Department of Physiology, Allegheny University of the Health Sciences, Philadelphia, Pennsylvania 19129
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ABSTRACT |
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The effects of
human cytomegalovirus (HCMV) infection on
Cl/HCO
3
exchanger activity in human lung fibroblasts (MRC-5 cells) were studied
using fluorescent, ion-sensitive dyes. The intracellular pH
(pHi) of mock- and HCMV-infected
cells bathed in a solution containing 5%
CO2-25 mM
HCO
3 were nearly the same. However,
replacement of external Cl
with gluconate caused an
H2DIDS-inhibitable (100 µM)
increase in the pHi of
HCMV-infected cells but not in mock-infected cells. Continuous exposure
to hyperosmotic external media containing CO2/HCO
3
caused the pHi of both cell types
to increase. The pHi remained
elevated in mock-infected cells. However, in HCMV-infected cells, the
pHi peaked and then recovered
toward control values. This pHi
recovery phase was completely blocked by 100 µM
H2DIDS. In the presence of
CO2/HCO
3, there was an H2DIDS-sensitive
component of net Cl
efflux
(external Cl
was
substituted with gluconate) that was less in mock- than in HCMV-infected cells. When nitrate was substituted for external Cl
(in the nominal absence
of
CO2/HCO
3),
the H2DIDS-sensitive net
Cl
efflux was much greater
from HCMV- than from mock-infected cells. In mock-infected cells,
H2DIDS-sensitive, net
Cl
efflux decreased as
pHi increased, whereas for
HCMV-infected cells, efflux increased as
pHi increased. All these results
are consistent with an HCMV-induced enhancement of
Cl
/HCO
3
exchanger activity.
cell volume; hydrogen 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid; sodium/hydrogen exchanger
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INTRODUCTION |
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CYTOMEGALY, THE ENLARGEMENT of the host cell, is a
major hallmark of human cytomegalovirus (HCMV) infection (see Fig. 1 in Ref. 1). Evidence suggests that cytomegaly and viral replication are
closely linked (25), and both may be linked to enhanced Na+ entry into the host cell (13).
We previously showed that 72 h after HCMV infection the activity of the
Na+/H+
exchanger (NHE) is enhanced in two ways (10):
1) the intracellular pH
(pHi) operating range for the
NHE is shifted toward higher, or more alkaline,
pHi values, and
2) in the absence of
CO2/HCO3, HCMV infection makes the NHE much more responsive to a challenge with
hyperosmotic solutions.
Many cells are able to homeostatically regulate their cell volume in
response to shrinkage by use of a combination of increased NHE and
Cl/HCO
3
exchanger activities. The increase of NHE activity due to cell
shrinkage tends to increase pHi.
In turn, the increase of pHi
stimulates
Cl
/HCO
3
exchanger activity. The enhanced
Na+ uptake (via the NHE) and
Cl
uptake (via the
Cl
/HCO
3
exchanger) result in an increased intracellular osmolyte content, which
causes net water entry. We previously suggested that the pathological
increase of host cell size (volume) caused by HCMV infection may be
due, at least in part, to the physiologically inappropriate activation
of such a pHi-linked mechanism
(10). Our recent finding of enhanced NHE activity after HCMV infection
is consistent with this hypothesis. The next obvious question is
whether HCMV infection also enhances
Cl
/HCO
3
exchanger activity of the host cell.
The anion exchanger or
Na+-independent
Cl/HCO
3
exchanger (hereafter referred to as the
Cl
/HCO
3
exchanger) has several distinctive properties permitting its functional
characterization. For instance, because it exchanges
Cl
for
HCO
3, removal of extracellular
Cl
will cause the transport
mechanism to mediate a net exchange of intracellular
Cl
for extracellular
HCO
3. This exchange will result in a
net loss of intracellular
Cl
as well as intracellular
alkalinization. Another characteristic of the
Cl
/HCO
3
exchanger is that it is inhibited by disulfonic acid stilbene
derivatives such as H2DIDS when
they are presented to the extracellular face of the cell membrane (6).
