1 Department of Physiology, Allegheny University of the Health Sciences, Philadelphia, Pennsylvania 19129; and 2 Instituto de Investigaciones Cardiológicas, Facultad de Medicina, Universidad de Buenos Aires, 1122 Buenos Aires, Argentina
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ABSTRACT |
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We examined the effects of human cytomegalovirus (HCMV)
infection on the
Na+-K+-Cl
cotransporter (NKCC) in a human fibroblast cell line. Using the Cl
-sensitive dye MQAE, we
showed that the mock-infected MRC-5 cells express a functional NKCC.
1) Intracellular
Cl
concentration
([Cl
]i)
was significantly reduced from 53.4 ± 3.4 mM to 35.1 ± 3.6 mM
following bumetanide treatment. 2)
Net Cl
efflux caused by
replacement of external Cl
with gluconate was bumetanide sensitive.
3) In
Cl
-depleted mock-infected
cells, the Cl
reuptake rate
(in HCO
3-free media) was reduced in
the absence of external Na+ and by
treatment with bumetanide. After HCMV infection, we found that although
[Cl
]i
increased progressively [24 h postexposure (PE), 65.2 ± 4.5 mM; 72 h PE, 80.4 ± 5.0 mM], the bumetanide and
Na+ sensitivities of
[Cl
]i
and net Cl
uptake and loss
were reduced by 24 h PE and abolished by 72 h PE. Western blots using
the NKCC-specific monoclonal antibody T4 showed an approximately
ninefold decrease in the amount of NKCC protein after 72 h of
infection. Thus HCMV infection resulted in the abolition of NKCC
function coincident with the severe reduction in the amount of NKCC
protein expressed.
bumetanide; intracellular chloride concentration, MRC-5 fibroblasts
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INTRODUCTION |
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HOST CELLS INFECTED WITH replicating human cytomegalovirus (HCMV) virions undergo a characteristic enlargement termed cytomegaly (e.g., Ref. 1). Despite the progress made in understanding the cascade of events required for host cell activation after HCMV infection, there is very limited information regarding the basis of the development of the host cell enlargement (1). However, evidence is accumulating to support the view that the enlargement could be due, at least in part, to an osmotically coupled uptake of water and inorganic ions. For example, the late infection phase during which cytomegaly develops is characterized by a sustained increase in Na+-K+-ATPase (Na+ pump) activity (12, 27) and in the number of Na+ pumps per cell (2). An important role for the Na+/H+ exchanger also seems likely in view of the findings of Fons et al. (12), who showed that HCMV replication can be substantially reduced by treating infected cells with amiloride, and those of Crowe et al. (11), who showed that HCMV infection caused a stimulation of Na+/H+ exchanger activity.
Usually, inorganic ion-driven increases of cell volume involve not only
Na+ but also an anion. The anion
most often involved is Cl.
In this regard, it is interesting that removal of external
Cl
from the incubation
media substantially reduced the effect of HCMV infection to increase
the number of ouabain binding sites (2). Furthermore, we recently
showed that
Cl
/HCO
3
exchanger activity is greatly increased in HCMV-infected cells
(21). By analogy with well-described cell volume
regulatory processes found in normal cells (e.g., Ref. 14), these
observations suggest that the combined activity of these two ion
transporters results in a net uptake of
Na+ and
Cl
. Most of the
Na+ is exchanged for
K+ via the enhanced activity of
the
Na+-K+-ATPase,
with the overall result being that the cells take up an isosmotic
solution of K+,
Na+, and
Cl
.
Such a mechanism would imply that as cell volume increased, so would
intracellular Cl
concentration (for instance, via uptake of a
high-Cl
, isosmotic fluid).
We recently showed that
[Cl
]i
increased 25-37 mM within 72 h after exposure to HCMV (21), a time
when host cell volume is estimated to have increased three- to
four-fold (2). However, only part of this
[Cl
]i
increase (~17 mM of 37 mM increase) was the result of the increased Cl
/HCO
3
exchanger activity, leaving unidentified the mechanism of about
one-half of the overall increase in
[Cl
]i
.
The combined activity of the
Na+/H+
exchanger and the
Cl/HCO
3
exchanger could, in principle, account for the observed volume increase
during cytomegaly. However, several groups have reported that, in
addition to the combined activity of the two exchangers mentioned
above, some cells may use a second process at the same time (e.g.,
Refs. 30, 32). Thus increased activity by
Na+-K+-Cl
cotransporter (NKCC, a
member of the SLC12 gene family) is an obvious candidate to account for
the remaining Cl
uptake.
