From the Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, 05508-900 São Paulo, Brazil
The molecular mechanisms by which Ca2+ and metal ions interact with the binding sites that modulate the tight junctions (TJs) have not been fully described. Metal ions were used as probes of these sites in the frog urinary bladder. Basolateral Ca2+ withdrawal induces the opening of the TJs, a process that is abruptly terminated when Ca2+ is readmitted, and is followed by a complete recovery of the TJ seal. Mg2+ and Ba2+ were incapable of keeping the TJ sealed or of inducing TJ recovery. In addition, Mg2+ causes a reversible concentration-dependent inhibition of the Ca2+-induced TJ recovery. The effects of extracellular Ca2+ manipulation on the TJs apparently is not mediated by changes of cytosolic Ca2+ concentration. The transition elements, Mn2+ and Cd2+, act as Ca2+ agonists. In the absence of Ca2+, they prevent TJ opening and almost immediately halt the process of TJ opening caused by Ca2+ withdrawal. In addition, Mn2+ promotes an almost complete recovery of the TJ seal. Cd2+, in spite of stabilizing the TJs in the closed state and halting TJ opening, does not promote TJ recovery, an effect that apparently results from a superimposed toxic effect that is markedly attenuated by the presence of Ca2+. The interruption of TJ opening caused by Ca2+, Cd2+, or Mn2+, and the stability they confer to the closed TJs, might result from the interaction of these ions with E-cadherin. Addition of La3+ (2 µM) to the basolateral Ca2+-containing solution causes an increase of TJ permeability that fully reverses when La3+ is removed. This effect of La3+, observed in the presence of Ca2+ (1 mM), indicates a high La3+ affinity for the Ca2+-binding sites. This ability of La3+ to open TJs in the presence of Ca2+ is a relevant aspect that must be considered when using La3+ in the evaluation of TJ permeability of epithelial and endothelial membranes, particularly when used during in vivo perfusion or in the absence of fixatives.
Key words: tight junction; calcium; cadmium; lanthanum; E-cadherinCa2+ is essential for cells to maintain intercellular contacts. When the extracellular Ca2+ is removed, the cell-
cell connections generally become loose and multicellular organizations are destroyed. A number of studies emphasize the role of extracellular Ca2+ on the stability
of mature tight junctions (TJs)1 in natural epithelia
(Sedar and Forte, 1964; Hays et al., 1965
; Galli et al.,
1976
; Meldolesi et al., 1978
; Pitelka et al., 1983
; Palant
et al., 1983
) and on the development of new TJs in cell cultures in confluence (Martinez-Palomo et al., 1980
;
Cereijido et al., 1980
, 1981
; González-Mariscal et al.,
1985
). The removal of extracellular Ca2+ causes the
opening of previously formed TJs and prevents de novo formation of TJs in confluent cell monolayers. Notwithstanding several studies addressing the role of extracellular Ca2+ in the dynamics of the TJs, major questions
are still pending. The relative importance of extracel-
lular (Gorodeski et al., 1997
; Contreras et al., 1992
;
González-Mariscal et al., 1990
) versus intracellular (Bhat
et al., 1993
; Jovov et al., 1994
; Stuart et al., 1994
) Ca2+
concentration on the control of TJs is not yet clearly
characterized. The cell adhesion molecule E-cadherin
(uvomorulin) (Gumbiner et al., 1988
), which is particularly rich at the zonula adhaerens (Boller et al., 1985
),
plays a key role as the extracellular Ca2+ binding molecule that modulates the formation and maintenance of
the epithelial junctional complex (Gumbiner et al.,
1988
). Ca2+ influences the conformation of E-cadherin
and stabilizes it in its adhesive state (Ringwald et al.,
1987
). In addition, the interaction of Ca2+ with E-cadherin is transduced across the cell membrane by a cascade of reactions involving phospholipase C, G proteins, protein kinase C, and calmodulin (Balda et al.,
1991
, 1993
). The structural and electrostatic mechanisms used by the Ca2+ binding sites of E-cadherin to
provide Ca2+ specificity are not yet fully understood, as
compared with the knowledge on the EF-hand-like sites
(Snyder et al., 1990
), in part because insufficient information is available regarding the ion specificity of the
Ca2+ binding sites. Only recently has the structure of
the epithelial cadherin domain responsible for selective cell adhesion been identified. The Ca2+ binding
site of the CAD1 domain of E-cadherin was inferred by nuclear magnetic resonance identification of the amino
acid residues whose backbone 13CO, 15N, or 1HN chemical shifts differed between Ca2+-bound and -free forms
(Overduin et al., 1995
). A negatively charged pocket is
formed by three sequences of residues with the side chains of the highly conserved Glu11, Glu69, and Asp100
well positioned to ligate Ca2+ (Overduin et al., 1995
).
