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Regulation and Modulation of pH in the Brain

Department of Physiology and Neuroscience, Department of Neurosurgery, New York University School of Medicine, New York, New York

ABSTRACT

I. INTRODUCTION

II. BUFFERING POWER

III. REGULATION OF INTRACELLULAR pH IN VERTEBRATE NEURONS

A. Neurons From Hippocampus

B. Neurons From Cerebral Cortex

C. Neurons From Cerebellum

D. Neurons From Spinal Cord

E. Medullary Neurons

F. Retinal Neurons and Photoreceptors

G. Sympathetic Neurons

H. Neoplastic Neural Cells

I. Synaptosomes

IV. REGULATION OF INTRACELLULAR pH IN GLIAL CELLS

A. pH Regulation in Nonmammalian Glial Cells

B. pH Regulation in Mammalian Astrocytes

1. Principal acid extrusion mechanisms in mammalian astrocytes

2. The astrocyte Na+-HCO3- cotransporter

3. Na+-independent acid extrusion in astrocytes

4. Cl-/HCO3- exchange in astrocytes

C. pH Regulation in Oligodendroglia

D. pH Regulation in Schwann Cells

E. pH Regulation in Glial Tumor Cells

F. pH Regulation and Volume Control in Glia

V. MOLECULAR IDENTIFICATION OF BRAIN ACID TRANSPORTERS

A. Na+/H+ Exchangers in Brain

B. HCO3- Transporters in Brain

1. The Cl-/HCO3- exchanger in the CNS

2. Na+-HCO3- cotransporters in the CNS

3. Na+-driven Cl-/HCO3- exchange in the CNS

VI. MODULATION OF INTRACELLULAR pH BY NEURONAL ACTIVITY

A. Modulation of pHi in Neurons

1. pHi shifts associated with depolarizing stimuli

2. pHi shifts associated with GABAA and glycine receptors

3. Additional modulators of neuronal pHi

B. Modulation of pHi in Glial Cells

VII. REGULATION AND MODULATION OF BRAIN EXTRACELLULAR pH

A. The pH of Interstitial Fluid

B. Changes in Interstitial pH Caused by Neural Activity

1. Buffering of brain interstitial fluid

2. Interstitial pH transients and CA

3. Nature of interstitial CA activity in brain

4. Bicarbonate-independent alkaline shifts

5. Bicarbonate-dependent alkaline shifts

6. Mechanisms of activity-dependent acid shifts

VIII. EFFECTS OF ACTIVITY-DEPENDENT pH SHIFTS ON NEURAL FUNCTION

A. Modulation of Function by Extracellular Alkaline Shifts

B. Modulation of Function by Activity-Dependent Acid Shifts

IX. CONCLUSION

	ABSTRACT

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	I. INTRODUCTION

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The importance of pH regulation to the viability of cells is widely recognized. The production of intracellular acid is a normal consequence of respiration and, if unchecked, can lead to a marked fall in intracellular pH, compromising vital functions. In this broad respect, the cells of the central nervous system (CNS) do not differ from those found in other tissues. Indeed, many of the mechanisms responsible for long-term "housekeeping" of hydrogen ions are similar.

The cellular diversity of the CNS is unique, however, and significant differences can be found in the complement of acid transport mechanisms expressed among subtypes of neurons and glial cells. This has been made apparent by physiological experiments on particular cells and by molecular studies that have revealed regional diversity in the expression of transporter subtypes.

The study of pH in the CNS is also distinguished by the occurrence of rapid increases or decreases in H⁺ that arise from electrical activity. These changes take place in time frames from milliseconds to minutes, involve neurons as well as glia, and occur in both the intracellular and extracellular compartments. Given numerous enzymes and ion channels that can be influenced by pH, a possible modulatory role of these pH transients has often been considered, particularly in relation to membrane potential and excitability. In this respect, the mechanisms that generate and regulate these pH changes are of considerable neurobiological interest.

The regulation of brain pH, the generation of activity-dependent pH changes, and their functional consequences have been the subject of earlier reviews (76, 80, 94). A comprehensive treatment of pH and brain function has also appeared in an excellent volume edited by Kaila and Ransom (152). The present contribution aims to provide an accessible, updated summary of this field and is consequently more circumscribed. To maintain reasonable limits, certain subjects have not received full coverage. Historical aspects and methodological issues have been described elsewhere (76, 149). The effect of pH on numerous enzymes and channels is an extensive subject that is also beyond the scope of this summary. With the exception of particularly notable studies, the reader is directed to various reviews of this topic (22, 211, 288, 298, 307, 319). Important pioneering work on pH regulation in the nervous system of invertebrates has been covered by several earlier contributions (76, 94, 259, 303). This review emphasizes pH studies in the mammalian CNS and ventures into invertebrate studies only when they are particularly relevant to the topic at hand.

	II. BUFFERING POWER

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The study of pH regulation requires the establishment of fundamental concepts of pH buffering and a consistent nomenclature. The definition and measurement of pH, the mechanisms of extracellular and intracellular buffering, and the chemical equilibria involved have been the subject of previous comprehensive reviews (55, 76, 259, 303). The purpose of this section is to highlight the basic concepts of buffering power relevant to physiological studies of pH, before addressing the means of intracellular and extracellular pH regulation in the nervous system.

The buffering power of aqueous solutions can be defined in a number of ways (157, 259). The most extensively used definition comes from the early work of Koppel and Spiro (172) and Van Slyke (323), where buffering power () is conceptualized as the concentration of added strong acid (or base) divided by the resulting change in pH. Thus

(1)

or

(2)

where A and B are the concentrations of strong acid or base, respectively. Accordingly, the buffering power (or buffering capacity) has units of concentration.

If one could analyze cytosol in the absence of organellar or metabolic influences, the buffering capacity would be determined by the summed contributions of the separate buffer species. In physiological studies, it is convenient to divide the total intracellular buffering capacity into the bicarbonate-dependent component and the nonbicarbonate or "intrinsic" buffering capacity, given the symbols _b and _i, respectively. The total intracellular buffering capacity (_T) is simply the sum of _b and _i.

The contribution to buffering provided by the CO₂-HCO₃^- equilibria (_b) can be approximated as

(3)

where [HCO₃^-]_i is the intracellular HCO₃^- concentration. The derivation of this formula assumes that cytosolic CO₂ is constant, due to its equilibration with extracellular CO₂, which serves as a fixed, infinite source. Such a system is said to be "open" with respect to CO₂. If one knows the system PCO₂ and the intracellular pH (pH_i), then [HCO₃^-]_i and _b can be calculated from rearrangement of the Henderson-Hasselbalch equation as

(4)

where PCO₂ is expressed in Torr and S is the solubility coefficient of CO₂ in mM/torr. It should be noted that the value of _b increases steeply as pH_i rises, being 2.5-fold greater at pH_i 7.4 versus 7.0.

