Cytosolic ionized Ca2+ modulates chemical hypoxia-induced hyperpermeability in intestinal epithelial monolayers

Naoki Unno, Shozo Baba, and Mitchell P. Fink

Department of Surgery, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215; and Second Department of Surgery, Hamamatsu University School of Medicine, Hamamatsu 431-3192, Japan

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

We reported previously that ATP depletion induced by glycolytic inhibition or cellular hypoxia increases the permeability of intestinal epithelial monolayers [N. Unno, M. J. Menconi, A. L. Salzman, M. Smith, S. Hagen, Y. Ge, R. M. Ezzell, and M. P. Fink. Am. J. Physiol. 270 (Gastrointest. Liver Physiol. 33): G1010-G1021, 1996]. In the present study, we examined the effects of the Ca2+ ionophore A-23187 or the intracellular ionized Ca2+ concentration ([Ca2+]i) chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM on the permeability of enterocytic (T84) monolayers depleted of ATP by metabolic inhibition. Permeability was assessed by measuring transepithelial electrical resistance and the transepithelial flux of fluorescein sulfonic acid. Although neither A-23187 nor BAPTA-AM affected ATP depletion, A-23187 augmented, whereas BAPTA-AM ameliorated, chemical hypoxia-induced hyperpermeability. BAPTA-AM ameliorated chemical hypoxia-induced cytoskeletal derangements. Monolayers subjected to chemical hypoxia but incubated in a low (i.e., 100 µM) [Ca2+] environment showed preservation of junctional integrity, whereas voltage-dependent Ca2+ channel blockers (NiCl2 or verapamil) failed to ameliorate chemical hypoxia-induced hyperpermeability. ATP depletion induces hyperpermeability in intestinal epithelial monolayers via a [Ca2+]i-dependent mechanism. Increased [Ca2+]i under these conditions reflects leakage of Ca2+ from the extracellular milieu via a mechanism unrelated to voltage-dependent Ca2+ channels.

1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester; adenosine 5'-triphosphate; fura 2; actin

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

THE INTESTINAL EPITHELIUM normally functions as a selective barrier, permitting the absorption of water, electrolytes, and nutrients but restricting the passage of larger and potentially toxic hydrophilic compounds (13). The permeability of the intestinal epithelium is not static but is dynamically regulated under both physiological and pathophysiological conditions (13). Previous studies have demonstrated that several factors are capable of modulating epithelial permeability to hydrophilic solutes, including various cytokines, e.g., interferon-gamma (15, 36) or interleukin-4 (5), hormones, e.g., insulin (19) or insulin-like growth factors (20), protein kinase C (31), and nitric oxide (28, 36).

Cellular ATP depletion induced by metabolic inhibition or cellular hypoxia is another factor that has been reported (3, 16) to increase the permeability of various types of epithelia. Even relatively modest degrees of ATP depletion, if maintained for a prolonged period of time, are associated with increases in the permeability of cultured enterocytic monolayers (35). The mechanism(s) whereby changes in cellular ATP content modulate epithelial permeability remain to be completely elucidated. It is known, however, that ATP depletion increases cytosolic ionized calcium concentration ([Ca2+]i) in endothelial (23) and epithelial (9, 18) cells. Moreover, elevation of [Ca2+]i induced by incubation of monolayers with the Ca2+ ionophore A-23187 has been shown to increase epithelial junctional permeability (7). Both ATP depletion (35) and elevation of [Ca2+]i (10) are known to cause fragmentation of F-actin and cytoskeletal derangements. Because the regulation of epithelial paracellular permeability has been closely linked to the function of the actin-based cytoskeleton (12), it seems plausible to hypothesize that ATP depletion, elevation of [Ca2+]i, and intestinal epithelial hyperpermeability are causally interrelated phenomena.

Prompted by the foregoing considerations, we used cultured T84 human enterocytic monolayers growing on permeable supports as an in vitro model to evaluate the interrelationships between ATP depletion and [Ca2+]i as determinants of intestinal epithelial permeability. The T84 cell line, derived from a human colon cancer, has been widely used to study various aspects of intestinal epithelial function, including the regulation of paracellular permeability (15). To induce ATP depletion, we subjected the cells to a form of "chemical hypoxia" by incubating the monolayers with the metabolic inhibitors 2-deoxyglucose (2-DG) and antimycin A (AA) in glucose-free medium.

