Role of calcium in tight junction between epithelial cells
formation
L. GONZALEZ-MARISCAL, R. G. CONTRERAS, J. J. BOLIVAR, A. PONCE, B. CHAVEZ DE RAMIREZ, AND M. CEREIJIDO Center of Research and Advanced Studies, Mexico City DF 07000; and Department of Physiology, Faculty of Medicine, Universidad National Autkoma de M&co, Mexico City DF 04510, Mexico
GONZALEZ-MARISCAL, L., R. G. CONTRERAS, J. J. BOL~VAR, A. PONCE, B. CHAVEZ DE RAMIREZ, AND M. CEREIJIDO. Role of calcium in tight junction formation between epithelial cells. Am. J. Physiol. 259 (Cell Physiol. 28): C978-C986, 1990.Upon transferring confluent monolayers of Madin-Darby canine kidney (MDCK) cells from a low-Ca2’ medium (1-5 PM) to one with 1.8 mM Ca2’ (Ca switch), tight junctions (TJs) assemble and seal, and transepithelial electrical resistance (TER) develops in 4-5 h, presumably through exocytotic fusion that incorporates junctional components to the surface membrane. In the present work we test this possibility and observe 1) that the Ca switch raises the cytosolic concentration of this ion; 2) that it also increases the membrane area by 22%; 3) that chloroquine, a drug which prevents exocytosis, blocks both the increase of surface membrane and the sealing of TJs; and 4) that if monolayers are not permanently switched to 1.8 mM Ca”, but are subject to a 15min pulse, cytosolic free Ca2’ concentration ([Ca”‘],) transiently increases but returns to low values (14 * 11 nM) and TER does not develop. Comparisons of the time course of TJ sealing with levels of [Ca2’],, as well as the relationship between these parameters and extracellular Ca2’ levels, suggest that this ion may act from the extracellular side or in a narrow intracellular domain in the close vicinity of the plasma membrane.
epithelial membranes; calcium concentration; sion; transepithelial electrical resistance
exocytotic
fu-
(TJs), which seal the interspace between epithelial cells, are very sensitive to Ca2+. Removal of this ion from the bathing solution opens the junctional complex between oxyntic cells of gastric glands (26) and pancreatic acinar cells (18). Its removal and restoration from solutions bathing monolayers of Madin-Darby canine kidney (MDCK) cells opens and reseals their TJs, a process that can be followed through the fall and recovery of the transepithelial electrical resistance (TER; Refs. 5, 6, 17). Calcium depletion provokes extensive endocytosis of the plasma membrane of mammary epithelial cells in culture, and some of the endocytic vesicles carry segments of junctional strands ( 24). Ca2’ is also involved in the assembly and sealing of newly formed TJs. Thus MDCK cells plated at confluence synthesize, assemble, and seal TJs in 12-16 h (46). Yet, if Ca2’ is not present, junctional components are nevertheless synthesized but seem to accumulate at the outermost cisternae of the Golgi, or even beyond this point, but without reaching the outer surface, and TER TIGHT
C978
remains negligible. Freeze-fracture replicas show that when Ca2+is added to monolayers under such conditions (“Ca switch”), junctional strands appear as early as 15 min, and the whole structure of the TJ is essentially completed in 4-5 h, coinciding with the development of TER (12, 13). On the basis that strands start to form in the position that they will occupy in mature TJs, and that further strands are developed in association with the first ones, we had previously suggested that junctional material coming with from the cytoplasm would be added directly to this region through a fusion of membrane vesicles, in a process which resembles an exocytotic fusion (13). The main purpose of the present work is to analyze the involvement of Ca2+ in this process. We explore the effect of extracellular Ca2+ on I) the intracellular concentration of this ion (measured with the fluorescent probe indo-l), 2) the incorporation of new surface membrane (measured through its electrical capacitance), and 3) the sealing of TJs (evaluated through the measurement of TER and the apical-tobasolateral flux of [3H]mannitol). We also tested the effect of drugs known to inhibit exocytosis on both the increase in membrane surface and the development of TER. METHODS
JUNCTIONS
0363-6143/90 $1.50 Copyright
0
Cell culture. Starter MDCK cultures were obtained from the American Type Culture Collection (MDCK, CCL 34). Upon arrival, cells were cloned, and all experiments reported in the present article were performed in cells of Clone 7, chosen because of its intense blistering activity when plated on nonpermeable supports. Cells were grown at 36.5OC in disposable plastic bottles (Costar 3250, Cambridge, MA) with a 95% air-5% CO2 atmosphere (VIP CO2 incubator 417, Lab Line Instruments, New Brunswick, NY) and 20 ml Dulbecco’s modified Eagle’s medium (DMEM; GIBCO 430-1600, Grand Island, NY) with 100 U/ml penicillin, 100 ,ug/ml streptomycin (GIBCO 600-5145), 0.8 U/ml insulin (Eli Lilly, Mexico), and 10% fetal calf serum (GIBCO 200-6170); in this paper this complete medium is referred to as normal calcium (NC) medium. Cells were harvested with trypsin-EDTA (In Vitro, Mexico) and plated on glass cover slips contained in 30-mm Petri culture dishes (Linbro Chemical, New Haven, CT) or on disks of Millipore
1990 the American
Physiological
Society
Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (137.092.098.118) on August 13, 2018. Copyright © 1990 American Physiological Society. All rights reserved.