In one study of cultured mammalian neurons, intracellular HCO₃^-, while present in significant concentration, was found to contribute little to _T (7). The basis of this observation was not clear. Faced with a rapid acid-base challenge, the rate of CO₂-HCO₃^- buffering would depend on the presence of carbonic anhydrase (see sect. VIIB, 1-3). It is plausible that insufficient activity of this enzyme limited the contribution of _b under these circumstances.

The major components of _i are associated with the buffering contribution of imidazole residues and phosphate (55). Determination of _i for a cell is not always straightforward. To measure _i, one might apply a known concentration of acid or base (in the absence of CO₂ and HCO₃^-) and measure the resulting change in pH_i. This procedure, however, would rarely give an accurate value, since the imposed pH_i change would be countered by plasma membrane acid transport in addition to cytosolic chemical buffering. Acid or base challenges are therefore typically made under conditions in which these transporters are inactive (e.g., under pharmacological blockade or ion substitution) or are minimally active (e.g., using a series of small base challenges). Under these conditions, the ratio of the acid (or base) load to the pH_i (Eq. 1 or 2) is then considered to be a measure of _i. Although values of _i obtained with such methods are useful in analyzing pH regulatory processes, they should be considered as no better than convenient operational measures, since they ignore the probable contributions of metabolism and organellar transport to the cytosolic H⁺ balance. _i has been operationally determined in this way for a number of neurons and glial cells and tends to falls in a range from 5 to 30 mM (76, 159), but may be as high as 40-50 mM in cells of neonatal brain (256). The value of _i can vary with pH_i and typically increases as pH_i falls (for examples, see Refs. 26, 35, 159).

Determination of the intracellular buffering capacity becomes critical when analyzing acid-base transport across the plasma membrane. The rate at which pH_i is changed by a transporter has quantitative relevance only in view of the buffering capacity that opposes the pH change. The molar rate of acid efflux from a cell (J) would therefore be given by

(5)

where J may be expressed as mM H⁺ extruded per unit time. A common mistake in studies of pH regulation is to compare values of dpH_i/dt instead of calculating J from

Equation 5. For example, if the rate of pH_i recovery from an acid load were the same in the presence and absence of CO₂-HCO₃^-, one could not assert that HCO₃^--dependent acid extrusion played no role. On the contrary, since _T is greater in the CO₂-HCO₃^--buffered media, one should conclude that the acid efflux rate (J) had increased and that HCO₃^--dependent acid extrusion was indeed present. In such studies, comparisons of J are best performed at the same baseline pH_i, since _b rises, and _i typically falls with elevation of pH_i.

	III. REGULATION OF INTRACELLULAR pH IN VERTEBRATE NEURONS

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The plasma membrane transport systems responsible for neuronal pH_i regulation were first investigated in large invertebrate cells. The modern period of this work was initiated by Roos, Boron, and Thomas, who have reviewed their classic studies (259, 303). The essential features of acid transport systems described in invertebrate neurons can also be found in vertebrate nerve cells. In the first investigation of a vertebrate neuron, the giant reticulospinal cells of the lamprey were found to eliminate acid loads by an amiloride-sensitive Na⁺/H⁺ exchanger and an Na⁺-, HCO₃^--, and Cl^--dependent acid extruder (75, 83). In subsequent studies of mammalian neurons, Na⁺/H⁺ exchange and Na⁺-dependent bicarbonate transport were frequently noted. In addition, some nerve cells were found to express a passive Cl^-/HCO₃^- exchanger, which served as an acid-loading mechanism during recovery from cytosolic alkalization. Significant differences in the function, expression, and pharmacological sensitivity of these transporters have been noted in the investigation of mammalian neurons. Details of these studies are provided below and are summarized in Tables 1 and 2.

A. Neurons From Hippocampus

Neurons derived from the hippocampus have been the subject of the most extensive studies of intracellular pH (pH_i) regulation in the mammalian nervous system. Cultured, fetal, rat hippocampal neurons were investigated by Raley-Susman et al. (250). A key finding was the presence of an Na⁺/H⁺ exchanger insensitive to amiloride and its derivatives. This transporter was the principal mechanism for maintenance of resting pH_i and for recovery from acid loads. Evidence for Na⁺/H⁺ exchange in these cells was based on the ion dependence of pH_i regulation in HCO₃^--free media. Thus an acidification was noted upon removal of external Na⁺, and acid extrusion was blocked in the absence of Na⁺, but could be supported by Li⁺. In HCO₃^--containing solutions, pH_i recovery from acid loads was slightly inhibited by the stilbene derivative DIDS and was Na⁺ dependent, suggesting that a form of Na⁺-linked HCO₃^- influx also contributed to net acid extrusion. Whether this mechanism was Cl^- dependent was not addressed.

Baseline pH_i of the fetal hippocampal neurons was not affected by removal of Cl^- or by application of DIDS, indicating that the fetal cells did not utilize a passive Cl^-/HCO₃^- exchanger as an acid-loading mechanism (250). This feature was confirmed in a later study of acutely isolated, fetal hippocampal neurons (251). In contrast, acutely isolated adult hippocampal neurons exhibited a pronounced alkalization upon removal of external Cl^-. In situ hybridization studies targeting the brain isoform of the Cl^-/HCO₃^- exchanger (see sect. VB3) were in agreement with these findings. Although the message could be identified in fetal brain (171, 251), there was a greater predominance in mature tissue (251).

Later studies of the acid extrusion mechanisms in fetal hippocampal neurons were undertaken by Baxter and Church (20). An Na⁺/H⁺ exchanger insensitive to ethylisopropylamiloride (EIPA) was noted, consistent with earlier data (250). However, in contrast to the previous studies, removal of Cl^- produced a DIDS-sensitive, Na⁺-independent alkalization, suggesting the presence of a passive Cl^-/HCO₃^- exchanger in the fetal-derived neuronal culture. Transition from HCO₃^--free (i.e., HEPES-buffered) to HCO₃^--containing solution induced a robust alkalization that was Na⁺ dependent and was blocked by DIDS, or by depletion of internal Cl^-. These observations suggested that fetal hippocampal neurons can extrude acid using both Na⁺/H⁺ exchange and an Na⁺-driven, Cl^-/HCO₃^- exchanger. The latter process appeared to play a greater role in pH_i regulation at room temperature. Thus transition from HEPES to HCO₃^- buffer induced an alkalization, or speeded recovery from acid loading, only at 18-22°C, and application of DIDS caused a fall in pH_i at this low temperature, but not at 37°C. At the warmer, physiological temperature, acid extrusion appeared to be dominated by Na⁺/H⁺ exchange.

