INTRODUCTION

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A number of viruses induce changes in membrane permeability to ions during infection as a result of gene expression (3). Rotavirus infection of cultured cells modifies the homeostasis of both mono- and divalent cations that appears to be mediated by the synthesis of viral proteins (6, 18). Rotavirus infection provokes an increase in Ca²⁺ permeation, intracellular [Ca²⁺] ([Ca²⁺]_i), and sequestered Ca²⁺ pools (17, 18). The changes in Ca²⁺ concentration do not seem to be the result of an effect on the Ca²⁺ pumps of the plasma membrane and the endoplasmic reticulum (ER) but rather seem to be the result of an increase of Ca²⁺ influx in excess of the regulatory capacity of the cell. Since the increase in permeability to Ca²⁺ appears long before a generalized modification of the permeability to other molecules, even those as small as ethidium bromide, this appears to be an early and primary effect of infection.

In a search for the viral protein related to the disturbances of Ca²⁺ homeostasis, Tian et al. (33, 34) studied the effects of the expression of individual recombinant proteins on the Ca²⁺ concentration in Spodoptera frugiperda cells (Sf9 cells). They found that only the expression of the product of gene 10, the nonstructural protein NSP4, induced a change in Ca²⁺ concentration in insect cells with no changes in permeability of the plasma membrane to Ca²⁺. Contrary to the observations of Michelangeli et al. (17, 18) they proposed that the increase in the cytosolic Ca²⁺ concentration brought about by rotavirus infection was not provoked by an increased Ca²⁺ entry. According to them, this would be due to the increase in ER membrane permeability to Ca²⁺ linked to the expression of NSP4 through a phospholipase C-independent pathway (33). In later work they showed that addition of exogenous NSP4 to HT29 cells induced Ca²⁺ release from inositol 1,4,5-triphosphate-sensitive stores and subsequent Ca²⁺ entry, depending on the binding of the protein (or a synthetic peptide) to a putative receptor on the plasma membrane (7, 27, 33). These effects were different from those obtained with Sf9 cells expressing NSP4.

In the present work we have characterized the permeability pathway induced by rotavirus infection after the beginning of viral protein synthesis and before the induction of cell lysis. The results indicate that infection of MA104 and HT29 cells activates a pathway of plasma membrane permeability to Ca²⁺. Several lines of evidence suggest that the pathway for the entry of Ca²⁺ has the characteristics of a Ca²⁺ channel rather than nonselective damage of the plasma membrane. Ca²⁺ entry showed an apparent saturation kinetics with respect to extracellular concentration changes. The entry of different cations followed a selectivity sequence, from Ba²⁺ to the impermeative La³⁺. The cation influx was secondarily inactivated by the intracellular cation concentration. The cation pathway was inhibited by La³⁺ and D600. La³⁺, Cr³⁺, and extracellular markers such as ethidium bromide as well as trypan blue did not permeate by this pathway.

	MATERIALS AND METHODS

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Cell cultures and virus infection. Fetal monkey kidney cells (MA104) were used in most experiments. In some cases, the colon carcinoma HT29 cells were used. Cells were grown and maintained as previously described (18). The OSU strain of rotavirus was used in all experiments. Infection was carried out for 1 h at 37°C. The inoculum was then removed, washed with phosphate-buffered saline, and further incubated with minimal essential medium (MEM) without fetal calf serum. Infectivity titers of the preparations used were measured by titration in microplates with an anti-VP6 monoclonal antibody (4B2D2) for immunohistochemical staining after methanol fixation (4). Cell infection was performed at a multiplicity of infection of around 20. Under these conditions, all cells in the monolayer were infected during the first cycle as ascertained by immunofluorescence of VP6.

