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Am J Physiol Cell Physiol 275: C1330-C1341, 1998;
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Vol. 275, Issue 5, C1330-C1341, November 1998

Na+-K+-Clminus cotransport in human fibroblasts is inhibited by cytomegalovirus infection

Lilia M. Maglova1, William E. Crowe1, Peter R. Smith1, Aníbal A. Altamirano2, and John M. Russell1

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

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

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

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

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-kappa B (NF-kappa B; Refs. 42, 43). Both known isoforms of the NKCC (NKCC1 and NKCC2) contain consensus recognition sites on their promoter for NF-kappa B (15, 31). In addition, HCMV-infected cells synthesize and secrete cytokines such as interleukin (IL)-6 and IL-1beta (33) as well as interferon (IFN)-gamma (5). IL-1beta 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-gamma , has been shown to inhibit NKCC transport activity and to reduce levels of [3H]bumetanide binding (10). In addition, IFN-gamma pretreatment prevented the stimulatory effects of IL-1beta 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.

    METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References

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 [Cl-]i, the following cell culture protocol was used. Three days after seeding on 6 × 24-mm glass coverslips, confluent MRC-5 cells were exposed for 1 h to a suspension containing either HCMV at a multiplicity of infection of ~5 plaque-forming units/cell or a mock-infecting, virus-free suspension (see Ref. 2 for details of mock infection). Twenty-four hours before the cells were used [48 h postexposure (PE) to HCMV], the FCS was reduced to 1% to minimize fluorescent dye loss (see Ref. 11).

For the Western blot studies that required a mixed microsomal membrane protein isolation, MRC-5 cells were plated on 150 × 25-mm petri dishes. The cells reached confluency in 7 days. The infection and mock-infection protocols were then the same as described above for the [Cl-]i experiments.

NKCC 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).

Figure 1 shows an experiment on a mock-infected cell illustrating the general protocol used to measure net Cl- efflux or uptake. Fluorescence readings (F) were obtained using wavelengths of 365 nm for excitation and 450 nm for emission (Fig. 1A). Cells were preincubated in the appropriate saline for 5 min (segment I). Segment II shows depletion of intracellular Cl- following the replacement of external Cl- with gluconate. In this particular case, the CO2/HCO-3 buffer was used because this greatly accelerates the rate of intracellular Cl- depletion in HCMV-infected cells (21). When the CO2/HCO-3 was replaced with HEPES (segment III), there was a small decrease in F. Segment IV illustrates how we determined the reuptake rates for Cl- by returning the external Cl- to the solution bathing cells previously depleted of cellular Cl-. The F0 value was then determined (segment V) by bathing the cells with a Cl--free (KNO3) solution containing 10 µM tributyltin and 10 µM nigericin (9, 16). Fluorescence readings were corrected for background (Fbkg) using a HEPES-buffered, KSCN solution containing 5 µM valinomycin to maximally quench the MQAE ion-sensitive signal (e.g., segment VI). The raw F values, corrected for Fbkg, could then be converted to F0/F values (Fig. 1B). For the experiments reported here, the rate of dye loss was minimal, averaging ~0.0001% per minute.


