RESUMO
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 (â¼50%) 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.
Assuntos
Arginina/administração & dosagem , Diabetes Mellitus Experimental/tratamento farmacológico , Suplementos Nutricionais , Hipoglicemiantes/administração & dosagem , Malus , Aloxano , Animais , Arginina/farmacocinética , Disponibilidade Biológica , Glicemia , Células Cultivadas , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/induzido quimicamente , Feminino , Alimentos Fortificados , Hipoglicemiantes/farmacocinética , Miócitos de Músculo Liso , RatosRESUMO
The interaction between lysine (Lys) and arginine (Arg) in the proximal intestinal region of Pacific bluefin tuna (Thunnus orientalis) was evaluated using the everted intestine method. This in vitro intestinal system has been shown to be an effective tool for studying the nutrient absorption without the need to handle the tuna fish in marine cages as needed for digestibility and amino acid (AA) absorption. We used a factorial design with two sets of variables: low and high Lys concentration (10 and 75 mM) and four different Arg concentrations (3, 10, 20, and 30 mM). Both amino acids were dissolved in marine Ringer solution with a basal amino acidic composition consisting of a tryptone solution (9 mg mL(-1)). No interaction was observed between the absorption of Lys and Arg during the first 10 min of the experiment when low concentration of Lys and Arg was used in the hydrolyzate solution. However, there seemed to be a positive effect on Lys absorption when both amino acids were at high concentrations (30 and 75 mM, respectively). This type of studies will led us to test different formulations and/or additives to better understand the efficiency of AA supplementation as an alternative to in situ studies that are difficult to follow to design with the Pacific Bluefin Tuna.
Assuntos
Arginina/farmacocinética , Absorção Intestinal/fisiologia , Lisina/farmacocinética , Atum/fisiologia , Análise de Variância , Animais , Arginina/metabolismo , Técnicas In Vitro , Lisina/metabolismo , México , Oceano Pacífico , Atum/metabolismoRESUMO
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.
Assuntos
Sistema y+L de Transporte de Aminoácidos/metabolismo , Plaquetas/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Hemostáticos/farmacologia , Ouabaína/farmacologia , Trombina/farmacologia , Uremia/metabolismo , Adulto , Arginina/farmacocinética , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Plaquetas/metabolismo , Glucose/farmacologia , Humanos , Técnicas In Vitro , Falência Renal Crônica/metabolismo , Lipopolissacarídeos/farmacologia , Potenciais da Membrana , Óxido Nítrico/metabolismo , Sódio/metabolismo , Trítio , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
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.
Assuntos
Arginina/farmacocinética , Falência Renal Crônica/sangue , Diálise Peritoneal Ambulatorial Contínua , Arginina/sangue , Transporte Biológico , Citrulina/sangue , Eritrócitos/metabolismo , Feminino , Humanos , Falência Renal Crônica/fisiopatologia , Lisina/sangue , Masculino , Pessoa de Meia-Idade , Ornitina/sangue , Uremia/sangue , Uremia/fisiopatologiaRESUMO
Insulin induces vasodilatation in human subjects and increases L-arginine transport and NO synthesis in human umbilical vein endothelial cells (HUVEC). Cell signalling events associated with insulin effects on activity and mRNA expression of the human cationic amino acid transporters 1 (hCAT-1) and 2B (hCAT-2B) are unknown. L-arginine transport and eNOS activity were determined in HUVEC exposed to insulin. mRNA levels for hCAT-1, hCAT-2B and eNOS were quantitated by real time RT-PCR and endothelial NO synthase (eNOS) protein was identified by Western blot analysis. Intracellular Ca2+, L-arginine and L-citrulline levels, L-[3H]citrulline formation from L-[(3)H]arginine, cGMP formation, nitrite level, ATP release and membrane potential were determined. Insulin increased L-arginine transport and the mRNA levels for hCAT-1 and hCAT-2B and eNOS expression and activity. Insulin also induced membrane hyperpolarization and increased intracellular Ca2+, L-[3H]citrulline, cGMP and nitrite formation. Insulin-mediated stimulation of the L-arginine/NO pathway is thus associated with increased hCAT-1 and hCAT-2B mRNA, and eNOS expression, via mechanisms involving membrane hyperpolarization, mitogen-activated protein kinases p42 and p44, phosphatidylinositol 3-kinase, NO and protein kinase C. We have characterized a cell signalling pathway by which hyperinsulinaemia could lead to vasodilatation in human subjects, and which could have implications in patients in whom plasma insulin levels are altered, such as in diabetes mellitus.