Excitation spectra of fura-2 in the presence of different cations. Excitation spectra of fura-2 were obtained at 37°C in a spectrofluorometer (Photon Technology International) equipped with a stirrer and temperature control. The quartz cuvette contained a solution of 10 µM fura-2 free acid, 10 mM HEPES (pH 7.2), and 100 mM KCl supplemented with a saturating concentration of the tested cation. The fluorescence spectrum of fura-2 was also determined in the absence of added cations, in the presence of 10 mM K₂H₂EGTA. The emission fluorescence was fixed at 510 nm. Figure 1 shows the fluorescence spectrum of fura-2 alone or complexed to the metal cations used in this study. Like binding of Ca²⁺, binding of Ba²⁺, Sr²⁺, and La³⁺ to fura-2 increases the fluorescence emission at 510 nm when excitation is at wavelengths of from 300 to 360 nm, with a maximum at around 340 nm (Fig. 1A). The isoemissive wavelengths, where fura-2 fluorescence becomes independent of cation concentration, were 356 nm for Ca²⁺ and La³⁺ and about 366 nm for Sr²⁺ and Ba²⁺ (uncorrected spectra). On the other hand, binding of Mn²⁺, Ni²⁺, and Co²⁺ to fura-2 quenched the fluorescence at all excitation wavelengths tested (Fig. 1B). Based on these spectra, we measured intracellular cation concentrations by using 340/380 nm ratios for Ca²⁺, Ba²⁺, Sr²⁺, and La³⁺ (see below) and measured the entry of Mn²⁺, Ni²⁺, and Co²⁺ as quenching of fura-2 fluorescence at 360 nm.

View larger version (19K): [in this window] [in a new window]   FIG. 1.   Fluorescence excitation spectra of fura-2 in the presence of metal cations. Fluorescence excitation spectra of fura-2 in the presence of saturating concentrations of Ca2+, Ba2+, Sr2+, and La3+ (A) or Ni2+, Co2+, and Mn2+ (B) are shown. Fluorescence emission was recorded at 510 nm. Under all conditions, the cuvette contained 1 mM cation in a solution of 10 µM fura-2 free acid, 10 mM HEPES (pH 7.2), and 100 mM KCl. The spectrum of fura-2 in the absence of any cation was obtained in the presence of 10 mM K2H2EGTA in the same medium to chelate traces of contaminant divalent cations. The y-axis scale in panel B has been increased by a factor of two.

Determination of intracellular Ca²⁺ and other divalent cation concentrations. The intracellular calcium concentration was measured by using the fluorescent indicator fura-2, which was incorporated intracellularly as its acetoxy-methyl ester (fura-2/AM). Cell monolayers were trypsinized, washed by centrifugation, and resuspended in MEM at an approximate concentration of 2 × 10⁶ cells/ml. Aliquots of the cell suspension were incubated with 10 µM fura-2/AM with pluronic (0.02%) in a medium containing 130 mM NaCl, 5 mM KCl, 1.0 mM CaCl₂, 1.0 mM MgCl₂, 11 mM glucose, 10.0 mM HEPES (pH 7.4), and 0.1% (wt/vol) albumin. Cell loading of fura-2 was carried out at 37°C for 30 min to enhance dye uptake. Under these conditions it may be possible that fura-2 was taken up and sequestered into intracellular compartments, leading to an overestimation of the [Ca²⁺]_i value. We have evaluated the contribution of this fraction by permeabilization with digitonin followed by solubilization with Triton X-100. The results show that the contribution of compartmentalized dye to the overall signal was not important under the loading conditions used for these cell types. Cells were washed twice by centrifugation to remove extracellular fluorophore and resuspended in 1.2 ml of the same medium without albumin. Fluorescence was measured at 37°C in a spectrofluorometer (Photon Technology International) equipped with a stirrer and temperature control. The excitation wavelengths were 340 and 380 nm, which were alternatively changed by computer control, allowing acquisition of one pair of data per second for experiments with fura-2. The emission wavelength was fixed at 510 nm. The ratio of the fluorescent signals measured at 340 and 380 nm was computer determined. The intracellular free Ca²⁺ concentration, [Ca²⁺]_i, was evaluated by using an apparent K_d for fura-2-Ca of 224 nM (8). The maximal fluorescence ratio (R_max) was determined by addition of digitonin (80 µg/ml) to permeabilize cells, and the minimal fluorescence ratio (R_min) was determined by the subsequent addition of 80 mM EGTA in 0.1 M Tris (pH 7.4) buffer. The intracellular [Ca²⁺] was calculated according to the equation described by Grynkiewicz et al. (8). Addition of EGTA (10 mM) to the cuvette containing fura-2-loaded cells did not change the baseline fluorescence, indicating that no significant dye leaked out during the measurement period.