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  		Fig. 1.   General protocol used to estimate changes of intracellular Cl- concentration ([Cl-]i). Experiment was designed to illustrate how we use signal from MQAE fluorescent dye to determine changes of [Cl-]i. In this example, we show results from mock-infected cells. After a period of loading with fluorescent dye, cells were bathed in control solution (segment I) to establish resting or control level of [Cl-]i. During segment II, all external Cl- was substituted with gluconate and Cl- depletion followed. Segment III represents changing buffer from CO2/HCO-3 to HEPES while maintaining external Cl- concentration at 0 mM. We then waited 15 min before proceeding with experiment. CO2/HCO-3 buffer was used to unload human cytomegalovirus (HCMV)-infected cells within a reasonable time because, unlike mock-infected cells, HCMV-infected cells have a significant Cl-/HCO-3 exchanger activity. In segment IV, we returned Cl- to external solution and followed reloading of intracellular Cl-. Segments V and VI show how, at end of each experiment, fluorescence signal was calibrated to define F0 and background F (Fbkg) (see text for details). A: emitted MQAE fluorescence (F) at 450 nm (when excited at 365 nm) expressed in arbitrary units. TBT/Nig, 10 µM tributyltin and 10 µM nigericin; KSCN/val, KSCN solution containing 5 µM valinomycin. B: MQAE fluorescence was converted to F0/F after F was corrected for Fbkg and F0 was experimentally determined using KSCN (see segment VI). C: [Cl-]i was calculated from F0/F values using experimentally determined Stern-Volmer constant. Rate constant for loss of intracellular Cl- (Cl- efflux rate constant) was then calculated from best monoexponential function fit to [Cl-]i vs. time relation (line through data points). D: instantaneous rate of net changes of [Cl-]i (d[Cl-]i/dt) was computed for both net Cl- efflux and uptake, and maximal d[Cl-]i/dt (d[Cl-]i/dtmax) is indicated by dashed line.

The F0/F values are directly proportional to [Cl-]i, the proportionality constant being the Stern-Volmer constant (KSV), according to the equation [Cl-]i = [(F0/F) - 1]/KSV. The KSV was determined in a separate set of experiments described previously (21). The KSV values we obtained (mock-infected cells, 25.7 M-1; 72-h PE HCMV-infected cells, 19.7 M-1) are in good agreement with those reported by others (e.g., Ref. 16) for other cell types. We used this relationship to convert the F0/F values to [Cl-]i values (Fig. 1C).

In many experiments, we quantitatively compared the effects of various treatments on the rate of net Cl- efflux. To do this, we fitted the [Cl-]i data vs. time (over period of 2-20 min after removal of external Cl-) to a monoexponential function, assuming that [Cl-]i asymptotically approaches 0 mM, to determine the rate constants (Fig. 1C; see Ref. 21). For experiments measuring the rate of net Cl- uptake, we calculated the instantaneous rate of change of [Cl-]i against time using the first order derivative (d[Cl-]i/dt; Fig. 1D) as we had no way of knowing the asymptotic value of [Cl-]i that each treatment would approach. The maximal d[Cl-]i/dt was used for all comparisons.

At concentrations >1 µM, bumetanide emits significant fluorescent light at 450 nm when excited at 365 nm (MQAE assay conditions). Therefore, in all experiments involving the use of bumetanide, the agent was present throughout the experiment and during the calibration procedure.

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/HCO-3-buffered solution contained (in mM) 123 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 10 glucose, and 25 NaHCO3; pH was adjusted to 7.4 by bubbling the solution with 5% CO2. The osmolality was 285 ± 5 mosmol/kgH2O. Cl--free CO2/HCO-3 solution had Cl- replaced with gluconate.

Bumetanide (Sigma, St. Louis, MO) was prepared as a 40 mM stock solution in ethanol and used at final concentrations of between 1 and 10 µM, as indicated. Tributyltin (Fluka, Milwaukee, WI) and nigericin (Sigma) 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) was also prepared as an ethanol stock solution (9 mM) and added to the saline solution to obtain a final concentration of 5 µM.

Gel 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 antibody alpha 5 (36), which is directed against the alpha -catalytic subunit of chicken Na+-K+-ATPase. Both antibodies were obtained from the Developmental Studies Hybridoma Bank (Iowa City, IA).

Cell 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-]i data were analyzed using Student's t-test.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

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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  		Fig. 2.   Effect of bumetanide on [Cl-]i. [Cl-]i was measured in mock-infected and 24- and 72-h postexposure (PE) HCMV-infected cells. Cells were bathed for 15 min in standard HEPES-buffered solution with 5 µM bumetanide or without bumetanide. Number of experiments for each treatment is shown in parentheses. [Cl-]i of mock-infected, control cells (53.4 ± 3.4 mM) was significantly lower than [Cl-]i of 24-h PE HCMV-infected cells (65.2 ± 4.5 mM, P < 0.05) and [Cl-]i of 72-h PE HCMV-infected cells (80.4.2 ± 5.0 mM, P < 0.001). Effect of bumetanide treatment to decrease [Cl-]i was statistically significant for mock-infected cells ([Cl-]i 35.1 ± 3.6 mM, P < 0.0001) and 24-h PE HCMV-infected cells ([Cl-]i 47.7 ± 1.7 mM, P < 0.0001) but not for 72-h PE HCMV-infected cells ([Cl-]i 70.7 ± 3.5 mM).

