Browsing by Author "Pearson, JD"

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    Cell signalling-mediating insulin increase of mRNA expression for cationic amino acid transporters-1 and-2 and membrane hyperpolarization in human umbilical vein endothelial cells
    (2004) González, M; Flores, C; Pearson, JD; Casanello, P; Sobrevia, L
    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 RTPCR and endothelial NO synthase (eNOS) protein was identified by Western blot analysis. Intracellular Ca2+, L-arginine and L-citrulline levels, L-[H-3]citrulline formation from L-[H-3]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-[H-3]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 hyper-insulinaemia 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.
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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.