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 (pH_i) operating range for the NHE is shifted toward higher, or more alkaline, pH_i 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/HCO₃ exchanger activities. The increase of NHE activity due to cell shrinkage tends to increase pH_i. In turn, the increase of pH_i stimulates Cl/HCO₃ exchanger activity. The enhanced Na⁺ uptake (via the NHE) and Cl uptake (via the Cl/HCO₃ 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 pH_i-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/HCO₃ exchanger activity of the host cell.

The anion exchanger or Na⁺-independent Cl/HCO₃ exchanger (hereafter referred to as the Cl/HCO₃ exchanger) has several distinctive properties permitting its functional characterization. For instance, because it exchanges Cl for HCO₃, removal of extracellular Cl will cause the transport mechanism to mediate a net exchange of intracellular Cl for extracellular HCO₃. This exchange will result in a net loss of intracellular Cl as well as intracellular alkalinization. Another characteristic of the Cl/HCO₃ 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/HCO₃ exchanger (28). However, this latter exchanger is inhibited as pH_i increases, whereas the Cl/HCO₃ exchanger is stimulated by alkaline pH_i (4, 24, 26, 32). Finally, this exchanger can exchange Cl for NO₃ (3, 14, 22), and this exchange, unlike that of Cl for HCO₃, will have no pH_i consequences, inasmuch as NO₃ 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/HCO₃ exchanger activity over the same time period it enhances NHE activity.

	METHODS

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Cell Cultures and HCMV Infection

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

The 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.

pH_i Measurements

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 (NO₃ 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 where K_SV is the Stern-Volmer constant.

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Calibration of N-(6-methoxyquinolyl)acetoethyl ester (MQAE) and determination of Stern-Volmer constant (KSV). A: top trace, uncorrected fluorescence output from a single glass coverslip on which MRC-5 cells were grown. At a, double-ionophore mixture of 10 µM tributyltin (TBT) and 10 µM nigericin (Nig) was added while cells were bathed in Cl-free solution (NO3 replaced Cl). Steady-state fluorescence under this condition is termed F0. At b, Cl concentration ([Cl]) of external fluid was changed from 0 to 75 mM; at c, it was returned to original Cl-free solution, at which time a new steady-state fluorescence (F'0) was obtained. In this manner we estimated amount of dye loss during procedure and applied an appropriate correction. MQAE dye loss was much faster from cells bathed in tributyltin + nigericin solution than in ionophore-free solutions; dye-leak corrections were not necessary for rest of this study. At d, background fluorescence was determined by switching to a solution containing 150 mM KSCN + 5 µM valinomycin (val). A series of such experiments were conducted using test solutions that contained 0-100 mM Cl. Bottom trace, F0/F corrected for dye loss and background as described above. B: individual F0/F data points obtained from a series of experiments similar to those shown in A plotted against [Cl] for mock- and human cytomegalovirus (HCMV)-infected cells (72 h after exposure). Slope of resulting line through these points gives KSV. For mock-infected cells, KSV = 25.7 ± 1.3 M1 (95% confidence limit = 23.0-28.5 M1); for HCMV-infected cells, KSV = 19.8 ± 1.2 M1 (95% confidence limit = 17.3-22.3 M1).

K_SV 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 NO₃ 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 K_SV was 25.7 and 19.7 M¹ 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 K_SV 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 K_SV 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 K_SV 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.

pH_i-Clamping Studies

All pooled data are means ± SE. Paired and unpaired t-tests were used, as appropriate, to test for statistical significance.

	RESULTS

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The present investigation studied two facets of Cl/HCO₃ exchange: its role in the determination of pH_i and its role in the determination of [Cl]_i.

Effect of CO₂/HCO₃ on Resting pH_i in Mockand HCMV-Infected Cells

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Effect of extracellular CO2/HCO3 on intracellular pH (pHi) of mock- and HCMV-infected cells (72 h after exposure). 2',7'-Bis(2-carboxyethyl)-5(6)-carboxyfluorescein technique was used to measure pHi. Each trace is collated average of 10-11 separate experiments. Lines are best fits through all data points actually collected. For clarity we show mean of every 6th data point with its associated SE. At arrow, external solution was changed from standard HEPES solution to standard HCO3 solution (5% CO2-25 mM HCO3). Steady-state pHi values before CO2/HCO3 treatment were 7.37 ± 0.02 (n = 11) and 7.53 ± 0.04 (n = 10) for mock- and HCMV-infected cells, respectively. Steady-state pHi values in CO2/HCO3 solution were 7.47 ± 0.02 (n = 11) and 7.45 ± 0.03 (n = 10) for mock- and HCMV-infected cells, respectively.