At concentrations of 10 µM or less, bumetanide is a potent and selective inhibitor of the NKCC (e.g., Ref. 13). To determine whether the NKCC played a role in determining the [Cl-]i, we exposed mock-infected and 24- and 72-h PE HCMV-infected cells to this inhibitor. Fifteen minutes of treatment with bumetanide (5 µM; identical results were obtained with 10 µM bumetanide; data not shown) reduced the [Cl-]i of mock-infected cells by 18.3 mM to 35.1 ± 3.6 mM (n = 9; P < 0.0001). Longer treatment with bumetanide did not result in a greater fall of [Cl-]i (data not shown). The same treatment reduced [Cl-]i in 24-h PE HCMV-infected cells by 17.5 mM, to 47.7 ± 1.7 mM (n = 8; P < 0.0005). Seventy-two hours after HCMV infection, bumetanide treatment had no statistically significant effect on [Cl-]i. Analysis of these data confirm our earlier observation that [Cl-]i increases following HCMV infection and extend it by showing that [Cl-]i has already significantly increased within 24 h of the onset of the infection. Further consideration of the data suggests that the NKCC plays an important role in the homeostatic maintenance of [Cl-]i in mock-infected and 24-h PE HCMV-infected cells. However, by 72 h PE, there is no evidence of an NKCC role in the maintenance of [Cl-]i, as the application of bumetanide had no significant effect on [Cl-]i.

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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  		Fig. 3.   Effect of bumetanide on decrease of [Cl-]i caused by bathing cells with a Cl--free solution. After an initial period of 15 min in standard HEPES solution, external bathing solution was switched to Cl--free HEPES solution (arrows). Filled symbols are control experiments and open symbols are from experiments in which 10 µM bumetanide was applied 15 min before removal of external Cl-. Lines through data points are best fits to a monoexponential function, assuming an asymptotic approach of [Cl-]i to 0 mM. A: effect of external Cl- removal with or without bumetanide in mock-infected cells. B: effect of external Cl- removal with or without bumetanide in 24-h PE HCMV-infected cells. C: effect of external Cl- removal with or without bumetanide in 72-h PE HCMV-infected cells. Data are from representative experiments. Calculated rate constants from at least 3 such experiments are collated and presented in Table 1.

We used a monoexponential function to fit the data points between 2 and 20 min after the external Cl- was replaced, assuming that [Cl-]i was asymptotically approaching 0 mM. Table 1 is a collation of these calculated rate constants, pooled and averaged for all the experiments we conducted using this protocol. These data show that the rate of Cl- loss by mock-infected cells was about twice that of HCMV-infected cells. However, ~50% of the rate of [Cl-]i decrease in mock-infected cells was due to a bumetanide-sensitive process, presumably the NKCC. In contrast, HCMV-infected cells had a substantially lower rate constant of decline of [Cl-]i than did mock-infected cells (cf. Ref. 21), and little of this decline is bumetanide sensitive.

                              
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  		Table 1.   Effects of HCMV infection and bumetanide on rate of decrease of [Cl-]i

We previously showed that, 72 h postinfection, the HCMV-infected cells have a ratio of cell volume to surface area that is estimated to be 1.44 times greater than mock-infected cells (11). One can obtain an index of the rates of net Cl- loss by the 72-h PE HCMV-infected cells relative to those of the mock-infected cells by multiplying the measured rate constants of the 72-h PE HCMV-infected cells by 1.44 (see Ref. 21). Doing this shows us that the rate of net Cl- loss by 72-h PE HCMV-infected cells in the presence of bumetanide is essentially the same as that observed in the mock-infected cells (0.086 min-1 vs. 0.08 min-1).