Assessment of membrane permeability to cations. (i) Step change of extracellular cation concentration. The relative permeabilities of mock- and virus-infected cells to cations were evaluated by imposing a step increase in the extracellular cation concentration and measuring the rate of change in the intracellular cation concentration during the first few seconds (18). This change is the result of net cation fluxes between the cytoplasm and outside and between the cytoplasm and cation-sequestering organelles. However, the elevation of intracellular cation concentration during the first few seconds (with a linear slope) should be a measurement of cation influx and hence of permeability to the cation. Membrane permeability to Mn²⁺, Ni²⁺, and Co²⁺ was measured by using cells loaded with fura-2. The entry of these cations was monitored by the initial quenching of intracellular fura-2 fluorescence at excitation and emission wavelengths of 360 and 510 nm, respectively. Results are expressed as relative fluorescence quenching with respect to the maximal quenching induced by the addition of digitonin (80 µg/ml).

(ii) ⁴⁵Ca²⁺ and ⁵¹Cr³⁺ uptake. Cell monolayers were grown to confluency in 24-well Linbro plates and infected as described above. At 4, 6, or 8 postinfection, maintenance medium was removed and replaced by 200 µl of MEM containing ⁴⁵Ca²⁺ (5 µCi/ml) or ⁵¹Cr³⁺ (50 µCi/ml). Uptake was stopped after 10 min by washing the cells four times by immersion in ice-cold phosphate-buffered saline. After drying, cells were dissolved with 0.25 ml of NaOH (0.1 N) and neutralized with HCl, and radioactivity was determined by liquid scintillation counting. As the number of cells per well was found to be rather constant, uptake values for single experiments are expressed as counts per minute per well.

Statistical analysis. In most cases, when appropriate, results of representative experiments of a series are shown. In addition, mean values ± standard errors of the means (SEMs) are tabulated for calculated parameters drawn from individual curves. The level of significance of the difference for values obtained for mock- and rotavirus-infected cells was calculated by the Mann-Whitney test. Differences were considered statistically significant when P was <0.05.

	RESULTS

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Rotavirus infection induces an increase of plasma membrane permeability to Ca²⁺. Figure 2 shows the effect of rotavirus infection on [Ca²⁺]_i and membrane permeability to Ca²⁺ in MA104 and HT29 cells at 7 h postinfection. The basal [Ca²⁺]_i in mock infected cells was around 100 nM for both cell lines. At 7 h postinfection, rotavirus infection had induced an increase in basal Ca²⁺ concentrations in both MA104 and HT29 cells, which attained a value two or three times higher than that in mock-infected cells.

View larger version (18K): [in this window] [in a new window]   FIG. 2.   Increase in intracellular Ca2+ concentration and plasma membrane permeability to Ca2+ in rotavirus-infected MA104 and HT29 cells. At 7 h postinfection, MA104 (A) and HT29 (B) monolayers were trypsinized, and cell suspensions were loaded with fura-2 for the measurement of [Ca2+]i (see Materials and Methods). Permeability to Ca2+ in rotavirus (V)- or mock (M)-infected cells was evaluated by the change in [Ca2+]i induced by the addition of 5 mM CaCl2 to the extracellular medium (arrow), which initially contained 1 mM Ca2+. Results of a representative experiment of a series of three for each cell type are shown.

View larger version (18K): [in this window] [in a new window]   FIG. 3.   Changes of intracellular Ca2+ concentration as a function of the magnitude of the extracellular Ca2+ increase. Intracellular Ca2+ concentration and permeability in rotavirus- and mock-infected MA104 (A) and HT29 (C) cells were determined at 7 h postinfection as detailed in the legend to Fig. 2. Membrane permeability to Ca2+ was evaluated by the change of [Ca2+]i induced by the addition of different Ca2+ concentrations (shown in parentheses, in millimolar) to the extracellular medium, which initially did not contain Ca2+, in rotavirus (V)- and mock (M)-infected cells. The insets in panels A and C show the first derivatives (d[Ca2+]i/dt) of these traces right after the Ca2+ pulse application (arrows). The computer traces for the derivatives have been offset to be able to observe the different curves. The peak value corresponds to the maximal rate of [Ca2+]i increase after Ca2+ addition (d[Ca2+]i/dtmax), which is plotted in panel B as a function of the magnitude of the extracellular step change for a range of Ca2+ concentrations from 2 to 30 mM in virus- and mock-infected MA104 cells. The data points from one experimental series were fitted to a hyperbolic curve by using Microcal Origin 4.00 software. See Table 1 for statistics.

