Hypoxia-induced regulation of nitric oxide synthase in cardiac endothelial cells and myocytes and the role of the PI3-K/PKB pathway.
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 PO(2) 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.
Strijdom
Hans
H
Department of Biomedical Sciences, Division of Medical Physiology, Faculty of Health Sciences, Stellenbosch University, Tygerberg, Republic of South Africa. jgstr@sun.ac.za
Friedrich
Sven O
SO
Hattingh
Suzél
S
Chamane
Nontuthuko
N
Lochner
Amanda
A
D000730:Androstadienes / Q000378:metabolism; D000818:Animals; D015687:Cell Hypoxia; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D004789:Enzyme Activation; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D008297:Male; D009206:Myocardium / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D000077191:Wortmannin
D000730:Androstadienes; D007527:Isoenzymes; D047428:Protein Kinase Inhibitors; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D000077191:Wortmannin
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