Noncultured hippocampal neurons were investigated by Schwiening and Boron (273). These studies utilized a specific population of pyramidal neurons, acutely isolated from the hippocampal CA1 region of 4- to 14-day-old rats. In the absence of HCO₃^-, the cells exhibited a low baseline pH_i (a mean of 6.81) and sluggish recovery from acid loads. In agreement with previous studies of fetal, hippocampal neurons (250), this pH_i recovery was inhibited by removal of Na⁺ (suggesting the presence of Na⁺/H⁺ exchange) and showed little sensitivity to amiloride. Unlike fetal hippocampal cultures, where Na⁺/H⁺ exchange was predominant (20), this mechanism played a minor role in acutely dissociated pyramidal neurons (in both the establishment of baseline pH_i and the extrusion of acid at 37°C). With addition of external HCO₃^-, a marked increase in baseline pH_i occurred. This HCO₃^--dependent acid extrusion required external Na⁺ as well as internal Cl^- and was sensitive to DIDS, indicating the operation of an Na⁺-driven Cl^-/HCO₃^- exchanger. It was notable that the demonstration of Cl^- dependence could not be achieved by incubation in Cl^--free media for up to 3 h. To adequately deplete intracellular Cl^-, repeated activation of the transporter in Cl^--free solution was required. These observations urge caution in the interpretation of experiments in which exposure to Cl^--free solution fails to alter pH_i regulation.

A study of pH_i regulation in both postnatal immature and mature CA1 neurons from rat was undertaken by Bevensee et al. (24). The basic properties of pH_i regulation were similar in the two age groups and featured an EIPA-insensitive Na⁺/H⁺ exchanger and a robust stimulation of acid extrusion by HCO₃^-, attributed to Na⁺-driven Cl^-/HCO₃^- exchange. A fraction of neurons exhibited a slow recovery from acid loads in the absence of external Na⁺, suggesting the presence of an H⁺ pump. Similar Na⁺-independent pH_i recovery was later noted in CA1 neurons acutely isolated from mouse (358). The principal difference between immature and mature CA1 neurons was related to the distribution of baseline pH_i. Acutely dissociated neurons derived from both age groups were found to have a wide range of initial pH_i values (6.3-7.7) in HCO₃^--free, HEPES-buffered media. Mature neurons displayed a bimodal distribution, falling into low pH_i (mean of 6.68) and high pH_i (mean of 7.32) categories. For high pH_i neurons, the relationship between pH_i and the acid extrusion rate was shifted in the alkaline direction and had a greater slope, both in the absence and presence of HCO₃^-.

The presence of a predominant Na⁺/H⁺ exchanger with low sensitivity to amiloride distinguishes hippocampal neurons from the great majority of cells. The nature of this amiloride resistance and its relationship to transporter isoforms remains unclear (see sect. VA). It is notable that acutely isolated CA1 neurons from mice displayed an Na⁺/H⁺ exchanger inhibited by the agent HOE 694 (358). However, the Na⁺/H⁺ exchanger of cultured fetal hippocampal neurons was insensitive to this drug (20).

B. Neurons From Cerebral Cortex

The regulation of pH_i in cortical neurons has been studied in cultured fetal cells obtained from rat (236) and mouse (245). In cultured fetal rat neurons, recovery from intracellular acid loads was partially inhibited by amiloride (or 5-N-methyl-N-isobutylamiloride) and was dependent on external Na⁺, indicating the operation of an Na⁺/H⁺ exchanger (236) [inhibition of acid extrusion by amiloride was also noted by Amos and Richards (8) in cultures of rat neocortical neurons]. Ou-Yang et al. (236) suggested the absence of HCO₃^--dependent acid extrusion in fetal neurons, based on the following: 1) the similarity of acid extrusion rates in HCO₃^--free and HCO₃^--containing solutions, 2) the failure of DIDS or Cl^- removal to inhibit recovery from acid loading, and 3) similar acid extrusion rates in the presence of amiloride versus amiloride plus DIDS. In contrast, recovery from alkaline loads was inhibited by DIDS and by the absence of Cl^-, suggesting the presence of a passive Cl^-/HCO₃^- exchanger.

The putative absence of HCO₃^--dependent acid extrusion in the cortical neurons studied by Ou-yang et al. (236) warrants comment. Although calculated acid efflux rates were similar in the presence and absence of HCO₃^-, analysis was highly dependent on accurate determination of the _i. It should also be noted that the method of acid loading in these studies was unorthodox, consisting of a transition from 5 to 15% CO₂ with compensatory increases in solution HCO₃^- (and decrease in Cl^-) to maintain a constant bath pH. Given the presence of a Cl^-/HCO₃^- exchanger in these cells, the changes in HCO₃^- and Cl^- could have confounded interpretation of the pH_i recoveries. This study also reported that addition of DIDS to amiloride-containing solution did not further reduce acid extrusion. How this experiment was carried out is uncertain, as a precipitate commonly forms when DIDS is combined with amiloride or one of its derivatives (unpublished observation). It is worth noting that like hippocampal neurons (273), the steady-state pH_i of the cultured cortical cells was higher in HCO₃^--containing media, which would be consistent with the presence of a HCO₃^--dependent acid extrusion mechanism.

In a later study of cultured fetal neurons from mouse cortex, it was suggested that cells utilized both Na⁺/H⁺ exchange and an HCO₃^--dependent acid extruder (245). After acid loading with the NH₄⁺ prepulse method (38) in HCO₃^--containing media, the pH_i recovery was markedly reduced by EIPA, indicating the operation of an Na⁺/H⁺ exchanger. The rates of pH_i recovery in the presence and absence of HCO₃^- (and during exposure to DIDS) were consistent with a HCO₃^--dependent component of acid efflux. However, the dpH_i/dt values were not converted to acid extrusion rates (Eq. 5), and therefore the relative contribution of Na⁺/H⁺ exchange versus HCO₃^--dependent processes remained unclear. The authors suggested that recovery from alkaline loads occurred by a DIDS-insensitive Cl^-/HCO₃^- exchanger. In addition, a passive H⁺ influx through voltage-gated H⁺ channels was proposed, since exposure to 1 mM Zn²⁺ slowed the recovery from alkaline loads. Such channels, however, would not be open near the resting membrane potential, as they typically activate near 0 mV. Moreover, there is no compelling evidence for their presence in vertebrate neurons (92).

C. Neurons From Cerebellum

Two studies of nerve cells cultured from rat cerebellum have been performed. Gaillard and Dupont (113) investigated Purkinje cells from neonatal (P0-P1) rats after 6-7 days in culture. In HCO₃^--free media, recovery from acid loading was abolished by withdrawal of external Na⁺ or by exposure to amiloride, indicating the operation of an Na⁺/H⁺ exchanger. In media containing HCO₃^-, recovery from acid loading was also completely blocked by amiloride, which suggested the absence of HCO₃^--dependent acid extrusion. The presence of passive Cl^-/HCO₃^- exchange was indicated by the presence of a DIDS-sensitive, Na⁺-independent alkalization upon removal of external Cl^-. The steady-state pH_i of these cells was consistent with this complement of transporters. Unlike hippocampal (273) and cortical neurons (236), the steady-state pH_i of the cultured Purkinje cells was lower in HCO₃^--containing (7.06) versus HCO₃^--free media (7.40). This finding is consistent with steady-state acid loading via Cl^-/HCO₃^- exchange and the absence of HCO₃^--dependent acid extrusion.