In addition, the homophilic specificity surface is also
sensitive to Ca2+ ligation through His79 and Met92, indicating that the Ca2+-induced conformational effect on
the homophilic specificity surface may reflect a mechanism by which Ca2+ levels regulate the adhesiveness of
cadherins (Overduin et al., 1995
).
Multiple factors (number, type, and geometry of
ligands, electrostatic interactions, cavity size and deformability of the site, dehydration of metal and ligand)
are among the variables that must be considered when
the metal ion selectivity of protein Ca2+ sites are analyzed (Snyder et al., 1990). The exclusion of Mg2+ from
many protein Ca2+ sites, for example, can be explained
in part by the fact that Mg2+ prefers a coordination
number of six and uses nitrogen as a ligand more frequently (Einspahar and Bugg, 1984
; Martin, 1984
), while coordination by seven oxygens is observed in protein
Ca2+ sites (Snyder et al., 1990
; Strynadka and James,
1989
).
In a previous study in the frog urinary bladder
(Lacaz-Vieira and Kachar, 1996), it was shown that apical Ca2+ may activate the TJ sealing mechanism, an effect that is not impaired by the presence of Ca2+ channel blockers (nifedipine, verapamil, Mn2+, or Cd2+) in
the apical solution, indicating that junction resealing in the frog urinary bladder does not depend on Ca2+ entering the cells through the apical membrane. Most likely, this effect results from Ca2+ entering partially disrupted
TJs, reaching the zonula adhaerens Ca2+ receptors (E-cadherins). It was also shown that protein kinase C plays a
significant role in the control of TJ assembly in the frog
urinary bladder since the PKC inhibitor (H7) and the
activator (diC8) markedly affect TJ recovery after they
are disrupted by apical hypertonicity.
The present study addresses the interactions of the metal ions with the binding sites that affect the function of the TJs in order to characterize their selectivity.
Urinary bladders of the frog Rana catesbeiana were used. Animals
were anesthetized by subcutaneous injection of a 2% solution of
3-aminobenzoic acid ethyl ester (methanesulfonate salt) (Sigma Chemical Co., St. Louis, MO) at a dose of 1 ml/100 g body wt. The abdominal cavity was opened, a cannula was passed through the cloaca, and the urinary bladder was inflated with 15-20 ml of
air according to the animal size. Plastic rings of 20-mm diameter were glued to the serosal surface of the bladder with ethylcyanoacrylate adhesive (Super Bonder; Loctite, Sáo Paulo, Brazil).
The fragment of tissue framed by the plastic ring was excised and
immersed in Ringer solution. Subsequently, it was mounted in a
modified Ussing's chamber (Castro et al., 1993), exposing an
area of 0.5 cm2. Hemichambers with a recessed rim filled with
high viscosity silicone grease (High Vacuum Grease; Dow-Corning Corp., Indianapolis, IN) prevented tissue edge damage
(Lacaz-Vieira, 1986
). Each chamber compartment was perfused
with a continuous flow of solution (up to 25 ml/min) driven by
gravity from reservoirs through plastic tubings. Unstirred layers
on the surfaces of the tissue were minimized by directing the incoming fluid towards the tissue surfaces. Each compartment was
drained through a spillway open to the atmosphere so that the
pressure inside each compartment was kept fairly constant at the
atmospheric level. Rapid solution changes were obtained without
interruption of voltage clamping by switching the inlet tubings at
their connections with the chamber.
