gp120-induced alterations of human astrocyte function: Na+/H+ exchange, K+ conductance, and glutamate flux

Holly K. Patton1, Zhen-Hong Zhou1, James K. Bubien1, Etty N. Benveniste2, and Dale J. Benos1

Departments of 1 Physiology & Biophysics and 2 Cell Biology, University of Alabama at Birmingham, Birmingham, Alabama 35294


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Many human immunodeficiency virus (HIV)-infected patients suffer from impaired neurological function and dementia. This facet of the disease has been termed acquired immunodeficiency syndrome (AIDS)-associated dementia complex (ADC). Several cell types, including astrocytes and neurons, are not productively infected by virus but are involved in ADC pathophysiology. Previous studies of rat astrocytes showed that an HIV coat protein (gp120) accelerated astrocyte Na+/H+ exchange and that the resultant intracellular alkalinization activated a pH-sensitive K+ conductance. The present experiments were conducted to determine whether gp120 affected human astrocytes in the same fashion. It was found that primary human astrocytes express a pH-sensitive K+ conductance that was activated on intracellular alkalinization. Also, gp120 treatment of whole cell clamped human astrocytes activated this conductance specifically. Furthermore, gp120 inhibited glutamate uptake by primary human astrocytes. These altered physiological processes could contribute to pathophysiological changes in HIV-infected brains. Because the gp120-induced cell physiological changes were partially inhibited by dimethylamiloride (an inhibitor of Na+/H+ exchange), our findings suggest that modification of human astrocyte Na+/H+ exchange activity may provide a means of addressing some of the neurological complications of HIV infection.

acquired immunodeficiency syndrome dementia complex; potassium channels; sodium/hydrogen exchanger


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

HUMAN IMMUNODEFICIENCY VIRUS (HIV)-associated dementia complex (ADC) affects >50% of individuals who have contracted the AIDS virus (21, 27). Clinically recognizable characteristics of ADC are cognitive, motor, and behavioral abnormalities. At autopsy, atrophy and other pathologies are prominent in the white matter and deep gray matter of the brain (27). Histopathological examination of the central nervous system (CNS) reveals multinucleated giant cells, astrogliosis, microgliosis, neural degeneration, myelin pallor, and breakdown of the blood-brain barrier.

The cellular pathophysiology does not appear to result solely from infection of a single cell type. The complex pathophysiology appears to result from abnormal cellular interactions, because HIV does not infect astrocytes or neurons (7, 31). In the CNS, HIV infects primarily cells of monocyte lineage such as microglia and macrophages (13). Because the primary cells of the CNS are not infected but do appear to be pathologically affected, the source of the pathophysiological changes must be indirect (i.e., not cellular destruction due to viral replication). Potential mediators of the CNS pathophysiology are HIV proteins such as coat proteins, inappropriate cytokines from infected immunological cells within the CNS, and alterations of nonneuronal cell functions (17, 22, 23). It has been shown that the HIV envelope coat protein gp120 is present in cerebral spinal fluid, making this protein a potential mediator of CNS pathophysiological changes in ADC (14).

gp120 alters glial cell function (22, 23) and induces activated macrophages to secrete eicosanoids, arachidonic acid, interleukin-2 (IL-2), and nitric oxide (10, 33). Eicosanoids have been shown to increase the release of glutamate (a neurotransmitter) and decrease its reuptake by astrocytes (16). In vitro application of gp120 to uninfected monocytes induced overexpression of proinflammatory cytokines such as IL-1beta , tumor necrosis factor-alpha (TNF-alpha ), platelet-activating factor (PAF), nitric oxide, and metabolites of phospholipase A2, such as arachidonic acid (15, 31). All of these compounds have the potential to produce CNS dysfunction as a consequence of their biological activity.