cA2+
EFFECT
ON
paper (Bedford, MA) with pores of 1.2 pm in diameter. Cells were usually between the 60- and 80th passage. Upon allowing 1 h for cell attachment, medium was discarded and monolayers were switched to fresh media. Ca switch. Once cells were left to attach for 1 h as described above, the resulting monolayers were washed three times with phosphate-buffered saline (PBS) without Ca2+ (GIBCO 410-1300) and transferred to minimal essential medium (GIBCO 410-3100) without Ca”‘. Determinations with a Ca”+-sensitive electrode detect 1-5 PM Ca2+; therefore, we refer to this medium as low calcium (LC). Twenty hours later the experimental protocol was started by changing the monolayers to NC medium. It was previously shown that after a Ca switch, cells polarize and make tight junctions on permeable and on impermeable supports (9, 13). Further experimental details are given in connection with each study. Membrane capacitance. Monolayers of cells cultured on cover slips were deposited on the glass bottom of a flat chamber fixed to the stage of an inverted Diavert microscope (Leitz, Wetzlar) equipped with Hoffman optics. Membrane capacitance was studied in the “whole cell” configuration described first by Hamill et al. (15) and in the particular case of MDCK cells by Bolivar and Cereijido (2). Recordings were made through a Dagan 8900 amplifier (Minneapolis, MN) with micropipettes pulled from borosilicate glass (Corning, 1.1-1.2 mm ID) using a vertical pipette puller (David Kopf Instruments 7000; Tujunga, CA). Micropipettes were covered with Sylgard and fire polished and had a resistance of 3-4 MQ. They were mounted in a pipette holder attached to a hydraulic micromanipulator (Narishige MF83, Tokyo, Japan). All recordings reported were obtained after seals of at least 10 GQ. Sealing and recordings were monitored with a Tektronix 5115 oscilloscope (Beaverton, OR). Currents were recorded through Ag-AgCl electrodes and filtered at 10 KHz. Signals were digitalized at 1 KHz and acquired and analyzed with a Compaq PC computer using a Fetchex program (Axon Instruments, Burlingame, CA). Stimulation and acquisition in whole cell clamp studies were made with a Clampex 1 program (Axon Instruments). Analyses were performed using a Clampan 1 program (Axon Instruments). Displays were made from pictures taken with a Polaroid camera (Cambridge, MA) from the screen of the oscilloscope. Capacitance measurements were performed at room temperature (ZO-25OC). The intracellular solution contained (in mM) 154 methanesulfonic acid, 10 NaOH, 141 KOH, 1.45 Ca(OH)2, 1.0 Mg(OH),, 2.3 ethylene glycolbis(P-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA), 10 glucose, and 10 N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES)-KOH (pH 7.4), with a “free-Ca” concentration of 3 x 10B7 M. This solution is referred to as K+ rich. The extracellular solution contained (in mM) 154 methanesulfonic acid, 146 NaOH, 5 KOH, 2.0 Ca(OH)2, 1.0 Mg(OH),, 10 glucose, and 10 HEPES-NaOH (pH 7.4). The volume of the bathing solution was 1.5 ml and was changed through perfusion. Measurements of TER. The degree of sealing of the tight junction was evaluated by measuring the electrical resistance across the monolayer. Disks were mounted
TIGHT
JUNCTIONS
c979
between two Lucite chambers of 1.0 ml on each side, with an exposed area of 0.23 cm”. Current was delivered via Ag-AgCl electrodes placed at 2.0 cm from the monolayer; the voltage deflection elicited was measured with a second set of electrodes placed at 1.0 mm from the membrane. The contribution of the Millipore filter and the bathing solution was subtracted, and all values reported correspond exclusively to the monolayers. A given disk was used only for a single determination and discarded to avoid leaks due to edge damage. Calcium activity measurements in solutions. Free calcium concentration was measured with a Ca”+-sensitive electrode made with a 1:l mixture of a cationic exchange resin (Fluka 9470, Buchs, Switzerland) with polyvinyl chloride diluted with tetrahydrofurane. A Beckman 71 pH meter (Fullerton, CA) in its voltage-measuring mode was used to calibrate the electrode and make the measurements. Electrodes had a Nernst potential of -28 mV per lo-fold concentration change, were linear in the range pCa 7.0-3.0, and deviated somewhat from linearity above pCa 7.0. Standard solutions were prepared according to Tsien and Rink (32). Calcium activity measurements in the cell. These measurements were performed with the calcium indicator indo-l acetoxymethyl ester (indo-l/AM; Molecular Probes, Eugene, OR). MDCK cells were plated on a 60mm tissue culture plate containing four to five glass cover slips (0.8 X 3 cm). After a 20-h incubation in LC or NC medium, confluent monolayers could be observed. Cover slips were then transferred to new culture plates with fresh medium containing 1 PM indo-l/AM previously dissolved in dimethyl sulfoxide (DMSO). The final concentration of DMSO in the plates did not exceed 0.1% (vol/vol). We added bovine serum albumin, 0.01% (wt/vol) ) t o cover slips loaded in LC medium. The cover slips were then incubated for 30 min at 37°C with continuous agitation in an orbital shaker. Monolayers were than washed seven times with a buffer [(in mM) 140 NaCl, 5 KCl, 1 MgC12, with or without 1.8 CaC12, 10 tris( hydroxymethyl)aminomethane (Tris), and 10 dextrose], previously passed (before adding Ca2+ and Mg2+) through a Chelex 100 column (Bio-Rad 731-6232, Richmond, CA). Cover slips were mounted in a Teflon stopper and placed inside plastic fluorometric cuvettes (Spectrocell, Oreland, PA) in a 30” angle with respect to the excitation source. The cuvette contained 3.0 ml of buffer with or without calcium depending on experimental needs. Fluorescence measurements were performed in a Perkin-Elmer LS-3B fluorometer (Norwalk, CT) equipped with a magnetic stirrer. The excitation/emission wavelengths used to monitor dye fluorescence were 345/405 nm. Cytosolic Ca2+ was calculated according to the following general formula: [Ca”‘], = &(F - F,i,)/ (F max - F), where & is the dissociation constant for Ca2’ binding (250 nM for indo-1). Briefly, after the initial fluorescence determination (F), ionomycin (Calbiochem, San Diego, CA) was added to a final concentration of 5 PM. This ionophore allows the Ca”+ buffer (1.8 mM) to penetrate and saturate the intracellularly trapped indo1 and thus to obtain maximum fluorescence (Fmax). In cells loaded in LC, CaC12 was added to a final concentration of 1.8 mM, after the F determination. Minimum
Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (137.092.098.118) on August 13, 2018. Copyright © 1990 American Physiological Society. All rights reserved.
