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(Circulation Research. 2003;92:821.)
© 2003 American Heart Association, Inc.

Editorials

Surviving Hypoxia

The Importance of Rafts, Anchors, and Fluidity

Aruni Bhatnagar

From the Division of Cardiology, Department of Medicine, University of Louisville, Louisville, Ky.

Correspondence to Aruni Bhatnagar, PhD, Division of Cardiology, Department of Medicine, Jewish Cardiovascular Research Center, 500 South Floyd St, University of Louisville, Louisville, KY 40202. E-mail aruni{at}louisville.edu

Key Words: hypoxia • adenosine • ecto-5'-nucleotidase • membrane fluidity • endothelium

Oxidative phosphorylation is the major energy-generating mechanism in aerobic cells. Although most cells can temporarily subsist on glycolysis, continued supply of oxygen is critical for maintaining high levels of mechanical, electrical, and metabolic activity. As a result, a wide range of biological responses of varying complexity have evolved to adapt to hypoxia and to optimize oxygen utilization. Currently, these responses are poorly understood. However, extant data provide a tantalizing glimpse into the rich diversity of these responses that range from the stimulation of signaling cascades and gene transcription events to the changes in excitability and blood flow.

Optimal function during hypoxia could be preserved either by reducing oxygen consumption or by optimizing oxygen delivery. Both these responses are triggered in hypoxic tissues and are mediated in part by adenosine. When oxygen is plentiful, adenosine is in the cells in the form of either ATP or ADP. However, during hypoxia, most cells release nucleotides into the extracellular space where the nucleotides are cleaved to adenosine by ecto-5'-nucleotidase (E5'N, CD73¹). Interstitial adenosine is either internalized (for nucleotide salvage) or it binds to one of its receptors (A₁, A_2A, A_2B and A₃) on the cell surface to induce a variety of tissue- and receptor-specific responses. Adenosine-initiated signaling pathways are generally protective and mitigate against the harmful effects of hypoxia.² During systemic hypoxia, for instance, adenosine plays an essential role in causing dilation of arterioles, which helps in maintaining oxygen delivery³; and in the heart, adenosine-mediated signaling triggers early ischemic preconditioning⁴ and establishes a delayed cardioprotective phenotype.⁵

The generation of adenosine during hypoxia is facilitated by an increase in the activity of E5'N.¹ The most straightforward way of stimulating E5'N under hypoxia is to increase its synthesis. Indeed, the promoter site of the E5'N gene contains binding sites for the transcription factors that are stimulated by hypoxia as well as adenosine.⁶ Thus, hypoxia-induced increase in adenosine could lead to an increase in cAMP (by stimulating A₂ receptors), which in turn could stimulate transcription via the cAMP-response element (CRE) on the E5'N promoter. Alternatively, the expression of E5'N could also be increased via hypoxia-inducible factor-1 (HIF-1)–dependent gene transcription (Figure). Transcriptional activation of E5'N by both these mechanisms has been described.⁶ However, in this issue of Circulation Research, Ledoux et al⁷ report yet another mechanism that regulates E5'N activity in endothelial cells. They demonstrate that prolonged hypoxia leads to an increase in E5'N activity. However, unlike previous reports, the increase in activity was not accompanied by an increase in E5'N protein or mRNA levels. The lack of transcriptional activation of E5'N is somewhat surprising because transcription factors such as HIF-1 and CREB are available to endothelial cells. Clearly, there are other mechanisms that regulate transcription activation of E5'N and these processes are not activated under the experimental conditions of the study.

View larger version (24K): [in this window] [in a new window]   Schematic representation of the potential mechanisms regulating E5'N activity. Hypoxia leads to an increase in AMP, which is then cleaved to adenosine by E5'N. Adenosine either binds to the adenosine receptors or is internalized via dipyridamole-sensitive carriers. Binding of adenosine to its receptor initiates multiple signaling events, of which the formation of cAMP could enhance the transcription of the E5'N gene. Transcriptional upregulation of E5'N could also be mediated by HIF. The activity of E5'N at the cell surface could also be enhanced by inhibiting endocytosis. As suggested by the studies of Ledoux et al,7,11 a decrease in isoprenylation of Rho-GTPase or an increase in saturated fatty acids in the membrane would prevent endocytosis, whereas cholesterol and mevalonate would decrease cell surface E5'N. Hypoxia would stimulate E5'N by increasing the palmitate content of the membrane. The activity and the surface expression of E5'N could also be enhanced upon PKC stimulation. Not all the pathways are expected to be operative at the same time or under the same set of conditions.

