Role of Na+/H+ exchanger during O2 deprivation in mouse CA1 neurons

Hang Yao1, Xiang-Qun Gu1, Robert M. Douglas1, and Gabriel G. Haddad1,2

1 Section of Respiratory Medicine, Department of Pediatrics, and 2 Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

To determine the role of membrane transporters in intracellular pH (pHi) regulation under conditions of low microenvironmental O2, we monitored pHi in isolated single CA1 neurons using the fluorescent indicator carboxyseminaphthorhodafluor-1 and confocal microscopy. After total O2 deprivation or anoxia (PO2 congruent  0 Torr), a large increase in pHi was seen in CA1 neurons in HEPES buffer, but a drop in pHi, albeit small, was observed in the presence of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. Ionic substitution and pharmacological experiments showed that the large anoxia-induced pHi increase in HEPES buffer was totally Na+ dependent and was blocked by HOE-694, strongly suggesting the activation of the Na+/H+ exchanger (NHE). Also, this pHi increase in HEPES buffer was significantly smaller in Na+/H+ exchanger isoform 1 (NHE1) null mutant CA1 neurons than in wild-type neurons, demonstrating that NHE1 is responsible for part of the pHi increase following anoxia. Both chelerythrine and H-89 partly blocked, and H-7 totally eliminated, this anoxia-induced pHi increase in the absence of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. We conclude that 1) O2 deprivation activates Na+/H+ exchange by enhancing protein kinase activity and 2) membrane proteins, such as NHE, actively participate in regulating pHi during low-O2 states in neurons.

hippocampus; transporter; anoxia; pH; sodium-hydrogen exchanger


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE REGULATION of intracellular pH (pHi) in neurons has been investigated fairly actively in the past several years, and it is clear now that this regulation is very complex. A number of membrane proteins that are relevant to this regulation are present and functional in neurons, and their role in various conditions is being delineated (3, 8, 20, 22-25, 27, 32).

Although we and others have been interested in understanding how neurons sense and respond to lack of oxygen, we still do not know how the various membrane proteins regulate pHi and how they participate in determining the pHi response to O2 deprivation. For example, we do not know whether the Na+/H+ exchanger (NHE) is stimulated or inactivated during hypoxia. It can be argued that a drop in pHi during hypoxia can activate this exchanger (1). However, a drop in extracellular pH may inhibit it (33). There are also other factors in the microenvironment that may have major effects on membrane proteins and pHi change. These include extracellular ions and neurotransmitters released from adjacent neurons and glia (14, 29). Hypoxia may, therefore, change pHi by affecting the function of membrane transporters, intracellular metabolism, and the microenvironment around cells.

To examine some of these mechanisms during O2 deprivation, we needed to simplify the system. In this work, we studied freshly dissociated single cells that were constantly perfused. We performed our experiments on CA1 neurons because we have considerable experience with them (3, 4, 11, 32). In addition, there are many studies in the literature using these neurons; hence, these studies could be helpful from a comparative point of view (12, 26). Our aim was then to investigate the role of neuronal membrane proteins involved in pHi regulation during O2 deprivation. Our hypothesis was that neuronal membrane proteins, such as NHE, play a critical role in the regulation of pHi during low-O2 states.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell preparation. B6SJL,+/swe (slow-wave epilepsy) mice were obtained from Jackson Laboratories (9). These heterozygous mice (+/swe) were mated in our institution, and the resulting homozygote Na+/H+ exchanger isoform 1 (NHE1) mutant (25%) and wild-type (25%) F1 mice progeny were used at the age of 21-30 days. The mice genotypes were confirmed by a PCR-based test. Hippocampi were removed and sliced into transverse sections of 400 µm in thickness. The slices were immediately transferred to a container with 25 ml of fresh, oxygenated, and slightly stirred HEPES buffer at room temperature. After 30 min of trypsin (0.08%) and 20 min of protease (0.05%) digestion, the slices were washed and left in the oxygenated solution. The CA1 region was then dissected out and triturated in a small volume (0.25 ml) of HEPES buffer. When chelerythrine chloride, H-89, or H-7 was used, cells were incubated with the inhibitor for 1 h before pHi was measured (16). These studies have been approved by the Yale Animal Care and Use Committee.

