RESUMO
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
Intrauterine growth retardation (IUGR) is associated with vascular complications leading to hypoxia and abnormal fetal development. The effect of IUGR on L-arginine transport and nitric oxide (NO) synthesis was investigated in cultures of human umbilical vein endothelial cells (HUVECs). IUGR was associated with membrane depolarization and reduced L-arginine transport (V(max)= 5.8+/-0.2 versus 3.3+/-0.1 pmol/microg protein per minute), with no significant changes in transport affinity (K(m)=159+/-15 versus 137+/-14 micromol/L). L-Arginine transport was trans-stimulated (8- to 9-fold) in cells from normal and IUGR pregnancies. IUGR was associated with reduced production of L-[3H]citrulline from L-[3H] arginine, lower nitrite and intracellular L-arginine, L-citrulline, and cGMP. IUGR decreased hCAT-1 and hCAT-2B mRNA, and increased eNOS mRNA and protein levels. IUGR-associated inhibition of L-arginine transport and NO synthesis, and membrane depolarization were reversed by the NO donor S-nitroso-N-acetyl-L,D-penicillamine. In summary, endothelium from fetuses with IUGR exhibit altered L-arginine transport and NO synthesis (L-arginine/NO pathway), reduced expression and activity of hCAT-1 and hCAT-2B and reduced eNOS activity. Alterations in L-arginine/NO pathway could be critical for the physiological processes involved in the etiology of IUGR in human pregnancies.
Assuntos
Transportador 2 de Aminoácidos Catiônicos/metabolismo , Endotélio Vascular/metabolismo , Retardo do Crescimento Fetal/metabolismo , Óxido Nítrico Sintase/metabolismo , Veias Umbilicais/metabolismo , Sistemas de Transporte de Aminoácidos Básicos , Arginina/análise , Arginina/sangue , Arginina/metabolismo , Transporte Biológico , Transportador 1 de Aminoácidos Catiônicos/genética , Transportador 1 de Aminoácidos Catiônicos/metabolismo , Transportador 2 de Aminoácidos Catiônicos/genética , Células Cultivadas , Citrulina/análise , Citrulina/sangue , Endotélio Vascular/enzimologia , Endotélio Vascular/fisiologia , Etilmaleimida/farmacologia , Feminino , Retardo do Crescimento Fetal/enzimologia , Retardo do Crescimento Fetal/genética , Regulação da Expressão Gênica , Humanos , Lisina/farmacologia , Potenciais da Membrana , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase Tipo III , Gravidez , RNA Mensageiro/biossíntese , Veias Umbilicais/citologia , Veias Umbilicais/enzimologiaRESUMO
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.