Studies of rat cerebellar granule cells (cultured from 7-day-old pups) revealed a similar behavior of steady-state pH_i (248). In HCO₃^--containing solution, pH_i was lower (7.27) compared with HCO₃^--free media (7.49). Although completely dependent on external Na⁺, the recovery of pH_i from acid loads was only partially blocked by amiloride. In roughly half the cells, this recovery exhibited a dependence on HCO₃^- (but was not blocked by stilbenes). Recovery from acid loading persisted after prolonged exposure to Cl^--free media, suggesting the possible operation of Na⁺-HCO₃^- cotransport in these cells. However, as noted by Schwiening and Boron (273), internal Cl^- is not readily depleted from neurons and may require repetitive acid loading in Cl^--free solution to be sufficiently eliminated from the cytoplasm. The presence of a passive Cl^-/HCO₃^- exchanger in a fraction of the granule cells was suggested by the observation of a reversible alkalization upon withdrawal of external Cl^- (however, this effect was not blocked by stilbenes). The lower steady-state pH_i noted in HCO₃^- media was consistent with the presence of a Cl^-/HCO₃^- exchanger that contributed to steady-state acid loading.

D. Neurons From Spinal Cord

Recently, pH_i regulation was studied in neurons cultured from ventral spinal cord of 14- to 15-day rat embryos (46). In HCO₃^--free media, acid extrusion was mediated by amiloride-sensitive Na⁺/H⁺ exchange. In the presence of HCO₃^-, recovery from acid loading was also significantly slowed by 1 mM amiloride (but was not blocked by 40 µM EIPA). Steady-state pH_i was reduced by DIDS, suggesting that a HCO₃^--dependent acid extruder contributed to the maintenance of baseline pH_i. Recovery from acid loads in HCO₃^--containing media was slowed by DIDS and abolished in Na⁺-free solutions. It was suggested that this recovery was mediated by an electrogenic Na⁺-HCO₃^- cotransporter, since a reversible alkalization was observed when external K⁺ was raised to 30 mM. Whether this alkalization was dependent on Na⁺ or HCO₃^- or could be evoked by other means of depolarization was not addressed, however. Thus experiments to more fully distinguish between Na⁺-driven Cl^-/HCO₃^- exchange and electrogenic Na⁺-HCO₃^- cotransport appear warranted. Removal of external Cl^- was found to elicit a reversible alkaline shift in the spinal cord neurons, suggesting the operation of a passive Cl^-/HCO₃^- exchanger. Baseline pH was lower in HCO₃^--containing media compared with HCO₃^--free, HEPES-buffered solution, consistent with steady-state acid loading via this mechanism.

E. Medullary Neurons

The neurons of the medulla oblongata are heterogeneous in form and function. Of particular interest in this region are the cells of the chemosensitive areas, which depolarize and fire in response to elevation of CO₂ or H⁺. The degree to which this response is triggered by a fall in pH_i, a fall in extracellular pH (pH_o), or elevation of CO₂ per se has been the subject of debate (218). Several recent studies have suggested that a fall in pH_i is the principal trigger for the chemosensitive response (110a, 342, 348). For such cells, therefore, the control of pH_i is a subject of great interest. The regulation of pH_i in chemosensitive neurons has recently been reviewed (249).

Putnam and colleagues (256-258) imaged pH_i from neurons of rat neonatal, medullary brain slices which had been incubated in BCECF. Neurons from two chemosensitive areas, nucleus of the solitary tract (NTS) and ventrolateral medulla (VLM), were compared with cells from the nonchemosensitive inferior olive (IO) and hypoglossal nucleus (HYP). In all cells, recovery from acid loading was completely abolished by amiloride but was insensitive to DIDS, suggesting that brain stem neurons utilize Na⁺/H⁺ exchange but not HCO₃^--dependent acid extrusion (257). In chemosensitive neurons, elevation of CO₂ induced a fall in pH_i that recovered only when bath pH was maintained at 7.48 (by simultaneous elevation of external HCO₃^- to 52 mM). In contrast, neurons from nonchemosensitive areas recovered when CO₂ was elevated, despite a fall in bath pH to 7.15 (at constant bath HCO₃^- of 26 mM). This difference was attributed to an Na⁺/H⁺ exchange mechanism in the chemosensitive neurons that was more readily inhibited by external H⁺ (256). Immunocytochemical and pharmacological data from chemosensitive neurons in organotypic culture have implicated the type 3 Na⁺/H⁺ exchanger isoform in these cells (348) (i.e., NHE-3; see sect. VA).

The presence of Cl^-/HCO₃^- exchange was suggested in VLM, HYP, and IO neurons, as a DIDS-sensitive alkalization was noted upon withdrawal of external Cl^-. This exchanger could mediate recovery from alkaline loads in neurons from all three of these regions; however, the Cl^-/HCO₃^- exchanger in VLM cells was inhibited by high bath pH (7.9), unlike the nonchemosensitive cells. In contrast, there was no apparent Cl^-/HCO₃^- exchanger in the chemosensitive NTS neurons, which accordingly, could not recover from alkaline loading (256).

F. Retinal Neurons and Photoreceptors

Early evidence for acid extrusion via Na⁺/H⁺ exchange was provided by Oakley and colleagues (160, 231) in studies of rod photoreceptors from toad. A characterization of pH_i regulatory mechanisms was later carried out in rods from frog (155) and salamander retina (264). Both amphibian rods utilized an amiloride-sensitive Na⁺/H⁺ exchanger. In addition, Na⁺-dependent acid extrusion was accelerated by HCO₃^- and inhibited by DIDS, suggesting the presence of an Na⁺-driven HCO₃^- transporter. Whether this mechanism was linked to Cl^- was not clear. Evidence for passive Cl^-/HCO₃^- exchange was found in both frog and salamander rods, as the withdrawal of Cl^- produced a rise in pH_i, which persisted in the absence of external Na⁺ in the case of frog rods (155). A study of retinal horizontal neurons from the skate also demonstrated the presence of amiloride-sensitive Na⁺/H⁺ exchange, and apparent HCO₃^--dependent acid extrusion, based on the inhibition of acid extrusion by DIDS (135).

Removal of external Cl^- had no effect on pH_i in the horizontal cells, suggesting that these neurons did not possess a passive Cl^-/HCO₃^- exchanger.

G. Sympathetic Neurons

One of the first studies of pH_i regulation in mammalian nerve cells utilized cultured rat sympathetic neurons (306). Acid extrusion was dependent on external Na⁺ and was inhibited by amiloride, providing clear evidence for Na⁺/H⁺ exchange. The role of HCO₃^--dependent acid extrusion in these cultured neurons was less certain. While addition of stilbenes did not affect recovery from acid loads, in some cases, pH_i recovery was stimulated by addition of 5-10 mM HCO₃^-.

H. Neoplastic Neural Cells

Neuroblastoma cells were among the first neural preparations in which Na⁺/H⁺ exchange was identified (21, 212). Acid extrusion via Na⁺/H⁺ exchange was subsequently reported in other studies of neural tumor cells (111, 204). Dickens et al. (100), studying neuroblastoma and pheochromocytoma (PC12) cells, noted the presence of Na⁺/H⁺ as well as Cl^-/HCO₃^- exchange. This paper also reported regional heterogeneity of pH_i, as the tips of extending neurites were more alkaline (by 0.2-0.3 pH units) than the cell body.