One of the functions generally attributed to the NKCC is that of
increasing the volume of cells that have shrunk below normal values
(e.g., Ref. 13). Also, the NKCC has been implicated in T lymphoblastoid
cell swelling as a result of human immunodeficiency virus (HIV)
infection (40). There is also evidence in some cells that the NKCC
participates in the moment-to-moment maintenance of normal cell volume
(e.g., Ref. 28). Thus upregulation of this ion transporter might be
reasonably expected to result in an increase of cell volume.
Beyond the unaccounted-for rise in
[Cl]i
(see above), there are several other reasons to suspect that HCMV
infection might affect the level of NKCC activity. HCMV infection
induces increased expression levels of cellular transcription factors
SP1 and nuclear factor-
B (NF-
B; Refs. 42, 43). Both
known isoforms of the NKCC (NKCC1 and NKCC2) contain consensus
recognition sites on their promoter for NF-
B (15, 31). In addition,
HCMV-infected cells synthesize and secrete cytokines such as
interleukin (IL)-6 and IL-1
(33) as well as interferon (IFN)-
(5). IL-1
and IL-6 have been shown to upregulate NKCC mRNA levels
and NKCC protein expression levels (35, 37) and functional activity
(35). At higher concentrations, IL-6 will inhibit NKCC activity (35). Conversely, another cytokine, IFN-
, has been shown to inhibit NKCC
transport activity and to reduce levels of
[3H]bumetanide binding
(10). In addition, IFN-
pretreatment prevented the stimulatory
effects of IL-1
treatment on NKCC protein expression levels (37). It
is also of interest that Nokta et al. (27) showed that the
ouabain-insensitive 86Rb uptake
(often found to be predominantly via the NKCC) was greatly reduced
within 24 h of HCMV infection, suggesting that the NKCC may have been
inhibited by the infection.
Thus it seemed highly possible that HCMV infection might alter NKCC activity. The present study was designed to determine what effects HCMV infection has on NKCC function and on levels of NKCC protein expression.
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METHODS |
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Cell culture and HCMV infection. Details of cell culture and HCMV infection protocols were presented previously (11). Briefly, a cell line (MRC-5; American Type Culture Collection) derived from human embryo lung fibroblasts, passages 22-28, 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 in air at 37°C. A stock of HCMV (strain AD169; originally a generous gift from Dr. T. Albrecht, Dept. of Microbiology, University of Texas Medical Branch, Galveston, TX) was generated in confluent MRC-5 cells (see Ref. 2 for more details).
For experiments measuring [ClNKCC transport activity measurements.
The transport activity of NKCC was assessed as the bumetanide- and
external Na+ concentration
([Na+]o)-sensitive
net movements of Cl either
into or out of the cells. We used the fluorescent dye N-(6-methoxyquinolyl)acetoethyl ester
(MQAE, Molecular Probes; Ref. 39) to measure the
[Cl
]i
as previously described (21). Briefly, cells grown on a glass coverslip
were loaded with MQAE by bathing them in a 10 mM solution of MQAE
(dissolved in MEM; 0% FCS) in the incubator (5%
CO2; 37°C) for 2-3 h. The
coverslip was then mounted in an SLM-Aminco spectrofluorometer (model
DMX-1000). Experiments were performed at room temperature to minimize
fluorescent dye loss. All experimental solutions contained 20 mM HEPES
(cf. Ref. 17) and had a constant osmolality of 285 mosmol/kgH2O (cf. Ref. 16).
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Standard solutions and reagents. Standard HEPES-buffered solution contained (in mM) 128 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 10 glucose, and 20 HEPES. In all HEPES-buffered solutions, pH was adjusted to 7.4 with N-methyl-D-glucamine (NMDG), and the osmolality was 285 ± 5 mosmol/kgH2O. For Na+-free HEPES solution, NaCl was replaced with NMDG chloride. NaCl-free HEPES solution contained (in mM) 120 NMDG gluconate, 5 potassium gluconate, 1 magnesium gluconate, 2 calcium gluconate, 10 glucose, and 20 HEPES (pH 7.4, osmolality 285 ± 5 mosmol/kgH2O).
