hco3− transporters and the Na+/H+ exchanger (NHE) contribute in a major way to maintenance of ionic and pH homeostasis in neurons. The study by Xue et al. (17) demonstrates that, in prolonged neuronal hypoxia, inhibition of HCO3− transporters by DIDS is protective and inhibition of NHE by either HOE 643 or T-162559 results in increased cell death. These observations have important implications for potential therapeutic interventions that could target upregulation of NHE activity in diseases marked by prolonged hypoxia. The protective role of NHE function in prolonged hypoxia contrasts markedly with a prosurvival response of NHE inhibition in certain models of myocardial ischemia or brain ischemia. This editorial focus includes a discussion of the data on NHE and HCO3− transporter inhibition in hypoxic neurons and in addition considers the differential contributions to cell fate decisions in hypoxia of mild or severe acidosis. In brain ischemia, for example, acidosis may contribute to neuronal death through elevations in oxidative stress, key modifications in prodeath signaling proteins, or potentially though increased intracellular Na+ and subsequent Ca2+ overload. Elevated reactive oxygen species (ROS) is known to be a major contributor to neuronal adaptations associated with chronic intermittent hypoxia. Chronic continuous hypoxia, by contrast, is not generally associated with ROS signaling, but interestingly some recent evidence points to some specific signaling roles for ROS in prolonged hypoxia, perhaps via hypoxia-inducible factor (HIF)-1. In chronic continuous hypoxia, in vivo, specific HCO3− transporters have been reported to be downregulated in brain perhaps as part of an energy-saving cellular survival response. The detrimental effects of inhibition of the NHE and the protective effects of inhibiting HCO3− transporters in prolonged neuronal hypoxia emphasize the critical importance of ionic and pH homeostasis in maintaining neuronal function and may lead to more comprehensive understanding of the role of pH balance in proapoptotic and prosurvival signaling.
While the mammalian brain does have some capability for depressing its metabolism upon oxygen deprivation, such protective mechanisms are known to fail readily in brain ischemia, leading to a series of events that are catastrophic for neurons, including ATP loss, membrane depolarization, and an uncontrolled release of excitatory neurotransmitters (12). Hypoxic injury in neurons is closely linked to a substantial Ca2+ overload that plays a central role in excitotoxic cell death (18). In brain tissue, hypoxia induces complex changes in extracellular and intracellular pH, and, under ischemic conditions, numerous studies report a decrease in intracellular pH. Cerebral ischemia is associated with metabolite build-up, resulting in a fall in both neuronal and glial pHi that may frequently render these cells more prone to injury. Central neurons are thus extremely sensitive to oxygen deprivation, and resulting alterations in ionic and pHi homeostasis may elicit cell injury and death.
The article by Xue et al. (17) provides important insights into the effects of hypoxia and acidosis on neurons in culture. This investigation demonstrates a prosurvival role for the NHE and a prodeath role for HCO3− transporters under conditions of prolonged hypoxic exposure.
THE NHE AND HCO3− TRANSPORTERS IN BRAIN
The ionic and pH homeostasis in neurones and glia is regulated to a major degree by two families of acid-base transporters, the NHE family and the HCO3−-dependent acid-base transport protein family. The latter family includes the Na+-independent Cl−/HCO3− anion exchanger (AE1-4), the Na+-dependent Cl−/HCO3− (NDCBE) exchanger, and a number of Na+-HCO3− cotransporters (NBC). The NHE subunit 1 (NHE1), NDCBE, AE3, and NBC are key contributors to acid-base regulation in neurons and glia. As emphasized in the study by Xue et al. (17) on cultured neurons subjected to long-term hypoxia, ionic disturbances involving modulated activity of the NHE and of members of the HCO3− transporter family may contribute in important ways to regulating levels of neuronal hypoxic injury. A key observation in this study is that the NHE plays a prosurvival role in neurones under conditions of prolonged hypoxia. The protective action of the NHE in hypoxic neurons is in direct contrast to its reported function in ischemic heart where NHE blockers are proposed for therapeutic interventions. In this investigation, pharmacological inhibition of HCO3− transporters using the reagent 4,4′-diisothiocyanatostilbene-2,2′-disulfonate (DIDS) prevented hypoxic cell death in cultured neurons and hippocampal slices.