Solutions. The HEPES-buffered solution contained (in mM) 125 NaCl, 3 KCl, 1.2 CaCl2, 1.2 MgSO4, 1.25 NaH2PO4, 30 HEPES, and 10 glucose. This solution was titrated to pH 7.38 at 35°C with NaOH. Na+ was removed from the solution by replacing NaCl and NaH2PO4 with N-methyl-D-glucamine (NMDG) and KH2PO4, respectively. Only 1.5 mM Na+ remained in the anoxia solution since dithionite is in the form of sodium salt. For the CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> solution, HEPES was replaced by 22 mM NaHCO3 and bubbled with 5% CO2 and 95% O2. The nigericin calibration solution contained (in mM) 105 KCl, 50 NMDG, 5 MgSO4, 10 glucose, and 30 HEPES. TNE buffer contained (in mM) 10 Tris (pH 7.5), 400 NaCl, 100 EDTA, and 0.6% SDS. TE buffer contained (in mM) 10 Tris (pH 8.0) and 10 EDTA. Cell-Tak was purchased from Collaborative Research (Bedford, MA), and carboxyseminaphthorhodafluor-1 (SNARF-1) was obtained from Molecular Probes (Eugene, OR). Nigericin, chelerythrine chloride, H-89, and H-7 were purchased from Sigma. HOE-694 was obtained as a gift from Dr. Hans-J. Lang (HMR/Hoechst Marion Roussel Chemical Research, Frankfurt, Germany).

Induction of hypoxia and anoxia. Hypoxia was induced by using a HEPES solution bubbled with 100% N2 for >4 h. Superfusate PO2 was monitored by a platinum wire electrode placed at the outflow end of the perfusion chamber and polarized at -0.8 V. The electrode was covered with a butyl acetate membrane to present a controlled diffusion barrier to oxygen. With the use of this hypoxia solution, PO2 was ~15-20 Torr when measured at the outflow end of the perfusion chamber. Anoxia was induced by adding 1.5 mM sodium dithionite into either 100% N2-bubbled HEPES buffer or 5% CO2 plus 95% N2-bubbled HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> solution. With dithionite, the PO2 was equal to 0 Torr when measured at the outflow end.

pHi measurements. Neurons were plated and allowed to settle down on a Cell-Tak-coated coverslip that was mounted on the bottom of a perfusion chamber. The chamber was then fixed on the stage of a Zeiss inverted microscope attached to a Bio-Rad MRC 600 laser confocal scanning unit. The cells were loaded with 10 µM of the acetoxymethyl ester form of SNARF-1 (prepared in dimethyl sulfoxide) for ~20 min. For consistency, as we have done previously (32), cells were considered for study if they were pyramidal in shape. The dye was excited at a wavelength of 514 nm, and the emission was detected by two photomultiplier tubes at two wavelengths (587 and 640 nm) for ratiometric analysis. One sample point was acquired every 30 s for both wavelengths. Ratios were obtained from these two fluorescence emission intensities, and the values were converted to pHi using a high-K+ nigericin calibration technique (6, 27, 30). Calibration experiments were done in wild-type and NHE1 mutant neurons, and no significant difference was found between them. Our detailed description of the calibration experiments has been previously published (32).

NHE1 mutant mice genotyping. Although the phenotype of the mutant mice was easily detected by their ataxic behavior, we performed genotyping on all presumed mutant mice to confirm the phenotype. The methods have been published elsewhere. In brief, genomic DNA was obtained from mice tails and used for PCR amplification with the primers 5'-TCGCCTCAGGAGTAGTGATGCG-3' (sense) and 5'-CGTCTTGTGCAGGGCATGA-3' (antisense), corresponding to base pairs 1397-1418 and 1800-1819 of mouse NHE1 cDNA sequence (accession no. U51112), respectively. DNA was subjected to the endonuclease Spe1 to differentiate between wild-type and homozygous mutant genotypes. Because of the nature of the Spe1 cleavage site, we obtained two bands in the mutant and one in the wild type.