We have demonstrated direct effects of gp120 on astrocyte function (9). It is possible that alterations in the function of astrocytes also adversely affect neurons and ultimately contribute to ADC. We have shown that exogenous application of gp120 increases rat astrocyte cytosolic pH by activating Na+/H+ exchange (3, 4), and we have also demonstrated that gp120 induces activation of a pH-sensitive potassium conductance in whole cell clamped rat astrocytes. This conductance could only be activated by gp120 when the pipette solutions were unbuffered. Buffering of the pipette solutions prevented any pH changes mediated by cell processes because of the overwhelming volume and correspondingly large buffering capacity. On the basis of this finding, it was hypothesized that the gp120-mediated activation of the K+ conductance was dependent on Na+/H+ exchange activity. Because there can be significant differences between species, and because ADC is a human problem, we retested this hypothesis in human astrocytes to assess directly whether gp120 alters K+ conductance in human astrocytes. This hypothesis is attractive because gp120-induced changes in astrocyte pH, K+ conductance, and glutamate regulation are cell physiological alterations that are consistent with the pathophysiological changes that eventually lead to the neuronal dysfunction observed in ADC. Primary human astrocytes were used for the present study to ensure the closest in vitro cell type to intact human brain tissue.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Procurement of human astrocytes. Normal brain tissue specimens were obtained from patients operated on for intractable epilepsy and were processed for tissue culture analysis as described previously (1, 2). Enriched astrocyte cultures were obtained ~30 days after plating by trypsinization (0.25% trypsin, 0.01% EDTA) and were monitored for glial fibrillary acidic protein (GFAP), an intracellular antigen unique to astrocytes (6). Primary astrocyte cultures were used at this time and were not passaged further. We have successfully used primary human astrocytes in a variety of studies (21, 32). All brain tissue was obtained in accordance with human tissue procurement procedures as approved by the University of Alabama at Birmingham (UAB) Institutional Review Board (April 22, 1998). SKMG-1 is an established human glioblastoma multiforme (GMB) cell line. These cells were also obtained from Dr. Y. G. Gillespie (Department of Surgery, Division of Neurosurgery, UAB) and have been described previously (11).

Electrophysiological methods. Cultured astrocytes were scraped from 35-mm culture dishes, washed with serum-free RPMI, and placed in a perfusion chamber mounted on an inverted microscope. Once the cells settled and adhered to the glass bottom of the chamber, gigaohm seals were formed between recording patch pipettes and the plasma membrane. The pipette solution contained 150 mM KCl and 1 mM EGTA, and the pH was adjusted to 7.0. The solutions were unbuffered. This was critical for whole cell current analysis because in previous experiments it was found that gp120 had no effect on astrocyte whole cell currents when the pipette solutions were buffered to pH 7.2 with 20 mM HEPES. The bath solution contained 150 mM NaCl, 6 mM HEPES, 1 mM EGTA, and 2 mM CaCl2, at a pH of 7.25.

After the cell-attached configuration was formed, the pipette potential was made 60 mV positive. This positive potential provided an inward driving force for the inward movement of potassium. Single-channel activity was recorded before and after superfusion with 13 nM gp120.

Ensemble whole cell currents were recorded using a voltage-clamp protocol that held the membrane potential at 0 mV for 1 s, with 800-ms test voltage clamps ranging from -100 to + 100 mV (in 20-mV increments), returning to the holding potential of 0 mV between each test potential. This voltage-clamp protocol prevented the activation of voltage-sensitive Na+ channels because the holding potential was depolarized. Once basal current levels were measured, the cells were superfused with 13 nM gp120. Typically, whole cell currents were recorded at 3, 5, and 10 min after superfusion with gp120. Membrane conductance was continuously monitored for the duration of each experiment by clamping the membrane potential between -60 and 0 or 0 and +60 mV between each set of test clamps.

Glutamate influx. SKMG astroglioma cells were plated and grown until confluent in Costar six-well plates. Once 80% confluent, the cells were washed and incubated in serum-free medium overnight. Before being used, the cells were washed twice with phosphate-buffered saline (PBS) and incubated for 10 min with either PBS, PBS + 13 nM gp120, or PBS + 13 nM gp120 + 10 µM dimethylamiloride (DMA). After 10 min, 2 µCi of [3H]glutamic acid were added. The cells were incubated for an additional 3 min. Ten microliters of supernatant were collected at 1 min for determination of the specific activity of the radioactive bath. After the 3-min incubation, the cells were washed four times with PBS and lysed with 1 ml of 1 M NaOH. Lysate (800 µl) was mixed with 5 ml of scintillation fluid. Subsequently, the radioactivity of the mixture was counted using a Hewlett-Packard scintillation counter. A Bio-Rad protein assay kit was used to determine the protein content of each well. The rate of glutamate uptake by the cells was calculated using the formula flux = dpm/specific activity × mg protein × time, where dpm is disintegrations per minute and time = 3 min.

RT-PCR for candidate Na+/H+ exchange proteins. Total RNA was isolated using the Trizol method according to the manufacturer's specifications (GIBCO BRL). Two micrograms of total RNA were reverse transcribed with Superscript II and oligo(dT) primers. Five microliters of the mixture containing the RT products were amplified using Taq polymerase, and degenerate primers were synthesized on the basis of the conserved transmembrane sequences from Na+/H+ exchangers (NHE1-4). The PCR products were visualized using a 1% agarose gel. After confirmation of the amplification of specific PCR products, 10 ng of the products were inserted into the TOPO TA cloning vector (Invitrogen). Transformation into One Shot cells (Invitrogen) was completed. Subsequently, the transformed cells were plated on ampicillin/X-Gal/isopropyl beta -d-thiogalactopyranoside and grown overnight. White colonies were selected and grown overnight in Luria broth.