These agents also inhibit the external
Na+-dependent
Cl
/HCO
3
exchanger (28). However, this latter exchanger is inhibited as
pHi increases, whereas the
Cl
/HCO
3
exchanger is stimulated by alkaline
pHi (4, 24, 26, 32). Finally, this
exchanger can exchange Cl
for NO
3 (3, 14, 22), and this
exchange, unlike that of Cl
for HCO
3, will have no
pHi consequences, inasmuch as
NO
3 is the salt of a strong acid
and, hence, is a very weak base. We exploited all these properties to
investigate whether HCMV infection increased
Cl
/HCO
3
exchanger activity over the same time period it enhances NHE activity.
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METHODS |
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Cell Cultures and HCMV Infection
Details of cell culture and HCMV infection protocols have been presented previously (10). Briefly, a cell line (MRC-5) derived from human embryo lung fibroblasts, passages 18-30, was cultured in MEM with Earle's salts, supplemented with 2 mM glutamine and 10% heat-inactivated FCS. The cells were grown in an incubator with a humidified atmosphere of 5% CO2-95% air at 37°C. A stock of HCMV (strain AD169) was generated in confluent MRC-5 cells (see Ref. 2 for more details).Three days after cells were seeded on 6 × 24-mm glass coverslips, confluent MRC-5 cells were exposed for 1 h to a suspension containing HCMV at a multiplicity of infection of approximately three plaque-forming units per cell or a mock-infecting, virus-free suspension (see Ref. 2 for details of mock infection). Two days after exposure to HCMV, the FCS was reduced to 1% (10).
Standard Solutions and Reagents
Standard HEPES-buffered solution contained (in mM) 128 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 10 glucose, and 20 HEPES. The pH was adjusted to 7.4 (at 37°C) with N-methyl-D-glucamine, and the osmolality was 285 ± 5 mosmol/kgH2O. The standard CO2/HCOThe high-K+ HEPES solution used in
the studies of pH dependence of
Cl efflux contained (in mM)
20 sodium gluconate, 20 potassium gluconate, 100 KNO3, 1 magnesium gluconate, 3 calcium gluconate, 10 glucose, and 20 HEPES, pH 7.4. Diethyl amiloride
(DEA; Molecular Probes, Corvallis, OR), a 5-amino-substituted
derivative of amiloride (19), was prepared as a 10 mM stock solution in
distilled water and used at a final concentration of 5 µM. This
concentration was sufficient to block virtually all NHE activity (10).
H2DIDS (Molecular Probes) was
added directly to the saline solution at a final concentration of 100 µM. Tributyltin (Fluka, Milwaukee, WI) and nigericin (Sigma Chemical,
St. Louis, MO) were made as 50 mM stock solutions in ethanol and added
directly to the saline solutions just before use to obtain a final
concentration of 10 µM. Valinomycin (Sigma Chemical) was also
prepared as an ethanol stock solution (9 mM) and added to the saline
solution to obtain a final concentration of 5 µM.
pHi Measurements
Our methods for measuring pHi with the pH-sensitive fluorescent probe 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF; Molecular Probes) are described in detail elsewhere (10). Briefly, cell populations were loaded with BCECF by exposure to 5 µM BCECF-AM, the permeant ester, for 10-30 min. An SLM-Aminco spectrofluorometer (model DMX-1000) was used to measure the dye fluorescence. The pH is proportional to the ratio of light emitted at 535 nm when BCECF is excited at two wavelengths (450 and 495 nm). The dye was calibrated intracellularly by use of the high-K+-nigericin technique (29). The pH calibration solution contained 130 mM potassium gluconate, 20 mM N-methyl-D-glucamine chloride, 2 mM MgCl2, 20 mM HEPES, and 10 µM nigericin. All pHi experiments were conducted at 37°C.Intracellular Cl Concentration
Measurements With Use of N-(6-Methoxyquinolyl)acetoethyl Ester
All MQAE experiments were performed in a spectrofluorometer at room
temperature to minimize the rate of dye loss from the cells. In
addition, the fluorescence at an intracellular
[Cl]
([Cl
]i)
of 0 mM (F0) was determined in
every experiment by use of the double-ionophore technique. This
involves bathing the cells with a
Cl
-free solution
(NO
3 replaced
Cl
) that contained 10 µM tributyltin (a
Cl
/OH
exchanger) and 10 µM nigericin (7, 20). At the end of every experiment, fluorescence readings were corrected for background by use
of an SCN
-containing
solution to maximally quench the MQAE ion-sensitive signal (Fig.