Effect of External Cl Removal on pH_i of Cells Exposed to CO₂/HCO₃

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Effect of external Cl replacement by gluconate on pHi of mock- and HCMV-infected cells (72 h after exposure). Cells were bathed in a fluid containing 5% CO2-25 mM HCO3. Each trace is collated average of 4-6 separate experiments. Lines are best fits through all data points actually collected. For clarity we show mean of every 6th data point with its associated SE. At arrow, solution was changed to one in which all Cl was replaced by gluconate. Steady-state pHi values before Cl replacement were 7.60 ± 0.06 (n = 4) and 7.51 ± 0.06 (n = 6) for mock- and HCMV-infected cells, respectively. At 5 min after external Cl replacement, pHi values were 7.50 ± 0.04 and 7.83 ± 0.15 for mock- and HCMV-infected cells, respectively. Average rate of increase of pHi for HCMV-infected cells was 0.11 ± 0.03 pH unit/min. In presence of 100 µM H2DIDS this increase was completely blocked (0.02 ± 0.03 pH unit/min, n = 4; not shown). After 5 min in Cl-free solution, pHi of HCMV-infected cells slowly decreases at a rate that is very similar to that of mock-infected cells. Large SE values in HCMV-infected cells reflect variability between experiments in activity of secondary acid-loading process. For both cell treatments, pHi returned to control values when extracellular Cl was returned to bathing solution (not shown).

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 pH_i change might be the result of net HCO₃ uptake in exchange for intracellular Cl mediated by the Cl/HCO₃ exchanger. We noted that after reaching a maximum the pH_i 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, pH_i 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 pH_i 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 pH_i. 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/HCO₃; in fact, a slight acidification was observed (10). The basis of this rapid, CO₂/HCO₃-dependent, DEA-, H₂DIDS-insensitive alkalinization is uncertain. However, consider that a reduction of cell volume would increase [HCO₃]_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 [HCO₃]_i relative to intracellular PCO₂, which would translate into a rise of pH_i.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Effects on pHi of mock- and HCMV-infected cells (72 h after exposure) of challenge with a hyperosmotic fluid. Cells were bathed in a fluid containing 5% CO2-25 mM HCO3. Each trace is collated average of several separate experiments. Lines are best fits through all data points actually collected. For clarity we show 3 data points with their associated SE. Initial pHi values of untreated mock- and HCMV-infected cells were the same: 7.43 ± 0.02 (n = 5) and 7.43 ± 0.02 (n = 11), respectively. At 1st arrow, solution was changed to one in which 78 mM NaCl had been added (t = 0), which increased osmolality to 146% of normal (hyper 146%). Left: top trace, effects of hyperosmotic challenge on pHi of an otherwise untreated mock-infected cell. pHi increased in 2 stages: an initial rapid shift of ~0.05 pH unit and a somewhat slower, but larger increase culminating in pHi = 0.202 ± 0.028 pH unit (n = 5) ~6 min after hyperosmotic solution was applied. Thereafter, pHi decreased very slowly. At 14 min after exposure to hyperosmotic fluid, pHi had decreased by 0.029 ± 0.014 pH unit, but this change was not statistically significant. Treatment of mock-infected cells with 100 µM H2DIDS (middle trace) did not cause any statistically significant change from control response. However, when 5 µM diethyl amiloride (DEA) was applied (bottom trace), all that remained of hyperosmotic effect was initial, small alkalinization. Right: effects of hyperosmotic challenge on pHi of an otherwise untreated HCMV-infected cell; protocol was same as that used for mock-infected cells (left). Exposure to 146% hyperosmotic, HCO3-containing fluid of an otherwise untreated HCMV-infected cell caused pHi to alkalinize. Increase of pHi occurred in 2 stages: an initial rapid phase of ~0.05 pH unit and a slower, but larger alkalinization culminating in a pHi = 0.14 ± 0.02 pH units (n = 11) ~6 min after beginning of exposure. Thereafter, a significant secondary decrease of pHi occurred 6-14 min after hyperosmotic fluid was applied (pHi = 0.074 ± 0.011, n = 11, P < 0.0001, paired t-test). When HCMV-infected cells were treated with 100 µM H2DIDS, pHi after 6 min of hyperosmotic challenge was increased even more than in HCMV-infected cells not treated with H2DIDS [0.223 ± 0.008 (n = 4) vs. 0.14 ± 0.02 (n = 11)], and these 2 values were significantly different (P < 0.05, unpaired t-test). In addition, secondary pHi decrease was abolished. When 5 µM DEA was applied, all that remained of hyperosmotic effect was initial, small alkalinization.