Statistics. Data are presented as means ± SD. Levels of significance were assessed using paired and unpaired forms of the Student's t-test. Differences in means were considered significant when P was <0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This study is based on pHi measurements in 152 CA1 neurons, which fulfilled our study criteria (32). In HEPES buffer, the mean steady-state pHi was 7.22 ± 0.24 (n = 66), and this increased to 7.39 ± 0.20 (n = 51) in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> solution.

Anoxia induces pHi changes in CA1 neurons. The pHi of each CA1 neuron was monitored before, during, and after applying anoxia. In the presence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, cells responded to anoxia with a slow and relatively small drop in pHi. However, in the absence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, cells responded with a dramatic pHi increase after the initiation of anoxia (Fig. 1A). The average decrease of pHi over 5 min of anoxia in CA1 neurons was 0.06 ± 0.11 pH units (n = 10) in the presence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, but the mean increase in HEPES buffer was 0.46 ± 0.13 pH units (n = 13, P < 0.001; Fig. 1B). Hypoxia (without the addition of dithionite) also caused an alkalinization in CA1 neurons in HEPES buffer. Although this alkalinization was significantly smaller (0.12 ± 0.07 pH units, n = 8, P < 0.05) than the alkalinization induced by anoxia, both hypoxia and anoxia (with dithionite) induced an increase in pHi. It would seem, then, that the increase in pHi did not result from a nonspecific action of dithionite.


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Fig. 1.   Anoxia-induced intracellular pH (pHi) change in CA1 neurons in the absence or presence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. A: 2 neurons with similar initial pHi are shown; one perfused with CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> solution showed a slight decrease in pHi when exposed to 5 min of anoxia, and the other was perfused with HEPES buffer and responded to the same anoxic stimulation with a dramatic increase in pHi. B: bar graph showing mean change of pHi induced by 5 min of anoxia in both the presence and absence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. Means are significantly different from each other.

The effect of Na+ removal on the anoxia-induced pHi increase in HEPES. NHE has been considered to play a very important role in the pHi regulation of central nervous system (CNS) neurons. To determine whether the increase in pHi with anoxia is dependent on Na+/H+ exchange, we first studied neurons in HEPES buffer in the presence or absence of Na+. The removal of Na+ caused an acidification in normoxic conditions (Fig. 2A), probably because of the inhibition of Na+-dependent acid extruders (such as NHE) or reversal of their activity. This acidification partly recovered, and this is most likely due to the activation of H+-ATPases on the cell membrane. After the pHi had reached a plateau in the absence of Na+, cells were exposed to anoxia. As seen in Fig. 2A, pHi did not increase, rather, it actually decreased. The mean pHi drop was 0.10 ± 0.10 (n = 7; Fig. 2, A and B), which contrasts with the response of cells bathed with HEPES containing Na+, demonstrating a major increase in pHi (0.46 ± 0.13, n = 13, P < 0.001). Therefore, in the absence of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, the anoxia-activated acid extrusion and increase in pHi was totally dependent on Na+, and the most likely candidate responsible for this acid extrusion is the NHE.


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Fig. 2.   Na+ removal eliminates the anoxia-induced pHi increase in HEPES. A: 5 min of anoxia caused a small acidification in a neuron in the absence of Na+. B: bar graph showing mean change of pHi induced by 5 min of anoxia in both the presence of Na+ (left; compare with Fig. 1) and its absence. Means are significantly different from each other.

The effect of HOE-694 on anoxia-induced pHi change. To be able to dissect out the role of the transporters that regulate pHi during O2 deprivation, we further examined the effect of the neuronal NHE blocker HOE-694 (32) on the anoxia-induced pHi changes in CA1 neurons in HEPES buffer. The anoxia-induced alkalinization was almost totally eliminated by 100 µM HOE-694 in HEPES buffer (Fig. 3A), and the pHi change was 0.02 ± 0.13 units (n = 7, P < 0.001 vs. control group; Fig. 3B, left).