Assuntos
Transportador 1 de Aminoácidos Catiônicos/genética , Transportador 2 de Aminoácidos Catiônicos/genética , Endotélio Vascular/fisiologia , Insulina/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Trifosfato de Adenosina/metabolismo , Sistemas de Transporte de Aminoácidos Básicos , Arginina/farmacocinética , Cálcio/metabolismo , Células Cultivadas , Endotélio Vascular/citologia , Humanos , Potenciais da Membrana/fisiologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo III , Oniocompostos/farmacocinética , Compostos Organofosforados/farmacocinética , Fosfatidilinositol 3-Quinases/metabolismo , Proteína Quinase C/metabolismo , RNA Mensageiro/metabolismo , Trítio , Veias Umbilicais/citologiaRESUMO
L-arginine transport is mediated by the cationic/neutral amino acid transport system y+L and cationic amino acid transporters y+/CATs in human umbilical vein endothelial cells (HUVECs). System y+/CATs activity may be rate-limiting for nitric oxide (NO) synthesis, but no reports have demonstrated system y+L involvement in NO synthesis in endothelium. We investigated the role of system y+L in NO synthesis in HUVECs. Transport of 1.5 microM L-arginine was inhibited (P < 0.05) by L-lysine (K(i), 1.4 micro M), L-leucine (K(i), 1.8 micro M) and L-phenylalanine (K(i), 4.1 microM), but was unaltered (P > 0.05) by L-alanine or L-cysteine. The system y+/CATs inhibitor, N-ethylmaleimide (NEM), did not alter 1.5 microM L-arginine transport, but inhibited (92 +/- 3 %) 100 microM L-arginine transport. L-arginine transport in the presence of NEM was saturable (V(max), 0.37 +/- 0.02 pmol (microg protein)(-1) min(-1); K(m), 1.5 +/- 0.3 microM) and competitively inhibited by L-leucine in the presence of Na+ (V(max), 0.49 +/- 0.06 pmol (microg protein)(-1) min(-1); K(m), 6.5 +/- 0.9 microM). HUVECs express SLC3A2/4F2hc, SLC7A7/4F2-lc2 and SLC7A6/4F2-lc3 genes encoding for the high-affinity transport system y+L. N(G)-Nitro-L-arginine methyl ester and L-leucine, but not NEM, inhibited NO synthesis in medium containing 1.5 microM L-arginine. Cells exposed to 25 mM D-glucose (24 h) exhibited reduced system y+L activity (V(max), 0.15 +/- 0.008 pmol (microg protein)(-1) min(-1); K(m), 1.4 +/- 0.3 microM) and NO synthesis. However, 25 mM D-glucose increased NO synthesis and L-arginine transport via system y+. Thus, L-arginine transport through system y+L plays a role in NO synthesis, which could be a determining factor in pathological conditions where the endothelial L-arginine-NO pathway is altered, such as in diabetes mellitus.