C980
cA2+
EFFECT
ON
TER
TJ
TIGHT
JUNCTIONS
(n
TER cm*) l
TJ (%I
(fbcm*)
to/01
800
80
80
600
60
60
100
40
20
0 I
1 1
TIME
IN Co+”
2 TIME in I,8
(he)
FIG. 1. Apical-to-basolateral [ 3H] mannitol flux (Left ordinate, 0), transepithelial electrical resistance (TER; middle ordinate, l ), and degree of junctional sealing (right ordinate, dashed line) as calculated with Eq. 1 at different times of a Ca switch (time 0). Monolayers plated on Millipore filters were mounted as a flat sheet between two Lucite chambers of 8.0 ml each and were vigorously stirred with magnetic bars. Area of exposed monolayer was 0.6 cm”. Each point corresponds to a single monolayer that was measured during 3 5-min periods at a given time after the Ca switch. Fluxes and TERs were recorded simultaneously in the same set of monolayers. TJ, tight junctions.
1
3 mM Cat+
I
1
4
5
(hrs)
FIG. 3. Intracellular Ca2+, TER, and degree of junctional sealing as a function of time during a Ca switch (symbols and definitions as in Fig. 2). Cells were plated at confluence in normal-calcium (NC) medium for 1.0 h, then switched to low-calcium medium; 20 h later cells were switched to NC medium.
TER (nacm2)
TJ (%)
500 100
------
1
/’
TER TJ cn - cm* 1 (%I
+ 1”“” 1’“”
5
400
200
C
2 3 0 7: 0 ”
80 300
150
60 200
100
40
2
2
50
100
20
0
3
z 0 I
I
#---al 6
I
5
EXTRACELLULAR
6
I
1
5
4
EXTRACELLULAR
0 3
pCatt
FIG. 2. Intracellular Ca2’ (left ordinate, 0), TER, and degree of junctional sealing 20 h after incubation in media with different Ca2’ activities. Cells were plated at confluence in normal calcium medium and 1 h later were switched to media with the pCa specified in the abscissa (symbols and definitions as in Fig. 1). They were loaded with indo-l/AM 30 min before each [Ca”‘], measurement. Parallel sets of monolayers were used for TER determination.
fluorescence (F,i,) was calculated after the addition of 2.0 mM MnC12 (F&, according to the following equation: Fmin = [1/6(F,,, - FM,) + FM,]. The autofluorescence of unloaded cells and all added reagents was determined and was negligible for indo-l-loaded cells. The contribution of external dye was determined by measuring the instantaneous decline in fluorescence upon addition of 5 mM EGTA and was found to be negligible. Certain cell types possessorganic anion transporters that promote the sequestration of indo-l into intracellular organelles and the secretion of this dye from the cell, and probenecid, an inhibitor of organic anion transport, blocks this effects. For this reason, we used probenecid (Sigma, St. Louis, MO) in our control studies. These
1
4
1
3
pCa++
FIG. 4. Activity of cytosolic Ca2+, TER, and percent of junctional sealing (symbols and definitions as in Fig. 2). Monolayers were incubated 20 h in low-calcium medium and then switched for an additional period of 5 h to media with the Ca2’ activities specified on the abscissa.
experiments (data not shown) indicated that 2.5 mM probenecid does not alter [Ca”‘], quantification, thus suggesting no dye leakage. Transepithelial flux of mannitol. Monolayers plated at confluence on Millipore filters as described above were mounted as a flat sheet between two Lucite chambers of 8.0 ml under continuous and vigorous stirring with two magnetic bars. D-[~-~H(N)] mannitol (Du Pont, Boston, MA) was added to the apical side (1.0 &i/ml), and periodic samples of 100 ~1 were taken from the opposite side and counted in a scintillation counter. TER was measured in the same set of monolayers at the end of each sampling period. Transmission electron microscopy (TEM). Monolayers were fixed with 2.5% glutaraldehyde after different times of incubation in LC or NC media. After washing in 0.1 M cacodylate buffer, cells were postfixed in 1% Os04 cacodylate buffer, dehydrated in ethanol, and embedded in Epon 812. Thin sections were cut with a diamond
Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (137.092.098.118) on August 13, 2018. Copyright © 1990 American Physiological Society. All rights reserved.
CA”+
EFFECT
(ll-
TER cm*)
- 600 - 500
ON TJ (%I 7100
- 80
- 400 - 60 - 300 - 40 - 200 -100 -0 1
I
0
1
1
2 TIME
1
1
3
4
in 1.8 mM Ca++
1
I
5
6
- 20
-0
(he)
FIG. 5. Effect of 1.0 mM La:‘+ on cytosolic Ca”’ activity, percent of junctional sealing during a Ca switch (symbols tions as in Fig. 2).
TER, and and defini-
600 : -c r 3 0 < 0
400
0
0 l
I
I
I
1
1
0
1
2
3
4
5
TIME
6. Effect of 10 PM verapamil a Ca switch. Dashed line, redrawn under control conditions.
FIG.
after Ca” TABLE
1. TER in monolayer
in 1.8 mM
(h)
of MDCK cells TER,
Control Verapamil (10 PM) TMB-8 (100 ,uM) BAPTA (25 ,uM)
Co++
on cytosolic Ca2’ activity from Fig. 3, represents cytosolic (0)
12- cm2
448k32 54Ok60 93t20 315t43
n
65 20 12 16
Values are means t SE; n, no. of observations. TER, transepithelial electrical resistance; TMB-8, 3,4,5-trimethoxybenzoic acid 8(diethylamino)octyl ester; BAPTA, 1,2-bis(2-aminophenoxy)ethaneN,N,N ‘,N ‘-tetraacetic acid.
knife, then examined and photographed in a Zeiss EM 10 electron microscope. Scanning electron microscopy. Cells were fixed with glutaraldehyde and dehydrated. Critical point drying from absolute ethanol into liquid CO2 was carried out in a Technics apparatus (Springfield, MA). Dried specimens affixed with double-sided tape to aluminum stubs were coated with gold in an ion sputter JFC-1000 JEOL evaporator (Japan). Specimens were examined in a JEOL JSM 35C scanning electron microscope at 15 KV.