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Fig. 3.   Effect of HOE-694 on the anoxia-induced pHi change in HEPES buffer. A: with 100 µM HOE-694 in HEPES buffer, 5 min of anoxia induced no change in pHi in contrast to that seen in the absence of that agent (compare B with Fig. 1). B: bar graph showing the mean changes of pHi from control (see Fig. 1) and the HOE-694 group. Means are significantly different from each other.

NHE1 is involved in the anoxia-activated acid-extrusion process. Because HOE-694 is a relatively nonspecific NHE blocker and since NHE1 is the most ubiquitous isoform in the CNS (18, 19), we took advantage of the NHE1 null mutant mouse to examine whether the anoxia-induced alkalinization in HEPES is the result of overactivation of NHE1. Figure 4A illustrates the pattern and amplitude of pHi changes following anoxia in neurons isolated from both wild-type and NHE1 mutant mice in HEPES solution. Compared with the response in the wild-type neuron, the mutant neuron showed a significantly slower and smaller pHi change following anoxia. The average anoxia-induced pHi increase in mutant neurons was 0.30 ± 0.13 (n = 10), and this was significantly smaller than that in wild-type neurons (0.46 ± 0.13 pH units, n = 13, P < 0.05; Fig. 4B). This result suggested that NHE1 is activated during anoxia and is only partly responsible for the anoxia-induced alkalinization seen in the absence of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. Presumably, other isoforms, such as NHE2, NHE4, and NHE5, might have also been activated. Another possible mechanism for the alkalinization is the activation of H+-ATPases.


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Fig. 4.   Anoxia-induced pHi increase was smaller in the Na+/H+ exchanger isoform 1 (NHE1) null mutant neurons. A: 2 neurons [one was a wild-type (WT) and the other a NHE1 mutant] with similar initial pHi. Both neurons were perfused with HEPES buffer and subjected to 5 min of anoxia. Although both neurons had a similar pattern in pHi change in response to anoxia, a smaller increase was seen in the mutant neuron. B: bar graph showing mean change of pHi induced by 5 min of anoxia in both NHE1 mutant and wild-type neurons. Means are significantly different from each other.

Kinase inhibition markedly attenuates the anoxia-activated acid extrusion. To understand how anoxia activates the NHE, we first pretreated cells with the rather nonspecific protein kinase inhibitor H-7 (60 µM) to determine whether reducing kinase activity can affect the anoxia-induced alkalinization. Figure 5A shows one example from these experiments. In HEPES solution, the H-7-pretreated cell responded to anoxia very little, if at all (0.006 ± 0.0324 pH units, n = 8; Fig. 5D). These data demonstrated that kinase activity was enhanced during anoxia, which, in turn, increased the activity of the NHE.


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Fig. 5.   Kinase inhibition attenuates the anoxia-activated acid extrusion process. A: the tested neurons were pretreated with 60 µM H-7 and then exposed to 5 min of anoxia in HEPES buffer. This cell showed a slight pHi decrease after anoxia instead of the increase seen in HEPES with anoxia in the absence of H-7 (see Fig. 1). B: pretreated neurons with 1.5 µM chelerythrine were exposed to 5 min of anoxia in HEPES buffer. In response to anoxia, this neuron increased pHi, but the amplitude was much smaller than in the untreated cells (see Fig. 1). C: pretreated cells with 30 µM H-89 were exposed to 5 min of anoxia in HEPES buffer. The response of this neuron to anoxia was still a pHi increase but the amplitude was much smaller (see Fig. 1). D: 4 bar graphs. The first from the left is the same as in Fig. 1, shown for comparative purposes; the rest are bars representing means of experiments in A-C. * Significantly different from control.