For the isolation of the cloned DNA, the cultures were separated by centrifugation. The pellet was resuspended in 100 µl of 50 mM glucose, 25 mM Tris · HCl, pH 8.0, and 10 mM EDTA. The mixture was incubated for 5 min at room temperature, and then 200 µl of 0.2 N NaOH and 1% SDS were added, and the mixture was again incubated for 5 min at room temperature. Next, 300 µl of 3.0 M K/5.0M acetate were added and the mixture was reincubated. Six hundred microliters of 5.0 M LiCl were added, and the mixture was then incubated for 5 min on ice. The reaction products were centrifuged for 10 min at 14,000 rpm, washed once with 70% EtOH, and resuspended in 50 µl of 5 µg/ml RNase A. The products were heated to 37°C for 30 min, packaged, and sent to the sequencing facility at Iowa State University for DNA sequencing.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

gp120-induced K+ conductance. Two types of human astrocytes [freshly isolated and continuously cultured (SKMG cell line)] were patch clamped in the cell-attached configuration. After formation of the gigaohm seals configuration, the cells were superfused with 13 nM gp120. Figure 1 shows the specific increase in single-channel activity induced by treatment with gp120. The pipette solutions contained 130 mM of K+, and the bath contained 130 mM Na+. Therefore, the inward current (downward) was carried by K+. The direct effect of pH on individual K+ channels was also tested in inside-out isolated membrane patches. Figure 2 shows the effect of increasing the pH of the bath solution from 6.6 to 7.4. Kinetic analysis indicated a threefold increase in single-channel activity produced (6% to 19%) by the increased pH. The conductance was calculated to be 130 pS from amplitude histogram analysis. These findings are similar to those reported previously for pH-sensitive K+ channels expressed by rat astrocytes (9).


View larger version (28K):
[in this window]
[in a new window]
 
Fig. 1.   Current records from cell-attached patches on SKMG (A) and freshly isolated human astrocytes (B). These records show that 13 nM gp120 induced K+ channel activation indirectly because the cell-attached configuration does not allow the peptide to come into direct contact with the channels being recorded.



View larger version (21K):
[in this window]
[in a new window]
 
Fig. 2.   A: single-channel currents recorded from an inside-out patch, showing typical channel activity (Po) when the bath solution (130 mM K-gluconate) was buffered to pH 6.6 (left) and increased activity when the bath solution was buffered to pH 7.4 (right). B: amplitude histograms showing a 1.3-pA increase in single-channel current when patch potential was increased by 10 mV, giving a single-channel conductance of 130 pS.

To assess directly whether potassium channels were activated by gp120, astrocytes were examined electrophysiologically in the whole cell patch-clamp configuration. For these experiments, the pipette solutions were unbuffered, because in rat astrocytes it was found that gp120 failed to activate any whole cell ionic conductances when the pipette solution was buffered to pH 7.2 (9). Figure 3 shows the time course of activation of human primary astrocyte conductance after exposure to 10 nM gp120. This experiment was repeated seven times, and gp120 activated the outward conductance in every repetition. Membrane conductances were measured as the chord between +100 mV to the reversal potential (outward conductance) and -100 mV to the reversal potential (inward conductance). In response to gp120, the outward conductance increased significantly from 1,028 ± 253 to 2,541 ± 698 pS/10 pF (P = 0.04, n = 7). There was no change in the inward conductance from a basal level of 628 ± 149 to 585 ± 158 pS/10 pF (P = 0.84, n = 7).


View larger version (34K):
[in this window]
[in a new window]
 
Fig. 3.   Current records from a whole cell clamped SKMG cultured human astrocyte showing gp120-mediated activation of a K+ conductance before (A) and after 1 (B), 3 (C), and 10 min (D) of treatment with 13 nM gp120. These results were typical of those observed in 7 separate experiments.

As an independent test of the hypothesis that the outward current increase was caused by increased intracellular pH, primary astrocytes were whole cell clamped and subsequently superfused with bath solution supplemented with 20 mM ammonium chloride. Figure 4 shows the increased outward currents induced by this treatment in a typical experiment. This treatment significantly (P = 0.003, n = 7) increased the outward conductance from a basal level of 515 ± 50 to 1,319 ± 221 pS/10 pF. Figure 5 shows the average changes in the current-voltage relations before and after treatment with gp120 or 20 mM NH4+. These findings, along with the single-channel data (Fig. 2), indicate that the current increase can be produced independent of exposure to gp120, as long as the cytoplasmic pH increases. These data support the hypothesis that the alteration of astrocyte K+ conductance is induced by alkalinization but does not directly require gp120.


View larger version (31K):
[in this window]
[in a new window]
 
Fig. 4.   Whole cell current records show basal current (A) and the current increase in response to treatment with 20 mM NH4Cl (B).