Solutions
Unless otherwise stated, the inner bathing solution was NaCl
Ringer's solution. The Ringer's compositions were (mM): NaCl Ringer: 115 NaCl, 2.5 KHCO3, and 1.0 CaCl2. Na2SO4 Ringer:
57.5 Na2SO4, 2.5 KHCO3, 1.0 CaSO4. NaCl HEPES Ringer: 115 NaCl, 2.5 KCl, 2.0 HEPES. All Ringer's solutions had their pH adjusted to 8.2 after aeration. The apical bathing fluids were simple
salt solutions, nonbuffered, prepared with glass-distilled water,
having pH ~6.0 and free Ca2+ concentration in the range of 1.5 × 1-7 and 2.0 × 1-7 M (Castro et al., 1993). In the beginning of
the experiments, the apical solution was 75 mM KCl.
Electrical Measurements
A conventional analog voltage clamp (DVC 1000; WPI, New Haven, CT) was used. Saturated calomel half-cells with 3 M KCl-agar bridges were used to measure the electrical potential difference across the bladder. Current was passed through Ag-AgCl 3 M KCl electrodes and 3 M KCl-agar bridges, adequately placed to deliver a uniform current density across the bladder. The clamping current was continuously recorded by a strip-chart recorder. Clamping current and voltage were also digitized through an analog-
to-digital converter (Digidata 1200 and Axotape 2.0; Axon Instruments Inc., Foster City, CA) and stored in a computer for further
processing. A digital Gaussian Filter (Colquhoun and Sigworth,
1983) was used to remove high frequency noise of the baseline of
all records used in the figures. This digital filter forms output values
i from input values
i by performing the arithmetic mean of
three consecutive current values, so that
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Isotopic Flux Measurements
14C-Sucrose (Amersham International, Little Chalfont, UK) was added to the apical or basolateral solution, and the fluid was recirculated with a peristaltic pump at a rate of 5 ml/min. An equilibration period of at least 20 min, during which the solution in the opposite compartment was continuously renewed, was allowed before the sampling in this compartment. During the sampling period, the flow of solution through the compartment was stopped and the solution was stirred by a plastic propeller driven by an electric motor. At the end of the collection interval, the solution was removed for radioactivity assay. Subsequently, the compartment was refilled and a new sampling period started.
TJ Blockade by the Selective Deposition of BaSO4
TJ blockade was induced according to a previously described
method (Castro et al., 1993). Tissues were bathed on the basolateral side by Na2SO4 Ringer's solution. To induce the blockade by
the selective deposition of BaSO4 in the TJs, the apical bathing
fluid was replaced by a solution of BaCl2 (50 mM) and a positive
clamping potential (+50 mV) was applied across the tissue to
force migration of Ba2+ and SO42+ against each other and the
formation of BaSO4 precipitate at the TJ level.
Chemicals
All chemicals were obtained from Sigma Chemical Co.
Statistics
The results are presented as mean ± SEM. Comparisons were
carried out using Student's paired t test. When more than two
groups were compared, significance was determined by two-way
analysis of variance followed by appropriate posttest comparison.
The P values cited include Bonferroni's correction (Neter and
Wasserman, 1974).
The experiments were carried out in short-circuited
frog urinary bladders bathed on the basolateral side by
NaCl-Ringer's solution (or a different Ringer's solution
according to the protocol), and on the apical side, in
most cases, by a simple solution of KCl (75 mM). The
absence of Na+ in the apical solution aimed at abolishing the short-circuit current as well as the role of transcellular Na+ conductance to the overall tissue electrical
conductance, so that changes in the transepithelial
electrical resistance (TER) reflected changes in the
electrical resistance of the tight junctions, as in other
tight epithelia (Jovov et al., 1994; Wills and Millinoff, 1990
).
Transepithelial Electrical Resistance
TER is shown in cm2, calculated from the deflections
of the clamping current induced by shifts of the clamping potential of 300 ms duration, ±1 mV amplitude at
15-s intervals, as TER =
Vt/
It, where
Vt and
It are
the changes in the electrical potential difference across
the tissue and clamping current, respectively. It, clamping current in µA/cm2. Positive (or inward) current
corresponds to the transport of positive charges across
the tissue, from the apical to the inner bathing solution. Vt, electrical potential difference across the tissue
(millivolts). The potential of the apical solution is referred to that of the inner solution.