    METHODS
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Methods
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Materials. DMEM, Ham's F-12 medium, penicillin, streptomycin, and FCS were obtained from GIBCO (Grand Island, NY). Fluorescein sulfonic acid (FS), rhodamine-phalloidin, fura 2-AM, and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM (33) were from Molecular Probes (Eugene, OR). The luciferin-luciferase reagent was purchased from LKB Wallac (Turku, Finland). All other reagents were from Sigma Chemical (St. Louis, MO), unless otherwise stated. Plasticware was from Costar (Cambridge, MA). T84 cells were obtained from American Type Culture Collection (Rockville, MD). Gas mixtures were obtained from Airco New England (Hingham, MA).

Cell culture. T84 cells were fed with a 1:1 mixture of DMEM and Ham's F-12 medium supplemented with HEPES (15 mM), NaHCO3 (15 mM), penicillin (40 µg/ml), ampicillin (8 µg/ml), and streptomycin (90 µg/ml) and titrated to pH 7.4. Cultures were maintained in a humidified 5% CO2 incubator at 37°C. The growth medium was replaced twice per week. Confluent monolayers growing in 75-cm2 tissue culture flasks were harvested with a solution of 0.1% trypsin and 1.0 mM EDTA in PBS and seeded onto collagen-coated Costar polycarbonate Transwell (12.0-mm diameter) membranes with a pore size of 3 µM. Cells seeded onto the Costar inserts were fed twice weekly. Inserts were used for experiments during the interval between 7 and 14 days after seeding. Unless otherwise specified, experiments were carried out in a HEPES-phosphate-buffered Ringer solution (HPBR) containing 135 mM NaCl, 5 mM KCl, 3.33 mM NaH2PO4, 0.83 mM Na2HPO4, 1 mM CaCl2, 1 mM MgCl2, 10 mM glucose, and 5 mM HEPES, pH 7.4. Chemical hypoxia was achieved by using modified HPBR in which the glucose was replaced with 10 mM 2-DG and to which 5 µM AA was added. The intracellular Ca2+ chelator BAPTA-AM was loaded into the cells during a 1-h preincubation period in feeding medium and then added to glucose-free HPBR at the desired concentration. The Ca2+ ionophore A-23187 (4 µM) was added at the beginning of the experiment.

Permeability measurements. The permeability of T84 monolayers was determined by measuring the transepithelial passage of FS (mol mass, 478 Da) or fluorescein isothiocyanate-dextran with an average molecular mass of 70,000 Da (FD70) as previously described (35). FS is a highly charged lipophobic moiety at physiological pH and, accordingly, is considered to be cell impermeant (11). FD70 is minimally charged at physiological pH so that permeation of cell monolayers via the paracellular pathway is unlikely to be affected by changes in the charge selectivity of the water-filled paracellular channel. FS (final concn, 200 µg/ml) or FD70 (final concn, 25 µg/ml) was added to the medium in the apical compartments of the Transwell chambers at the beginning of the experiments. After the desired period of incubation, samples of the media from the apical and basolateral compartments were diluted in PBS (pH 7.40) and assayed for FS or FD70 concentration using a Perkin-Elmer LS50 fluorescence spectrophotometer at an excitation wavelength of 492 nm (slit width, 2.5 nm), an emission wavelength of 515 nm (slit width, 10 nm), and an integration time of 10 s. The permeability of the monolayers was expressed as a clearance (C; in nl · h-1 · cm-2), according to the equation C = Faright-arrow b · [tracer]a-1 · S-1, where Faright-arrow b is the flux of the fluorescent probe from the apical to the basolateral compartment (light units/h), [tracer]a is the concentration of FS or FD70 in the apical chamber medium at the beginning of the incubation period (light units/nl), and S is the surface area of the monolayer (in cm2). Simultaneous controls were performed with each experiment.