Standard CO2/HCOGel electrophoresis and Western blotting. For Western blot analysis, mixed microsomal membranes were isolated as follows from confluent MRC-5 cells that were either mock- or HCMV-infected (72 h PE) following the method of Sun et al. (34). Cells were washed two times with ice-cold PBS (pH 7.4) and collected by centrifugation at 3,000 rpm for 10 min at 4°C. The pellet was resuspended in homogenization buffer containing (in mM) 25 Tris, 2 MgCl2, 1 EDTA, 20 µM leupeptin, and 1 phenylmethylsulfonyl fluoride (PMSF) (pH 7.4) and was sonicated at 4°C (SON-IM-1 sonicator; Heat Systems, Farmingdale, NY). After removal of cellular debris by 4 min of centrifugation at 3,000 rpm, the supernatant was centrifuged at 100,000 g for 30 min. The resulting crude membrane preparation was resuspended in membrane buffer containing (in mM) 2.9 Tris, 0.29 EDTA, 20 µM leupeptin, and 1 PMSF (pH 7.4). Protein content for each preparation was determined by the Lowry assay using Bio-Rad DC protein assay (Bio-Rad). We observed that the amount of protein per 150 × 25-mm petri dish obtained from HCMV-infected cells was 1.76 ± 0.42 (n = 3) times the amount obtained from an identical dish on which mock-infected cells were grown. This is despite the fact that, by actual cell count, the plates containing HCMV-infected cells had only 75% as many cells as the plates containing mock-infected cells (see below).
For Western blotting, membrane protein samples and prestained molecular mass markers (Bio-Rad) were denatured in SDS reducing buffer (2% SDS, 1.5% dithiothrietol, 62 mM Tris · HCl, pH 6.8, 10% glycerol, 0.012% bromphenol blue) and were heated at 70-80°C for 4 min. The samples were then electrophoretically separated on 7.5% SDS gels (Mini-PROTEAN II, Bio-Rad), and the resolved proteins were electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes for 1 h (100 V, 4°C). The blots were incubated overnight in blocking buffer (Western-Light Plus kit, Tropix) at 4°C. The blots were subsequently incubated for 1 h at room temperature with the monoclonal antibody. After three washes with blocking buffer (Western-Light Plus kit), the blots were incubated for 30 min at room temperature with biotinylated secondary antibody (goat anti-mouse IgG-IgM, 1:10,000 dilution), followed by two or three washes with blocking buffer to remove unbound secondary antibody. The PVDF membrane was further treated for 20 min with alkaline phosphatase-conjugated streptavidin, washed in blocking buffer, and treated with assay buffer (Western-Light Plus kit) before it was immersed for 5 min in chemiluminescent CSPD substrate for the alkaline phosphatase. X-ray film (Fuji-RX) was exposed to the PVDF membrane between 30 s and 3 min. Two different antibodies were used in this study. For detection of the NKCC, we used the monoclonal antibody T4, which was developed against the carboxy-terminal 310 amino acids of the human colonic NKCC (NKCC1) but recognizes both NKCC1 and NKCC2 isoforms (10). For detection of the Na+ pump or Na+-K+-ATPase, we used the monoclonal antibodyCell counting. To quantitatively evaluate the results of the Western blotting experiments, it was necessary to determine the number of cells per plate in mock- and HCMV-infected cells grown to confluence. Cells were grown with the same seeding, culturing, and infection conditions previously described for either spectrofluorometric or Western blot studies. At 72 h PE, they were fixed using 10% Formalin for 24 h. The Formalin was removed, and the cells were stained by exposure to 0.03% methylene blue for 24 h. The fixed and stained cells were washed with water and photographed using a Nikon camera mounted to a Nikon microscope. Final magnification was ×16. At the same time, a micrometer grid was photographed to permit calculation of the size of the photographic field, which was 1.23 mm2. The culture dishes had a diameter of 35 mm and a surface area of 962 mm2, so one photographic field represented 1/782 of the entire dish. We sampled three photographic fields of each culture dish. In three separate determinations, we found that the number of HCMV-infected cells per dish was 74.1 ± 4.9% the number of mock-infected cells per dish.
Data are representative or are presented as means ± SE. The [Cl ![]() |
RESULTS |
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Effect of bumetanide on
[Cl]i.
We previously reported that the
[Cl
]i
of the host MRC-5 cells increased dramatically 72 h PE to HCMV. The
increase was noted in the absence as well as in the presence of
CO2/HCO
3 (21). The focus of the present study was to characterize the effects of
HCMV infection on NKCC activity; therefore, we omitted CO2/HCO
3
from the bathing solution. Cells were bathed with the HEPES-buffered
standard solution for 15 min before determination of
[Cl
]i.
The results of these studies are seen in Fig.
2, which illustrates that there is
progressive increase in
[Cl
]i
as the HCMV infection progresses. Thus the
[Cl
]i
of mock-infected cells was 53.4 ± 3.4 mM
(n = 12). HCMV-infection resulted in a
statistically significant increase of
[Cl
]i
relative to the mock-infected cells (24 h PE, 65.2 ± 4.5 mM, n = 9, P < 0.05; 72 h PE, 80.4 ± 5.0 mM, n = 22, P < 0.001).