View larger version (12K):
[in this window]
[in a new window]
 
Fig. 5.   A: current-voltage relations before () and after 10-min exposure to 13 nM gp120 (). B: current-voltage relations before () and after treatment with 20 mM NH4Cl ().

gp120 does not alter astrocyte whole cell current in the absence of extracellular Na+. After formation of the whole cell configuration on SKMG astrocytes, the bath Na+ was replaced with the impermeant monovalent cation N-methyl-D-glucamine (NMDG). This organic cation does not flow through ion channels and is not transported by the Na+/H+ exchanger (NHE). These experiments were carried out to establish whether the indirect activation of astrocyte K+ channels was dependent on Na+. Figure 6A shows that no current was activated by gp120 when the bath Na+ was replaced with NMDG. However, in the same cell the outward current was activated (after gp120 treatment) when the bath NMDG was replaced with Na+ (Fig. 6B). These findings are consistent with the hypothesis that gp120 exerts direct effects on Na+/H+ exchange, thereby increasing cytosolic pH, and activating a pH-sensitive potassium conductance.


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 6.   Difference currents (i.e., the current before treatment subtracted from the current after treatment) showing that gp120 failed to activate any current when the bath solution contained N-methyl-D-glucamine (A). In the same cell, replacement of the bath Na+ induced the activation of outward K+ currents after gp120 treatment (B).

DMA partially inhibits gp120-mediated astrocyte K+ conductance. Astrocytes were whole cell clamped and superfused with 10 µM DMA. This amiloride analog inhibits Na+/H+ exchange (25). The efficacy of DMA is greatest for NHE1 (IC50 ~ 0.1 µM). However, DMA is somewhat less effective at inhibiting other NHE isoforms. For example, the IC50 for DMA is 11-14 µM for the inhibition of NHE3 (25). In whole cell clamped astrocytes pretreated with 10 µM DMA, subsequent treatment with 13 nM gp120 activated the K+ conductance. However, the activation was slow and incomplete, suggesting some inhibition of Na+/H+ exchange. The partial inhibition was confirmed by washout of the DMA. As shown in Fig. 7, washout of the DMA resulted in a rapid, more specific activation of the outward K+ conductance.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 7.   Three sequential sets of current records from a single whole cell-clamped SKMG astrocyte. A: basal current in the presence of 10 µM dimethylamiloride (DMA). B: on superfusion with 13 nM gp120, there was a clear increase in the outward conductance. C: the conductance increased further on washout of the DMA, indicating a partial inhibition of the gp120-stimulated effect.

Unique human astrocyte NHE protein. Because the effects of gp120 on astrocyte whole cell currents were dependent on Na+, and because gp120 had no effect on astrocyte Na+ conductance itself, it was hypothesized that alterations in Na+/H+ exchange could mediate the effects on K+ conductance by altering cytosolic pH. This hypothesis was supported by the observation that gp120 increased cytosolic pH in intact rat astrocytes (3, 4). Moreover, it was shown that gp120 failed to alter K+ currents in whole cell clamped rat astrocytes when the pipette solution was buffered to pH 7.2 with HEPES (9) but increased the K+ conductance when the pipette solutions were left unbuffered (9).

Western blot analysis of human astrocyte membrane proteins using antibodies directed against NHE1-4 yielded negative results. These findings suggested that if these NHE isoforms were expressed by human astrocytes, the expression level was below the level of detection by Western blot. Others have found that NHE1 was the predominant isoform expressed by rat hippocampal astrocytes (29). Also, Bevensee et al. (5) suggested the expression of additional NHE isoforms based on altered amiloride sensitivity. These findings leave open the possibility that additional undescribed isoforms may be expressed by human astrocytes. To test the possibility that human astrocytes expressed an NHE isoform that did not react with the available anti-NHE antibodies, degenerate primers to transmembrane sequences of known NHEs were constructed and used in reverse transcriptase polymerase chain reactions to amplify NHE mRNA purified from SKMG cultured human astrocytes. These reactions yielded a specific band of 519 bp (Fig. 8). The band was subcloned and sequenced to determine whether it bore any sequence similarity to known human NHE proteins. A sequence comparison yielded a positive correlation with a sequence submitted by Grafham on August 4, 1998 (GenBank accession no. AL022165). At the amino acid level, the Grafham sequence was 63% identical to the sequence of NHE2 and NHE6. At the amino acid level, the sequence we obtained from SKMG astrocytes (termed NHEX-H) was 68% identical to the Grafham sequence (Table 1).


View larger version (40K):
[in this window]
[in a new window]
 
Fig. 8.   RT-PCR products from SKMG cell mRNA using degenerate primers for membrane spanning segments of Na+/H+ exchange proteins. Left lane: standard size markers (phiX 174 DNA1 Hac III, Promega). Right lane: the 519-bp band from which the Na+/H+ sequence was obtained.