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.

If removal of external Na+ reduces [Cl-]i by preventing Cl- uptake via the NKCC, leaving only NKCC-mediated Cl- efflux, then treatment with bumetanide should reduce or abolish net Cl- loss. If 10 µM bumetanide was present when external Na+ was removed, the rate of net Cl- loss was reduced by ~90% in mock-infected cells (Table 1). Of the remaining external Na+-dependent [Cl-]i loss in the 24-h PE HCMV-infected cells, about two-thirds was blocked by treatment with 10 µM bumetanide, whereas bumetanide had no measurable effect in the 72-h PE HCMV-infected cells.

Table 1 permits a comparison between the effects of removing external Cl- and removing external Na+. Note that in the mock-infected cells the magnitude of the bumetanide-sensitive component of the rate constants for net Cl- loss was about the same for both treatments and that nearly all the [Na+]o-dependent Cl- loss was prevented by bumetanide.

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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  		Fig. 4.   Effect of bumetanide on net Cl- uptake. Cells were first depleted of intracellular Cl- by exposure for 15-35 min to Cl--free (gluconate-substituted, CO2/HCO-3-buffered) solution, followed by 15-min exposure to Cl--free, HEPES-buffered solution ± 10 µM bumetanide. At arrows, external Cl- was returned either without (filled symbols) or with bumetanide (open symbols). Each trace is composite of a number of individual experiments indicated as means ± SE of data taken every minute. A: mock-infected cells (control, n = 3; bumetanide-treated, n = 3). B: 24-h PE HCMV-infected cells (control, n = 4; bumetanide-treated, n = 2). C: 72-h PE HCMV-infected cells (control, n = 5; bumetanide-treated, n = 3).

When Cl- was returned to the external solution in the absence of bumetanide, [Cl-]i increased in mock-infected (Fig. 4A) as well as in 24-h PE (Fig. 4B) and 72-h PE (Fig. 4C) HCMV-infected cells. The rate of [Cl-]i recovery was much faster for mock-infected cells than for HCMV-infected cells. In cells treated with 10 µM bumetanide for 15 min before and after the return of extracellular Cl-, the recovery of [Cl-]i by mock-infected cells was much more inhibited than that of the HCMV-infected cells.

We calculated the first order derivative to determine the rate of [Cl-]i increase (i.e., d[Cl-]i/dt) after the external Cl- was returned. The maximal rates of the [Cl-]i increase in the absence and presence of 10 µM bumetanide are summarized in Table 2. This maximal rate occurred ~2 min (range 1.6-2.6 min) after the external Cl- was returned to the bathing solution. In qualitative agreement with the net Cl- efflux results (see Table 1), Table 2 shows that mock-infected cells have a substantially higher overall Cl- uptake rate as well as a larger bumetanide-sensitive component of the Cl- uptake.

                              
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  		Table 2.   Effects of HCMV infection, bumetanide, and [Na+]o on rate of increase of [Cl-]i by Cl--depleted cells

The forgoing results were obtained using 10 µM bumetanide. Essentially identical results were obtained when another series of cells were treated with 1 µM bumetanide (data not shown). This result suggests an IC50 for bumetanide for MRC-5 cells below 1 µM, a value in good agreement with the generally accepted range of published bumetanide IC50 values for inhibition of the NKCC (e.g., Ref. 13).

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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  		Fig. 5.   Dependence of net Cl- uptake on external Na+. Cells were first depleted of intracellular Cl- by exposure for 15-35 min to Cl--free (gluconate-substituted), CO2/HCO-3-buffered solution, followed by 15-min exposure to Cl--free, HEPES-buffered solution. At arrows, external Cl- was returned either with (filled symbols) or without (open symbols) extracellular Na+. A: mock-infected cells (control, n = 5; Na+-free, n = 4). B: 24-h PE HCMV-infected cells (control, n = 5; Na+-free, n = 5). C: 72-h PE HCMV-infected cells (control, n = 5; Na+-free, n = 3).