TIGHT
C981
JUNCTIONS
Results are expressed as means t SD (n is the number of observations). RESULTS
In the figures illustrating the relationship between cytosolic Ca2+ and TER, we also represent the fraction of sealed TJs (fc) as calculated by the following equation fc = [MR,
- &)lI[M&
- fW1
(1)
where R,, Rt, and R, are the values of TER before, during, and after complete sealing of the TJs. Figure 1 shows the relationship between this parameter, TER, and the apical-to-basolateral flux of [3H]mannitol. It may be observed that the flux of this substance sharply drops to a basal level coinciding with the sealing of TJs around the first hour of Ca switch. Figure 2 shows the effect of extracellular Ca2+ on the intracellular concentration of this ion (open circles), on TER (filled circles), and on the degree of junction formation (dashed line) of monolayers of MDCK cells that have been plated at confluence for 1 h in NC medium, then transferred to media with the concentration of Ca2+ specified in the abscissa, and incubated for 20 h in this medium. It may be noticed that the establishment of TJ depends critically on the presence of Ca2+. However, because the cytosolic concentration of this ion varies as a function of the external one, the side of the membrane where the ion is required may not be elucidated. As mentioned in the introduction, if monolayers are left for 20 h in 1-5 PM Ca2’ (LC) junctions do not form, an observation that is confirmed by the information in Fig. 2. Yet, when these monolayers are transferred to media with 1.8 mM Ca2+ (NC) at the 20th hour (Ca switch) junctions develop with a relatively fast kinetics (13). Figure 3 shows an experiment designed to study the evolution of cytosolic Ca2+ (open circles) during a Ca switch. It may be seen that there is a dramatic increase followed by a sharp drop that reaches a minimum in -15 min and then increases slowly to concentrations similar to those found in control monolayers that were continuously incubated in NC [14l t 15 (n = 6) vs. 154 t 8 nM (n = 15)]. If monolayers were not permanently switched to NC, but returned to LC 15 min after the switch, cytosolic Ca2+ falls back to low values [ 14 t 11 16) at 4.5 h] and TER does not develop. nM(n= The next step was to determine the minimum Ca”+ level in the cytoplasm compatible with junction formation during a Ca switch. Accordingly, after a 20-h incubation in LC media we switched the monolayers to media having a variety of pCa. Figure 4 shows that monolayers bathed in 0.1 mM Ca”+ have 96% of their TJs sealed. Interestingly enough, at this extracellular concentration cytosolic Ca”+ remains low, suggesting that it is not necessarily an increase in this parameter that triggers junction formation. To explore these possibilities we tried to interfere with the penetration of Ca”+ with La”’ and verapamil. La”’ (1.0 mM) drastically reduces the influx of Ca”+ in MDCK cells (unpublished results). In the presence of La3’ the cytosolic concentration of Ca2+ increases at a much slower rate than in control conditions (compare Figs. 3
Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (137.092.098.118) on August 13, 2018. Copyright © 1990 American Physiological Society. All rights reserved.
C982
cA2+
EFFECT
ON TIGHT
JUNCTIONS
FIG. 7. A: scanning electron micrograph of early attachment to a Millipore filter of 3 Madin-Darby canine kidney (MDCK) cells that had been previously harvested with trypsin-EDTA. B and C: transmission electron micrograph of monolayers of MDCK cells that 1 h after plating have been incubated for 20 h in low-calcium and normal-calcium media, respectively. Arrowhead in B points to a vesicular compartment; arrowheads in C point to intercellular spaces.
and 5). La3+ also abolishes the initial peak of Ca2+ concentration observed in Fig. 3, suggesting that it is due to the fast penetration of this ion. Data in Fig. 5 show that in spite of the low cytosolic Ca2+ cells manage to assemble and seal their TJs, albeit at a somewhat lower speed. La3+ does not substitute for Ca2+ in the triggering
of junction formation, because if instead of switching the monolayers to Ca2+ they are switched to a medium containing 1 mM La3+ they develop a TER of only 7 + 1 G. cm2 (n = 6) (vs. 449 of control monolayers, Fig. 2). Verapamil, a drug which blocks Ca2+ penetration through voltage-sensitive Ca channels (31), does not
Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (137.092.098.118) on August 13, 2018. Copyright © 1990 American Physiological Society. All rights reserved.
CA”+
I
I
1
I
6
5
4
3
L 7
EFFECT
EXTRACELLULAR
ON
pCa++
8. Membrane surface of MDCK as a function of the activity of Ca” in the incubating medium. Cells were harvested with trypsinEDTA and suspended in medium containing EGTA to adjust free Ca2’ to desired concentration. pCa was measured with a Ca2+-sensitive electrode. At the extremely low concentrations on the Left, only a small proportion of the cells attached to the substrate. These remained rounded and isolated. Membrane surface was evaluated through its capacitance 20 h after plating. FIG.
