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
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ABSTRACT |
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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
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
hippocampus; transporter; anoxia; pH; sodium-hydrogen exchanger
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INTRODUCTION |
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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.
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MATERIALS AND METHODS |
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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
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
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.
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RESULTS |
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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
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
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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
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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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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
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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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DISCUSSION |
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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
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
Na+/H+
exchange is activated during anoxia.
In the mammalian CNS, the NHE is the major
HCO
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 |
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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.
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FOOTNOTES |
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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.
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