We calculated the derivative of the increase of [Cl-]i as a function of time after the external Cl- was returned in the presence and in the absence of extracellular Na+. In this series, the peak rate also occurred ~2 min (range 1.8-2.3 min) after the external Cl- was returned to the bathing solution. The collated and averaged results are presented in Table 2. Once again, we see that mock-infected cells have a substantially higher overall Cl- uptake rate as well as a higher [Na+]o-sensitive rate of Cl- uptake. Table 2 compares the peak rates of Cl- uptake for both the bumetanide treatment and external Na+ studies. It shows that both treatments have nearly identical effects on the rates of net Cl- uptake. This supports the view that they both are acting on the NKCC. These results further reinforce the pattern that the mock-infected cells have much more functional NKCC activity and that HCMV infection greatly reduces this activity at 24 h PE and abolishes it by 72 h PE.

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).

This antibody recognized an abundant polypeptide centered at ~175 kDa in mock-infected cells (Fig. 6A, lane 1). However, when the same amount of protein (30 µg) from the 72-h PE HCMV-infected cells was probed with antibody, two polypeptides were faintly recognized; one at 175 kDa and the other ~ 130 kDa (Fig. 6A, lane 2). Whether this latter polypeptide represents a proteolytic fragment is unknown. To further estimate the difference in the levels of NKCC protein expressed, we compared the immunoreactivity of 10 µg protein from mock-infected cells (Fig. 6A, lane 3) with 60, 80, and 100 µg protein from HCMV-infected cells (Fig. 6A, lanes 4-6). Even with 100 µg of protein, the 175-kDa band remained very faint.


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  		Fig. 6.   Western blot analysis using T4 antibody against Na+-K+-Cl- cotransporter. A total of 3 separate membrane preparations were used to obtain following results. A: Western blot (representative of 3 repetitions) using T4 at a 1:1,000 dilution (~0.4 mg/ml) detected a band of 175 kDa. Lanes 1 and 2, 30 µg of crude membranes from mock-infected and 72-h PE HCMV-infected cells, respectively. Lane 3, 10 µg of membranes from mock-infected cells. Lanes 4-6, 72-h PE HCMV-infected cells loaded with 60, 80, and 100 µg, respectively. B: density dependence on amount of protein load. Western blots (n = 2 for each cell type) were performed using a 1:1,000 dilution of monoclonal antibody T4 while varying amount of protein applied. Band densities at ~175 kDa were further analyzed using an ISee digital imaging system (Inovision) and plotted in arbitrary units (arb u) of optical density (OD) against protein load of mock-infected cells (1-30 µg) and HCMV-infected cells (30-120 µg). Lines through data represent best fits of all data in this linear region of film. Linear regression parameters of these lines are as follows: mock-infected cells, slope = 22.5 ± 3.7 OD units/µg, r2 = 0.96; HCMV-infected cells, slope = 1.12 ± 0.07 OD units/µg, r2 = 0.99.

Scanning densitometry was used to quantitatively compare the abundance of the protein isolated from the mock- and HCMV-infected cells. To ensure that the band densities were within the linear range of the X-ray film, we varied the amount of protein loaded on the electrophoresis gel (Fig. 6B). Figure 6B shows that when >30 µg of protein from mock-infected cells were applied, the resultant density signal saturated. We used linear regression analysis to determine the linear region of the density vs. protein concentration relationship. We found that for the mock-infected cells, the linear portion had a slope of 22.5 optical density (OD) units/µg protein, whereas for the HCMV-infected cells, the slope of the linear region was 1.12 OD units/µg protein. Thus, by comparing the slopes, we see that the density per milligram of the 175-kDa band was 20.1 times greater in mock-infected cells than in HCMV-infected cells. In an alternative approach, we kept the amount of protein applied to the electrophoresis gel constant and varied the T4 antibody dilution over the range 1:1,000 to 1:20,000. We obtained similar results (data not shown).