n
5
18
II 8 IO
15
f
pco.02
t < 0.601
1
u
0 1
I 041'
1
20
1
1
I
25 TIME
(hours)
9. Membrane area of MDCK cells as measured with the membrane capacitance method during the Ca-switch procedure. Cells were harvested and plated at confluence at time 0. One hour later the resulting monolayers were transferred to low-calcium (LC) medium. Twenty hours later they were switched to normal-calcium (NC) medium. Membrane surface was measured as membrane capacitance in cells that were plated in LC medium for
formation
L. GONZALEZ-MARISCAL, R. G. CONTRERAS, J. J. BOLIVAR, A. PONCE, B. CHAVEZ DE RAMIREZ, AND M. CEREIJIDO Center of Research and Advanced Studies, Mexico City DF 07000; and Department of Physiology, Faculty of Medicine, Universidad National Autkoma de M&co, Mexico City DF 04510, Mexico
GONZALEZ-MARISCAL, L., R. G. CONTRERAS, J. J. BOL~VAR, A. PONCE, B. CHAVEZ DE RAMIREZ, AND M. CEREIJIDO. Role of calcium in tight junction formation between epithelial cells. Am. J. Physiol. 259 (Cell Physiol. 28): C978-C986, 1990.Upon transferring confluent monolayers of Madin-Darby canine kidney (MDCK) cells from a low-Ca2’ medium (1-5 PM) to one with 1.8 mM Ca2’ (Ca switch), tight junctions (TJs) assemble and seal, and transepithelial electrical resistance (TER) develops in 4-5 h, presumably through exocytotic fusion that incorporates junctional components to the surface membrane. In the present work we test this possibility and observe 1) that the Ca switch raises the cytosolic concentration of this ion; 2) that it also increases the membrane area by 22%; 3) that chloroquine, a drug which prevents exocytosis, blocks both the increase of surface membrane and the sealing of TJs; and 4) that if monolayers are not permanently switched to 1.8 mM Ca”, but are subject to a 15min pulse, cytosolic free Ca2’ concentration ([Ca”‘],) transiently increases but returns to low values (14 * 11 nM) and TER does not develop. Comparisons of the time course of TJ sealing with levels of [Ca2’],, as well as the relationship between these parameters and extracellular Ca2’ levels, suggest that this ion may act from the extracellular side or in a narrow intracellular domain in the close vicinity of the plasma membrane.
epithelial membranes; calcium concentration; sion; transepithelial electrical resistance
exocytotic
fu-
(TJs), which seal the interspace between epithelial cells, are very sensitive to Ca2+. Removal of this ion from the bathing solution opens the junctional complex between oxyntic cells of gastric glands (26) and pancreatic acinar cells (18). Its removal and restoration from solutions bathing monolayers of Madin-Darby canine kidney (MDCK) cells opens and reseals their TJs, a process that can be followed through the fall and recovery of the transepithelial electrical resistance (TER; Refs. 5, 6, 17). Calcium depletion provokes extensive endocytosis of the plasma membrane of mammary epithelial cells in culture, and some of the endocytic vesicles carry segments of junctional strands ( 24). Ca2’ is also involved in the assembly and sealing of newly formed TJs. Thus MDCK cells plated at confluence synthesize, assemble, and seal TJs in 12-16 h (46). Yet, if Ca2’ is not present, junctional components are nevertheless synthesized but seem to accumulate at the outermost cisternae of the Golgi, or even beyond this point, but without reaching the outer surface, and TER TIGHT
C978
remains negligible. Freeze-fracture replicas show that when Ca2+is added to monolayers under such conditions (“Ca switch”), junctional strands appear as early as 15 min, and the whole structure of the TJ is essentially completed in 4-5 h, coinciding with the development of TER (12, 13). On the basis that strands start to form in the position that they will occupy in mature TJs, and that further strands are developed in association with the first ones, we had previously suggested that junctional material coming with from the cytoplasm would be added directly to this region through a fusion of membrane vesicles, in a process which resembles an exocytotic fusion (13). The main purpose of the present work is to analyze the involvement of Ca2+ in this process. We explore the effect of extracellular Ca2+ on I) the intracellular concentration of this ion (measured with the fluorescent probe indo-l), 2) the incorporation of new surface membrane (measured through its electrical capacitance), and 3) the sealing of TJs (evaluated through the measurement of TER and the apical-tobasolateral flux of [3H]mannitol). We also tested the effect of drugs known to inhibit exocytosis on both the increase in membrane surface and the development of TER. METHODS
JUNCTIONS
0363-6143/90 $1.50 Copyright
0
Cell culture. Starter MDCK cultures were obtained from the American Type Culture Collection (MDCK, CCL 34). Upon arrival, cells were cloned, and all experiments reported in the present article were performed in cells of Clone 7, chosen because of its intense blistering activity when plated on nonpermeable supports. Cells were grown at 36.5OC in disposable plastic bottles (Costar 3250, Cambridge, MA) with a 95% air-5% CO2 atmosphere (VIP CO2 incubator 417, Lab Line Instruments, New Brunswick, NY) and 20 ml Dulbecco’s modified Eagle’s medium (DMEM; GIBCO 430-1600, Grand Island, NY) with 100 U/ml penicillin, 100 ,ug/ml streptomycin (GIBCO 600-5145), 0.8 U/ml insulin (Eli Lilly, Mexico), and 10% fetal calf serum (GIBCO 200-6170); in this paper this complete medium is referred to as normal calcium (NC) medium. Cells were harvested with trypsin-EDTA (In Vitro, Mexico) and plated on glass cover slips contained in 30-mm Petri culture dishes (Linbro Chemical, New Haven, CT) or on disks of Millipore
1990 the American
Physiological
Society
Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (137.092.098.118) on August 13, 2018. Copyright © 1990 American Physiological Society. All rights reserved.