The general protocol consisted in analyzing the interactions of metal ions with the binding sites that affect the TJ permeability according to a Ca2+-switch assay that consisted of a two-step process: (a) increase of
TJ permeability, characterized by a drop in TER, was induced by removing Ca2+ from the basolateral solution.
(b) TJ recovery, characterized by return of TER to initial values, was achieved by the reintroduction of Ca2+
into the basolateral solution. Small, short-term perturbations of the TJs were induced to prevent or minimize
tardy regulatory responses that might complicate the
results. This is exemplified by the fact that the rate of
Ca2+-induced TJ recovery depends on the degree of TJ
opening, which, in turn, depends on the time the bladders were without Ca2+, in agreement with observations
in MDCK (Martinez-Palomo et al., 1980) and A6 (Jovov
et al., 1994
) cell monolayers. To cope with this problem, the drop of TER in response to Ca2+ withdrawal
was normally terminated by the readmission of Ca2+ to
the basolateral fluid when TER reached values close to
250
cm2. The experiments were carried out, unless
specified, with nominally Ca2+-free apical solution. The
presence of Ca2+ in the apical solution is not essential
for stability of TJs in A6 cell monolayers (Jovov et al.,
1994
) or in the frog urinary bladder (Lacaz-Vieira and
Kachar, 1996
).
Effect of Basolateral Ca2+ on TER
Ca2+ removal from the basolateral solution (NaCl,
Na2SO4, or NaCl HEPES Ringer's solution) induces, after a lag time (generally between 30 s and 3 min), a
pronounced drop of TER. Once started, the decline of
TER shows a rapid progress. Return to Ca2+ promptly
stops the decline of TER and triggers a full recovery. In
the example of Fig. 1 A, the onset of TER decline has a
lag time of 90 s and TER drops to 2% of the initial
value in 160 s. Mean values of TER for a group of eight
bladders bathed on the apical side by 75 mM KCl and
by NaCl Ringer's solution on the basolateral side are:
initial condition, 11,729.5 ± 1,532.5 cm2; 120 s after
Ca2+ removal from the basolateral medium, 207.3 ± 32.3
cm2; after full recovery of TER in response to
the reintroduction of Ca2+ into the basolateral medium, 11,567.1 ± 1,667.0
cm2. The changes in short-circuit current (SCC) that take place in conjunction
with the changes in TER result from the movement of ions (mostly Na+ and K+) along the paracellular pathway driven by their concentration differences in the
bathing solutions.
A stepwise reduction of basolateral Ca2+ concentration (by addition of EGTA) does not result in a decrease of TER until [Ca2+]bl reaches values in the range
of 70-100 µM. A further decrease in concentration resulted in a pronounced decline of TER. The dependence of steady state values of TER on [Ca2+]bl is sigmoidal and conforms with the Hill equation (Rodwell, 1996): TER[Ca] = TER/(1 + [Km/[Ca]]n), with a Km
value of 62 ± 28 µM and a Hill coefficient (n) of 8.6 ± 0.9, indicating a steep dependence of TER on [Ca2+]bl.
TER[Ca] is the value of TER at any given serosal Ca2+
concentration; TER is the value of TER at 1 mM serosal
Ca2+ concentration; Km is the serosal Ca2+ concentration that reduces TER to 50% of the value at normal Ca2+ concentration.
The drop of TER that follows basal Ca2+ withdrawal is
caused by a decrease of TJ permeability since it is accompanied by a significant increase of tissue permeability to 14C-sucrose that fully reverses upon reintroduction of Ca2+. The sucrose influx (Jin), which reflects
the magnitude of the paracellular permeability, increased from (a) 0.65 ± 0.06 pmol cm2 min
1 in the
control condition (75 mM KCl on the apical side and
NaCl Ringer's on the basolateral side) to (b) 3.74 ± 0.09 pmol cm
2 min
1, 5 min after Ca2+ removal from
the basolateral solution, and returned to a steady value
of (c) 0.68 ± 0.07 pmol cm
2 min
1 10 min after addition of Ca2+ to the basolateral solution. Statistical comparison: a-b, P < 0.01; a-c, P = NS (n = 6).