Transepithelial resistance measurements. Measurements of transepithelial resistance (TER) were performed in culture medium using monolayers grown on 12-mm inserts with a surface area of 1.0 cm2. To measure TER, 100-µA current pulses (1 s) were passed via Ag-AgCl electrodes. The resultant voltage deflections were detected using a separate pair of Ag-AgCl electrodes and a pulse height analyzer (model 558C-5, University of Iowa, Iowa City, IA). Fluid resistance was subtracted, and the net resistance was expressed as ohms per square centimeter.

ATP assay. Cellular ATP levels were determined using the luciferin-luciferase method as previously described (35). At desired time points, cells were harvested by scraping. ATP was extracted with ice-cold 2% trichloroacetic acid and 2 mM EDTA. After sonication, ATP content was measured by a luciferin-luciferase chemiluminescence assay. As an internal calibration, a known quantity of an ATP standard was added to the samples and the corresponding bioluminescence was measured. ATP data were calculated as nanomoles of ATP per well and expressed as a percentage of the value measured in monolayers incubated simultaneously with standard medium.

Measurements of [Ca2+]i in T84 cells. Measurement of [Ca2+]i was carried out using a method modified from that described previously by Denning et al. (6). The cells were grown on glass coverslips and bathed in HPBR. The cells were loaded with fura 2 by incubation with 10 µM (final concn) fura 2-AM and 0.05% (final concentration) Pluronic F127 for 60 min at 37°C. The glass coverslips were placed in a triangular glass cuvette (Starna Cells, Atascadero, CA) positioned in a SPEX DM3000CM spectrofluorometer. The cells were excited alternatively at wavelengths of 340 and 380 nM, and the emission fluorescence intensity was monitored at 505 nM. Background values for unloaded cells were determined from values obtained from the raw data. [Ca2+]i was determined from the ratio of the fluorescence intensities induced by excitation at the two wavelengths (i.e., F340/F380) after subtraction of background fluorescence. The maximal fluorescence ratio was determined 15-20 min after adding 20 µM ionomycin and 40 µM digitonin in the presence of 2 mM CaCl2. The minimum fluorescence ratio was determined 20-30 min after replacement of the bath solution with Ca2+-free HPBR containing 20 mM EGTA and 20 µM ionomycin. [Ca2+]i values were calculated by software provided with the SPEX DM3000CM, using a previously reported equation and the Ca2+ dissociation constant for fura 2 (2.8 × 10-7 M) (8). For experiments in which the cells were subjected to chemical hypoxia, the medium was changed to modified HPBR buffer in which 10 M glucose was replaced with 10 mM 2-DG and 5 µM AA was added.

Fluorescence microscopy. T84 cells grown on 12-mm Transwell membranes were fixed in 4% paraformaldehyde in PBS (pH 7.4). After fixation, the membranes were washed twice in PBS (5 min each), extracted with 0.1% Triton X-100 in PBS for 2 min, and washed twice (5 min each) in PBS. For localization of filamentous actin (F-actin), the membranes were cut out of the chambers with a razor blade and stained for 30 min with rhodamine-phalloidin diluted 1:50 in PBS and washed twice (15 min each) in PBS. The membranes were then placed on a microscope slide, and (to reduce bleaching of fluorescence) a drop of 1 mg/ml p-phenylenediamine in a 9:1 mixture of glycerol and PBS (pH 8.5) was added to each membrane before application of a coverslip and sealing with nail polish. All specimens were examined using a laser confocal imaging system (Bio-Rad MRC 600) attached to a Zeiss Axiovert microscope with a Zeiss Plan-Neofluor oil immersion ×100 objective (Carl Zeiss, Thornwood, NY).