cA2+
EFFECT
ON
paper (Bedford, MA) with pores of 1.2 pm in diameter. Cells were usually between the 60- and 80th passage. Upon allowing 1 h for cell attachment, medium was discarded and monolayers were switched to fresh media. Ca switch. Once cells were left to attach for 1 h as described above, the resulting monolayers were washed three times with phosphate-buffered saline (PBS) without Ca2+ (GIBCO 410-1300) and transferred to minimal essential medium (GIBCO 410-3100) without Ca”‘. Determinations with a Ca”+-sensitive electrode detect 1-5 PM Ca2+; therefore, we refer to this medium as low calcium (LC). Twenty hours later the experimental protocol was started by changing the monolayers to NC medium. It was previously shown that after a Ca switch, cells polarize and make tight junctions on permeable and on impermeable supports (9, 13). Further experimental details are given in connection with each study. Membrane capacitance. Monolayers of cells cultured on cover slips were deposited on the glass bottom of a flat chamber fixed to the stage of an inverted Diavert microscope (Leitz, Wetzlar) equipped with Hoffman optics. Membrane capacitance was studied in the “whole cell” configuration described first by Hamill et al. (15) and in the particular case of MDCK cells by Bolivar and Cereijido (2). Recordings were made through a Dagan 8900 amplifier (Minneapolis, MN) with micropipettes pulled from borosilicate glass (Corning, 1.1-1.2 mm ID) using a vertical pipette puller (David Kopf Instruments 7000; Tujunga, CA). Micropipettes were covered with Sylgard and fire polished and had a resistance of 3-4 MQ. They were mounted in a pipette holder attached to a hydraulic micromanipulator (Narishige MF83, Tokyo, Japan). All recordings reported were obtained after seals of at least 10 GQ. Sealing and recordings were monitored with a Tektronix 5115 oscilloscope (Beaverton, OR). Currents were recorded through Ag-AgCl electrodes and filtered at 10 KHz. Signals were digitalized at 1 KHz and acquired and analyzed with a Compaq PC computer using a Fetchex program (Axon Instruments, Burlingame, CA). Stimulation and acquisition in whole cell clamp studies were made with a Clampex 1 program (Axon Instruments). Analyses were performed using a Clampan 1 program (Axon Instruments). Displays were made from pictures taken with a Polaroid camera (Cambridge, MA) from the screen of the oscilloscope. Capacitance measurements were performed at room temperature (ZO-25OC). The intracellular solution contained (in mM) 154 methanesulfonic acid, 10 NaOH, 141 KOH, 1.45 Ca(OH)2, 1.0 Mg(OH),, 2.3 ethylene glycolbis(P-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA), 10 glucose, and 10 N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES)-KOH (pH 7.4), with a “free-Ca” concentration of 3 x 10B7 M. This solution is referred to as K+ rich. The extracellular solution contained (in mM) 154 methanesulfonic acid, 146 NaOH, 5 KOH, 2.0 Ca(OH)2, 1.0 Mg(OH),, 10 glucose, and 10 HEPES-NaOH (pH 7.4). The volume of the bathing solution was 1.5 ml and was changed through perfusion. Measurements of TER. The degree of sealing of the tight junction was evaluated by measuring the electrical resistance across the monolayer. Disks were mounted
TIGHT
JUNCTIONS
c979
between two Lucite chambers of 1.0 ml on each side, with an exposed area of 0.23 cm”. Current was delivered via Ag-AgCl electrodes placed at 2.0 cm from the monolayer; the voltage deflection elicited was measured with a second set of electrodes placed at 1.0 mm from the membrane. The contribution of the Millipore filter and the bathing solution was subtracted, and all values reported correspond exclusively to the monolayers. A given disk was used only for a single determination and discarded to avoid leaks due to edge damage. Calcium activity measurements in solutions. Free calcium concentration was measured with a Ca”+-sensitive electrode made with a 1:l mixture of a cationic exchange resin (Fluka 9470, Buchs, Switzerland) with polyvinyl chloride diluted with tetrahydrofurane. A Beckman 71 pH meter (Fullerton, CA) in its voltage-measuring mode was used to calibrate the electrode and make the measurements. Electrodes had a Nernst potential of -28 mV per lo-fold concentration change, were linear in the range pCa 7.0-3.0, and deviated somewhat from linearity above pCa 7.0. Standard solutions were prepared according to Tsien and Rink (32). Calcium activity measurements in the cell. These measurements were performed with the calcium indicator indo-l acetoxymethyl ester (indo-l/AM; Molecular Probes, Eugene, OR). MDCK cells were plated on a 60mm tissue culture plate containing four to five glass cover slips (0.8 X 3 cm). After a 20-h incubation in LC or NC medium, confluent monolayers could be observed. Cover slips were then transferred to new culture plates with fresh medium containing 1 PM indo-l/AM previously dissolved in dimethyl sulfoxide (DMSO). The final concentration of DMSO in the plates did not exceed 0.1% (vol/vol). We added bovine serum albumin, 0.01% (wt/vol) ) t o cover slips loaded in LC medium. The cover slips were then incubated for 30 min at 37°C with continuous agitation in an orbital shaker. Monolayers were than washed seven times with a buffer [(in mM) 140 NaCl, 5 KCl, 1 MgC12, with or without 1.8 CaC12, 10 tris( hydroxymethyl)aminomethane (Tris), and 10 dextrose], previously passed (before adding Ca2+ and Mg2+) through a Chelex 100 column (Bio-Rad 731-6232, Richmond, CA). Cover slips were mounted in a Teflon stopper and placed inside plastic fluorometric cuvettes (Spectrocell, Oreland, PA) in a 30” angle with respect to the excitation source. The cuvette contained 3.0 ml of buffer with or without calcium depending on experimental needs. Fluorescence measurements were performed in a Perkin-Elmer LS-3B fluorometer (Norwalk, CT) equipped with a magnetic stirrer. The excitation/emission wavelengths used to monitor dye fluorescence were 345/405 nm. Cytosolic Ca2+ was calculated according to the following general formula: [Ca”‘], = &(F - F,i,)/ (F max - F), where & is the dissociation constant for Ca2’ binding (250 nM for indo-1). Briefly, after the initial fluorescence determination (F), ionomycin (Calbiochem, San Diego, CA) was added to a final concentration of 5 PM. This ionophore allows the Ca”+ buffer (1.8 mM) to penetrate and saturate the intracellularly trapped indo1 and thus to obtain maximum fluorescence (Fmax). In cells loaded in LC, CaC12 was added to a final concentration of 1.8 mM, after the F determination. Minimum
Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (137.092.098.118) on August 13, 2018. Copyright © 1990 American Physiological Society. All rights reserved.
