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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

  • Cell and Molecular Physiology
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Abstract

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-[3H]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.

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Acknowledgements

Supported by Fondo Nacional de Ciencia y Tecnología (FONDECYT 1030781, 1030607, 7030004, 7030109) and The Wellcome Trust (UK). C.F. holds CONICYT-PhD (Chile) fellowship. We thank Miss Alexandra Almeida for excellent secretarial assistance and the midwives of Hospital Clínico of the Pontificia Universidad Católica de Chile labour ward for supply of umbilical cords.

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  1. Carlos Flores

    Present address: Universidad Austral de Chile, Valdivia, Chile

Authors and Affiliations

  1. Cellular and Molecular Physiology Laboratory (CMPL), Department of Obstetrics and Gynaecology, Medical Research Centre (CIM), School of Medicine, Faculty of Medicine, Pontificia Universidad Católica de Chile, P.O. Box 114-D, Santiago, Chile

    Marcelo González, Carlos Flores, Paola Casanello & Luis Sobrevia

  2. Centre for Cardiovascular Biology and Medicine, King’s College London, Guy’s Campus, London, SE1 1UL, UK

    Jeremy D. Pearson

  3. Department of Pathophysiology, Program of Physiology, Biomedical Sciences Institute (ICBM), Faculty of Medicine, Universidad de Chile, Santiago, Chile

    Paola Casanello

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Correspondence to Luis Sobrevia.

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González, M., Flores, C., Pearson, J.D. et al. 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. Pflugers Arch - Eur J Physiol 448, 383–394 (2004). https://doi.org/10.1007/s00424-004-1261-x

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