I. Synaptosomes

There is wide consensus that acid extrusion from brain synaptosomes is mediated by amiloride-sensitive Na⁺/H⁺ exchange (146, 216, 265, 268). It has been reported that there is no HCO₃^--dependent acid extrusion in synaptosomes (216, 265). This finding was based on similar rates of recovery from acidosis in the presence and absence of HCO₃^- and lack of sensitivity to stilbene derivatives. However, an identical dpH/dt in the presence of HCO₃^- would imply a greater rate of acid efflux, since intracellular buffering capacity should be elevated in the presence of CO₂ and HCO₃^-. Therefore, the possibility of HCO₃^--dependent acid extrusion in these preparations cannot be excluded. A rise in pH_i was noted in synaptosomes exposed to elevated external K⁺ (265) [although in an earlier study (254), elevated external K⁺ did not produce a similar rise in pH_i]. This apparent depolarization-induced alkalization (DIA) persisted in the absence of HCO₃^- and Na⁺, suggesting that it was not attributable to electrogenic Na⁺-HCO₃^- cotransport. The cause of the DIA was not elucidated.

Synaptosomal acid transport linked to a Cl^- flux was suggested by one study in which withdrawal of external Cl^- caused a DIDS-sensitive rise in pH_i. This alkalization, however, was not dependent on HCO₃^-, suggesting the presence of a Cl^--H⁺ cotransport mechanism (195). A similar putative Cl^--H⁺ cotransporter (or equivalently, a Cl^-/OH^- exchanger) has been noted in cardiac myocytes (183).

	IV. REGULATION OF INTRACELLULAR pH IN GLIAL CELLS

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The pH regulatory mechanisms of glial cells are comprised of transport systems very similar to those found in neurons. Physiological studies suggest that glia differ from neurons and from one another in their expression of Na⁺-linked bicarbonate transporters. Electrogenic Na⁺-HCO₃^- cotransporters appear to be especially prevalent among glial cells. Details elaborated below are summarized for glial cells according to cell type in Table 3.

A. pH Regulation in Nonmammalian Glial Cells

The first detailed investigations of glial pH_i were conducted on invertebrate cells which could withstand long-term penetration with pH-sensitive microelectrodes. Pioneering work on the giant glial cells of the leech demonstrated the presence of at least three acid extrusion mechanisms: an Na⁺/H⁺ exchanger, an Na⁺-driven Cl^-/HCO₃^- exchanger, and an electrogenic Na⁺-HCO₃^- co-transporter (95, 96, 98, 293). Later work indicated the additional presence of a passive Cl^-/HCO₃^- exchanger in these cells (294). Evidence for Na⁺/H⁺ exchange (11) and electrogenic Na⁺-HCO₃^- cotransport (12) was also reported for glial cells of the mudpuppy optic nerve. This work is well covered by the review of Deitmer and Rose (94).

B. pH Regulation in Mammalian Astrocytes

Studies of intracellular pH in mammalian glia have focused mainly on astrocytes. With a few exceptions, these studies have made use of pH-sensitive fluorescent dyes rather than pH microelectrodes. Almost all investigations have been performed on cultured astrocytes. Generally, the mechanisms of acid transport appear similar to those found in the invertebrate glia; however, some notable differences exist among mammalian astrocyte studies.

1. Principal acid extrusion mechanisms in mammalian astrocytes

The presence of Na⁺/H⁺ exchange in mammalian astrocytes is well established and uniformly supported. Kimelberg and colleagues (163, 166) provided early evidence for Na⁺/H⁺ exchange in primary astrocyte cultures based on detection of Na⁺-dependent extracellular pH shifts. The use of amiloride or its derivatives to acidify baseline pH_i or inhibit pH_i recovery from acid loads served as the principal evidence for Na⁺/H⁺ exchange in later astrocyte studies (26, 53, 87, 205, 247, 277). The presence of an Na⁺/H⁺ exchanger in mammalian astrocytes has also been confirmed by immunocytochemical studies which revealed the presence of the amiloride-sensitive NHE-1 isoform of this transporter (247) (see sect. VA).

In some studies, the amiloride analog EIPA appeared ineffective against the astrocyte Na⁺/H⁺ exchanger (44) or caused a paradoxical increase in pH_i (45, 247). Bevensee et al. (26) reported that while amiloride and EIPA could inhibit acid extrusion with similar efficacy at low pH_i, EIPA appeared less effective near baseline pH_i. Moreover, a fall in pH_i was consistently produced by amiloride but not by EIPA. The basis of these observations remains unclear.

Inhibition of Na⁺/H⁺ exchange by low external pH was described by Aronson et al. (10) in their studies of the renal transporter. A similar property was noted by Mellergard and Siesjo (205) who found that cultured astrocytes failed to extrude acid in bicarbonate-free media when the external pH was <6.9. This feature has significant implications for the role of astrocyte Na⁺/H⁺ exchange in normal versus pathological settings. While culture studies show a significant contribution of this transporter when astrocyte pH_i approaches 6.0 (26), such a low cytosolic pH would probably only arise in vivo under ischemic conditions, where it would be accompanied by a prominent fall in pH_o, in the range of 6.2-6.9 (174-176, 215, 220, 332). Given the inhibition of this transporter by external H⁺, it seems that the astrocyte Na⁺/H⁺ exchanger would never operate within the pH_i range that caused its maximum activation in tissue culture. In fact, under conditions which simulated the external ion shifts, acidosis and hypoxia of ischemic brain, the pH_i of cultured astrocytes fell to 6.7-6.8 and exhibited little or no recovery of pH_i over a 20- to 30-min period (28).

Virtually all studies agree on the presence of Na⁺-and HCO₃^--linked acid transport in astrocytes. Differences exist regarding the role of Na⁺-driven Cl^-/HCO₃^- exchange and electrogenic Na⁺-HCO₃^- cotransport. Using mouse cortical astrocytes, Chow et al. (87) reported that recovery from acid loads was not Cl^- dependent, and therefore suggested that a form of Na⁺-HCO₃^- cotransport was involved. In contrast, Mellergard et al. (203) noted diminished acid extrusion after withdrawal of Cl^- and suggested that their cultured rat cortical astrocytes utilized Na⁺-driven Cl^-/HCO₃^- exchange, but not Na⁺-HCO₃^- cotransport. Curiously, they reported a DIA, a phenomenon typically associated with an electrogenic Na⁺-HCO₃^- cotransporter (see below). Shrode and Putnam (277) provided strong support for both Na⁺-driven Cl^-/HCO₃^- exchange and electrogenic Na⁺-HCO₃^- cotransport in their cultured rat cortical astrocytes. A later study of cultured cerebellar astrocytes also found evidence for both exchangers (167). A detailed examination of pH_i regulation in cultured rat hippocampal astrocytes was conducted by Bevensee et al. (23, 26). Using a fluorescent Cl^--sensitive dye to confirm the depletion of cytosolic Cl^-, these authors were able to convincingly demonstrate that HCO₃^--linked acid extrusion did not depend upon Cl^- and, accordingly, was attributed to Na⁺-HCO₃^- cotransport.