To circumvent a conceivable objection that sucrose
flux measurements, which involve long periods of time,
might not provide a clear indication that the initial
drop of TER in response to basolateral Ca2+ withdrawal
results from an increase of TJ permeability, additional experiments were performed in which open TJs were
blocked by the selective deposition of BaSO4 (Castro et
al., 1993). The urinary bladders were bathed on the basolateral side by a sulfate-containing solution (Na2SO4
Ringer's, see MATERIAL AND METHODS) to cause precipitation of BaSO4 in the open TJs when BaCl2 is added to the apical compartment. As soon as TER decreased in
response to Ca2+ withdrawal from the basolateral fluid,
the addition of Ba2+ to the apical solution leads to a
prompt and marked increase of TER that results from
the blockade of the permeabilized TJs by precipitation
of BaSO4 (Fig. 2). In a control group of bladders bathed by NaCl Ringer's, no effect was observed in response to the addition of Ba2+ to the apical solution,
excluding the possibility that the increase of TER
caused by apical Ba2+ resulted from the blockade of a
transcellular pathway involving K channels (Van Driessche
and Zeiske, 1980
). The experiments with Ba2+ provide
strong evidence that the early drop of TER associated with basolateral Ca2+ withdrawal results from a relaxation of the TJ seal.
Role of Cytosolic Ca2+ Concentration on TER Responses to Changes in Extracellular Ca2+ Concentration
To ascertain the contribution of cytosolic Ca2+ concentration on TER responses to changes in extracellular Ca2+ concentration, two experimental approaches were used.
BAPTA-AM.The epithelial cells were loaded with Ca2+ chelator by incubating tissues (n = 5) with the cell-permeant BAPTA-AM ester (10 µM) on both sides for 20 min. No effect was observed on TER. The chelator was then removed together with Ca2+ from the basolateral fluid, leading to a drop of TER that fully recovered upon Ca2+ return to the basolateral fluid. The fact that in the presence of an intracellular Ca2+ chelator, the introduction of Ca2+ into the basolateral fluid triggers TJ recovery is a strong argument in favor of an extracellular effect of Ca2+, most certainly at the level of E-cadherin.
Ionophore A-23187.Two different protocols were tested. In one (n = 3), the experiments were performed with an apical solution containing 75 mM KCl, 1 mM Ca2+, and 3 µM A23187. The presence of the ionophore in the apical solution caused only a small decrease of TER that soon stabilized. A subsequent removal of basolateral Ca2+ induced a reduction of TER similar to that shown in Fig. 1 A. Recovery was obtained by reintroducing Ca2+ into the basolateral fluid. In another group of experiments (n = 5), the apical solution was 75 mM KCl plus the ionophore (3 µM). TJs were opened by removal of basolateral Ca2+, and then the apical solution was replaced by another containing, in addition to the ionophore, 1 mM Ca2+. Upon addition of Ca2+ to the apical solution, a transient reduction of TER that lasted 1-2 min was observed, followed by a subsequent decline of TER. Return to Ca2+ in the basolateral solution then triggered a complete recovery of TER. These experiments suggest that Ca2+ entering the cells through the pathways created by the ionophore may transiently trigger TER recovery. However, a complete and stable recovery of the TJ seal was only obtained upon addition of Ca2+ to the basolateral solution.
Divalent Metal Ions
These experiments aimed to appraise the degree of interaction of divalent cations with the basolateral Ca2+ sites that affect the sealing of the TJs. Two different aspects were analyzed: (a) the ability of the metal ion to prevent the opening of the TJs when the metal ion replaced the basolateral Ca2+, and (b) the ability of the metal ion to induce the resealing of TJs previously opened by the removal of basolateral Ca2+.
Alkaline earth metals as controls: Mg2+ and Ba2+.The equimolar substitution of basolateral Ca2+ by Mg2+ causes a drop in TER similar to that induced by the removal of Ca2+ from the basolateral fluid. Return to Ca2+ leads to a full recovery of TER (Fig. 1 B). A similar behavior is observed in response to the substitution of the basolateral Ca2+ by Ba2 (Fig. 1 C). These results indicate that Mg2+ and Ba2+ are ineffective in maintaining the TJ seal in the frog urinary bladder. Experiments (not shown) also indicate that TJs previously opened by Ca2+ withdrawal do not close in response to addition of Mg2+ or Ba2+ (1 mM) to the basolateral medium. Higher concentrations up to 10 mM were tested without effect.