1

1

La³⁺ and D600 block Ca²⁺ entry stimulated by infection. As lanthanum is a well-known inorganic calcium channel blocker, we have tested its effects on Ca²⁺ entry. As shown in Fig. 4A, addition of 100 µM LaCl₃ to mock- or virus-infected MA104 cells did not alter fura-2 fluorescence, indicating that this cation did not enter these cells. Addition of 5 mM Ca²⁺ to virus- and mock-infected cells in the presence of La³⁺ did not induce the intracellular Ca²⁺ rise observed in controls. These results indicate that extracellular La³⁺ blocked calcium entry in both mock- and virus-infected cells. Similar results were obtained with HT29 cells (Fig. 4C). Removal of extracellular La³⁺ resulted in the complete reversal of the inhibitory effect (not shown). The blockade of Ca²⁺ influx by La³⁺ was concentration dependent, affecting the initial rate of Ca²⁺ entry (d[Ca²⁺]_i/dt) and the maximal concentration attained after the change (Fig. 4B), with a calculated 50% inhibitory concentration of 13.8 µM (Fig. 4B, inset).

View larger version (15K): [in this window] [in a new window]   FIG. 4.   La3+ blocks Ca2+ entry induced by rotavirus infection. MA104 and HT29 cell monolayers were trypsinized at 7 h postinfection and loaded with fura-2. The cells were resuspended in 1 ml of medium containing 1 mM Ca2+. (A) Effect of La3+ on Ca2+ entry in rotavirus (a and c)- and mock (b and d)-infected cells. Addition of 100 µM LaCl3 (c and d) and 5 mM Ca2+ (a, b, c, and d) to cell suspensions is indicated by arrows. (B) Dose-response curve for inhibition of Ca2+ entry by La3+ in rotavirus-infected MA104 cells. La3+ concentrations (in micromolar) are indicated at the right of each curve. The inset corresponds to the peak rate of change in [Ca2+]i during the first 15 s following addition of extracellular Ca2+ (5 mM) in the presence of different La3+ concentrations (d[Ca2+]i/dtmax), calculated as described for Fig. 3. (C) Inhibition of Ca2+ entry by La3+ in rotavirus-infected HT29 cells. La3+ (100 µM) was added at time zero, and 5 mM Ca2+ was added to the medium for both untreated and La3+-treated infected cells. Results of a representative experiment of a series of three are shown in each case. IC50, 50% inhibitory concentration.

View larger version (14K): [in this window] [in a new window]   FIG. 5.   Metoxyverapamil (D600) inhibits Ca2+ entry induced by rotavirus infection. MA104 and HT29 cell monolayers were trypsinized at 7 h postinfection and loaded with fura-2. (A) Cells were preincubated for 30 min with 160 µM D600 (during fura-2 loading), and then the cells were washed and resuspended in 1 ml of medium containing 1 mM Ca2+ and 160 µM D600 and the free Ca2+ concentration was measured. Addition of 5 mM Ca2+ to evaluate membrane permeability is indicated by an arrow. (B) Effect of different concentrations of D600 (added at the time indicated by the arrow) on the change in [Ca2+]i induced by the addition of 5 mM Ca2+. The inset corresponds to peak values of the first derivatives (d[Ca2+]i/dtmax), calculated as described for Fig. 3, plotted as a function of D600 concentration. The experimental points are joined by straight lines. (C) Inhibition of Ca2+ entry by D600 in rotavirus-infected HT29 cells. Addition of 80 µM D600 and 5 mM Ca2+ is indicated by arrows. Results of a single experiment representative of two are shown in each case. V, virus-infected cells; M, mock-infected cells; IC50, 50% inhibitory concentration.

Permeability of the plasma membrane to metal cations in virus-infected MA104 cells. Having demonstrated a Ca²⁺ permeability pathway induced by infection, we studied its selectivity for other divalent cations. For this purpose, we took advantage of the changes in fluorescence of fura-2 upon binding to various metal cations. Figure 6 shows the comparison of the changes in intracellular Ca²⁺, Ba²⁺, and Sr²⁺ concentrations induced by an extracellular cation pulse (5 mM) in suspensions of mock- and virus-infected MA104 cells preincubated in the absence of nominal free Ca²⁺. The intracellular concentrations presented in the curves correspond to the change induced by the pulse and were calculated by using the respective K_ds for the cations. The basal level before addition of the pulse, corresponding to the intracellular free Ca²⁺ concentration, was subtracted from the curve.