Differences in the identification of acid extrusion mechanisms among astrocytes could be attributed to a number of factors. Discrepancies may arise due to use of low Cl^- media to test for Cl^--dependent transport. Unless depletion of internal Cl^- can be assured, failure to inhibit acid transport after Cl^- removal cannot be considered convincing (whereas inhibition of transport after Cl^- withdrawal clearly supports Cl^- dependence) (277). Differences in data may also originate from altered expression of transporters, arising from varied culture conditions, species of origin (e.g., mouse vs. rat) or brain region (e.g., cortex, vs. cerebellum vs. hippocampus).

2. The astrocyte Na⁺-HCO₃^- cotransporter

The Na⁺-HCO₃^- cotransporter of astrocytes has received considerable attention due to its electrogenic character and the resulting dependence on membrane potential. A form of this transport mechanism was first described in renal proximal tubule (37), and with the exception of one study (203), its expression in mammalian astrocytes is agreed upon. Among glia, the presence of this transporter was first reported in invertebrates, where Na⁺-HCO₃^--dependent, electrogenic acid extrusion (13, 96), and a membrane potential-driven shifts in pH_i were described (98).

The first clue to the presence of this transporter in mammalian astrocytes came from the observation of a depolarization-induced alkalization (DIA) in rat cortical astrocytes studied in vivo. During stimulation of surrounding cortex, a rapid rise in astrocyte pH_i was correlated with a depolarization of the astrocyte membrane, attributed to elevation of extracellular K⁺ concentration ([K⁺]_o) (81). Addition of Ba²⁺ prevented both the activity-evoked depolarization (17) and the cytosolic alkalization, despite a similar evoked rise in [K⁺]_o (82).

Data consistent with electrogenic Na⁺-HCO₃^- cotransport was subsequently reported in cultured astrocytes (23, 50, 53, 232, 277), reactive astrocytes in gliotic brain slices (128, 129), and acutely isolated astrocytes from retina (221, 223, 224). This body of evidence is formed by two related observations. The first is the occurrence of a hyperpolarization (or an outward current) upon transition from bicarbonate-free to bicarbonate-containing media, which may be inhibited by DIDS or related stilbene derivatives (53, 221, 224, 232). A potential diffi-culty with this criterion is that the presence of a bicarbonate conductance could give rise to a similar shift in membrane potential (or membrane current under voltage clamp). In a novel, alternative approach, Bevensee et al. (23) withdrew external sodium to reverse the transporter and demonstrated a stilbene-inhibited depolarization.

The second observation favoring electrogenic Na⁺-HCO₃^- cotransport has been the presence of a DIA in astrocytes. This rise in pH_i is typically Na⁺ and HCO₃^- dependent and is often blocked by stilbenes (50, 129, 222, 223, 240). In gliotic brain slices, however, an Na⁺-independent component of the DIA and an insensitivity to stilbenes were noted in reactive astrocytes (129).

The dependence of the transport direction upon voltage is governed by the difference between membrane potential and the equilibrium potential for a Na⁺-HCO₃^- cotransporter (E_NBC). The general relation for E_NBC is given by

(6)

where n is the net HCO₃^--Na⁺ transport ratio (37, 96). The preponderance of evidence suggests that the astrocyte mechanism has a stoichiometry of 2:1 (23, 50, 232) and that the net change in transport elicited by membrane depolarization is an influx of Na⁺ and HCO₃^-.

For most astrocytes, values of E_NBC would be on the order of -80 mV (76), and given a membrane potential of similar magnitude, the transporter would normally be close to equilibrium. Some astrocytes have membrane potentials of -50 mV or less, however (32, 287). For these cells, the energetics of transport may favor a large, steady-state influx of Na⁺ and HCO₃^- (provided E_NBC was maintained at a more negative value). However, little is known about how E_NBC or transporter stoichiometry varies among astrocyte subtypes.

A notable exception comes from the work of Newman (221, 222, 224) who investigated Na⁺-HCO₃^- transport in acutely dissociated Muller cells of the salamander retina. These specialized glia are sometimes considered to be astrocyte variants due to their expression of glial fibrillary acidic protein (27). Muller cells were found to have a transporter with a reversal potential near 0 mV, consistent with a stoichiometry of one Na⁺ per three HCO₃^-. As such, the energetics would favor the efflux of these ions at normal negative membrane potentials. Physiological studies found the transporter predominantly localized to the distal end foot process which normally abuts the vitreous humor. This led to the suggestion that the transporter is specialized to shuttle metabolically produced CO₂/HCO₃^- out of the retina (221). In later studies, evidence for Na⁺-HCO₃^- cotransport was also found in Muller cells and astrocytes of rat retina (223).

Numerous questions about localization, function, and role of astrocytic Na⁺-HCO₃^- cotransporters remain unsettled. One issue is whether a DIA can always be completely attributed to this mechanism, since this alkalizing response, albeit diminished, can sometimes occur in HCO₃^--free media (42, 44, 50, 98, 204, 239). These observations might be explained by a mechanism with a high affinity for HCO₃^-, capable of utilizing the small concentrations of this ion generated from metabolically derived CO₂ (97). Whether this carrier actually utilizes HCO₃^- or CO₃^2- in the transport mechanism has not been settled, however (see sect. VIIB6).

From the functional standpoint, Na⁺-HCO₃^- cotrans-port appears capable of net acid extrusion in response to cytosolic acidification, as well as the modulation of transmembrane pH, in response to changes in membrane potential. The latter property may be a normal and unique feature of astrocytes, since neural activity rapidly depolarizes glial cells as a result of [K⁺]_o elevation (132, 161, 234, 253, 284). The astrocyte Na⁺-HCO₃^- cotransporter may therefore play a role in the modulation of both intracellular and interstitial pH (see sects. VIB and VIIB6).

3. Na⁺-independent acid extrusion in astrocytes

While the ability of astrocytes to extrude acid loads appears to be mainly due to a combination of Na⁺/H⁺ exchange and Na⁺-linked HCO₃^- transport, reports of Na⁺-independent transport have appeared. Recording with pH microelectrodes from cultured mouse astrocytes in HCO₃^--free media, Walz (353) noted a recovery from acute acid loads that was unaffected by either amiloride or withdrawal of external Na⁺. This result stands in marked contrast to a large body of evidence indicating that amiloride-sensitive Na⁺/H⁺ exchange is the principal acid extrusion mechanism of astrocytes in the absence of HCO₃^- (see above). However, it may be noted that penetration of the mouse astrocytes with microelectrodes was made possible after rounding the cells by several hours of exposure to 1 mM dibutyryl cAMP (339). In response to this treatment, it is plausible that changes in transporter expression occurred. Downregulation of the Na⁺/H⁺ exchanger and the expression of a plasmalemmal H⁺-ATPase could account for these results.