Statistics. Data are expressed as means ± SE. Statistical comparisons were performed using the two-tailed Student's t-test for unpaired data and one- or two-way analysis of variance for repeated measures followed by Duncan's test, as appropriate. The null hypothesis was rejected for P < 0.05.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Effect of chemical hypoxia on cellular ATP levels. Intracellular levels of ATP decreased rapidly when T84 cells were incubated in glucose-free HPBR containing 10 mM 2-DG and 5 µM AA [Glu(-)2-DG/AA], declining to <5% of the control value within 60 min (Fig. 1). In contrast, ATP levels remained relatively constant over the same period of observation when T84 cells were incubated in standard (glucose-containing) HPBR medium. The decrease in intracellular ATP content induced by chemical hypoxia was not affected by the presence of either A-23187 (4 µM) or BAPTA-AM (10 µM) in the medium, a finding that suggests these agents had no effects on cellular energy metabolism, at least under the imposed condition of acute metabolic inhibition.


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Fig. 1.   ATP levels in T84 cells subjected to "chemical hypoxia" in the presence or absence of the Ca2+ ionophore A-23187 or the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM. Chemical hypoxia was induced by incubation with 10 mM 2-deoxyglucose (2-DG) and 5 µM antimycin A (AA) in glucose-free buffer (chemical hypoxia). We studied the following 4 conditions: incubation in glucose-containing control medium (control; black-square); chemical hypoxia [Glu(-)2-DG/AA; bullet ]; chemical hypoxia in the presence of 10 µM BAPTA-AM [Glu(-)2-DG/AA + BAPTA; black-triangle]; and chemical hypoxia in the presence of 4 µM A-23187 [Glu(-)2-DG/AA + A-23187; black-lozenge ]. Cellular ATP contents are expressed as %average value recorded for simultaneously run controls incubated in glucose-containing growth medium. Results are means ± SE; n = 4 for each condition. * P < 0.01 vs. time-matched values for monolayers subjected to chemical hypoxia in the presence or absence of A-23187 or BAPTA-AM.

Effect of chemical hypoxia on permselectivity of T84 monolayers. The apical compartments of Transwell chambers containing T84 cells were loaded with either FS or FD70, and the monolayers were subjected to either chemical hypoxia or incubated with control (glucose-containing) medium. Permeability was assessed after 60 and 120 min of incubation with Glu(-)2-DG/AA or the control medium. As shown in Fig. 2A, the clearances of FS were 463 ± 10 and 2,658 ± 353 nl · cm-2 · h-1 after 60 min of incubation under control or chemical hypoxia conditions, respectively (P < 0.01). After 60 min incubation in control medium or Glu(-)2-DG/AA medium, clearances of FD70 were 11.7 ± 6.3 and 13.3 ± 3.2 nl · cm-2 · h-1, respectively (P = not significant). After 120 min, FS clearances were 625 ± 131 and 3,003 ± 317 nl · cm-2 · h-1 under control and chemical hypoxia conditions, respectively (P < 0.01; Fig. 2B). After 120 min, FD70 clearances were 18.3 ± 3.5 and 197.5 ± 42.3 nl · cm-2 · h-1 for monolayers incubated under control or chemical hypoxia conditions, respectively (P < 0.01). These results indicate that monolayers maintained permselectivity for 60 min despite the imposition of chemical hypoxia but tended to lose size selectivity after 120 min of incubation under ATP-depleting conditions.


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Fig. 2.   Comparison of effect of chemical hypoxia on permeability of T84 monolayers to 2 different probes with markedly different molecular sizes. Fluorescein sulfonic acid (FS) (mol mass, 478 Da) or fluorescein isothiocyanate-dextran with an average molecular mass of 70,000 Da (FD70) was added to the apical compartments of bicameral diffusion chambers at the beginning of incubation with Glu(-)2-DG/AA medium. After 60 (A) and 120 min (B) of incubation, concentrations of FS (filled bars) or FD70 (hatched bars) were determined in the basolateral compartment, and permeability was calculated as described in METHODS. NS, not significant.

Effect of chemical hypoxia on [Ca2+]i. Changes in [Ca2+]i induced by chemical hypoxia in T84 cells grown to confluence on glass coverslips were assessed using fluorescence microscopy and the Ca2+-sensitive dye fura 2. As shown in Fig. 3, induction of chemical hypoxia evoked a time-dependent increase in [Ca2+]i. At 60 min, [Ca2+]i was ~500 nM in cells incubated with Glu(-)2-DG/AA medium. The increase in [Ca2+]i induced by chemical hypoxia was exacerbated by the addition of A-23187 (4 µM) to the medium. In contrast, the increase in [Ca2+]i induced by metabolic inhibition was ameliorated by the addition of BAPTA-AM (10 µM) to the medium. In T84 cells incubated under control conditions, [Ca2+]i remained relatively constant at ~50-80 nM for up to 90 min.