Depending on the model system, pharmacological blocking of the NHE may be either prosurvival or death inducing. For example, in extensive investigations in models of myocardial ischemia, it has been reported that NHE1 inhibition in ischemia with reperfusion results in potent myocardial protection (2). This prosurvival effect of NHE1 inhibition appears to be mediated by a decrease in intracellular Na+ that consequently prevents intracellular Ca2+ overload arising via reverse-mode Na+/Ca2+ exchange. In a two-vessel occlusion model of cerebral ischemia-reperfusion injury, 5-(N-ethyl-N-ispropyl)amiloride, an Na+/H+ exchange inhibitor, resulted in decreased CA1 pyramidal neuron loss (15). Although the previously mentioned studies point to NHE inhibition as protective, a common theme in such investigations is the necessity for reperfusion to reveal such a role for NHE inhibitors. Critically, in a number of cell types, including renal tubular epithelial cells and cerebellar granule neurons, inhibition of NHE contributes in an important way to activation of specific cell death pathways.
HCO3− transporters may be classified as either Na+-coupled HCO3− transporters or AE anion exchangers, which are Cl−-HCO3− exchangers. It has been demonstrated previously that a DIDS-sensitive Na+- and HCO3−-dependent mechanism, possibly a Na+/HCO3− cotransporter, in addition to a NHE act to maintain steady-state pHi in cultured mouse cortical neurons. pHi recovery after acid loading is dependent primarily on a NHE and a DIDS-sensitive Na+-dependent Cl−/HCO3− exchanger. In the study by Xue et al. (17), DIDS, which is capable of inhibiting HCO3− transporters and Cl− channels, was protective against neuronal injury in cortical neuronal cultures and in hippocampal slices. The results from this investigation point strongly toward a role for the DIDS inhibition of a HCO3− transporter in addition to the inhibition of the NHE by either HOE 643 or T-162559 in pHi regulation in prolonged hypoxia. Because of the protective nature of the DIDS-induced inhibition of HCO3− transporters in prolonged neuronal hypoxia, the DIDS-sensitive mechanism is proposed by the authors as a potential therapeutic target.
The role of HCO3− transporters in brain has been investigated recently in vivo using a mouse model of prolonged hypoxia (4). In mice subjected to continuous chronic hypoxia over 2–4 wk, expression levels in brain for two electroneutral Na+-coupled HCO3− transporters NBCn1 and NCBE were found to be decreased significantly. These HCO3− transporters act as acid extruders and thus elicit a net uptake of HCO3− and raise pHi. The depletion in NBCn1 and NCBE in hypoxia would be likely to result in lower steady-state pHi. This downregulation of NBCn1 and NCBE expression may be an energy-saving strategy for the cell, with the decreased pHi representing a biproduct of this strategy. A major theory of hypoxic adaptations derives from evidence that cells respond to oxygen deprivation by downregulating ATP generation as well as ATP-consuming processes (8, 9). Thus, for example, in hypoxia-tolerant cells, “channel arrest” involves a downregulation of ion pumping mechanisms that comprise a major component of cellular ATP utilization. In responding to chronic hypoxic stress, there is evidence for a global cellular strategy involving a generalized decrease in protein synthesis combined with preferential gene expression and selective translation of only specific messages (8). Ma and Haddad (13) have proposed that there is a hierarchy of proteins that may selectively be maintained or decreased in hypoxia, depending on cellular requirements. In the study by Chen et al. (4), the reported depletion in levels of electroneutral HCO3− transporters in chronic hypoxia was out of proportion to a generalized decrease in protein levels and may therefore represent part of a selective and critical regulatory step in maintaining ionic and pH homeostasis in the brain.
IMPORTANT DISTINCTIONS BETWEEN CHRONIC CONTINUOUS HYPOXIA AND CHRONIC INTERMITTENT HYPOXIA AS MODEL SYSTEMS
Chronic continuous hypoxia (CCH) is distinguished from chronic intermittent hypoxia (CIH) by virtue of the latter's clear use of ROS in the signaling processes associated with the resulting cellular adaptations. Thus CCH has been seen as a factor in certain physiological adaptations, including embryonic development and tissue alterations associated with living at high altitudes as well as in a variety of disease conditions, including anemia and ischemia. CCH results in a near doubling of capillary density in the brain, a process that is regulated by HIF-1 and angiopoietin-2 (10). By contrast, CIH may be associated with such diseases as obstructive sleep apnea/hyponea (or hypoventilation) syndrome, and, in rats, CIH is related to a reversible defect in memory and motor function. A recent study related to brain acidemia in a mouse model of CIH has demonstrated decreased expression of NHE subunits and of AE3 anion exchanger and of NBC, especially in cerebellum and hippocampus (5). It was proposed that this decrease in acid-extruding proteins would contribute to the susceptibility of neurons to damage from acidosis and that these detrimental effects were, in part, linked to signaling that involves the increased ROS levels that are characteristic of the CIH model.