1A).
This solution contained 150 mM K+,
150 mM SCN
, 20 mM HEPES, pH
7.4, and 5 µM valinomycin (20).
[Cl
]i
is directly proportional to the ratio
F0/F (where F is the fluorescence
reading corrected for background; see above) according to the following
equation
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KSV was
experimentally determined for mock- and HCMV-infected cells (Fig.
1B). The double-ionophore technique
(see above) was used to completely deplete the cells of
Cl, and the fluorescence
intensity was noted as F0. Then,
in the continuous presence of the ionophores, the cells were exposed to
an external solution with a known
[Cl
], and the
assumption was made that, in the presence of the ionophores, extracellular
[Cl
]
([Cl
]o)
was equal to
[Cl
]i.
Finally, the cells were exposed to the KSCN solution to obtain the
maximally quenched fluorescent signal (Fig.
1A). This procedure was repeated
over a range of
[Cl
]o
values, with a new coverslip of cells used for each
[Cl
]o.
The
[Cl
]i
calibration solutions were made by mixing
high-K+ HEPES solution with
Cl
or
NO
3 as the anion (see
Standard Solutions and Reagents).
After correction for dye leakage (Fig. 1A, bottom trace), F0/F
values at each
[Cl
]i
were determined. The resultant
F0/F values are plotted in Fig. 1B, and
KSV was 25.7 and
19.7 M
1 for the mock- and
HCMV-infected cells, respectively. As noted in the legend of Fig.
1B, the fits of the slopes of the
lines used to determine the
KSV indicate
that, with 95% confidence, the two cell treatments indeed have
different values. The basis for the difference is unknown, but given
the profound effects of the virus on the host cell, it is not
surprising that such a difference might exist. If, however, there is no
difference between the
KSV values for
mock- and HCMV-infected cells, the apparent increase of
[Cl
]i
caused by HCMV infection would be 30% less than we report here. Both
values of KSV are in reasonable agreement with
those reported by other workers for other cell types (20). The efflux
rate constants were calculated from the experimental data with use of a
monoexponential function and with the assumption that
[Cl
]i
asymtotically approached 0 mM.
pHi-Clamping Studies
To vary pHi over the range 6.4-7.8, we used the high-K+-nigericin technique (29). Briefly, we exposed cells to a nigericin solution similar to that described above for calibration of the pHi indicator BCECF, except all the ClAll pooled data are means ± SE. Paired and unpaired t-tests were used, as appropriate, to test for statistical significance.
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RESULTS |
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The present investigation studied two facets of
Cl/HCO
3
exchange: its role in the determination of
pHi and its role in the
determination of
[Cl
]i.
Effect of
CO2/HCO3
on Resting pHi in Mockand
HCMV-Infected Cells
|
Effect of External Cl Removal on
pHi of Cells Exposed to
CO2/HCO
3
|
In contrast, the HCMV-infected cells responded by alkalinizing ~0.3
pH unit within 5 min of external
Cl removal. This
alkalinization could be prevented by pretreating the cells with 100 µM H2DIDS (data not shown),
which suggests that this pHi
change might be the result of net HCO
3 uptake in exchange for intracellular
Cl
mediated by the
Cl
/HCO
3
exchanger. We noted that after reaching a maximum the
pHi of HCMV-infected cells
decreased. Whether this secondary acidification is related to the
acidification observed in mock-infected cells is unknown. On return of
Cl
to the external
solution, pHi returned to
pretreatment values (not shown). Characterization of the
Cl
-dependent acidification
processes in mock- and HCMV-infected cells awaits further
investigation.
Effect of
CO2/HCO3
on the pHi Response to a Hyperosmotic
Challenge
Figure 4 shows the results of increasing
the osmolality of the standard
CO2/HCO3
bathing solution to 146% of the normal, control osmolality. For the
mock- and HCMV-infected cells, hyperosmotic challenge resulted in an
alkalinization of pHi. This
alkalinization appeared to have two components: an initial, small, but
relatively rapid, DEA- and
H2DIDS-insensitive alkalinization and a somewhat slower, but much larger, DEA-sensitive alkalinization. The DEA-insensitive alkalinization was not observed in the absence of
CO2/HCO
3;
in fact, a slight acidification was observed (10). The basis of this
rapid,
CO2/HCO
3-dependent, DEA-, H2DIDS-insensitive
alkalinization is uncertain. However, consider that a reduction of cell
volume would increase
[HCO
3]i as well as intracellular PCO2.