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Fig. 3.   Representative tracings showing changes in intracellular Ca2+ concentration ([Ca2+]i) after induction of chemical hypoxia in T84 cells in the presence or absence of 10 µM BAPTA-AM or 4 µM A-23187. [Ca2+]i was measured using the Ca2+-sensitive fluorescent dye fura 2. After induction of chemical hypoxia in the absence of BAPTA or A-23187, [Ca2+]i increased progressively. Addition of A-23187 further augmented the increase in [Ca2+]i induced by chemical hypoxia, whereas addition of BAPTA-AM blunted the increase in [Ca2+]i.

Effect of A-23187 or BAPTA-AM on chemical hypoxia-induced derangements in barrier function. The TER of T84 monolayers decreased rapidly after the induction of chemical hypoxia (Fig. 4A). Similarly, permeability to the hydrophilic probe FS increased rapidly when the cells were incubated with Glu(-)2-DG/AA medium (Fig. 4B). The addition of A-23187 (4 µM) potentiated the decrease in TER and the increase in FS clearance induced by chemical hypoxia. In contrast, the addition of BAPTA-AM (10 µM) significantly ameliorated the derangements in barrier function induced by Glu(-)2-DG/AA medium.


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Fig. 4.   Effect of chemical hypoxia on transepithelial electrical resistance (TER; A) and permeability to FS (B) of T84 monolayers. Conditions studied are as described in Fig. 1 legend. black-square, Control; bullet , Glu(-)2-DG/AA; black-triangle, Glu(-)2-DG/AA + BAPTA; black-lozenge , Glu(-)2-DG/AA + A-23187. Results are means ± SE; n = 4 for each condition. * P < 0.01 vs. time-matched value for control monolayers incubated with glucose-containing buffer.

Reversibility of chemical hypoxia-induced barrier dysfunction. T84 cells were incubated with Glu(-)2-DG/AA medium for 60 or 120 min. Then the metabolic inhibitors were removed by washing, and the cells were incubated for another 60 min in control (glucose-containing) medium. When the cells were subjected to chemical hypoxia for only 60 min, TER decreased dramatically to ~50% of the baseline value during the period of metabolic inhibition but recovered to ~90% of the normal level after washout of 2-DG and AA and restoration of normal glucose-containing medium (Fig. 5). However, when cells were metabolically inhibited for 120 min, TER failed to recover during the reversal period. When monolayers were subjected to 60 min of metabolic inhibition in the presence of A-23187 (4 µM), TER decreased dramatically during the chemical hypoxia period and similarly failed to recover after washout of 2-DG and AA (data not shown).


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Fig. 5.   Reversibility of decrease in TER induced by subjecting T84 monolayers to chemical hypoxia. After monolayers were incubated for 60 min under conditions leading to development of chemical hypoxia, glucose-free medium containing 10 mM 2-DG and 5 µM antimycin A was replaced with standard growth medium. Results are means of 8 replicates.

Fluorescence microscopy. We previously showed that ATP depletion causes redistribution of F-actin filaments in cultured intestinal epithelial monolayers (35). In the present study, we compared the distribution of F-actin in T84 monolayers subjected to 90 min of chemical hypoxia in the presence or absence of BAPTA-AM (10 µM). Figure 6A shows the distribution of F-actin stained with rhodamine-phalloidin in T84 cells incubated under control conditions. When the cells were subjected to chemical hypoxia, perijunctional actin filaments were fragmented and the continuity of F-actin distribution at points of cell-to-cell attachment was lost (Fig. 6B). However, when T84 cells were subjected to chemical hypoxia in the presence of BAPTA-AM, the normal distribution of F-actin was reasonably well preserved (Fig. 6C).