Transition elements: Cd2+ and Mn2+.The equimolar substitution of basolateral Ca2+ by Cd2+ (Fig. 3 A) or Mn2+
(Fig. 4) does not lead, as observed for the alkaline
earth metals, to a reduction of TER, indicating that
these transition elements show a Ca2+ agonistic effect
in short term experiments, characterized by their ability to keep the TJs closed in the absence of basolateral Ca2+. A subsequent withdrawal of Cd2+ (Fig. 3 A) or of
Mn2+ (Fig. 4) triggers junction opening, indicated by a
decline of TER that follows a time course comparable
with that observed in response to basolateral Ca2+
removal. TJ opening that follows Cd2+ removal is
promptly halted upon reintroduction of Ca2+ (Fig. 3 A)
or even Cd2+ into the basolateral solution. Recovery is,
however, incomplete even in response to Ca2+, suggesting a residual, apparently toxic effect of Cd2+. This
toxic effect of Cd2+ is greatly reduced or even eliminated if Ca2+ is also present, as shown in Fig. 3 B. Tissues exposed for several minutes to basolateral Cd2+ (1 mM) in the presence of a normal basolateral Ca2+ concentration behave, after Cd2+ removal, as control tissues not exposed to Cd2+.
The effect of basolateral Mn2+ (Fig. 4) is different from that of Cd2+ since, in addition to promptly halting the decline of TER, Mn2+ leads to a slower but well characterized recovery of TER, a process that is accelerated and reaches completion if Mn2+ is replaced by Ca2+.
Apical Ca2+ May Reach the Binding Sites that Affect the TJs
The results presented so far show reversible changes of
TER due to manipulation of basolateral Ca2+ in the absence of apical Ca2+. Similar results can be obtained in
the presence of 1 mM Ca2+ in the apical solution.
Higher concentrations of apical Ca2+, however, may
curb the increase of TJ permeability that results from
basolateral Ca2+ withdrawal. Thus, the presence of 10 mM Ca2+ in the apical solution markedly depresses
(Fig. 5 A) or even abolishes (Fig. 5 B) TER decrease in
response to basolateral Ca2+ withdrawal. A subsequent
removal of apical Ca2+ speeds up (Fig. 5 A) or triggers
(Fig. 5 B) a TER decrease that had been blocked by the
high apical Ca2+ concentration. TJ permeability increase induced by withdrawal of basolateral Ca2+ stops
promptly and reverts almost completely in response to
addition of 10 mM Ca2+ to the apical solution (Fig. 5
B). Ca2+ channel blockers (Nifedipine, 1 and 3 µM;
Verapamil, 0.3 mM) added to the apical solution have
no influence on the effect of a high apical Ca2+ concentration, confirming previous findings (Lacaz-Vieira and Kachar, 1996) that the effect of apical Ca2+ is not mediated by Ca2+ entering the cells through apical Ca2+
channels. In conclusion, these results support the notion that apical Ca2+, crossing the open TJs, may reach
the binding sites affecting TJ permeability. When a sufficient Ca2+ concentration is present in the apical solution, diffusion through normally closed TJs may be sufficient to raise the Ca2+ concentration at the binding
sites as to overcome the withdrawal of Ca2+ from the
basolateral solution.
Mg2+ Competes with Ca2+ for the Binding Sites
These experiments use the fact just described that
open TJs allow access of apical ions to the binding sites
that affect the TJs. The presence of Mg2+ in the apical
solution causes a concentration-dependent inhibition of TER recovery in response to Ca2+. The effect of apical Mg2+ starts to be noticed at apical concentrations at
or above 5 mM. Fig. 6 shows an example in which 20 mM Mg2+ in the apical bathing medium practically
abolishes the recovery of TER that occurs in response
to the reintroduction of basolateral Ca2+. The subsequent withdrawal of Mg2+ from the apical solution triggers the TJ recovery process. These results show that
Mg2+ reversibly competes with Ca2+ for the binding sites
that control the TJ. It is interesting to observe that a high
apical Mg2+ concentration inhibits the Ca2+-induced
recovery of TER, but not the ability Ca2+ has to halt the
decrease of TER (Fig. 6).