View larger version (21K): [in this window] [in a new window]   FIG. 6.   Comparison of the permeabilities of the plasma membrane to Ca2+, Ba2+, and Sr2+ in mock- and rotavirus-infected MA104 cells. At 7 h postinfection, the intracellular cation concentration in mock (A)- or rotavirus (B)-infected MA104 cells loaded with fura-2 was measured. The cells were suspended in a medium (1 ml) without added Ca2+ prior to the experiment. Cations were added to the cuvette at a final concentration of 5 mM at the time indicated by the arrows. The intracellular cation concentration was calculated by using fura-2 fluorescence ratios monitored at 340- and 380-nm excitation wavelengths and by using 224 nM, 2.4 µM, and 5.2 µM as Kds for Ca2+, Ba2+, and Sr2+, respectively, and the Rmax was determined by addition of digitonin in the presence of the cation. Variations in cation concentration are shown and were calculated by subtracting the initial basal value, just before the addition of the cation. The insets show the first derivatives of these traces (d[Ca2+]i/dtmax) right after cation addition (arrows). The computer traces for the derivatives have been offset to be able to observe the different curves. The peak value corresponds to the maximal rate of intracellular concentration change after cation addition. The experiments shown were performed with the same batch of cells for all ions and both mock- and virus-infected cells. See Table 2 for statistics.

View larger version (18K): [in this window] [in a new window]   FIG. 7.   Comparison of the permeabilities of the plasma membrane to Mn2+, Ni2+, and Co2+ in rotavirus- and mock-infected MA104 cells. At 7 h postinfection, MA104 cells were trypsinized, loaded with fura-2, washed, and resuspended in medium (1 ml) without added Ca2+. Cations were added to the cuvette at a final concentration of 5 mM (5 µl of a 1 M solution) at the times indicated by the arrows. Mn2+, Ni2+, and Co2+ entry was measured by the quenching of fluorescence with an excitation wavelength of 356 nm, the isoemissive wavelength for Ca2+, and an emission wavelength of 510 nm. The curves represent fluorescence relative to total quenching attained by the addition of digitonin to saturate fura-2 with the indicated quenching cation. The insets show the first derivatives of these traces right after the cation pulse application (arrows). The computer traces for the derivatives have been offset to be able to observe the different curves. The peak value corresponds to the maximal rate of intracellular fluorescence change after cation addition. The experiments shown were performed with the same batch of cells for all ions and for both mock (M)- and virus (V)-infected cells. Results of a representative experiment of a series of four are shown.

View larger version (22K): [in this window] [in a new window]   FIG. 8.   Effect of La3+ on Ba2+ and Mn2+ entry in mock- and virus-infected MA104 cells. MA104 cell monolayers were trypsinized at 7 h postinfection, loaded with fura-2, washed, and resuspended in 1 ml of medium without added Ca2+ (nominally Ca2+ free). (A) Inhibition of Ba2+ entry by La3+ in mock- and virus-infected cells. Addition of 100 µM LaCl3 (c and d) and 5 mM Ba2+ (a, b, c, and d) to rotavirus (a and c)- and mock (b and d)-infected MA104 cells is indicated by the arrows. The variation in cation concentration is shown and was calculated by subtracting the initial basal value, just before the addition of the cation. (B) Inhibition of Mn2+ entry by La3+ in virus- and mock-infected cells. Experiments were performed as described for panel A. The addition of 100 µM LaCl3 (c and d) and 5 mM Mn2+ (a, b, c, and d) is indicated by the arrows. Maximal quenching was attained by addition of 10 µM digitonin in all cases.

View larger version (15K): [in this window] [in a new window]   FIG. 9.   Effect of La3+ on the regulation of Ca2+ and Ba2+ intracellular concentrations in rotavirus-infected HT29 cells. HT29 cell monolayers were trypsinized at 7 h postinfection and loaded with fura-2. The cells were washed and resuspended in 1 ml of medium containing 1 mM Ca2+. Arrows indicate the time of addition of 5 mM CaCl2 (A) or BaCl2 (B) and of 100 µM LaCl3 (A and B). Variations in cation concentrations are shown and were calculated by subtracting the initial basal value, just before the addition of the cation. Results of a representative experiment in a series of two are shown.

	DISCUSSION

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We have previously reported that during the course of rotavirus infection of MA104 cells, there was a progressive increase in membrane permeability to molecules of increasing size. At early times after initiation of viral protein synthesis (4 to 6 h postinfection), permeability to monovalent cations such as Na⁺ and K⁺, as well as Ca²⁺, increased. Later, after 8 h postinfection, molecules which are normally impermeative in an uninfected cell, such as ethidium bromide and trypan blue, entered (17, 18).