Pappas and Ransom (239) were able to uncover a plasmalemmal vacuolar H⁺-ATPase in cultured rat hippocampal astrocytes that made a small contribution to acid extrusion. This transporter was identified in HCO₃^--free media by a small, bafilomycin-sensitive component of acid extrusion that remained in the absence of external Na⁺ or the presence of amiloride. In agreement with these findings, V-type ATPase mRNA has been isolated from cultured astrocytes (246). However, in view of the small component of acid extrusion attributed to this mechanism, its normal function is unclear. In the hippocampal astrocyte study, addition of bafilomycin caused a small intracellular acidification, suggesting a role in the maintenance of steady-state pH_i (239). In cultured cortical astrocytes, however, the H⁺-ATPase inhibitors bafilomycin and concanamycin did not affect baseline pH_i (5).

4. Cl^-/HCO₃^- exchange in astrocytes

The presence of a Cl^-/HCO₃^- exchanger in astrocytes was suggested in early studies of astrocyte Cl^- fluxes by Kimelberg et al. (163). In most subsequent reports, the expression of this transporter was noted by the observation of a cytosolic alkalization upon removal of external Cl^-. This alkalosis was reported in cerebellar (53) and cortical astrocytes (203), where it was inhibited by DIDS. In contrast, in hippocampal astrocytes, withdrawal of Cl^- did not alter pH_i notably, and it was suggested that this transporter was not prominent in these cells (23). Shrode and Putnam (277) cautioned that alkalization upon withdrawal of external Cl^- could be mediated by reversal of a Na⁺-coupled Cl^-/HCO₃^- exchanger. To convincingly demonstrate the presence of passive Cl^-/HCO₃^- exchange in cortical astrocytes, these investigators showed that removal of external Cl^- produced a DIDS-sensitive intracellular alkalization that persisted in the absence of Na⁺. This process appeared to be active only at an elevated baseline pH_i, suggesting a role in the recovery from alkaline loads.

C. pH Regulation in Oligodendroglia

A detailed study of pH_i regulation in oligodendrocytes from mouse spinal cord was conducted by Kettenmann and Schlue (162). These cells displayed a high baseline pH_i, ranging from 7.2-7.9 in HCO₃^--containing media of pH 7.4. In the absence of HCO₃^-, pH_i was lower, and recovery from acid loading occurred via a typical amiloride-sensitive Na⁺/H⁺ exchange mechanism. In HCO₃^--containing media, an additional acid extruder was identified that was Na⁺ dependent and apparently not linked to Cl^-. The observation of a DIA (upon elevation of [K⁺]_o) suggested that this mechanism could be an electrogenic Na⁺-HCO₃^- cotransporter; however, this process was unaffected by stilbenes or furosemide. The presence of a Cl^-/HCO₃^- exchanger in these oligodendrocytes was uncertain. Removal of external Cl^- resulted in a higher baseline pH_i, consistent with passive Cl^-/HCO₃^- exchange; however, the removal of HCO₃^- had no complementary effect on Cl^- transport (138).

Further studies of oligodendroglia were carried out by Gaillard and colleagues (41, 42) using cultured cerebellar cells. In mature oligodendrocytes, baseline pH_i was 7.04 in HCO₃^--containing media (a value considerably lower than that of cultured spinal oligodendrocytes) and was unaffected by stilbenes or Cl^- removal, suggesting the absence of a Cl^-/HCO₃^- exchanger. In oligodendrocyte precursors, however, a DIDS-sensitive, passive Cl^-/HCO₃^- exchanger was identified. Steady-state acid loading by Cl^-/HCO₃^- exchange accounted for a lower baseline pH_i of the precursor cells, which averaged 6.88 (41). The differences in pH_i noted during oligodendrocyte development may be related to the events leading to termination of cell division, as has been suggested for rat astrocytes (241).

In HCO₃^--free media, both mature cerebellar oligodendrocytes and precursor cells extruded acid via an amiloride-sensitive Na⁺/H⁺ exchanger (41, 42). In the presence of HCO₃^-, an electrogenic Na⁺-HCO₃^- cotransporter was also identified. This transporter was unaffected by DIDS as was noted earlier for spinal cord oligodendrocytes (162). Analysis of the rate of alkalization and the calculated HCO₃^- influx induced by graded elevations of external K⁺ suggested a reversal potential close to -60 mV, consistent with an Na⁺-HCO₃^- transport stoichiometry of 1:3 (42). Since the resting potential of these cells was also near -60 mV, it was suggested that this transporter was normally close to equilibrium.

D. pH Regulation in Schwann Cells

Primary cultures of Schwann cells from rat sciatic nerve were studied by Nakhoul et al. (217). In addition to a classic, amiloride-sensitive Na⁺/H⁺ exchanger, these cells exhibited a passive Cl^-/HCO₃^- exchanger, revealed by the presence of an Na⁺-independent alkalization upon removal of external Cl^-. The Cl^-/HCO₃^- exchanger of the Schwann cells was unaffected by DIDS. These experiments did not address whether an Na⁺-linked HCO₃^--dependent acid extrusion mechanism was present.

A role for pH_i in the proliferation of Schwann cells was suggested in an early study in which mitogenic stimulation resulted in an apparent rise in pH_i (267). This alkaline shift was associated with the Na⁺/H⁺ exchanger. Inhibition of Na⁺/H⁺ exchange after addition of a mitogen significantly reduced the degree of subsequent mitosis.

E. pH Regulation in Glial Tumor Cells

The regulation of pH_i was studied in C6 glioma cells by Shrode and Putnam (277) in conjunction with their investigation of rat cortical astrocytes. The glioma cells shared two acid extrusion mechanisms with the astrocytes: a conventional, amiloride-sensitive Na⁺/H⁺ exchanger and a DIDS-sensitive, Na⁺-driven Cl^-/HCO₃^- exchanger. Unlike the astrocytes, the glioma cells did not have an electrogenic Na⁺-HCO₃^- cotransporter, as they displayed no depolarization-induced alkalization upon elevation of external K⁺. A second study of C6 glioma cells (utilizing NMR spectroscopy) also presented evidence for Na⁺/H⁺ exchange and HCO₃^- transport (111). A comparison of pH_i regulation in glioma cell lines (C6 and human U-251, U-118, and U-87) versus primary cortical astrocyte cultures (199) reported a higher steady-state pH_i in the gliomas (also noted by Shrode and Putnam for C6 cells) (277) and a higher rate of recovery from acid loads. These effects were attributed to enhanced activity of the Na⁺/H⁺ exchanger in the glioma cell lines, which was not attributed to genetic alterations. Disparity in the efficacy or expression of the HCO₃^--dependent acid transporters was also noted, as the astrocytes rapidly alkalinized upon introduction of CO₂-HCO₃^-, whereas the glioma cells underwent a marked acidification.