Although mock- and HCMV-infected cells exhibited a secondary, DEA-sensitive alkalinization, the peak pH_i 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 pH_i ~6 min after the onset of the hyperosmotic challenge. After that peak, however, there were significant differences between the pH_i behaviors of the two cell treatments in the continued presence of the hyperosmotic challenge. The pH_i of mock-infected cells changed very little over the next 8 min (Fig. 4). However, the pH_i of the HCMV-infected cells decreased ~0.1 pH unit (Fig. 4) over this same period. This "recovery" of pH_i 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 pH_i 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

View this table: [in this window] [in a new window]   Table 1.   Steady-state intracellular Cl concentration measured with MQAE

Effect of HCMV Infection on Net Cl Efflux in CO₂/HCO₃-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 pH_i is at its new steady state and that [HCO₃]_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/HCO₃, Cl is lost about twice as fast from mock-infected cells as from HCMV-infected cells and 2) in the absence of CO₂/HCO₃, treatment with 100 µM H₂DIDS does not reduce the rate of Cl loss by either cell treatment (in fact, some stimulation was noted).

View larger version (24K): [in this window] [in a new window]   Fig. 5.   Effect on [Cl]i of replacing extracellular Cl with gluconate without and with 100 µM H2DIDS in nominal absence of CO2 and HCO3 in mock- (A) and HCMV-infected cells 72 h after exposure (B). [Cl]i was measured using MQAE. Each trace is from a representative experiment. For clarity we show every 6th data point. At arrow, superfusing solution was changed from standard HEPES solution to an identical solution in which all Cl was replaced with gluconate. Lines through data points after arrow represent best fit to an exponential equation. Rate constants were 0.118 and 0.150 min1 for control and H2DIDS-treated mock-infected cells, respectively, and 0.047 and 0.055 min1 for control and H2DIDS-treated HCMV-infected cells, respectively.

View this table: [in this window] [in a new window]   Table 2.   Cl loss into gluconate-containing media

Effect of HCMV Infection on Net Cl Efflux in CO₂/HCO₃-Containing Media

View larger version (21K): [in this window] [in a new window]   Fig. 6.   Effect on [Cl]i of replacing extracellular Cl with gluconate in presence of CO2 and HCO3 without and with 100 µM H2DIDS in mock- (A) and HCMV-infected cells 72 h after exposure (B). [Cl]i was measured using MQAE. Each trace is from a representative experiment. For clarity we show every 6th data point. At arrow, superfusing solution was changed from standard CO2/HCO3 solution to an identical solution in which all Cl was replaced with gluconate. Lines through data points after arrow represent best fit to an exponential equation. Rate constants were 0.103 and 0.104 min1 for control and H2DIDS-treated mock-infected cells, respectively, and 0.107 and 0.045 min1 for control and H2DIDS-treated HCMV-infected cells, respectively.

H₂DIDS-Sensitive Net Cl Efflux Stimulated by External NO₃

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View larger version (23K): [in this window] [in a new window]   Fig. 7.   Effect on [Cl]i of replacing external Cl with NO3 in mock- (A) and HCMV-infected cells 72 h after exposure (B) with and without 100 µM H2DIDS. Each trace is collated average of 5-10 experiments. For clarity we show mean of every 6th data point with its associated SE. At arrow, solution was changed from standard HEPES (Cl) to standard HEPES (NO3). [Cl]i was measured using MQAE. Lines through data points after arrow represent best fit to an exponential equation. Rate constants are 0.161 ± 0.011 (n = 10) and 0.134 ± 0.012 min1 (n = 6) for control and H2DIDS-treated mock-infected cells, respectively, and 0.182 ± 0.013 (n = 6) and 0.0.083 ± 0.004 min1 (n = 5) for control and H2DIDS-treated HCMV-infected cells, respectively.