The permeability pathway for Ca²⁺ in infected cells was studied by using step changes of extracellular Ca²⁺ concentration. Studies using fluorescent indicators have shown that the initial transient increase in [Ca²⁺]_i following the extracellular Ca²⁺ change reflects the influx of Ca²⁺ from the external compartment (31). The fast increase in [Ca²⁺]_i in response to Ca²⁺ addition appeared to be directly related to the influx pathway, since it varied with the driving force for Ca²⁺ entry. The elevation of the apparent V_max for Ca²⁺ entry in the virus-infected cell without a change in the apparent affinity constant for Ca²⁺ may suggest that infection induced the activation of a cellular Ca²⁺ channel.

The selectivity of the Ca²⁺ entry pathway induced by rotavirus infection was characterized by using a set of cations that are known to permeate different Ca²⁺ channels in other systems (12, 23, 31). We took advantage of the ability of fura-2 to complex to metal cations other than Ca²⁺ and the possibility of calculating actual intracellular concentrations with the known K_ds (16). The Ca²⁺ pathway in both mock- and rotavirus-infected cells was permeable to other divalent cations, such as Ba²⁺, Sr²⁺, Mn²⁺, and Co²⁺. However, it was poorly permeable to Ni²⁺ and impermeable to the trivalent cations La³⁺ and Cr³⁺. These characteristics are common to numerous divalent cation channels in both excitable and nonexcitable cells (10, 14, 19, 24, 31). The influx pathway exhibited an apparent sequence of Sr²⁺  Ba²⁺ > Ca²⁺, which may reflect the actual permeability sequence. No quantitative comparison was possible with the quenching cations Mn²⁺ and Co²⁺, since determination of concentrations was not feasible. In all cases rotavirus-infected cells showed a much higher permeability than mock-infected ones.

The intracellular concentrations of all permeative cations increased upon addition of an extracellular pulse according to a similar temporal pattern. After an initially fast (1 to 2 min) elevation, the increase of intracellular concentration attained a new rate characterized by a smaller slope, in both mock- and virus-infected cells. The second phase should be the result of a balance between cation influx and efflux, both of which are subject to regulation. The rate of influx can be modulated by a change in the intracellular cation concentration which may lead to a closure of the pathway (10, 24). At the same time, regulatory mechanisms governing the efflux would be activated. When we compared the slopes of the second phase, that for Ba²⁺ was much higher than that for Ca²⁺. It has been shown that Ba²⁺ does not replace Ca²⁺ as a substrate for the Ca²⁺-ATPases of the ER or the plasma membrane or for the Na⁺/Ca²⁺ exchanger (35). The results obtained with the addition of La³⁺ after the pulse of Ba²⁺ and Ca²⁺ further support this interpretation. La³⁺ added after the peak of the response to an extracellular Ca²⁺ pulse induced a decrease in fluorescence. Since La³⁺ blocks cation influx, this decrease should reveal the removal of excess Ca²⁺ from the cytosol by regulatory mechanisms. In the case of Ba²⁺, La³⁺ blocked the entry and the concentration remained constant. Excess Ba²⁺ that entered before the addition of La³⁺ was not removed from the cytoplasm by the pumps. Therefore, the change in the kinetics of [Ba²⁺]_i should reflect a time-dependent inhibition of the influx pathway, as has been described for other systems (31). In the case of Ca²⁺, reduction in the influx pathway as well as activation of the efflux component results in regulation of [Ca²⁺]_i to a new steady state.

Calcium channel blockers are commonly used to characterize the nature of Ca²⁺ pathways. The majority of normal epithelial cells do not contain voltage-gated calcium channels. However, L-type Ca²⁺ channels have been detected in renal cells of the proximal and distal tubules and Henle's loop (25, 26, 28, 37), and malignant transformation seems to induce the expression of this type of channel in the pancreatic tumor cell line AR4-2J (2, 5). Also, Ca²⁺ influx induced by carbachol is inhibited by verapamil in the intestinal carcinoma HT29 cell line (21). We do not know if mock-infected cells contain a channel sensitive to D600. The Ca²⁺ influx pathway in rotavirus-infected cells was partially inhibited by D600. We cannot claim at this point that an L-type Ca²⁺ channel is involved in the Ca²⁺ influx pathway induced by rotavirus, because the concentrations of D600 required to cause inhibition are higher than those required for the inhibition of L-type channels. However, infection may have induced the expression or activation of such a channel. Preliminary results obtained by using the patch clamp technique with MA104 cells suggest that infection activates a voltage-dependent Ca²⁺ channel (24a). If this is confirmed, infection may have induced depolarization by increasing the permeability of the cell membrane to Na⁺ and K⁺ and thereby increasing a subsequent Ca²⁺ influx (6).