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External
[Cl]-dependent net
intracellular Cl
loss.
We previously demonstrated that in
CO2/HCO
3-free
solutions the rate of net intracellular
Cl
loss into
Cl
-free
(gluconate-substituted) solution is significantly reduced by 72 h of
HCMV infection (21). In the present study, we examined the effect of
bumetanide on the rate of net intracellular
Cl
loss caused by bathing
the cells in a Cl
-free
solution (gluconate substituted for
Cl
;
CO2/HCO
3-free).
These experiments were performed to obtain further evidence for the
functional correlates of the expression of the NKCC protein in our
cells. In addition, we wanted to know whether the HCMV-induced
reduction in the rate of Cl
loss was detectable within 24 h PE. Figure
3 shows six different representative
examples of the effects on
[Cl
]i
of replacing extracellular
Cl
with gluconate. These
experiments were performed on mock-transfected and 24- and 72-h PE
HCMV-infected cells in the absence and presence of 10 µM bumetanide.
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External Na+
removal causes net loss of intracellular
Cl.
If the bumetanide-sensitive net loss of intracellular
Cl
is the result of net
Cl
efflux through the NKCC,
then removal of external Na+ ought
to similarly cause a fall of
[Cl
]i.
Table 1 gives the average rate constants for the net decrease of
[Cl
]i
caused by removing external Na+
(NMDG replacement) for each cell treatment. In a pattern similar to
that noted for Cl
loss into
Cl
-free solutions, the rate
of
[Na+]o-dependent
decline of
[Cl
]i
was greatest in the mock-infected cells, it was much smaller at 24 h
PE, and by 72 h PE it was nearly zero.
Net Cl uptake is inhibited by
bumetanide.
Cells were depleted of Cl
by exposing them to
Cl
-free,
CO2/HCO
3-containing
solution while continually monitoring [Cl
]i
for 20-30 min. Then, the external solution was changed to a HEPES-buffered Cl
-free
solution for an additional 15 min. The
CO2/HCO
3-containing solution was used because
Cl
/HCO
3
exchange permits a rapid intracellular Cl
depletion for the
HCMV-infected cells (see METHODS and
Fig. 1; also see Ref. 21). As seen in Fig.
4, such treatment reduced [Cl
]i
to very near 0 mM in mock-infected cells and 24-h PE HCMV-infected cells but could not completely deplete cellular
Cl
from the 72-h PE
HCMV-infected cells. The reason or reasons for this are unclear but
could, in principle, be related to a Donnan-like effect resulting from
the presence of positively charged intracellular macromolecules and the
inevitable dilution of other anions caused by the increase in cell
volume and
[Cl
]i.
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Effect of removal of external
Na+ on
Cl uptake.
For this series of studies, we returned the external
Cl
either in the presence
of normal
[Na+]o
or in the complete absence of external
Na+ (NMDG replacement). Figure 5
shows that the rate and extent of net
Cl
reuptake were
substantially faster in the presence of external Na+ for mock-infected cells,
somewhat less dependent on Na+ for
24-h PE HCMV-infected cells, and insensitive to external Na+ removal in 72-h PE
HCMV-infected cells.
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NKCC protein expression in mock-infected and 72-h PE HCMV-infected
MRC-5 cells.
The preceding functional characterization of the
NKCC-mediated fluxes strongly suggests that mock-infected cells
functionally express the NKCC and that the cotransporter plays a major
role in the maintenance of intracellular
Cl homeostasis in
mock-infected MRC-5 cells. Our results further suggest that the
functional activity of the NKCC rapidly decreases after HCMV infection
and is, for all practical purposes, no longer present in 72-h PE
HCMV-infected cells. One possible explanation for this observation is
that HCMV infection progressively decreases the amount of expressed
NKCC protein in the plasma membrane. To test this hypothesis, we
performed a Western blot analysis on mock-infected and 72-h PE
HCMV-infected cells using the NKCC-specific monoclonal antibody T4.
This antibody recognizes a denatured NKCC polypeptide with a molecular
mass in the range 130-195 kDa, depending on the level of
glycosylation (20).