C980
cA2+
EFFECT
ON
TER
TJ
TIGHT
JUNCTIONS
(n
TER cm*) l
TJ (%I
(fbcm*)
to/01
800
80
80
600
60
60
100
40
20
0 I
1 1
TIME
IN Co+”
2 TIME in I,8
(he)
FIG. 1. Apical-to-basolateral [ 3H] mannitol flux (Left ordinate, 0), transepithelial electrical resistance (TER; middle ordinate, l ), and degree of junctional sealing (right ordinate, dashed line) as calculated with Eq. 1 at different times of a Ca switch (time 0). Monolayers plated on Millipore filters were mounted as a flat sheet between two Lucite chambers of 8.0 ml each and were vigorously stirred with magnetic bars. Area of exposed monolayer was 0.6 cm”. Each point corresponds to a single monolayer that was measured during 3 5-min periods at a given time after the Ca switch. Fluxes and TERs were recorded simultaneously in the same set of monolayers. TJ, tight junctions.
1
3 mM Cat+
I
1
4
5
(hrs)
FIG. 3. Intracellular Ca2+, TER, and degree of junctional sealing as a function of time during a Ca switch (symbols and definitions as in Fig. 2). Cells were plated at confluence in normal-calcium (NC) medium for 1.0 h, then switched to low-calcium medium; 20 h later cells were switched to NC medium.
TER (nacm2)
TJ (%)
500 100
------
1
/’
TER TJ cn - cm* 1 (%I
+ 1”“” 1’“”
5
400
200
C
2 3 0 7: 0 ”
80 300
150
60 200
100
40
2
2
50
100
20
0
3
z 0 I
I
#---al 6
I
5
EXTRACELLULAR
6
I
1
5
4
EXTRACELLULAR
0 3
pCatt
FIG. 2. Intracellular Ca2’ (left ordinate, 0), TER, and degree of junctional sealing 20 h after incubation in media with different Ca2’ activities. Cells were plated at confluence in normal calcium medium and 1 h later were switched to media with the pCa specified in the abscissa (symbols and definitions as in Fig. 1). They were loaded with indo-l/AM 30 min before each [Ca”‘], measurement. Parallel sets of monolayers were used for TER determination.
fluorescence (F,i,) was calculated after the addition of 2.0 mM MnC12 (F&, according to the following equation: Fmin = [1/6(F,,, - FM,) + FM,]. The autofluorescence of unloaded cells and all added reagents was determined and was negligible for indo-l-loaded cells. The contribution of external dye was determined by measuring the instantaneous decline in fluorescence upon addition of 5 mM EGTA and was found to be negligible. Certain cell types possessorganic anion transporters that promote the sequestration of indo-l into intracellular organelles and the secretion of this dye from the cell, and probenecid, an inhibitor of organic anion transport, blocks this effects. For this reason, we used probenecid (Sigma, St. Louis, MO) in our control studies. These
1
4
1
3
pCa++
FIG. 4. Activity of cytosolic Ca2+, TER, and percent of junctional sealing (symbols and definitions as in Fig. 2). Monolayers were incubated 20 h in low-calcium medium and then switched for an additional period of 5 h to media with the Ca2’ activities specified on the abscissa.
experiments (data not shown) indicated that 2.5 mM probenecid does not alter [Ca”‘], quantification, thus suggesting no dye leakage. Transepithelial flux of mannitol. Monolayers plated at confluence on Millipore filters as described above were mounted as a flat sheet between two Lucite chambers of 8.0 ml under continuous and vigorous stirring with two magnetic bars. D-[~-~H(N)] mannitol (Du Pont, Boston, MA) was added to the apical side (1.0 &i/ml), and periodic samples of 100 ~1 were taken from the opposite side and counted in a scintillation counter. TER was measured in the same set of monolayers at the end of each sampling period. Transmission electron microscopy (TEM). Monolayers were fixed with 2.5% glutaraldehyde after different times of incubation in LC or NC media. After washing in 0.1 M cacodylate buffer, cells were postfixed in 1% Os04 cacodylate buffer, dehydrated in ethanol, and embedded in Epon 812. Thin sections were cut with a diamond
Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (137.092.098.118) on August 13, 2018. Copyright © 1990 American Physiological Society. All rights reserved.
CA”+
EFFECT
(ll-
TER cm*)
- 600 - 500
ON TJ (%I 7100
- 80
- 400 - 60 - 300 - 40 - 200 -100 -0 1
I
0
1
1
2 TIME
1
1
3
4
in 1.8 mM Ca++
1
I
5
6
- 20
-0
(he)
FIG. 5. Effect of 1.0 mM La:‘+ on cytosolic Ca”’ activity, percent of junctional sealing during a Ca switch (symbols tions as in Fig. 2).
TER, and and defini-
600 : -c r 3 0 < 0
400
0
0 l
I
I
I
1
1
0
1
2
3
4
5
TIME
6. Effect of 10 PM verapamil a Ca switch. Dashed line, redrawn under control conditions.
FIG.
after Ca” TABLE
1. TER in monolayer
in 1.8 mM
(h)
of MDCK cells TER,
Control Verapamil (10 PM) TMB-8 (100 ,uM) BAPTA (25 ,uM)
Co++
on cytosolic Ca2’ activity from Fig. 3, represents cytosolic (0)
12- cm2
448k32 54Ok60 93t20 315t43
n
65 20 12 16
Values are means t SE; n, no. of observations. TER, transepithelial electrical resistance; TMB-8, 3,4,5-trimethoxybenzoic acid 8(diethylamino)octyl ester; BAPTA, 1,2-bis(2-aminophenoxy)ethaneN,N,N ‘,N ‘-tetraacetic acid.
knife, then examined and photographed in a Zeiss EM 10 electron microscope. Scanning electron microscopy. Cells were fixed with glutaraldehyde and dehydrated. Critical point drying from absolute ethanol into liquid CO2 was carried out in a Technics apparatus (Springfield, MA). Dried specimens affixed with double-sided tape to aluminum stubs were coated with gold in an ion sputter JFC-1000 JEOL evaporator (Japan). Specimens were examined in a JEOL JSM 35C scanning electron microscope at 15 KV.