Because CO2 will rapidly reequilibrate across the cell membrane, the net effect of the cell
volume reduction would be an increase of
[HCO
3]i relative to intracellular PCO2, which
would translate into a rise of
pHi.
|
Although mock- and HCMV-infected cells exhibited a secondary, DEA-sensitive alkalinization, the peak pHi reached by the mock-infected cells was somewhat greater than that reached by the HCMV-infected cells (~0.2 vs. 0.14 pH unit; not significant). In addition, Fig. 4 shows that mock- and HCMV-infected cells reached this peak pHi ~6 min after the onset of the hyperosmotic challenge. After that peak, however, there were significant differences between the pHi behaviors of the two cell treatments in the continued presence of the hyperosmotic challenge. The pHi of mock-infected cells changed very little over the next 8 min (Fig. 4). However, the pHi of the HCMV-infected cells decreased ~0.1 pH unit (Fig. 4) over this same period. This "recovery" of pHi in the HCMV-infected cells was statistically significant (P < 0.0001, paired t-test) and was abolished by treatment with 100 µM H2DIDS (Fig. 4, 6 min). In addition, treatment with H2DIDS caused the peak pHi to be significantly more alkaline for HCMV-infected cells (P < 0.05, unpaired t-test). As noted above, DEA treatment prevented the slow, secondary phase of alkalinization for both mock- and HCMV-infected cells.
Effect of
CO2/HCO3
on
[Cl
]i
in Mockand HCMV-Infected Cells
|
Effect of HCMV Infection on Net Cl
Efflux in
CO2/HCO
3-Free
Media
Cells were bathed with a nominally
CO2/HCO3-free
medium for at least 15 min before removal of extracellular Cl
. This ensures that
pHi is at its new steady state and
that
[HCO
3]i
0 mM. Figure 5 shows that, under these
conditions, when extracellular Cl
is replaced (with
gluconate), the
[Cl
]i
of HCMV-infected cells decreased much more slowly than that of
mock-infected cells. The data points between 2 and 20 min after the
Cl
replacement were fitted
with a single-exponential function with the assumption that the
[Cl
]i
asymptotically approaches 0 mM. The resultant calculated rate constants
are summarized in Table 2. We previously
showed a 1.44 times greater cell volume-to-plasma membrane surface area
ratio of HCMV- than mock-infected cells (10). To meaningfully compare the relative rates of Cl
movement of the mock- and HCMV-infected cells, this morphological effect needs to be taken into consideration. To facilitate this comparison of the relative rates of
Cl
movement, we have
multiplied the experimentally determined rate constants in Table 2 by a
normalized volume-to-surface area ratio. This ratio is defined as 1 for
the mock-infected cells and is therefore equal to 1.44 for
HCMV-infected cells 72 h after exposure (10). After applying this
correction (Table 2), we see that 1)
in the absence of
CO2/HCO
3,
Cl
is lost about twice as
fast from mock-infected cells as from HCMV-infected cells and
2) in the absence of
CO2/HCO
3, treatment with 100 µM H2DIDS
does not reduce the rate of
Cl
loss by either cell
treatment (in fact, some stimulation was noted).
|
|
Effect of HCMV Infection on Net Cl
Efflux in
CO2/HCO
3-Containing
Media
|
H2DIDS-Sensitive Net
Cl Efflux Stimulated by External
NO
3
|
Figure 7B, in contrast, shows the
effect of the same protocols on HCMV-infected cells 72 h after
infection. In six such experiments, NO3 replacement resulted in a loss
of intracellular Cl
with a
rate constant of 0.182 ± 0.013 min
1. Treatment with 100 µM H2DIDS reduced the rate
constant by 54%, to 0.083 ± 0.004 min
1. In three experiments
we examined the effects of NO
3 replacement on cells infected 24 h before their assay. In this case the
control rate constant was 0.152 ± 0.005 min
1, and after
H2DIDS treatment it decreased
35%, to 0.099 ± 0.017 min
1. The fact that the net
Cl
efflux caused by
NO
3 substitution was much greater
in the HCMV- than in the mock-infected cells and that Cl
loss in the
HCMV-infected cells was largely prevented by
H2DIDS treatment suggests that
NO
3 exchanges with
Cl
via the
Cl
/HCO
3
exchange mechanism and that this mechanism is much more active in the
HCMV- than in the mock-infected cells.
