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Hypoxia-induced regulation of nitric oxide synthase in cardiac endothelial cells and myocytes and the role of the PI3-K/PKB pathway

Molecular and Cellular Biochemistry volume 321, pages 23–35 (2009)Cite this article

Abstract

The roles of endothelial nitric oxide synthase (eNOS), and its putative association with protein kinase B (PKB), and inducible nitric oxide synthase (iNOS) are not well characterized in hypoxic cardiac cells and there is a lack of studies that measure nitric oxide (NO) directly. Objective To measure NO production in cardiomyocytes and cardiac microvascular endothelial cells (CMECs) under baseline and hypoxic conditions and to evaluate the expression, regulation and activation of eNOS, iNOS and PKB. The effect of PI3-K/PKB inhibition on NO production and eNOS expression/activation was also investigated. Methods Adult rat cardiomyocytes and rat CMECs were made hypoxic by cell pelleting and low PO2 incubation. Intracellular NO was measured by FACS analysis of DAF-2/DA fluorescence, and eNOS, iNOS and PKB were evaluated by Western blotting or flow cytometry. Upstream PKB inhibition was achieved with wortmannin. Results (1) NO levels increased in both cell types after exposure to hypoxia. (2) In hypoxic CMECs, eNOS was upregulated and activated, no iNOS expression was observed and PKB was activated. (3) In myocytes, hypoxia did not affect eNOS expression, but increased its activation. Activated PKB also increased during hypoxia. FACS analysis showed increased iNOS in hypoxic myocytes. (4) Wortmannin resulted in decreased hypoxia-induced NO production and reduced activated eNOS levels. Conclusions Cardiomyocytes and CMECs show increased NO production during hypoxia. eNOS seems to be the main NOS isoform involved as source of the increased NO generation, although there may be a role for iNOS and other non-eNOS sources of NO in the hypoxic myocytes. Hypoxia-induced PKB and eNOS activation occurred simultaneously in both cell types, and the PI3-K/PKB pathway was associated with hypoxia-induced NO production via eNOS activation.

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Acknowledgments

This study was sponsored in part by the Medical Research Council (MRC) of South Africa, National Research Fund (NRF) of South Africa and the Harry Crossley Foundation, University of Stellenbosch.

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Authors and Affiliations

  1. Department of Biomedical Sciences, Division of Medical Physiology, Faculty of Health Sciences, Stellenbosch University, P.O. Box 19063, Tygerberg, 7505, Republic of South Africa

    Hans Strijdom, Sven O. Friedrich, Suzél Hattingh, Nontuthuko Chamane & Amanda Lochner

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  1. Hans Strijdom
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  2. Sven O. Friedrich
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  3. Suzél Hattingh
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Correspondence to Hans Strijdom.

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Strijdom, H., Friedrich, S.O., Hattingh, S. et al. Hypoxia-induced regulation of nitric oxide synthase in cardiac endothelial cells and myocytes and the role of the PI3-K/PKB pathway. Mol Cell Biochem 321, 23–35 (2009). https://doi.org/10.1007/s11010-008-9906-2

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Keywords

  • Nitric oxide
  • eNOS
  • iNOS
  • PKB
  • Hypoxia
  • Cardiomyocytes
  • Cardiac endothelial cells