                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Amino acid sequence comparison of NHEX-H and GRAF-1

This sequence most closely resembles the sequence of NHE2 and bears little resemblance to that of NHE1. On the basis of the negative Western blot results for well-established NHE isoforms, it was hypothesized that the activity of this currently unique NHE isoform was potentially the protein affected primarily by gp120.

gp120 inhibits glutamate uptake by astrocytes. Glutamate transport into astrocytes is an Na+-dependent process. Also, the glutamate cotransport protein is sensitive to pH, operating at reduced efficacy when the pH at the cytosolic face is increased (28). gp120-mediated stimulation of Na+/H+ exchange would tend to reduce the transmembrane Na+ gradient, providing less driving force for Na+-dependent glutamate influx. Also, gp120-mediated stimulation of Na+/H+ exchange would elevate the cytosolic pH (1, 2). Therefore, by two synergistic mechanisms, gp120 should reduce glutamate uptake by astrocytes treated with the HIV coat protein. We tested this hypothesis directly by measuring glutamate uptake by human astrocytes. Figure 9 shows that gp120 did inhibit [3H]glutamate uptake, and the inhibition was partially prevented by pretreatment with the NHE inhibitor DMA. These findings are consistent with the hypothesis that gp120 stimulates astrocyte Na+/H+ exchange, and this stimulation affects Na+- and H+-dependent processes such as glutamate uptake and membrane K+ conductance.


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 9.   Glutamate uptake was significantly reduced (34% of basal, n = 6, P < 0.001) when the cells were pretreated with 13 nM gp120 alone. The gp120-mediated reduction in glutamate uptake was attenuated when DMA was added to the cultures before gp120 treatment (54% of basal and 142% of uptake after gp120 alone, n = 23, P = 0.003 vs. basal and P < 0.001 vs. gp120 alone).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The findings presented in this study address several issues. First, they demonstrate directly that the HIV coat protein gp120 is a biologically active compound on exposure to human CNS cells. Second, these findings provide functional evidence to support the hypothesis that gp120-mediated alterations in astrocyte cell physiology can produce abnormalities that could play a role in the pathogenesis of ADC. Third, and possibly most important, is the finding that gp120-mediated stimulation of Na+/H+ exchange can directly alter the chemical gradient driving Na+ via the exchanger and the electrochemical Na+ driving force. These studies show that another consequence can be alterations in astrocyte K+ conductance. The findings presented here link three processes and demonstrate the counterbalancing effects of ion channels and solute-driven transporters such as NHEs, the Na+/glutamate cotransporter, and K+ channels.

Single-channel recordings in the cell-attached configuration demonstrated activation of putative K+ channels by gp120. The significance of this finding is that the activation of these ion channels was indirect. In the cell-attached patch configuration, all of the channels that are recorded are within a patch of membrane surrounded by a gigaohm seal to the patch pipette tip. This seal prevents leak of electrical currents and also prevents any components of the bath solution from coming into direct contact with the ion channels within the patch. Therefore, if a compound such as gp120 is introduced into the bath solution and effects on channels within the cell-attached patch are observed, these effects must have been produced by changes in the cytosolic environment or cellular signal transduction pathways. In this configuration, the extracellular faces of the channels are surrounded by the gigaohm-sealed pipette and membrane patch, and the cytosolic faces are sequestered from the bath solution by the plasma membrane. It is possible that gp120 could be transported into the cells and potentially affect ion channels directly from the cytosolic side. However, this possibility is unlikely in astrocytes, because astrocytes are not infected by HIV. Therefore, the most likely explanation for the mechanism whereby gp120 treatment activated ion channels in cell-attached patches is that it influences some other astrocyte cell surface protein, which in turn results in a cascade of events intracellularly that has one manifestation: the activation of K+ channels. From studies using BCECF to measure cell pH in intact rat (8) and human astrocytes, it is known that gp120 induced a rapid and substantial alkalinization (3, 4). Thus the most likely candidate for a cell surface protein that has the ability to alter cell pH is an NHE protein. It should be noted that this effect appears to be relatively specific for the NHE protein expressed by astrocytes, because gp120 fails to induce pH changes in other cell types such as microglia and macrophages (3, 4) as well as lymphocytes and renal mesangial cells (unpublished observations).

In whole cell clamped astrocytes, gp120 treatment specifically induced the activation of outward currents. The ionic conditions were such that the outward current could only be carried by K+ or Cl-. Tests to assess whether Cl- could be the charge carrier (i.e., a bath solution of Na+ gluconate to remove Cl-) produced the same results, thereby eliminating extracellular Cl- as a potential charge carrier for the outward current activated by gp120.