IS THERE A ROLE FOR ROS SIGNALING IN PROLONGED HYPOXIA?
Although ROS signaling is not regarded as central to CCH, it may be that a role for ROS signaling in prolonged hypoxia has not been precluded, and, for example, HIF-1 activation in hypoxia is regulated by redox mechanisms in the dopaminergic PC-12 cell line (1). In examination of potential hypoxia-sensing mechanisms using a cardiac myocyte model, Chandel and Shumacker (3) have proposed a key sensing role for elevated cellular ROS based on initial studies on hypoxic cardiac myocytes (3, 6). This elevated cellular ROS is postulated to occur in hypoxia as a result of a decreased reduction of O2 to H2O by cytochrome oxidase, resulting in a release of electrons upstream at complex III of the mitochondrial electron transport chain and consequently generation of superoxide (6). Although this cardiac myocyte study was based on 1–2 h of hypoxia (a relatively short duration of hypoxia), a number of related investigations illustrate the potential for a key contribution of ROS generation in prolonged hypoxia. For example, in kidney-derived HEK293 cells subjected to prolonged hypoxia, mitochondrial ROS generation induced a HIF-1-independent depletion in cellular glutathione levels (14). In heart and skeletal muscle, ROS generation in chronic hypoxia may perform a protective function through modulation of Ca2+ signaling, leading to an increased mitochondrial bioenergetic capacity (7).
Under hypoxic conditions, does acidosis promote cell death or cell survival?
In the mouse model of continuous chronic hypoxia, the reported decrease in expression of HCO3− transporters may represent an energy-saving strategy, and the result may be a reduced pHi and a decreased capacity of cells to respond to changes in intracellular acidity (4). In neuronal cultures subjected to prolonged hypoxia, acidosis would again be damaging. Under hypoxic conditions, pharmacological inhibition of the acid extruder NHE1 would increase intracellular acidification and in so doing contribute to neuronal damage and, potentially, enhancement of apoptotic processes (17).
The nature of the contributions of acidosis to neuronal cell death in hypoxic conditions is likely to depend in part on the degree of the acidosis, with mild acidosis in some instances being seen as protective. In a model of focal brain ischemia, acidosis induced by hypercarbic ventilation resulted in decreased infarct volume relative to nonacidotic treatment, implicating moderate acidosis (optimally at brain pH 6.8) as protective against ischemic damage (11). In several in vitro neuronal models, reduced pH is protective against ischemia and against glutamate. Reduced pHo decreases N-methyl-d-glucamine (NMDA)-mediated Ca2+ entry, resulting in hypoxic neuronal protection in vitro (11, 18).
In common with the effects mentioned previously on ischemic heart, augmented intracellular acidification in ischemic brain could activate NHE function, causing a damaging increase in intracellular Na+ (11). Free radical formation may also be a potential mechanism for ischemic damage augmented by lowered pH, with such an effect occurring via iron delocalization and the Fenton reaction. In a model of focal brain ischemia, tissue damage was augmented by preexisting hyperglycemia with a greater than twofold elevation in free radical production (16, 11). In contrast to the NMDA receptor actions on in vitro neuronal cultures mentioned above, cell death mediated by the AMPA/kainate receptor contribution to ischemic cell death in neuronal cultures is augmented by acidosis. Lowered pHo was reported to increase AMPA-kainate receptor-induced neurotoxicity in cultured neocortical cells (11).
In summary, the article by Xue et al. (17) is important in terms of demonstrating a protective effect on hypoxic neurons of DIDS-mediated inhibition of HCO3− transporters and an injurious effect of inhibiting NHE1 by either HOE 643 or T-162559. The prosurvival role of NHE1 and the prodeath role of HCO3− transporters are indicative of the critical importance of maintaining ionic and pH homeostasis as a strategy for neuronal hypoxic survival. This study helps to open the way to a more comprehensive understanding of the relationship between hypoxia with acidosis and key signaling events associated with hypoxic adaptation, such as HIF-1 responses and stress kinase activation. It is very likely as the field progresses further that we will soon be able to incorporate the respective contributions of these ion exchangers and transporters into a characterization of critical death processes that may involve different apoptosis pathway components, including Bcl-2 family members, endonucleases, and caspases.
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