Mitochondria as signaling organelles in the vascular endothelium.
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Journal: 2006/June - Proceedings of the National Academy of Sciences of the United States of America
ISSN: 0027-8424
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Vascular endothelial cells are highly glycolytic and consume relatively low amounts of oxygen (O(2)) compared with other cells. We have confirmed that oxidative phosphorylation is not the main source of ATP generation in these cells. We also show that at a low O(2) concentration (<1%) endogenous NO plays a key role in preventing the accumulation of the alpha-subunit of hypoxia-inducible factor 1. At higher O(2) concentrations (1-3%) NO facilitates the production of mitochondrial reactive oxygen species. This production activates the AMP-activated protein kinase by a mechanism independent of nucleotide concentrations. Thus, the primary role of mitochondria in vascular endothelial cells may not be to generate ATP but, under the control of NO, to act as signaling organelles using either O(2) or O(2)-derived species as signaling molecules. Diversion of O(2) away from endothelial cell mitochondria by NO might also facilitate oxygenation of vascular smooth muscle cells.
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Proc Natl Acad Sci U S A 103(14): 5379-5384
PMID: 16565215

Mitochondria as signaling organelles in the vascular endothelium

Wolfson Institute for Biomedical Research, University College London, Cruciform Building, Gower Street, London WC1E 6AE, United Kingdom
To whom correspondence should be addressed. E-mail: ku.ca.lcu@adacnom.s

Contributed by Salvador Moncada, February 8, 2006

.
*M.Q. and S.L.C. contributed equally to this work.

Author contributions: M.Q., S.L.C., and S.M. designed research; M.Q. and S.L.C. performed research; A.G. contributed new reagents/analytic tools; M.Q., S.L.C., and A.G. analyzed data; and M.Q., S.L.C., and S.M. wrote the paper.

Copyright © 2006 by The National Academy of Sciences of the USA

Abstract

Vascular endothelial cells are highly glycolytic and consume relatively low amounts of oxygen (O2) compared with other cells. We have confirmed that oxidative phosphorylation is not the main source of ATP generation in these cells. We also show that at a low O2 concentration (<1%) endogenous NO plays a key role in preventing the accumulation of the α-subunit of hypoxia-inducible factor 1. At higher O2 concentrations (1–3%) NO facilitates the production of mitochondrial reactive oxygen species. This production activates the AMP-activated protein kinase by a mechanism independent of nucleotide concentrations. Thus, the primary role of mitochondria in vascular endothelial cells may not be to generate ATP but, under the control of NO, to act as signaling organelles using either O2 or O2-derived species as signaling molecules. Diversion of O2 away from endothelial cell mitochondria by NO might also facilitate oxygenation of vascular smooth muscle cells.

Keywords: AMP-activated protein kinase, hypoxia-inducible factor 1α, hypoxia, nitric oxide
Abstract

Endogenously synthesized nitric oxide (NO) is a highly diffusible gas that has a variety of physiological functions, some of which are mediated by activation of the soluble guanylate cyclase enzyme (1). In the last decade cytochrome c oxidase, the terminal enzyme in the mitochondrial electron transport chain, has also been identified as a target of the action of NO (2–4). Acting on the latter enzyme, NO can regulate cellular oxygen (O2) consumption (5) and the mitochondrial redox state, facilitating the release of free radicals, which act as a signaling mechanism (6). Furthermore, inhibition of mitochondrial O2 consumption by NO leads to a situation in which, though O2 might be available, cells and tissues are unable to use it. This phenomenon has been termed “metabolic hypoxia,” a condition that differs from true hypoxia in which O2 availability is insufficient (5). In metabolic hypoxia there is also a redistribution of O2 away from mitochondria toward nonrespiratory O2-dependent targets (7). Inhibition of cell respiration by NO is also known to activate glycolysis in some cells through a mechanism involving activation of 6-phosphofructo-2-kinase (8).

Although we have previously demonstrated that endogenous NO regulates O2 consumption in vascular endothelial cells and other cell types (RAW246.7) (6, 9), the consequences of this effect have yet to be studied in detail. Of particular interest are the consequences in vascular endothelial cells, which have been known for some time to be glycolytic (10) and to possess high concentrations of constitutive NO (9).

To investigate the bioenergetic and signaling consequences of the action of NO on cytochrome c oxidase in vascular endothelial cells, we have studied the behavior of two key transduction mechanisms involved in the response to hypoxia and in the regulation of the bioenergetic status of the cell, namely hypoxia-inducible factor 1 (HIF-1) (11–13) and AMP-activated protein kinase (AMPK) (14, 15). Our results suggest that, in human endothelial cells, mitochondria under the control of NO regulate the activity of both HIF-1 and AMPK in a manner consistent with a role as signaling organelles, independent of their bioenergetic functions.

Effects of glycolytic and mitochondrial inhibitors on [ATP], at different O2 concentrations. Treatments were carried out for 2 h at the indicated O2 concentration before [ATP] was determined by chemiluminescence. For details of procedures, see Materials and Methods. Data represent the mean ± SEM of at least four independent experiments. 2DG, 20 mM; rotenone, 0.5 μM, antimycin, 0.5 μM.

Effects of glycolytic and ETC inhibitors on AMP:ATP ratios at different O2 concentrations. Treatments were carried out for 2 h before samples were processed. Each individual sample was neutralized, centrifuged, and filtered before HPLC separation. For further details of procedures, see Materials and Methods. Data represent the mean ± SEM of at least three independent experiments. 2DG, 20 mM; rotenone, 0.5 μM.

Acknowledgments

We thank Annie Higgs and Jorge Erusalimsky for their critical reading of this article. M.Q. [formerly a Ph.D. student at the University of Valencia (Valencia, Spain)] is the recipient of a fellowship from the Ministerio de Sanidad y Consumo of Spain. S.L.C. is sponsored by a grant from the Wellcome Trust. S.M. is the recipient of a grant from the Medical Research Council.

Acknowledgments

Abbreviations

AMPKAMP-activated protein kinase
HIF-1hypoxia-inducible factor 1
HUVEChuman umbilical vein endothelial cell
DHEdihydroethidine
eNOSendothelial NO synthase
ROSreactive oxygen species
l-NMMAN-monomethyl-l-arginine
2DG2-deoxy-d-glucose.
Abbreviations

Footnotes

Conflict of interest statement: No conflicts declared.

Footnotes

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