Assuntos
Sistema y+L de Transporte de Aminoácidos/metabolismo , Arginina/farmacocinética , Endotélio Vascular/metabolismo , Óxido Nítrico/biossíntese , Veias Umbilicais/metabolismo , Sistemas de Transporte de Aminoácidos Básicos/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Transporte Biológico Ativo/fisiologia , Células Cultivadas , Humanos , CinéticaRESUMO
BACKGROUND: Obstructive nephropathy leads to tubulointerstitial fibrosis and loss of renal function. Nitric oxide has been shown to have antifibrotic properties. We examined nitric oxide synthase (NOS) activity and expression in kidneys from children who underwent surgery release of unilateral ureteropelvic junction (UPJ) obstruction in relation to clinical and histologic parameters. METHODS: NOS activity and the expression of NOS isoforms measured at the mRNA level by reverse transcription-polymerase chain reaction (RT-PCR) assay were determined in tissue obtained by biopsy from obstructed kidneys of 18 children at the time of pyeloplasty. Tissue from kidneys removed because of various malignancies were issued as control. RESULTS: A significant increase in calcium/calmodulin-independent NOS activity (iNOS) and iNOS mRNA expression was found in the medulla of obstructed kidneys. Calcium/calmodulin-dependent NOS activity (cNOS) and endothelial (eNOS) mRNA, by contrast, were increased in the cortex from obstructed kidneys. A role of tumor necrosis factor-alpha (TNF-alpha) on enhanced iNOS was suggested by the finding of increased urine levels in obstructed pelvis. Increased interstitium macrophage number, by immunolabeling of CD68, was related to the delay in obstruction release and to decreased glomerular filtration rate (GFR) at surgery. A positive linear relationship was found between cNOS activity in cortex and creatinine clearance. The degree of interstitial fibrosis correlated negatively with cNOS activity in cortex. CONCLUSION: In kidneys from children with UPJ obstruction an increased activity and expression of iNOS in medulla and cNOS-dependent eNOS in cortex were demonstrated. A role of cNOS in modulating GFR and interstitial fibrosis can be suggested. Prolonged UPJ obstruction would lead to a worsened prognosis on renal injury.
Assuntos
Rim/enzimologia , Óxido Nítrico Sintase/metabolismo , Obstrução Ureteral/metabolismo , Arginina/farmacocinética , Criança , Pré-Escolar , Citrulina/metabolismo , Feminino , Regulação Enzimológica da Expressão Gênica , Humanos , Imuno-Histoquímica , Lactente , Masculino , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase Tipo II , Óxido Nítrico Sintase Tipo III , RNA Mensageiro/metabolismo , TrítioRESUMO
D-glucose infusion and gestational diabetes induce vasodilatation in humans and increase L-arginine transport and nitric oxide (NO) synthesis in human umbilical vein endothelial cells. High D-glucose (25 mmol/L, 2 minutes) induced membrane hyperpolarization and an increase of L-arginine transport (V(max) 6.1+/-0.7 versus 4.4+/-0.1 pmol/ microg protein per minute) with no change in transport affinity (K(m) 105+/-9 versus 111+/-16 micromol/L). L-[3H]citrulline formation and intracellular cGMP, but not intracellular Ca2+, were increased by high D-glucose. The effects of D-glucose were mimicked by levcromakalim (ATP-sensitive K+ channel blocker), paralleled by p42/p44(mapk) and Ser(1177)-endothelial NO synthase phosphorylation, inhibited by N(G)-nitro-L-arginine methyl ester (L-NAME; NO synthesis inhibitor), glibenclamide (ATP-sensitive K+ channel blocker), KT-5823 (protein kinase G inhibitor), PD-98059 (mitogen-activated protein kinase kinase 1/2 inhibitor), and wortmannin (phosphatidylinositol 3-kinase inhibitor), but they were unaffected by calphostin C (protein kinase C inhibitor). Elevated D-glucose did not alter superoxide dismutase activity. Our findings demonstrate that the human fetal endothelial L-arginine/NO signaling pathway is rapidly activated by elevated D-glucose via NO and p42/44(mapk). This could be determinant in pathologies in which rapid fluctuations of plasma D-glucose may occur and may underlie the reported vasodilatation in early stages of diabetes mellitus.