It is not surprising that an elevation of cellular pH activates a K+ conductance in astrocytes. There are numerous examples of K+ channels that activate when the pH at their cytosolic face is increased: ROMK channels (24, 30), cardiac ATP-sensitive K+ channels (12), and renal pH-sensitive K+ channels (19, 29). In each of these cases there may be specific tissue or cell function-related reasons for the expression of pH-sensitive K+ channels. Likewise, the expression of pH-sensitive K+ channels by astrocytes appears to be related to their function of maintaining an appropriate extracellular environment in the CNS.

In these experiments, gp120 was used to alter the transport characteristics of astrocyte plasma membranes. Direct measurement showed a reduction in Na+-dependent glutamate uptake. This is a potentially pathological alteration, because it has been shown recently that "acute disruption of transporter activity immediately results in a significant accumulation of extracellular glutamate," at least in brain slices (20). Abnormally high extracellular levels of this neurotransmitter could certainly play a role in the production of at least some the symptoms of ADC. Also, direct measurements of brain pH by nuclear magnetic resonance show focal areas of abnormally alkaline pH within the brain in advanced cases of ADC (26). These macroscopic changes are also consistent with the in vitro astrocyte cell physiological abnormalities induced by gp120 in the present experiments.

Perhaps the most significant finding of the present study was the indirect induction of outward K+ currents by gp120 treatment. This finding provides some insight into the interactions and balance among Na+-coupled transport, energy metabolism, and ion channels in the astrocyte plasma membrane. It also helps to define a role for pH-sensitive K+ channels in astrocytes. As H+ is produced by oxidative metabolism, fuel for the reactions must be imported. One source is Na+-coupled glucose cotransport. This process tends to increase cell Na+. The H+ is removed via Na+/H+ exchange. This also would tend to increase cell Na+. Add to these basic cell functions the periodic reuptake of glutamate subsequent to local neuronal activity. This also is accomplished by cotransport of glutamate with Na+. All of these processes utilize the energy inherent in the electrochemical gradient for Na+ and tend to dissipate the Na+ concentration gradient somewhat. Normally this Na+ influx is balanced by the activity of the Na+-K+-ATPase. This process removes cellular Na+ but increases intracellular K+. Thus a mechanism must be present that allows K+ to flow outward down its electrochemical gradient at negative membrane potentials. One mechanism that can permit such a K+ efflux is an increase in the outward rectification of K+ channels, nascent in the plasma membrane. Such a change in the biophysical characteristics of K+ channels allows for easier K+ efflux if the membrane potential is less negative than the equilibrium potential for K+. A model that summarizes these processes is shown in Fig. 10.


View larger version (25K):
[in this window]
[in a new window]
 
Fig. 10.   Model of an astrocyte plasma membrane showing Na+-coupled cotransport processes that control glutamate reuptake, H+ production, and the removal of cell Na+. These processes must function in balance to achieve a steady state. During periods of high Na+-coupled influx such as during glutamate reuptake, pH-sensitive K+ channels may open to maintain the Na+ driving force.

When astrocytes are exposed to gp120, the normal membrane and metabolic homeostasis becomes altered because of the inappropriate increase in Na+/H+ exchange. Under these abnormal conditions, the effect of the increased cell pH was observed when the cells were voltage clamped. However, the increased K+ conductance was only observed at positive membrane potentials. Because it is not known whether gp120 depolarizes astrocytes in situ, we do not know for sure whether these in vitro alterations in astrocyte K+ conductance play any significant role in the pathophysiology of ADC. Nonetheless, these changes are consistent with the hypothesis that an altered extracellular environment (specifically, an increase in extracellular K+) can lead to Ca2+-induced neurotoxicity (13, 14).

The gp120-induced alterations in human astrocyte cell physiology, demonstrated directly by these experiments, are consistent with the hypothesis that astrocyte dysfunction plays a role in ADC. The gp120-induced reduction in glutamate uptake is potentially the most significant abnormality, because excess glutamate is neurotoxic. The alterations in cell physiology are also consistent with nuclear magnetic resonance imaging findings from the brains of individuals with ADC. These findings show focal areas of elevated pH (26). The flow of cerebral spinal fluid through the brain is slow, and therefore the clearance of any local gp120 is much slower than in the general circulation. The same is true for glutamate, which is one reason why astrocyte membrane function must be maintained and be closely regulated. If gp120 alters astrocyte membrane function in vivo, problems such as ADC are possible.


    ACKNOWLEDGEMENTS

This work was supported by National Institutes of Health Grants MH-50421 (to D. J. Benos), MH-55795 (to E. N. Benveniste), and DK-52789 (to J. K. Bubien). J. K. Bubien is an Established Investigator of the American Heart Association.