To meaningfully compare the relative abundance of the NKCC in mock- and HCMV-infected cells from these immunoblot studies, we needed to normalize the amount of protein to the number of cells represented by that protein. We have shown that the number of cells on a 72-h PE HCMV-infected dish is ~75% the number on an identical mock-infected dish (see METHODS). Furthermore, a dish of HCMV-infected cells produced 1.76 times the amount of microsomal membrane protein produced by the mock-infected cells (by Lowry assay). Thus 1 µg of protein from the HCMV-infected cells represents only 0.75/1.76 = 0.43 the number of cells represented by 1 µg of protein from mock-infected cells. Using this figure to normalize the T4 binding to a per-cell basis, our results indicate that mock-infected cells have 8.6 times more expressed NKCC than do the 72-h PE HCMV-infected cells.

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 alpha 5, which recognizes the alpha -subunit of the Na+-K+-ATPase (36), can be used to estimate the abundance of the Na+ pump using the same approach that we used to estimate the effect of HCMV infection on the NKCC (see above). We found that the alpha 5 antibody recognized a single polypeptide centered at ~95 kDa in both mock- and HCMV-infected cells (Fig. 7A, lanes 1 and 2). An equivalent load of total protein (30 µg) resulted in a more intense band in 72-h PE HCMV-infected than in mock-infected MRC-5 cells. Furthermore, comparing the band densities of 20 µg protein isolated from HCMV-infected cells (Fig. 7A, lane 6) to those of the 20, 40, and 60 µg of protein isolated from mock-infected cells (lanes 3-5), it is clear that HCMV-infected cells express more Na+-K+-ATPase than do the mock-infected cells. This is in good agreement with our earlier work using [3H]ouabain binding (2).


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  		Fig. 7.   Western block analysis using alpha 5 antibody against alpha -subunit of Na+-K+-ATPase. A total of 3 separate membrane preparations were used to obtain following results. A: Western blot (representative of 3 repetitions) using alpha 5 antibody at a 1:500 dilution (~0.8 µg/ml) detected a band at 95 kDa. Lanes 1 and 2, 30 µg crude membranes from mock-infected and 72-h PE HCMV-infected cells, respectively. Lane 6, 20 µg crude membranes from 72-h PE HCMV-infected cells. Lanes 3-5, crude membranes from mock-infected cells loaded at 20, 40, and 60 µg, respectively. Bands noted at 70 kDa are nonspecific. B: density dependence on amount of protein load. Western blots (n = 2 for both cell types) were performed using a 1:500 dilution of monoclonal antibody alpha 5 while varying amount of protein applied from mock-infected cells (10-90 µg) and 72-h PE HCMV-infected cells (20-75 µg). Band densities at ~95 kDa were further analyzed as described for Fig. 6, and results were plotted as arbitrary OD units against protein load. Data points within linear region of film were fitted with a linear regression program. Linear regression parameters of these lines are as follows: mock-infected cells, slope = 0.35 ± 0.06, r2 = 0.96; HCMV-infected cells, slope = 1.06 ± 0.13, r2 = 0.98.

When the amount of protein was varied at a constant antibody dilution (1:500; Fig. 7B), fitting the linear portions of the relationships revealed that the density of the band derived from HCMV-infected cells was threefold greater on a per-microgram basis than that from the mock-infected cells. Correcting this value for the number of cells represented by 1 µg of protein (see preceding section for this correction) shows that an HCMV-infected cell expressed ~6.9 times as much Na+ pump protein as did mock-infected cells. This result is in reasonable agreement with our earlier estimate of the increase in the number of ouabain binding sites (~4-fold; Ref. 2). In an alternative approach, we held the amount of protein applied to the electrophoresis gel constant while varying the dilution of the antibody over the range 1:100 to 1:5,000. We obtained similar results (data not shown).