Assuntos
Arginina/metabolismo , Endotélio Vascular/metabolismo , Glucose/farmacologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Óxido Nítrico/metabolismo , Sistemas de Transporte de Aminoácidos Básicos , Arginina/farmacocinética , Transporte Biológico/efeitos dos fármacos , Transportador 1 de Aminoácidos Catiônicos/genética , Transportador 1 de Aminoácidos Catiônicos/metabolismo , Transportador 2 de Aminoácidos Catiônicos/genética , Transportador 2 de Aminoácidos Catiônicos/metabolismo , Células Cultivadas , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Humanos , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Oniocompostos/farmacocinética , Compostos Organofosforados/farmacocinética , Técnicas de Patch-Clamp , Bloqueadores dos Canais de Potássio/farmacologia , Proteína Quinase C/metabolismo , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Superóxido Dismutase/metabolismo , Veias Umbilicais/citologia , alfa-Tocoferol/farmacologiaRESUMO
One of the limiting steps in the regulation of nitric oxide (NO) synthesis is the availability of its precursor, L-arginine, which depends on the presence of a specific uptake system. A characterization of the L-arginine uptake mechanism in the golden hamster retina was performed. This mechanism was stereospecific, saturable, and monophasic, with an apparent of 56.1 +/- 2.0 microM and a maximum velocity of 36.0 +/- 2.8 pmol/mg prot/min. The basic amino acids L-lysine and L-ornithine but not D-arginine or the nitric oxide synthase inhibitors, N(omega)-nitro-L-arginine methyl ester and N(omega)-nitro-L-arginine impaired L-arginine influx. Preincubation with L-lysine for 1 h prior to the transport assay significantly stimulated L-arginine uptake. Saturation studies of L-arginine uptake performed at 12.00 and 24.00 h indicated a higher value of Vmax at midnight than at midday. When the hamsters were placed under constant darkness or constant light for 48 h and killed at equivalent time points, representing subjective day and subjective night, the differences in L-arginine influx disappeared. Semiquantitative RT-PCR analysis showed that the levels of mRNAs for both CAT-1 and CAT-2B were significantly higher at midnight than at midday. L-Arginine significantly increased cGMP accumulation in a time-dependent manner, with maximal effects during the night. Based on these results, it might be presumed that hamster retinal L-arginine uptake is regulated by the photic stimulus.
Assuntos
Arginina/farmacocinética , Transportador 1 de Aminoácidos Catiônicos/metabolismo , Retina/metabolismo , Animais , Transportador 1 de Aminoácidos Catiônicos/genética , Transportador 2 de Aminoácidos Catiônicos/genética , Transportador 2 de Aminoácidos Catiônicos/metabolismo , Cricetinae , GMP Cíclico/metabolismo , Expressão Gênica/fisiologia , Masculino , Mesocricetus , Estimulação Luminosa , Fotoperíodo , RNA Mensageiro/análise , Transdução de Sinais/fisiologia , TrítioRESUMO
The availability of L-arginine is of pivotal importance for the synthesis of nitric oxide, a signaling molecule in the CNS. Here we show the presence of a high-affinity L-arginine uptake system (Km of 4.4 +/- 0.5 microM and a Vmax of 26.0 +/- 0.9 fmol/well/min) in cultured chick retinal cells. Different compounds, such as N(G)-mono-methyl-L-arginine and L-lysine, were able to inhibit the uptake that was also inhibited 60-70% in the absence of sodium and/or calcium ions. No trans stimulation was observed when cells were preloaded with L-lysine. The data indicate that the L-arginine uptake in cultured retinal cells is partially mediated by the y+ system, but has a great contribution of the B(0,+) system. Autoradiographic studies revealed that the uptake is predominant in glial cells and can also be detected in neurons, whereas immunocytochemistry of nitric oxide synthase and L-citrulline showed that the enzyme is present in neurons and photoreceptors, but not in glial cells. L-[3H]Arginine is released from purified glial cultures incubated with high concentrations of potassium in the extracellular medium. Moreover, the amino acid released from preloaded glial cells was taken up by purified neuronal cultures. These results indicate that L-arginine released from glial cells is taken up by neurons and used as substrate for the synthesis of nitric oxide.