TIGHT
C981
JUNCTIONS
Results are expressed as means t SD (n is the number of observations). RESULTS
In the figures illustrating the relationship between cytosolic Ca2+ and TER, we also represent the fraction of sealed TJs (fc) as calculated by the following equation fc = [MR,
- &)lI[M&
- fW1
(1)
where R,, Rt, and R, are the values of TER before, during, and after complete sealing of the TJs. Figure 1 shows the relationship between this parameter, TER, and the apical-to-basolateral flux of [3H]mannitol. It may be observed that the flux of this substance sharply drops to a basal level coinciding with the sealing of TJs around the first hour of Ca switch. Figure 2 shows the effect of extracellular Ca2+ on the intracellular concentration of this ion (open circles), on TER (filled circles), and on the degree of junction formation (dashed line) of monolayers of MDCK cells that have been plated at confluence for 1 h in NC medium, then transferred to media with the concentration of Ca2+ specified in the abscissa, and incubated for 20 h in this medium. It may be noticed that the establishment of TJ depends critically on the presence of Ca2+. However, because the cytosolic concentration of this ion varies as a function of the external one, the side of the membrane where the ion is required may not be elucidated. As mentioned in the introduction, if monolayers are left for 20 h in 1-5 PM Ca2’ (LC) junctions do not form, an observation that is confirmed by the information in Fig. 2. Yet, when these monolayers are transferred to media with 1.8 mM Ca2+ (NC) at the 20th hour (Ca switch) junctions develop with a relatively fast kinetics (13). Figure 3 shows an experiment designed to study the evolution of cytosolic Ca2+ (open circles) during a Ca switch. It may be seen that there is a dramatic increase followed by a sharp drop that reaches a minimum in -15 min and then increases slowly to concentrations similar to those found in control monolayers that were continuously incubated in NC [14l t 15 (n = 6) vs. 154 t 8 nM (n = 15)]. If monolayers were not permanently switched to NC, but returned to LC 15 min after the switch, cytosolic Ca2+ falls back to low values [ 14 t 11 16) at 4.5 h] and TER does not develop. nM(n= The next step was to determine the minimum Ca”+ level in the cytoplasm compatible with junction formation during a Ca switch. Accordingly, after a 20-h incubation in LC media we switched the monolayers to media having a variety of pCa. Figure 4 shows that monolayers bathed in 0.1 mM Ca”+ have 96% of their TJs sealed. Interestingly enough, at this extracellular concentration cytosolic Ca”+ remains low, suggesting that it is not necessarily an increase in this parameter that triggers junction formation. To explore these possibilities we tried to interfere with the penetration of Ca”+ with La”’ and verapamil. La”’ (1.0 mM) drastically reduces the influx of Ca”+ in MDCK cells (unpublished results). In the presence of La3’ the cytosolic concentration of Ca2+ increases at a much slower rate than in control conditions (compare Figs. 3
Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (137.092.098.118) on August 13, 2018. Copyright © 1990 American Physiological Society. All rights reserved.
C982
cA2+
EFFECT
ON TIGHT
JUNCTIONS
FIG. 7. A: scanning electron micrograph of early attachment to a Millipore filter of 3 Madin-Darby canine kidney (MDCK) cells that had been previously harvested with trypsin-EDTA. B and C: transmission electron micrograph of monolayers of MDCK cells that 1 h after plating have been incubated for 20 h in low-calcium and normal-calcium media, respectively. Arrowhead in B points to a vesicular compartment; arrowheads in C point to intercellular spaces.
and 5). La3+ also abolishes the initial peak of Ca2+ concentration observed in Fig. 3, suggesting that it is due to the fast penetration of this ion. Data in Fig. 5 show that in spite of the low cytosolic Ca2+ cells manage to assemble and seal their TJs, albeit at a somewhat lower speed. La3+ does not substitute for Ca2+ in the triggering
of junction formation, because if instead of switching the monolayers to Ca2+ they are switched to a medium containing 1 mM La3+ they develop a TER of only 7 + 1 G. cm2 (n = 6) (vs. 449 of control monolayers, Fig. 2). Verapamil, a drug which blocks Ca2+ penetration through voltage-sensitive Ca channels (31), does not
Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (137.092.098.118) on August 13, 2018. Copyright © 1990 American Physiological Society. All rights reserved.
CA”+
I
I
1
I
6
5
4
3
L 7
EFFECT
EXTRACELLULAR
ON
pCa++
8. Membrane surface of MDCK as a function of the activity of Ca” in the incubating medium. Cells were harvested with trypsinEDTA and suspended in medium containing EGTA to adjust free Ca2’ to desired concentration. pCa was measured with a Ca2+-sensitive electrode. At the extremely low concentrations on the Left, only a small proportion of the cells attached to the substrate. These remained rounded and isolated. Membrane surface was evaluated through its capacitance 20 h after plating. FIG.
n
5
18
II 8 IO
15
f
pco.02
t < 0.601
1
u
0 1
I 041'
1
20
1
1
I
25 TIME
(hours)
9. Membrane area of MDCK cells as measured with the membrane capacitance method during the Ca-switch procedure. Cells were harvested and plated at confluence at time 0. One hour later the resulting monolayers were transferred to low-calcium (LC) medium. Twenty hours later they were switched to normal-calcium (NC) medium. Membrane surface was measured as membrane capacitance in cells that were plated in LC medium for