To investigate the possible role of protein kinase C (PKC) or protein kinase A (PKA) in the modulation of pHi by NHE during anoxia, we pretreated neurons with either chelerythrine (1.5 µM), a PKC inhibitor, or H-89 (30 µM), a PKA inhibitor, and examined their response to anoxia. Figure 5B shows an example of the pHi measurement of chelerythrine-pretreated CA1 neurons. In HEPES solution and in the presence of this blocker, although pHi increased (0.12 ± 0.20 pH units, n = 6), the change was significantly smaller than for the untreated group (0.46 ± 0.13 pH units, n = 13, P < 0.001; Fig. 5D). These results suggested that the activation of PKC was at least partly responsible for the anoxia-induced pHi increase. Figure 5C also shows only a slight pHi increase in another experiment of a neuron pretreated with H-89 and followed by anoxia. The mean anoxia-induced pHi change was very small (0.05 ± 0.06 pH units, n = 6) compared with the control group (0.46 ± 0.13 pH units, n = 13, P < 0.001; Fig. 5D).


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Because it has been well demonstrated that pHi is reduced in cells during hypoxia/ischemia in vivo (12, 26), it would seem reasonable to assume that anaerobic metabolism plays an important role in lowering pHi. However, the role of various neuronal exchangers and transporters in pHi regulation during low-O2 conditions has not been well studied. One major reason for trying to understand the role of such membrane proteins during hypoxia is that there is already evidence from work in heart muscles and from our previous work on neurons that Na+/H+ exchange and Na+ loading play an important role in the pathogenesis of hypoxic or ischemic neuronal injury (7, 8, 15, 17). It is important to mention here that our current studies showed that pHi had a seemingly paradoxical change during anoxia, i.e., an increase in pHi during anoxia in HEPES solution in the absence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. Clearly, however, we do not suggest that pHi increases during anoxia in vivo. We should highlight two issues in this regard: 1) the studies in the literature that showed a pHi decrease during anoxia or hypoxia were done in the presence of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, unlike our experiments in which we used both solutions containing or lacking CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, and 2) by studying the effect of anoxia in HEPES as well as in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, we have been able to uncover the response of some membrane transporters in low-O2 environments.

Effect of anoxia on the regulation of pHi. One of our major findings in this paper is that O2 deprivation induced an alkalinization in the absence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. To ascertain that this increase in pHi during anoxia is not a nonspecific effect of the O2 scavenger that we used (dithionite), we performed two types of experiments. In the first, we exposed neurons to a hypoxic solution, a solution that was bubbled with nitrogen only, with no dithionite. Although the cells responded with a smaller pHi increase in this hypoxic solution than in anoxia, this increase in pHi in the absence of dithionite supports the idea that the increase in pHi is not related to the O2 scavenger per se but to the lowering of PO2. In the second type of experiment, we used another pH-sensitive dye, 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF), with the same cells and obtained similar data (data not shown), indicating that the increase in pHi in anoxia is not dependent on interactions between SNARF-1 and dithionite.

It is reasonable to assume that the changes in pHi that are observed in vivo during low-O2 states at any one time are the net result of the simultaneous activation (or inactivation) of a variety of cellular processes. Because physiological solutions are often used, i.e., CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-containing solutions, the role of certain exchangers, such as the NHE, may not be readily observed. Similarly, the differences in the response of CA1 cells when CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> solution is used or is omitted provided us with the idea that HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-dependent acid loaders and acid extruders might be involved. Another potential reason for the differences between in vivo and in vitro situations is related to the control over the microenvironment. The same consideration may apply to cultured preparations. For example, the accumulation of H+ in the extracellular space might affect the activity of NHE (33) in vivo or cultured neurons more than in our freshly dissociated neurons, since we control the composition of the perfusate throughout the experiments, such as the ion concentration and extracellular pH, to tease apart some of the mechanisms that are operative. Indeed, in their recent work, Diarra et al. (10) have shown an anoxia-induced acidification in HEPES buffer in cultured neurons, although a dramatic alkalinization was seen after the reapplication of O2. The major difference between our preparation and theirs may be related to the presence of neuronal connectivity and synaptic activity among cultured neurons that may constitute a different microenvironment.