La3+ Causes TJ Opening in the Presence of Ca2+
These experiments were carried out in NaCl HEPES
Ringer's solution to prevent precipitation of La3+ in bicarbonate Ringer's solution due to the formation of
poorly soluble lanthanum bicarbonate. Addition of
La3+ (2 µM, as La(NO3)3) to the basolateral Ringer's
solution (in the presence of Ca2+) causes a drop of
TER (Fig. 7 A) with lag phases and time courses similar
to those observed in response to basolateral Ca2+ withdrawal (Fig. 7 B). A subsequent removal of La3+ from
the basolateral solution promotes a recovery of TER
similar to what is observed when Ca2+ is readmitted to
the basolateral solution after being previously removed.
Higher concentrations of La3+ (>1 mM) may slow the
quick recovery that follows its removal. This effect of
basolateral La3+ on TER, observed in the presence of a
normal Ca2+ concentration, characterizes a Ca2+ antagonistic effect of La3+.
Addition of La3+ (2 µM as La(NO3)3) to the apical solution (75 mM KCl) causes no effect on TER as it does
when added to the basolateral solution. This indicates
that the normally closed TJs of the urinary bladder are
sufficiently restrictive to hamper the movement of the
trivalent La3+ ion, preventing it from reaching the Ca2+
binding sites that, as previously seen, are readily accessible to La3+ from the basolateral aspect of the tissue. After La3+ removal from the apical bathing fluid (Fig. 8),
tissues respond to the withdrawal of basolateral Ca2+ as
a fresh tissue. Conversely, the addition of La3+ (2 µM)
to the apical solution after the permeability of the TJs had been increased in response to basolateral Ca2+ removal has two distinct effects (Fig. 8 B): (a) apical La3+
promptly terminates the process of TER decrease, and
(b) its presence in the apical compartment blocks TER
recovery that takes place in response to basolateral
Ca2+ addition. The first effect resembles that of Ca2+,
Cd2+, and Mn2+. The second indicates that open TJs
permit La3+ to enter from the apical side and reach the
Ca2+ binding sites that control TJ function, acting as if
La3+ had been added to the basolateral fluid.
The present study deals with the interactions of metal ions with the extracellular Ca2+-binding sites that modulate the TJs in the frog urinary bladder. Focus was addressed to the early events associated with TJ opening and closing.
The dependence of TER, which reflects the degree
of permeability of the TJs on basolateral Ca2+ concentration was evaluated to characterize the dependence
of the TJ regulatory system of the frog urinary bladder
on the external Ca2+ concentration. The steep dependence of TER on [Ca2+]bl, with a Km of 62 ± 28 µM and
a Hill coefficient of 8.6 ± 0.9 (n = 5), indicates a high
Ca2+ affinity of the extracellular Ca2+ sites and is in
agreement with other tissues, such as MDCK (González-Mariscal et al., 1990) and A6 (Jovov et al., 1994
) cell
monolayers.
The results with the intracellular Ca2+ chelator indicates that a rise of intracellular Ca2+ concentration is
not a critical step in the resealing of TJs induced by
raising the basolateral Ca2+ concentration. Nonetheless, it cannot be ruled out that a sudden increase of cytosolic Ca2+ concentration may activate, at least transiently, the mechanism of TJ sealing. However, a complete and stable recovery of the TJ seal was only
obtained upon addition of Ca2+ to the basolateral solution. Our findings are in agreement with observations
in monolayers of human cervical cell line CaSki, where
the effects of extracellular Ca2+ on TJ permeability
were found not to be mediated by mobilization of cytosolic Ca2+ (Wild et al., 1997). On the other hand, the
observations with the Ca2+ ionophore recalls the findings in A6 cell monolayers, where an increase of cytosolic Ca2+ concentration induced by the ionophore
A-23187 caused recovery of the TJ seal (Jovov et al.,
1994
).
