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 |
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 |
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-1
, tumor necrosis factor-
(TNF-
), 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 |
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
-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 |
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.
|
|
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 |
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 |
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
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