Membrane biology of RBCs may have a role in the negative clinical effects associated with PRBC transfusion.

The biological effect of pharmacological treatment on dimethylaminohydrolases (DDAH-1) and cationic amino acid transporter-1 (CAT-1) expression in patients with acute congestive heart failure.

AIM: Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide (NO) which plays an important role in controlling vascular tone and regulates the contractile properties of cardiac myocytes. The aim of this study was to investigate the effect of pharmacological treatment on symmetric dimethylarginine (SDMA), ADMA and arginine plasma concentrations in patients with acute congestive heart failure (ACHF) through the evaluation of type-1 system cationic amino acid transporter-1/type 1 dimethylarginine dimethylaminohydrolases-1 (CAT-1/DDAH-1). METHODS AND RESULTS: 25 hospitalized cardiology patients with symptomatic acute congestive HF (NYHA Class III-IV) and impaired left ventricular (LV) function (ejection fraction<35%) were included in the study. ADMA, SDMA, and arginine plasma concentrations were assessed before and after pharmacological treatment by high performance liquid chromatography. All patients received an adequate pharmacological treatment for ACHF. ADMA and SDMA plasma levels were significantly higher after pharmacological treatment respect to baseline values (pre-treatment) (0.75 vs 0.48; 1.31 vs 1.03; p<0.01). Arginine plasma concentration was significantly lower after therapy respect to baseline values (0.78 vs 0.99; p<0.01). This is associated more with the modulation of DDAH-1 protein than with of CAT-1 system transport. CONCLUSIONS: In patients with ACHF, acute renal impairment function and the modulation of metabolism and extracellular transport by the DDAH-1/CAT-1 system determine high ADMA and SDMA levels after therapy for acute congestive heart failure.

Characterization of cationic amino acid transporters (hCATs) 1 and 2 in human skin.

In the present study, we characterized the distribution of human cationic amino acid transporters 1 (hCAT1) and 2 (hCAT2) in healthy skin and compared it to psoriatic skin lesions by means of immunohistochemistry. Moreover, we tested the hypothesis that L -arginine and L -ornithine influence the expression and synthesis of hCAT1 and hCAT2 in cell culture experiments by means of real-time-PCR and Western blot. Immunohistochemical comparison between healthy and psoriatic skin revealed a decreased amount of hCAT1, especially in the stratum granulosum of psoriatic skin; the distribution pattern of hCAT2 was not significantly affected in psoriatic skin. Cell culture experiments showed that supraphysiological concentrations of 15 mM L -arginine (72 h) lead to a significant increase of the hCAT1-mRNA and protein expression, whereas other concentrations had no significant influence. In contrast, L -arginine concentrations of 2 mM led to a significant increase of the hCAT2B mRNA-expression after 24 h. However, 48 and 72 h revealed no significant changes and high concentrations (15 mM L -arginine) led to a significant downregulation of the hCAT2B transporter over all time points analyzed. L -ornithine had no effect on the hCAT1 expression of mRNA and protein level. On the other hand the expression of hCAT2B was significantly up regulated at a 5-mM concentration of L -ornithine at all analyzed time points. Other concentrations had no effect. For the first time, the findings yield data about hCAT1 and hCAT2 on protein-level and suggest that L -arginine is a worthwhile object of studies, which investigated L -arginine as a possible therapeutic agent to reduce psoriatic symptoms.

The acute phase response alters cationic amino acid transporter expression in growing chickens (Gallus gallus domesticus).

The effect of an acute phase response (APR) on cationic amino acid transporter (CAT1-3) mRNA expression in liver, muscle, bursa and thymus was determined in broiler strain chickens. The APR was initiated by injecting Salmonella typhimurium lipopolysaccharide subcutaneously (LPS; 1 mg/kg bw). In Experiment 1, CAT1-3 mRNA expression was determined at multiple time points following LPS administration. LPS increased bursa and liver total and high affinity CAT mRNA expression (P<0.05) and transiently increased pectoralis total CAT mRNA expression (P<0.05). Total CAT mRNA expression in the thymus decreased 7.7-fold from 0 to 8 h after LPS injection (P<0.05). In Experiment 2, fasted chicks were uninjected or LPS-injected. LPS increased total and high affinity CAT mRNA 2-fold in both the bursa and liver (P<0.05) and did not change thymus total and high affinity CAT mRNA expression (P>0.05). LPS increased liver weight only (P<0.05) and did not alter the plasma lysine and arginine concentration (P>0.05). In Experiments 3 and 4, thymocyte proliferation and total protein content were dependent upon the media lysine concentration (P<0.001). The inability of the thymus to compete for lysine and arginine during the APR may limit the ability of thymocytes to develop during infections.

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.