It has been proposed that the increased Ca²⁺ uptake in virus-infected cells could be secondary to depletion of ER stores through the so-called capacitative pathway induced by the expression of NSP4 (7, 27, 33). However, evidence argues against this possibility: (i) the emptying of Ca²⁺ from the ER would not be compatible with rotavirus maturation and stability (17, 29); (ii) there is an increase in radioactive Ca²⁺ pools, sensitive to thapsigargin, in rotavirus-infected cells (17, 18); (iii) depletion of the stores by thapsigargin in mock-infected cells provoked a capacitative entry of Ca²⁺ much smaller than that induced by rotavirus infection (17); and (iv) the cation selectivity profile of the capacitative channel, Ca²⁺ > Ba²⁺ = Sr²⁺, is not exhibited by the rotavirus-activated pathway (24). Therefore, the Ca²⁺ pathway activated during rotavirus infection appears to be different from the capacitative one. A possible explanation for the discrepancy is that NSP4 expression in Sf9 cells may have effects on ER calcium different from those induced by the expression of the entire genome in infected mammalian cell lines. However, at later times postinfection, when cytoplasmic Ca²⁺ has already increased and pool depletion may have occurred, both mechanisms may be operative.

Several lines of evidence suggest that the pathway for the entry of Ca²⁺ has the characteristics of a channel rather than of unspecific damage: (i) apparent saturation kinetics with respect to extracellular concentration; (ii) entry of cations following an apparent selectivity sequence, from Ba²⁺ to the impermeative La³⁺; (iii) inactivation of the influx induced by the cation; (iv) inhibition of Ca²⁺ influx by La³⁺ and D600; and (v) lack of permeability of the pathway to La³⁺, Cr³⁺, and extracellular markers such as ethidium bromide as well as trypan blue. Although a cellular Ca²⁺ channel preexisting in the cell might be activated by rotavirus infection, it is also possible that a viral protein synthesized during infection and inserted in the membrane acts as a Ca²⁺ channel. Viral proteins in other systems, such as the M2 protein of influenza A virus, function as ion channels (15).

Changes in the permeability of the plasma membrane to Ca²⁺ have been shown to exist in other viral infections and may be a general mechanism for the induction of cytotoxicity. An increase in calcium permeation and/or cytosolic concentration has been found to occur as a result of infection by cytomegalovirus (22), measles and vaccinia viruses (30, 32), coxsackievirus (36), and poliovirus (11). In some cases this could be linked to viral gene expression (22, 32). The expression of recombinant viral proteins such as the A38L protein of vaccinia virus (30), the 2B protein of cosxackievirus (36), or the 2BC protein of poliovirus (1) induced alterations in intracellular Ca²⁺ homeostasis. In the case of coxsackievirus, it appears that Ca²⁺ entry is linked to depletion of ER stores (36). Also, poliovirus infection activates phospholipase C, inositol 1,4,5-triphosphate synthesis, and probably the capacitative entry of Ca²⁺ (1, 9). The external addition of individual viral proteins like gp120 and gp160 of human immunodeficiency virus also potentiates agonist-induced Ca²⁺ entry (13). The sensitivity of this permeability to verapamil and inhibitors of the dihydropyridine family may suggest the participation of voltage-gated channels in some systems (11, 13, 32).

Regardless of the molecular mechanism of Ca²⁺ entry, alterations of membrane permeability to Ca²⁺ of the rotavirus-infected cell could have important physiological consequences. Increases in [Ca²⁺]_i during rotavirus infection have been associated with cytotoxicity in MA104 cells (17, 18, 24a). Also, the expression of NSP4 in mammalian cells, using a vaccinia virus vector, induced cytotoxicity (20). This effect may have resulted from an increase in [Ca²⁺]_i. Whether the cytotoxic effects of NSP4 expression and those provoked by increases in [Ca²⁺]_i are related is not yet known. The viral protein involved in the chain of events leading to the activation of permeability to Ca²⁺ during infection of cultured cells remains to be defined.