Browsing by Author "Sobrevia, L"

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    Equilibrative nucleoside transporter 2 is expressed in human umbilical vein endothelium, but is not involved in the inhibition of adenosine transport induced by hyperglycaemia
    (W B SAUNDERS CO LTD, 2005) Aguayo, C; Casado, J; Gonzalez, M; Pearson, JD; San Martin, R; Casanello, P; Pastor Anglada, M; Sobrevia, L
    Human equilibrative, Na+-independent nucleoside transport is mediated by membrane proteins sensitive (system es, hENT1) or insensitive (system ei, hENT2) to nitrobenzylthioinosine (NBMPR). Gestational diabetes and elevated extracellular concentrations of D-glucose reduce adenosine transport in human umbilical vein endothelium (HUVEC). We studied hENT2 and hENT1 expression in HUVEC, and the effect of D-glucose on their activity and expression in HUVEC preincubated with 25 MM D-glucose (24 h). hENT2 and hENT1 mRNA were quantified by real-time reverse transcription polymerase chain reaction, and their proteins were detected by Western blotting. hENT2 and hENT1 proteins are co-expressed in HUVEC and are located at the plasma membrane, however, hENT2 was mainly cytoplasmatic and perinuclear in location. D-Glucose reduced hENT1 and hENT2 mRNA expression, but only hENT1 protein abundance at the plasma membrane. Adenosine transport was inhibited by D-glucose and NMBPR (1 mu M) in intact cells and membrane vesicles. Hypoxanthine inhibited adenosine transport in the absence or in the presence of 1 mu M NBMPR. D-Glucose reduced NBMPR maximal binding in intact cells, membrane vesicles, and plasma membrane fractions. In conclusion, the present study demonstrates that hENT2 and hENT1 are co-expressed in HUVEC, and even when adenosine transport is also mediated by hENT2, the hENT2-mediated transport activity is not involved in the D-glucose-induced down-regulation of total adenosine transport.
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    Nitric oxide synthesis requires activity of the cationic and neutral amino acid transport system y(+)L in human umbilical vein endothelium
    (WILEY, 2003) Arancibia Garavilla, Y; Toledo, F; Casanello, P; Sobrevia, L
    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 muM L-arginine was inhibited (P < 0.05) by L-lysine (K-i, 1.4 muM), L-leucine (K-i,1.8 mum) and L-phenylalanine (K-i, 4.1 muM), 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 muM L-arginine transport, but inhibited (92 +/- 3 %) 100 muM L-arginine transport. L-Arginine transport in the presence of NEM was saturable (V-max, 0.37 +/- 0.02 pmol (mug protein)(-1) min(-1); K-m, 1.5 +/- 0.3 muM) and competitively inhibited by L-leucine in the presence of Na+ (V-max, 0.49 +/- 0.06 pmol (mug protein)(-1) min(-1); K-m, 6.5 +/- 0.9 muM). 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 muM L-arginine. Cells exposed to 25 MM D-glucose (24 h) exhibited reduced system y(+)L activity (V-max, 0.15 +/- 0.008 pmol (mug protein)(-1) min(-1); K-m, 1.4 +/- 0.3 muM) and NO synthesis. However, 25 HIM 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.
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    Role of adenosine transport in gestational diabetes-induced L-arginine transport and nitric oxide synthesis in human umbilical vein endothelium
    (WILEY, 2004) Vasquez, G; Sanhueza, F; Vasquez, R; Gonzalez, M; San Martin, R; Casanello, P; Sobrevia, L
    Gestational diabetes is associated with increased L-arginine transport and nitric oxide (NO) synthesis, and reduced adenosine transport in human umbilical vein endothelial cells (HUVEC). Adenosine increases endothelial L-arginine/NO pathway via A(2) purinoceptors in HUVEC from normal pregnancies. It is unknown whether the effect of gestational diabetes is associated with activation of these purinoceptors or altered expression of human cationic amino acid transporter 1 (hCAT-1) or human equilibrative nucleoside transporter I (hENT1), or endothelial NO synthase (eNOS) in HUVEC. Cells were isolated from normal or gestational diabetic pregnancies and cultured up to passage 2. Gestational diabetes increased hCAT-1 mRNA expression (2.4-fold) and activity, eNOS mRNA (2.3-fold), protein level (2.1-fold), and phosphorylation (3.8-fold), but reduced hENT1 mRNA expression (32%) and activity. Gestational diabetes increased extracellular adenosine (2.7 mum), and intracellular L-arginine (1.9 mm) and L-citrulline (0.7 mm) levels compared with normal cells (0.05 mum, 0.89 mm, 0.35 mm, respectively). Incubation of HUVEC from normal pregnancies with 1 mum nitrobenzylthioinosine (NBMPR) mimicked the effect of gestational diabetes, but NBMPR was ineffective in diabetic cells. Gestational diabetes and NBMPR effects involved eNOS, PKC and P42/44(mapk) activation, and were blocked by the A(2a) purinoceptor antagonist ZM-241385. Thus, gestational diabetes increases the L-arginine/NO pathway involving activation of mitogen-activated protein (MAP) kinases, protein kinase C (PKC) and NO cell signalling cascades following activation of A(2a) purinoceptors by extracellular adenosine. A functional relationship is proposed between adenosine transport and modulation Of L-arginine transport and NO synthesis in HUVEC, which could be determinant in regulating vascular reactivity in diabetes mellitus.