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

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 [Cl-]i of mock-infected cells and of 24-h PE HCMV-infected cells. However, the [Cl-]i of 72-h PE HCMV-infected cells was essentially unaffected by bumetanide. Table 1 shows that, in the presence of bumetanide, both mock-infected and 72-h PE HCMV-infected cells lose intracellular Cl- with about the same rate constant (0.08 min-1 vs. 0.06 min-1, respectively). Furthermore, when the rate constant for intracellular Cl- loss by the HCMV-infected cells is corrected for the increased ratio of cell volume to surface area that occurs as a result of the cytomegaly, the indexed rate constant for the 72-h PE HCMV-infected cells increases to 0.086 min-1. Even if there is some uncertainty about the exact magnitude of this indexing factor, it is clear that, in the presence of bumetanide, both mock- and HCMV-infected cells have the same level of non-NKCC-mediated Cl- permeability as the mock-infected cells. Thus the lack of effect of bumetanide on the [Cl-]i observed with the 72-h PE HCMV-infected cells reflects a decreased NKCC activity and is not the result of cell membrane impermeability to Cl-.

The effects of HCMV infection on NKCC activity were further assessed by studying the effects of bumetanide treatment and external Na+ removal on the rates of net loss and net uptake of Cl-. The general pattern that emerged was that the mock-infected cells had the greatest level of bumetanide-sensitive or [Na+]o-dependent net fluxes; the 24-h PE HCMV-infected cells exhibited ~35% of the activity observed in the mock-infected cells, whereas the 72-h PE HCMV-infected cells had between 2 and 20% of the activity of the mock-infected cells (e.g., Tables 1 and 2). Thus HCMV infection has already begun to reduce NKCC activity within the first 24 h of infection, which is well before cytomegaly begins to develop.

The results of the functional studies just described correlate very well with the Western blot results using the T4 antibody. Together, these two approaches provide independent lines of evidence for HCMV-induced downregulation of the NKCC. The Western blot studies showed that 72 h of infection with HCMV reduced the amount of NKCC protein detectable by the T4 antibody to ~12% of that found in mock-infected cells.

The HCMV-induced increase in Na+ pump abundance determined by either [3H]ouabain binding or alpha 5 antibody binding are in quite good agreement, considering the difference in techniques. These data provide an index for HCMV-induced host cell enlargement. It is therefore reasonable to use alpha 5 antibody binding as a reference for comparison of the effects of HCMV infection on other membrane transport proteins. When the relative abundance of NKCC levels are normalized to alpha 5 antibody binding levels, it can be seen that HCMV infection reduces NKCC density to ~2% of that observed in mock-infected cells. This makes it likely that the severe reduction of NKCC functional activity at 72 h PE was caused by a significant reduction of the density of the NKCC protein in the cell membrane.

HCMV inhibits NKCC expression. The virus, or products stimulated by viral infection, may interfere with NKCC gene transcription. IFN-gamma 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-gamma 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-gamma , IL-6, or another cytokine. In this regard, it may be of interest that several IFNs, including IFN-gamma , IFN-beta , and IFN-alpha , 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.

Another possibility is that the high [Cl-]i itself may contribute to downregulation of the NKCC both functionally and at the levels of DNA translation and transcription and, hence, of protein expression. It is now well established that a rise in [Cl-]i can functionally inhibit the NKCC (e.g., Ref. 6). This effect is believed to result from a reduction of phosphorylation of the NKCC protein (e.g., Ref. 19). Several observations by others suggest that viral and host cell processes may be differentially affected by intracellular ionic composition, including the [Cl-]i. For instance, Carrasco and Smith (8) showed that in a cell-free system, protein synthesis will occur under ionic conditions unfavorable for host cell protein synthesis. High [Cl-]i has been reported to reduce protein synthesis by interfering with mRNA binding to ribosomes (41). In addition, high concentrations of Cl- have been reported to reduce host cell DNA polymerase activity while enhancing HCMV DNA polymerase activity (26). Replacement of potassium glutamate with potassium chloride has been reported to dramatically suppress certain protein-DNA interactions in vivo (18). Thus the virally directed increase in [Cl-]i we have reported (Fig. 2; also see Ref. 21) may play an important role in reducing the expression of cellular proteins such as the NKCC protein while favoring the expression of viral proteins.