    FOOTNOTES

Address for reprint requests and other correspondence: D. J. Benos, Dept. of Physiology and Biophysics, MCLM 704, 1530 3rd Ave. South, Birmingham, Alabama 35294 (E-mail: benos{at}physiology.uab.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 17 August 1999; accepted in final form 20 March 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Barnum, SR, Jones JL, and Benveniste EN. Interferon-gamma regulation of C3 gene expression in astroglioma cells. J Neuroimmunol 38: 275-282, 1992[ISI][Medline].

2.   Barnum, SR, Jones JL, and Benveniste EN. Interleukin-1 and tumor necrosis factor mediated regulation of C3 gene expression in human astroglioma cells. Glia 7: 225-236, 1993[ISI][Medline].

3.   Benos, DJH, Hahn BH, Bubien JK, Ghosh SK, Mashburn NA, Chaikin MA, Shaw GM, and Benveniste EN. Envelope glycoprotein gp120 of human immunodeficiency virus type 1 alters ion transport in astrocytes: implications for AIDS dementia complex. Proc Natl Acad Sci USA 91: 494-498, 1994[Abstract].

4.   Benos, DJ, McPherson S, Hahn BH, Chaikin MA, and Benveniste EN. Cytokines and HIV envelope glycoprotein gp120 stimulate Na+/H+ exchange in astrocytes. J Biol Chem 269: 13811-13816, 1994[Abstract/Free Full Text].

5.   Bevensee, MO, Weed RA, and Boron WF. Intracellular pH regulation in cultured astrocytes from rat hippocampus. I. Role of HCO3-. J Gen Physiol 110: 453-465, 1997[Abstract/Free Full Text].

6.   Bignami, A, Eng LF, Dahl D, and Uyeda CY. Localization of the glial fibrillary acidic protein in astrocytes by immunofluorescence. Brain Res 43: 429-435, 1972[ISI][Medline].

7.   Blumberg, BM, Gelbard HA, and Epstein LG. HIV-1 infection of the developing nervous system: central role of astrocytes in the pathogenesis. Virus Res 32: 253-267, 1994[ISI][Medline].

8.   Boyarsky, G, Ransom B, Schlue WR, Davis MB, and Boron WF. Intracellular pH regulation in single cultured astrocytes from rat forebrain. Glia 8: 241-248, 1993[ISI][Medline].

9.   Bubien, J, Benveniste EN, and Benos DJ. HIV-gp120 activates large-conductance apamin-sensitive potassium channels in rat astrocytes. Am J Physiol Cell Physiol 268: C1440-C1449, 1995[Abstract/Free Full Text].

10.   Bukrinsky, M, Nottet HSLM, Schmidtmayerova H, Dubrovsky L, Flanagan CR, Mullins ME, Lipton SA, and Gendelman HE. Regulation of nitric oxide synthase activity in human immunodeficiency virus type 1 (HIV-1)-infected monocytes: implication for HIV-associated neurological disease. J Exp Med 181: 735-745, 1995[Abstract].

11.   Cairncross, JG, Mattes MJ, Beresford HR, Albino AP, Houghton AN, Lloyd KO, and Old LJ. Cell surface antigens of human astrocytoma defined by mouse monoclonal antibodies: identification of astrocytoma subsets. Proc Natl Acad Sci USA 79: 5641-5645, 1982[Abstract].

12.   Cuevas, J, Bassett AL, Cameron JS, Furukawa T, Myerburg RJ, and Kimura S. Effect of H+ on ATP-regulated K+ channels in feline ventricular myocytes. Am J Physiol Heart Circ Physiol 261: H755-H761, 1991[Abstract/Free Full Text].

13.   Dickson, DW. Macrophages in the HIV CNS disease: microglia as reservoirs and perpetrators of HIV CNS disease. In: The Neurology of AIDS, edited by Gendelman HE, Lipton SA, Epstein L, and Swindells S.. New York: Chapman and Hall, 1998.

14.   Dreyer, EB, Kaiser PK, Offermann JT, and Lipton SA. HIV-1 coat protein neurotoxicity prevented by calcium channel antagonists. Science 248: 364-367, 1990[ISI][Medline].

15.   Gelbard, HA, Nottet HS, Swindells S, Jett M, Dzenko KA, Genis P, White R, Wang L, Choi YB, Zhang D, Lipton SA, Tourtellotte WW, Epstein LG, and Gendelman HE. Platelet-activating factor: a candidate human immunodeficiency virus type 1-induced neurotoxin. J Virol 68: 4628-4635, 1994[Abstract].

16.   Genis, P, Jett M, Bernton EW, Boyle T, Gelbard HA, Dzenko K, Keane RW, Resnick L, Mizrachi Y, Volsky DJ, Epstein LG, and Gendelman HE. Cytokines and arachidonic metabolites produced during human immunodeficiency virus (HIV)-infected macrophage-astroglia interactions: implications for the neuropathogenesis of HIV disease. J Exp Med 176: 1703-1718, 1992[Abstract].