Na+/H+ exchange is activated during anoxia. In the mammalian CNS, the NHE is the major HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-independent acid extruder and, so far, six isoforms of its gene family have been identified. However, it has previously been shown that NHE1 is the most ubiquitously expressed (2, 19, 21) in the CNS. Although hippocampal neurons do not have the full complement of NHE isoforms (19), our current data demonstrate that the anoxia-induced pHi increase could be caused by the activation of NHE. Although NHE1 is a major membrane protein in the CNS, other isoforms of NHE could also be involved. HOE-694 eliminated the anoxia-induced pHi increase completely, but the fact that we still had a substantial rise in pHi in the NHE1 mutant during anoxia argues for the lack of specificity of HOE-694 for NHE1 activity. This lack of specificity of HOE-694 is not unlike what was found in the pancreatic duct (18). Indeed, the NHE1 mutant cells that we used in this study had a smaller increase in pHi during anoxia than wild-type cells. Because the anoxia-induced alkalinization in HEPES buffer is totally Na+ dependent, we believe that other isoforms of NHE, such as isoforms 2, 4, and 5 (2, 5, 9), could have been activated in the mutant cells and could have been responsible for the pHi increase in NHE1 mutant neurons.

How NHE activity increases during hypoxia has not been totally delineated. Sheldon and Church (28) have reported the involvement of PKA in anoxia-induced pHi change in CA1 neurons. In this paper, we have evidence that NHEs are activated during anoxia, at least partly, because of the upstream activation of PKA and PKC. The interesting finding in our system is that anoxia seems to activate both kinases, A and C, since H-7 and both H-89 and chelerythrine block, one at a time, the major increase in pHi in HEPES solutions. We do not know, however, how the lack of O2 activates protein kinases. There are a number of possible mediators, with one being an increase in cytosolic Ca2+ concentration, which can lead to increased kinase activity and activation of NHE (31).

Physiological significance. We believe that our observations in this paper put into perspective the physiological importance of membrane proteins in regulating pHi during low-O2 states. Indeed, the increased levels of protons inside neurons during these states are a result of many cellular processes besides anaerobic metabolism, which had been thought to be the major or sole source of intracellular acidosis. This acidosis is most likely due to the net result of intracellular buffering capacity (which may be different during anoxia), intracellular Ca2+ levels, the activity and the level of firing of neurons, which certainly changes during hypoxia (13), how disturbed the ionic homeostasis is (25), and the initial pHi of these neurons, which determines the activity of membrane proteins at the outset. Last, as we have found in this work, the activity of membrane acid loaders or extruders will be important in determining the pHi level. For example, the NHEs are incriminated in our studies during anoxia. How they get activated is at present unknown but may be related, as we have shown, to kinase activation. Indeed, through manipulations of various solutions and blockers, we have been able to demonstrate that this particular exchanger is important during O2 deprivation.

In summary, we show in this work that the major pH regulators in hippocampal neurons, the NHEs, play a key role in keeping the homeostasis of intracellular acid base balanced in neurons after O2 deprivation. Our study demonstrates the active role of membrane transporters that regulate pHi in neurons as a function of O2 level in their microenvironment.


    ACKNOWLEDGEMENTS

The authors thank Drs. David F. Donnelly and Samuel K. Agulian for advice and assistance in making oxygen microelectrodes and Drs. Mark O. Bevensee and Patrice Bouyer for assistance in performing validation experiments with BCECF and for helpful discussions. We also thank Dr. Hang-J. Lang for the generous gift of HOE-694.


    FOOTNOTES

This work was supported by National Institutes of Health Grants P01-HD-32573 and NS-35918 (to G. G. Haddad).

Address for reprint requests and other correspondence: G. G. Haddad, Yale Univ. School of Medicine, Dept. of Pediatrics, Section of Respiratory Medicine, Fitkin Memorial Pavilion, Rm. 506, 333 Cedar St., New Haven, CT 06510 (E-mail: gabriel.haddad{at}yale.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 9 November 2000; accepted in final form 25 May 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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