In addition to transcriptional induction, the activity of E5'N could be increased by posttranslational modification by protein kinase C (PKC). Although specific mechanisms remain unclear, stimulation of PKC by phorbol esters, ₁-adrenergic receptors, nitric oxide, or ischemia has been shown to stimulate E5'N.⁸ Additionally, E5'N could be activated by promoting its translocation to the cell surface, a phenomenon that could also partly account for the stimulatory effect of PKC. The E5'N protein is anchored to the membrane via glycosyl phosphatidyl inositol (GPI) bound to a serine residue at its C-terminal end.¹ Under basal conditions, less than 50% of the protein is present at the cell surface while the remainder is associated with cytoplasmic membranes. There is a continuous exchange of the protein between the cell surface and cytoplasmic membranes⁹ and hence a decrease in the protein endocytosis or increased delivery to the surface could stimulate ectoplasmic E5'N activity (Figure). Ledoux et al⁷ suggest that although PKC has no role in their system, hypoxia increases the expression of E5'N at the plasma membrane by diminishing the rate of E5'N endocytosis. This suggestion is based on the observation that the amount of E5'N tagged by a radio-iodinated antibody that disappeared after 18 hours of incubation was lower under hypoxic than aerobic conditions. Because GPI-anchored proteins preferentially bind to cholesterol-rich lipid rafts or detergent-resistant membrane (DRM) domains,¹⁰ a decrease in membrane cholesterol could also prevent endocytosis and stimulate E5'N. Indeed, previous studies by the same group of investigators have shown that chelation of cholesterol attenuates E5'N activation, and that E5'N endocytosis could be diminished upon treatment of HMG-CoA inhibitors.¹¹ The decrease in endocytosis upon treatment with lovastatin was not due to cholesterol depletion, but due to inhibition of isoprenoid synthesis, which decreases isoprenylation of Rho-GTPases, and in turn, polymerization of actin. However, in the present study,⁷ hypoxia did not affect E5'N localization in DRM or actin organization, and the effects of hypoxia were not reversed by activating Rho-GTPase, indicating that mechanisms other than increased association with lipid rafts need to be invoked.

One of the earliest responses of cardiovascular tissues to hypoxia is a decrease in fatty acid oxidation due to the inability of hypoxic tissues to support ß-oxidation. This would lead to an increase in fatty acid content, which in turn could affect the fatty acid composition of the membrane. Alternatively, hypoxia could activate a host of phospholipases, which could support remodeling of membrane phospholipids. In their experiments, Ledoux et al⁷ noted an increase in the saturated fatty acid content in the membranes of endothelial cells exposed to hypoxia, although the amounts of phospholipids and free cholesterol were not affected. Because the increase was relatively specific for palmitate, it appears that the changes in the membrane fatty acids are due to selective remodeling of the membrane during hypoxia rather than a general gain of fatty acids due to inhibition of ß-oxidation. Based on these observations, Ledoux et al⁷ propose that hypoxia-induced enrichment of the membrane in saturated fatty acids decreases endocytosis by diminishing membrane fluidity.

The idea that membrane fluidity could regulate the function of membrane-bound proteins and their localization or endocytosis has been invoked in many other contexts, although how this happens is not altogether clear. Discrete locations of high cholesterol:phospholipids in lipid rafts have been shown to selectively retain GPI-anchored proteins to the cell surface. In most phospholipids, saturated fatty acids occupy the sn-1 position. Therefore, for the level of saturation to increase, the 16:0 and 18:0 fatty acids are expected to primarily replace 18:1, which is the only unsaturated fatty acid at the sn-1 position. This is consistent with the observation that the hypoxic increase in the16:0 content was accompanied by a decrease in 18:1 with no change in the primarily sn-2 fatty acids such as 20:4. Hence, the hypoxic cells appear to undergo extensive remodeling at the sn-1 position perhaps mediated by an unknown phospholipase A₁. The net result of this remodeling will be a decrease in the membrane fluidity and a decrease in endocytosis. Another interesting possibility is that membranes with a higher content of saturated fatty acids are better able to stabilize DRM so that increasing the content of saturated fatty acids prevents the spontaneous destabilization and endocytosis of these domains. Alternatively, changes in the membrane fluidity could affect the steps preceding endocytosis or membrane fusion. Regardless of the specifics, the suggestion that phospholipid remodeling could chronically mediate hypoxic signaling has wide-ranging implications. Not only does this provide for a novel mechanism of hypoxic adaptation of endothelial cells, but further elucidation of this phenomenon could help in understanding several other processes as diverse as myocardial preconditioning, vasodilation, neuronal adaptation, induction of sleep, and the immune modulation of neuronal signaling and vascular perfusion.

Cellular responses to oxygen involve direct sensing via "oxygen sensors," which trigger changes in intracellular signaling that lead to an acute response such as changes in membrane currents and contractility, and, if hypoxia is chronic, to adaptation.¹² Direct sensors (eg, K⁺ channels, NADPH oxidase) have the advantage of a first-order response; however, responses driven and established by intracellular signaling are higher-order responses that involve several steps subject to multiple regulatory influences. Learning from the intricate responses of kinases and phosphatases, it appears that such higher-order responses could be very specific and effective. Indeed, activation of E5'N by palmitate offers a new paradigm by which changes in membrane fluidity could trigger signaling responses that in turn could facilitate adaptation to the hypoxic state. Clearly, extensive work is required to sort out the details of this phenomenon and to establish its significance to the hypoxic and ischemic responses of the endothelium in vivo.

Footnotes

The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.

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