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Western blot studies on the Na+ pump in mock- and HCMV-infected MRC-5 cells. We previously measured the amount of [3H]ouabain binding in mock- and HCMV-infected cells (2) to estimate the effect of HCMV infection on Na+ pump activity. Because HCMV infection does not greatly affect the density of Na+ pumps (2), we used the number of Na+ pumps as an index of membrane surface area (11). This is because comparing differences in specific host cell protein levels between mock- and HCMV-infected cells is complex. As infection decreases the number of cells, the host cell enlarges, and as the infection progresses, an increasing fraction of the total protein is of viral origin. Our earlier findings (2) suggested that Na+ pump density (in relation to cell surface area) was little affected by HCMV infection. Therefore, if it could be shown by Western blot analysis that the Na+ pump abundance had increased as expected from the [3H]ouabain results, it would provide a useful reference protein for the comparison of the effect of HCMV infection on other host cell transport proteins such as the NKCC.
The monoclonal antibody
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DISCUSSION |
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HCMV infection reduces NKCC activity. The present work shows that mock-infected MRC-5 human fibroblasts not only express the NKCC protein in their membranes but also have a functional NKCC. It further shows that infection of the MRC-5 cells with HCMV results in a large reduction of NKCC activity as well as a large reduction of NKCC protein. The functional downregulation of the cotransporter was evident as early as 24 h PE.
Treatment with bumetanide, a relatively specific inhibitor of the NKCC in concentrations at or below 10 µM (13), resulted in a significant reduction of the [ClHCMV inhibits NKCC expression.
The virus, or products stimulated by viral infection, may interfere
with NKCC gene transcription. IFN- in T84 cells (10) and high levels
of IL-6 in endothelial cells (35) have been demonstrated to
functionally downregulate NKCC activity. HCMV-infected fibroblasts have
been shown to upregulate IFN-
and IL-6 mRNAs as well as the
expression of the proteins themselves (5, 33). Thus the reduction of
NKCC protein expression and function may be an effect of IFN-
, IL-6,
or another cytokine. In this regard, it may be of interest that several
IFNs, including IFN-
, IFN-
, and IFN-
, have been shown to
stimulate the
Na+/H+
exchanger (4, 24). This might explain why the
Na+/H+
exchanger is stimulated at the same time as the NKCC is downregulated.
What is basis of the increased
[Cl]i
caused by HCMV infection?
Maglova et al. (21) reported that 72 h PE, HCMV infection increased the
[Cl
]i
of MRC-5 cells bathed in HCO
3 saline
by ~37 mM. When the cells were bathed in HEPES saline, the
[Cl
]i
still increased by ~27 mM. Our present results confirm this latter
increase of
[Cl
]i
after 72 h of HCMV infection and extend it by showing that the increase
has already begun within 24 h of the infection, when the
[Cl
]i
had increased from 53.4 mM to 65.2 mM, an increase of ~12
mM.
HCMV reduces NKCC activity while upregulating
Na+/H+
exchanger and
Cl/HCO
3
exchanger activities.
Why does the virus downregulate the NKCC at the same time it is
upregulating the
Na+/H+
exchanger and the
Cl
/HCO
3
exchanger activities (21)? Both mechanisms import
Na+ and
Cl
, and it is reasonable to
assume that most of the imported
Na+ is exchanged for
K+ via the simultaneously
upregulated Na+ pump (Fig. 7; see
Refs. 2, 12, 27). Hence, both mechanisms would presumably result in the
net uptake of isosmotic K+ + Na+ + Cl
solution. An obvious
difference between the two approaches is that the NKCC mechanism
directly imports K+ in addition to
the K+ exchanged for
Na+, leading to the possibility
that this mechanism would result in a higher
[K+]i
than the combined
Na+/H+
exchanger and
Cl
/HCO
3
exchanger mechanism. However, as long as the
Na+ pump exchanges most of the
imported Na+ for
K+, this difference is unlikely to
be important.
Current summary of effects of HCMV on ion transport pathways.
The combined results from several laboratories show that HCMV affects a
variety of ion transporters. The effects include stimulation of the
Na+ pump (e.g., Refs. 1, 2, 27),
stimulation of the
Na+/H+
exchanger (11), inhibition of a
Na+ and stimulation of a
K+ channel (3), and stimulation of
the
Cl/HCO
3
exchanger (21). Our present results add the nearly complete loss of the
NKCC to this lengthening list of HCMV effects on ion transport
mechanisms.
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ACKNOWLEDGEMENTS |
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We acknowledge the excellent technical assistance of Charles Rassier, Junying Chen, and Xiyin Chen.
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FOOTNOTES |
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This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-11946 to J. M. Russell.
Some of these results were presented in abstract form (22, 23).
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 Lane, Philadelphia, PA 19129.
Received 18 May 1998; accepted in final form 27 July 1998.
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