17.   Glass, J, and Johnson R. Human immunodeficiency virus and the brain. Annu Rev Neurosci 19: 1-26, 1996[ISI][Medline].

19.   Ikeda, M, Murata M, Miyoshi T, Tamba K, Muto S, Imai M, and Suzuki M. Transcriptional activation of RACTK1 K+ channel gene by apical alkalization in renal cortical collecting duct cells. J Clin Invest 98: 474-481, 1996[Abstract/Free Full Text].

20.   Jabaudon, D, Shimamoto K, Yasuda-Kamatani Y, Scanziani M, Gahwiler B, and Gerber U. Inhibition of uptake unmasks rapid extracellular turnover of glutamate of nonvesicular origin. Proc Natl Acad Sci USA 96: 8733-8738, 1999[Abstract/Free Full Text].

21.   Levy, J, Shimabukuro J, Hollander H, Mills J, and Kaminsky L. Isolation of AIDS-associated retroviruses from cerebrospinal fluid and brain of patients with neurological symptoms. Lancet 2: 586-588, 1985[ISI][Medline].

22.   Lipton, S, and Rosenberg P. Excitatory amino acids as a final common pathway for neurologic disorders. N Engl J Med 330: 613-622, 1994[Free Full Text].

23.   Lipton, SA, and Gendelman HE. Seminars in medicine of the Beth Israel Hospital, Boston. Dementia associated with acquired immunodeficiency syndrome. N Engl J Med 33: 934-940, 1995.

24.   McNicholas, CM, MacGregor GG, Islas LD, Yang Y, Hebert SC, and Giebisch G. pH-dependent modulation of the cloned renal K+ channel, ROMK. Am J Physiol Renal Physiol 275: F972-F981, 1998[Abstract/Free Full Text].

25.   Noel, J, and Pouyssegur J. Hormonal regulation, pharmacology, and membrane sorting of vertebrate Na+/H+ exchanger isoforms. Am J Physiol Cell Physiol 268: C283-C296, 1995[Abstract/Free Full Text].

26.   Patton, HK, Benveniste EN, Chu W-J, and Benos DJ. pH and metabolite changes in cerebellum of HIV-infected individuals (Abstract). FASEB J 13: A851, 1999[ISI].

27.   Petito, CK, Cho ES, Lemann W, Navia BA, and Price RW. Neuropathology of acquired immunodeficiency syndrome (AIDS): an autopsy review. J Neuropathol Exp Neurol 45: 635-646, 1986[ISI][Medline].

28.   Pizzonia, JH, Ransom BR, and Pappas CA. Characterization of Na+/H+ exchange activity in cultured rat hippocampal astrocytes. J Neurosci Res 44: 191-198, 1996[ISI][Medline].

29.   Reyes, R, Duprat F, Lesage F, Fink M, Salinas M, Farman N, and Lazdunski M. Cloning and expression of a novel pH-sensitive two pore domain K+ channel from human kidney. J Biol Chem 273: 30863-30869, 1998[Abstract/Free Full Text].

30.   Schulte, U, Hahn H, Wiesinger H, Ruppersberg JP, and Fakler B. pH-dependent gating of Romk (Kir1.1) channels involves conformational changes in both N and C termini. J Biol Chem 273: 34575-34579, 1998[Abstract/Free Full Text].

31.   Tardieu, M, Hery C, Peudenier S, Boespglug O, and Mantagnier L. Human immunodeficiency virus type 1-infected monocytic cells can destroy human neural cells after cell-to-cell adhesion. Ann Neurol 32: 11-17, 1992[ISI][Medline].

32.   Van Wagoner, N, Oh J-W, Repovic P, and Benveniste EN. Interleukin-6 (IL-6) production by astrocytes: autocrine regulation by IL-6 and the soluble IL-6 receptor. J Neurosci 19: 5236-5244, 1999[Abstract/Free Full Text].

33.   Wahl, SM, Allen JB, McCartney-Francis N, Morganti-Kossman MC, Kossman T, Ellingsworth L, Mai UE, Mergenhagen SE, and Orenstein JM. Macrophage- and astrocyte-derived transforming growth factor beta  as a mediator of central nervous system dysfunction in acquired immune deficiency syndrome. J Exp Med 173: 981-991, 1991[Abstract].


Am J Physiol Cell Physiol 279(3):C700-C708
0363-6143/00 $5.00 Copyright © 2000 the American Physiological Society




This Article
Abstract
Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Search for citing articles in:
ISI Web of Science (10)
Google Scholar
Articles by Patton, H. K.
Articles by Benos, D. J.
Articles citing this Article
PubMed
PubMed Citation
Articles by Patton, H. K.
Articles by Benos, D. J.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online