Figure 8 compares the
H2DIDS-sensitive rate constants
for Cl loss in exchange for
NO
3 in mock- and HCMV-infected (24 and 72 h after exposure) cells. It indicates that HCMV induces an
infection time-dependent increase in the rate constants for H2DIDS-sensitive
Cl
efflux. Thus, within 24 h of exposure to the virus, the activity of the
H2DIDS-sensitive pathway,
presumably the
Cl
/HCO
3
exchanger, is almost doubled, and it increases further between 24 and
72 h after exposure. Application of the normalized volume-to-surface
area ratio correction for the 72-h-postexposure cells shows that HCMV
infection increased the index for the rate of
Cl
loss for
H2DIDS-sensitive
Cl
by ~5.3 fold, from
~0.027 to 0.143.
|
pHi Sensitivity of
H2DIDS Net
Cl Efflux
Figure 9 shows the results of varying the
pHi (accomplished by varying
pHo; see
METHODS) on the rate constant of net
Cl efflux caused by
replacing external Cl
with
NO
3. In the case of the
mock-infected cells, the
H2DIDS-sensitive component of the
net Cl
efflux decreased as
pHi was increased. However, in
HCMV-infected cells the
H2DIDS-sensitive component was
more than doubled as pHi was
increased from 6.4 to 7.8. Thus
Cl
/NO
3
exchange in HCMV-infected cells displays a
pHi sensitivity suggestive of its
being mediated by a
Cl
/HCO
3
exchanger.
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DISCUSSION |
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HCMV Infection Causes Increased
Cl/HCO
3
Exchanger Activity
pHi consequences.
We have obtained several results that lead us to the conclusion that
HCMV infection results in a significant enhancement of Cl/HCO
3
exchanger activity. Removal of external Cl
creates a favorable
thermodynamic gradient for the outward net movement of cellular
Cl
. If this outward net
movement is obligatorily linked to an inward net movement of
HCO
3 via the
Cl
/HCO
3
exchanger, an H2DIDS-sensitive
intracellular alkalinization will result. When we removed external
Cl
in the presence of
CO2/HCO
3,
the pHi of HCMV-infected MRC-5
cells (72 h after exposure) became ~0.3-0.5 pH unit more alkaline (Fig. 3). In contrast, the
pHi of mock-infected cells is made
acidic by the same treatment (Fig. 3). The alkalinization induced in
the HCMV-infected cells by external
Cl
removal was prevented by
treatment with 100 µM H2DIDS, a
well-known inhibitor of the
Cl
/HCO
3
exchanger (6). Thus these observations regarding the effects of
removing external Cl
on
pHi are consistent with the view
that HCMV infection strongly enhances
Cl
/HCO
3
exchanger activity in MRC-5 cells.
[Cl]i
consequences.
Further evidence that HCMV enhances the
Cl
/HCO
3
exchanger mechanism comes from studies on
[Cl
]i.
Using MQAE, we found that the steady-state
[Cl
]i
of HCMV-infected cells bathed in
CO2/HCO
3-containing solution was 63% higher than that of mock-infected cells (Table 1).
Part, but not all, of this difference can be attributed to HCO
3-dependent mechanisms. Thus
treatment with H2DIDS (Figs.
6B and
7B) reduced the resting
[Cl
]i
for HCMV-infected cells. In addition, bathing mock- and HCMV-infected cells in HEPES-buffered solutions resulted in a reduction of
[Cl
]i
(Table 1). For mock-infected cells the decrease was relatively small,
declining from 58 to 53 mM; for HCMV-infected cells,
[Cl
]i
fell from 95 to 78 mM. In keeping with our
pHi results, the apparent
contribution of a
CO2/HCO
3-dependent process is much greater for the HCMV- than for the mock-infected cells.