The cell-free studies mentioned above that show that high salt favors viral protein synthesis over that of host cell protein have often used quite high concentrations of salts. However, in an intact cell under physiological conditions, there is a limited degree to which salt levels can be raised. This is because animal cells will remain in osmotic equilibrium with their external fluid, i.e., 285 mosmol/kgH2O. Therefore, the sum of the intracellular concentrations of the three major ions, K+, Na+, and Cl-, cannot exceed 285 mM, and in fact the upper limit would be expected to be somewhat less than that (due to other necessary osmotically active substances). Thus, in intact cells, it may not be possible to achieve the clear-cut effects reported from studies on cell-free systems. Nevertheless, by increasing the [Cl-]i by ~50%, as reported here, the HCMV infection may bias the protein synthetic machinery in favor of viral proteins.

Finally, there is the possibility of virally mediated posttranslational modification of the NKCC. It is possible that a virally induced proteolytic activity might result in the reduction of NKCC activity and protein expression we observed.

Cell swelling is also known to inhibit the cotransporter (e.g., Ref. 13), and might be considered as a reason for the loss of NKCC activity following HCMV infection. However, we show that this loss is already quite prominent by 24 h PE, well before the cell volume increases (1, 2).

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.

The present results clearly show that the non-HCO-3-dependent increase of [Cl-]i noted by Maglova et al. (21) cannot be caused by enhanced NKCC activity in the HCMV-infected cells. Nor do the present results comparing the effects on net Cl- uptake of bumetanide treatment and Na+-free treatment (see Fig. 5 and Table 2) point to an enhanced Na+-Cl- cotransport process as being the cause for the increased [Cl-]i. We have suggested (21) that the HCMV-infected cells might be substantially depolarized relative to the mock-infected cells. Even in the absence of any active uptake of Cl-, membrane depolarization coupled with a voltage-sensitive pathway for Cl- transmembrane movement would result in an increase of [Cl-]i. Therefore, the higher [Cl-]i may be the combined result of an enhanced Cl-/HCO-3 exchanger activity and a depolarized membrane potential.

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.

It may be important that cells infected with HCMV can enter the cell cycle but are arrested in late G1 phase (7). Whether this may be related to the observation that inhibition of the NKCC can block cell proliferation by preventing cells from making the transition from the G1 to the S phase of the cell cycle (29) remains to be demonstrated.

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.

To date, the results of ion transport studies studying HCMV (a DNA virus) contrast in several important ways from those reported from studies of several RNA viruses, including HIV. HCMV infection enhances the activity of the Na+/H+ exchanger, which is an acid-extruding mechanism. In contrast, HIV infection has been reported to decrease the resting intracellular pH of the host cells (25), implying (though not specifically demonstrating) the downregulation of some acid-extruding mechanism by HIV (although see Ref. 4). Sindbis virus (an alpha -RNA virus) has been shown to decrease Na+-K+-ATPase activity (38), and our group has shown that HCMV upregulates this pump (e.g., see Fig. 7 and Refs. 2, 27). Finally, Voss et al. (40) presented evidence that HIV-infected cells have enhanced NKCC activity, in sharp contrast to our present results. Thus it seems highly likely that although numerous viruses may have profound effects on ion movements, the particular effects differ quite significantly among the various viruses. This ought not to be surprising given the different strategies used by different viruses for their reproduction and host cell lethality.

In conclusion, we have demonstrated that HCMV infection significantly reduces the activity and the apparent expression of NKCC in human fibroblasts. Despite the loss of this means of active Cl- uptake, HCMV infection results in a significant increase of [Cl-]i.

    ACKNOWLEDGEMENTS

We acknowledge the excellent technical assistance of Charles Rassier, Junying Chen, and Xiyin Chen.

    FOOTNOTES

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.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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Am J Physiol Cell Physiol 275(5):C1330-C1341
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