A study by Volk et al. (331) provided evidence for a vacuolar H⁺-ATPase in C6 glioma cells, based on a fall in pH_i observed after application of H⁺-ATPase inhibitors. A voltage-clamp study of bafilomycin-sensitive currents from C6 glioma cells provided additional data supporting the presence of an electrogenic plasmalemmal H⁺-ATPase in these cells (246). In a comparison of vacuolar ATPase mRNA in C6 cells, primary astrocyte cultures, and immortalized astrocytes, no differences in levels were noted (246). However, an equivalence in H⁺-ATPase message may not imply similar activity of the transporter in glioma cells and astrocytes. Indeed, significant differences between these cells have been noted in this regard. For example, in the study of Volk et al. (331), the pronounced fall in the steady-state pH_i during application of H⁺-ATPase inhibitors suggested a particularly prominent role for this mechanism in C6 cells. In contrast, H⁺-ATPase inhibitors had little or no effect on baseline pH_i in cultured astrocytes (5, 239).

F. pH Regulation and Volume Control in Glia

The activation of acid transport mechanisms can lead to a net transmembrane flux of osmoles, resulting in changes in cell volume. Linkages between volume regulation of glial cells and the regulation of cytosolic pH can therefore be anticipated. For example, activation of Na⁺/H⁺ exchange can lead to glial swelling, owing to the accumulation of Na⁺ (143a). In addition, the regulatory response to changes in osmolarity involving taurine transport can have immediate, direct effects on pH_i (234a). Due to the role of glial swelling in clinical brain edema, this subject has received considerable attention in literature beyond the scope of the present review. For an overview of this topic, the contribution of Kempski et al. (161a) may be consulted.

	V. MOLECULAR IDENTIFICATION OF BRAIN ACID TRANSPORTERS

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Molecular correlates of several acid transport mechanisms have been identified in the mammalian CNS. Functional characterization in expression systems has served to confirm the physiological identity of these transporters. Localization of particular mRNAs and immunostaining of specific proteins have provided insights into possible regional functions of these molecules. Details provided below are summarized in Table 4.

A. Na⁺/H⁺ Exchangers in Brain

The gene family of Na⁺/H⁺ exchangers identified in vertebrate tissues consists of at least seven members, commonly denoted NHE-1 through NHE-7. Deduced amino acid sequences have indicated similarity in membrane-spanning domains and distinct putative regulatory domains in the cytosolic regions (54, 226, 336). The first member cloned was the human growth factor-activated NHE-1 (266), an abundant isoform thought to play a housekeeping role in maintenance of steady-state pH_i.

NHE-1 mRNA is widespread in the mammalian CNS (191, 235). Immunocytochemical analysis has also identified the protein in assays of whole tissue (105, 247) and astrocyte cultures (247). Transcripts, localized by Northern analysis and in situ hybridization, revealed a marked presence in hippocampus, periamygdaloid cortex, and cerebellum (191). A general increase in message (191) and protein (105) was noted in postnatal cortex. This observation may account for the lower resting pH_i and the slower recovery from acid loading reported in fetal versus adult hippocampal neurons (24). However, NHE-1 is typically sensitive to amiloride (or its analogs) (226), a feature at odds with its predominance in hippocampus, where neuronal Na⁺/H⁺ exchange has little or no sensitivity to these agents (250, 273). It is undetermined whether this characteristic of CA1 pyramidal neurons is due to cell-specific alterations of NHE-1 or the presence of an alternative NHE isoform.

mRNA for other NHE isoforms has been reported in the mammalian CNS. NHE-2 message was noted in whole brain (343), and later localized to cerebral cortex and brain stem (105, 191), where the mRNA level was apparently one-tenth that of NHE-1 (191). Message for NHE-3, however, was limited to Purkinje cells of the cerebellum (191) (but has been noted in medullary chemosensitive neurons in organotypic cultures, Ref. 348).

An in situ hybridization study of NHE-4 reported a predominance of this isoform in hippocampus (30). Compared with NHE-1, -2 and -3, the sensitivity of NHE-4 to amiloride was low. NHE-4 may therefore appear to be a candidate for the neuronal transporter in hippocampus. However, concentrations of amiloride that had no effect on acid extrusion in hippocampal neurons (250, 273) could inhibit NHE-4 activity by 50-60% when expressed in fibroblasts (30). Moreover, NHE-4 could not be activated by acid loading under isosmolar conditions. An increase in acid extrusion required hyperosmolar media, suggesting its activation was linked to changes in cytoskeletal elements brought about by cell shrinkage (31). If NHE-4 displayed similar properties in hippocampal neurons, it could not be responsible for the normal extrusion of H⁺ by these cells. Later studies raise additional questions about the role of NHE-4 in hippocampus, as it was found to be principally located in cerebral cortex and brain stem-diencephalon (105, 191).

Cloning and expression of the NHE-5 isoform in PS120 cells revealed a functional Na⁺/H⁺ exchanger with a sensitivity to EIPA. In situ hybridization demonstrated strong localization to the hippocampal dentate gyrus, with lower levels noted in the CA1 fields and cerebral cortex (14). In separate studies, cloning of NHE-5 and expression in fibroblasts (15), or Chinese hamster ovary cells (292), also yielded a functional Na⁺/H⁺ exchanger. Less specific localization to hippocampus and other regions was noted by Northern analysis (15).

The NHE-6 isoform has a high degree of sequence identity to a mitochondrial Na⁺/H⁺ exchanger of yeast, termed NHA2. Its abundant expression in mitochondria-rich tissues, including brain, increased suspicion that it also was a mitochondrial Na⁺/H⁺ exchanger (230). Recent studies have not confirmed a mitochondrial localization, however, and have provided evidence that the transporter is targeted to endoplasmic reticulum (209), or endosomes (47). NHE-7 is also in organelles, localized to the trans-Golgi network (229a).

The function of individual NHE isoforms in brain is unclear, with the possible exception of NHE-1. It is likely that the principal role of NHE-1 in the CNS is the maintenance of steady-state pH_i and the recovery from cytosolic acid loads. This is supported by recent studies of a spontaneous slow-wave epilepsy mutant mouse (89, 358). The autosomal recessive defect of these animals arises from a null allele of NHE-1 localized to chromosome four and is associated with ataxic gait, absence and tonic-clonic seizures, and selective neuronal death in brain stem and cerebellum (89). In a study of pH_i regulation in HEPES-buffered media, acutely dissociated CA1 pyramidal neurons from mutant mice displayed a more acidic state pH_i and a lower rate of acid extrusion compared with cells from wild-type animals. In some instances, recovery from acid loading was virtually absent in the neurons from mutant animals (358).

The roles of NHE-2 through NHE-7 are more subject to conjecture. Given the well-established utilization of Na⁺/H⁺ exchange in cell volume regulation (336), this function appears plausible, particularly for NHE-4 (30). Regulation of pH within organellar compartments appears likely in the case of NHE-6 (47, 209, 230) and NHE-7 (229a).

B. HCO₃^- Transporters in Brain

The transport processes that regulate pH_i through the transmembrane flux of bicarbonate fall within the bicarbonate transporter superfamily. Identified and cloned species correspond to the physiologically well-characterized categories of Cl^-/HCO₃^- exchange, Na⁺-HCO₃^- cotransport, and Na⁺-driven Cl^-/HCO₃^- exchange. Subspecies of all three transporter classes have been identified in the CNS.