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 min¹. Treatment with 100 µM H₂DIDS reduced the rate constant by 54%, to 0.083 ± 0.004 min¹. In three experiments we examined the effects of NO₃ replacement on cells infected 24 h before their assay. In this case the control rate constant was 0.152 ± 0.005 min¹, and after H₂DIDS treatment it decreased 35%, to 0.099 ± 0.017 min¹. The fact that the net Cl efflux caused by NO₃ 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 NO₃ exchanges with Cl via the Cl/HCO₃ 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 NO₃ 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/HCO₃ 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. 

View larger version (14K): [in this window] [in a new window]   Fig. 8.   H2DIDS-sensitive Cl/NO3 exchange efflux rate constants. Experiments identical to those illustrated in Fig. 7 were performed on mock- and HCMV-infected cells after 24 and 72 h of infection (24h and 72h PE). Each bar represents difference between average of at least 3 control and H2DIDS experiments for each of 3 cell treatments.

pH_i Sensitivity of H₂DIDS Net Cl Efflux

Figure 9 shows the results of varying the pH_i (accomplished by varying pH_o; see METHODS) on the rate constant of net Cl efflux caused by replacing external Cl with NO₃. In the case of the mock-infected cells, the H₂DIDS-sensitive component of the net Cl efflux decreased as pH_i was increased. However, in HCMV-infected cells the H₂DIDS-sensitive component was more than doubled as pH_i was increased from 6.4 to 7.8. Thus Cl/NO₃ exchange in HCMV-infected cells displays a pH_i sensitivity suggestive of its being mediated by a Cl/HCO₃ exchanger.

View larger version (18K): [in this window] [in a new window]   Fig. 9.   pHi dependence of net Cl efflux via Cl/NO3 exchange. High-K+-nigericin technique was used to "clamp" pHi to 6.4-7.8. External Cl was completely replaced with NO3. An exponential efflux rate constant was determined from fall of [Cl]i by use of nonlinear least-squares curve fits to data points obtained 5-25 min after solution was changed. This was done in absence and presence of 100 µM H2DIDS. A: results of individual experiments on mock-infected cells. Lines (solid and dashed) are linear best fits to rate constant vs. pHi data. B: results of individual experiments on HCMV-infected cells (72 h after exposure). Lines are best fits to a 2nd-order regression in case of H2DIDS data and a 3rd-order regression for control data. Bottom: pH dependence of H2DIDS-sensitive rate constant of net Cl efflux caused by NO3 replacement of Cl. Lines were determined as difference between lines in A and B for net Cl efflux under control and H2DIDS conditions. pHo, extracellular pH.

	DISCUSSION

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HCMV Infection Causes Increased Cl/HCO₃ Exchanger Activity

pH_i consequences. We have obtained several results that lead us to the conclusion that HCMV infection results in a significant enhancement of Cl/HCO₃ 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 HCO₃ via the Cl/HCO₃ exchanger, an H₂DIDS-sensitive intracellular alkalinization will result. When we removed external Cl in the presence of CO₂/HCO₃, the pH_i of HCMV-infected MRC-5 cells (72 h after exposure) became ~0.3-0.5 pH unit more alkaline (Fig. 3). In contrast, the pH_i 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/HCO₃ exchanger (6). Thus these observations regarding the effects of removing external Cl on pH_i are consistent with the view that HCMV infection strongly enhances Cl/HCO₃ exchanger activity in MRC-5 cells.

[Cl]_i consequences. Further evidence that HCMV enhances the Cl/HCO₃ 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/HCO₃-containing solution was 63% higher than that of mock-infected cells (Table 1). Part, but not all, of this difference can be attributed to HCO₃-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 pH_i results, the apparent contribution of a CO₂/HCO₃-dependent process is much greater for the HCMV- than for the mock-infected cells. However, even in the nominal absence of CO₂/HCO₃, the two cell treatments have significantly different [Cl]_i.