However, even in the nominal absence of
CO2/HCO
3, the two cell treatments have significantly different
[Cl
]i.
Effects of HCMV Infection of Other
Cl Transport Pathways
For the HCMV-infected cells,
Cl/HCO
3
exchange is an important means of
Cl
transport into and out
of the cell. In the absence of
CO2/HCO
3, Cl
efflux from
HCMV-infected cells is extremely slow, despite the fact that
[Cl
]i
is much higher than in the mock-infected cells. This may suggest that
HCMV infection has decreased the electrochemical driving force (by
causing membrane potential depolarization; see below). That would cause
decreased net Cl
movement
through channels. Alternatively, HCMV infection might result in
inhibition of the Cl
channels.
Why Is
[Cl]i
So High in HCMV-Infected Cells?
In addition to effects on possible intracellular
Cl-loading
processes discussed above, HCMV infection might reduce the membrane resting potential so that the higher
[Cl
]i
would result from some voltage-sensitive pathway for
Cl
transmembrane movement.
Finally, HCMV infection might reduce the activity of
Cl
transport processes,
which would lower
[Cl
]i,
such as
K+-Cl
cotransport and/or
Cl
channels. Final
resolution of the basis of the high
[Cl
]i
in HCMV-infected cells awaits further studies.
CO2/HCO3
Treatment Induces NHE Osmosensitivity in Mock-Infected Cells
In addition to the presence of
CO2/HCO3,
there is another difference in the experimental conditions of the two
sets of studies. The earlier study used sucrose to increase the
osmolality, whereas we used NaCl in the present study. However, the
difference in the osmolyte used to increase the external fluid osmolality is unlikely to explain the difference. Control experiments performed in the presence of
CO2/HCO
3
showed that increasing the osmolality of the solution bathing
mock-infected cells with sucrose also stimulated a DEA-sensitive
intracellular alkalinization (not shown). Thus we must consider that it
is the presence of
CO2/HCO
3
that has, in some way, changed or greatly enhanced the response of the
NHE to the hyperosmotic challenge.
Changes of
[Cl]i
have been shown to play a modulatory role in the activity of the NHE
(16, 27). However, our results (Table 1) show only a modest increase of
[Cl
]i
in the mock-infected cells caused by bathing in
CO2/HCO
3 solution. Chen and Boron (8) suggested that there could be an
extracellular HCO
3 "receptor"
that activates the NHE in a way to make it much more
sensitive to cell volume changes. A clear resolution of this
intriguing effect on the behavior of the NHE of changing from a
CO2/HCO
3-free to a
CO2/HCO
3containing
fluid awaits further study.
HCMV Infection Causes Concurrent Increases in Activities of NHE
and
Cl/HCO
3
Exchange
As discussed above, our present results provide strong evidence that
HCMV infection, in addition to its effects on NHE activity, also
substantially increases the activity of the
Cl/HCO
3
exchanger. The combination of increased activity of these two ion
exchangers, coupled by the NHE-induced pHi alkalinization, has often been
demonstrated to effect an increase in cell volume after cell shrinkage
(18, 24). It is possible that their combined increased activity may be
responsible, at least in part, for the cell swelling so characteristic
of HCMV infection. In support of that view is the fact that the
increase in
Cl
/HCO
3
exchanger activity is already apparent 24 h after exposure to the virus
(Fig. 8), well in advance of the increase of cell size that begins at
~36-48 h after exposure (1).
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ACKNOWLEDGEMENTS |
---|
We gratefully acknowledge the excellent technical assistance of Joshua C. Russell, Charles Rassier, and Kenneth Wilson.
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FOOTNOTES |
---|
This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-11946 (J. M. Russell).
Preliminary results have been presented in abstract form (9, 11, 12, 23).
Present address of A. A. Altamirano: Dept. de Microbiología, Facultad de Medicina, Universidad de Buenos Aires, Paraguay 2155, 1121 Buenos Aires, Argentina.
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. §1734 solely to indicate this fact.
Address for reprint requests: J. M. Russell, Dept. of Physiology, Allegheny University of the Health Sciences, 2900 Queen Ln., Philadelphia, PA 19129.
Received 6 February 1998; accepted in final form 20 April 1998.
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