The result of a prolonged extracellular Ca2+ withdrawal has been described as causing a progressive
disarray of the TJ structure in natural epithelia or in
cell-cultured monolayers. In short-term experiments,
however, Ca2+ removal is not accompanied by gross distortions of freeze-fracture images (Martinez-Palomo
and Erlij, 1975; Martinez-Palomo et al., 1980
; Lacaz-Vieira
and Kachar, 1996
), indicating that the rapid phase of
TER drop after Ca2+ withdrawal might result from subtle alterations of the TJs structure not detectable by
conventional methods.
The finding that the alkaline earth metals, Mg2+ and
Ba2+, used as controls, were ineffective both in keeping
the TJs closed and inducing the resealing of previously
opened TJs is in consonance with findings in MDCK
cell monolayers where Mg2+ and Ba2+ were also ineffective in promoting junction resealing (Martinez-Palomo et al., 1980; Contreras et al., 1992
).
The transition elements, Mn2+ and Cd2+, behave as
Ca2+ agonists since, in the absence of basolateral Ca2+,
they promote stability of the TJs and halt almost instantly the TJ opening process triggered by basolateral
Ca2+ removal. These effects of Mn2+ and Cd2+, which
resemble the action of Ca2+, might result from their interaction with E-cadherin molecules that are mostly
concentrated in the zonula adhaerens (Boller et al.,
1985) and are known to be involved in the assembly of
the junctional complex (Gumbiner et al., 1988
). In addition, Mn2+ promotes an almost complete recovery of
TER, closely resembling the effect of extracellular
Ca2+. This effect of Mn2+ is in harmony with the observation in the toad urinary bladder, where Mn2+ and
Sr2+ were able to revert the rapid fall of TER that takes
place when Ca2+ was withdrawn from the medium (Lipson et al., 1965
). In contrast, in the bullfrog gastric mucosa, Sr2+ does not promote recovery of the junctional
seal (Forte and Nauss, 1963
); in MDCK cell monolayers, only Ca2+ was effective in triggering TJ formation
during a Ca2+ switch; Mg2+, Ba2+, Mn2+, and Cd2+ were
ineffective (Contreras et al., 1992
). These results show that major differences can be found among tissues and,
for the sake of consistency, one given tissue must be
thoroughly studied.
E-cadherin, in addition to its affinity for Ca2+ (Ringwald et al., 1987), also binds Cd2+, as can be inferred
from binding experiments with E-CAD1, a recombinant 145-residue polypeptide that corresponds to one
of the extracellular Ca2+-binding regions of E-cadherin
(Prozialeck et al., 1996
). In contrast to our experiments, where Cd2+ stabilizes the TJs in the closed state
and interrupts the TJ opening process that is triggered
by basolateral Ca2+ removal, recent studies show that
Cd2+ can selectively damage the TJs between LLC-PK1
cells (Prozialeck et al., 1995
) and human proximal tubule cells (Hazen Martin et al., 1993
) and causes disruption of the TJ-associated microfilaments in rat Sertoli cells (Hew et al., 1993
). It is conceivable that this
discrepancy might result from the duration of Cd2+
contact with the preparation. In our case, brief tissue
exposures to Cd2+ might have prevented the onset of
major toxic effect, which might have been the cause of
TJ disruption in other structures. Consequently, our
short-term experiments apparently permit us to dissociate an agonistic effect of Cd2+ on the TJs from a less
specific toxic effect.
How could Cd2+ stabilize the TJs in closed state, halt
the TJ opening process but, at the same time, be unable to promote recovery to the TJ seal? A reasonable
interpretation would be that Cd2+ acts as a Ca2+ agonist
but, in addition, it presents toxic side effects that develop at a slower pace, preventing the recovery of the
TJ seal. The possibility cannot be discarded that Cd2+
(as well as Ca2+ and Mn2+), in addition to interacting
with E-cadherin, acts by bridging junctional sites at the
level of the TJs themselves. This interpretation is supported by experiments in MDCK cell monolayers prefixed with glutaraldehyde, where Ca2+ removal still
caused a pronounced drop of TER (Martinez-Palomo et al., 1980). In disharmony, however, is the observation that in junctional complex-enriched fractions
from mouse liver, Ca2+ chelation with EGTA does not
disrupt the negative-stained images of zonulae occludentes (Stevenson and Goodenough, 1984
).
The fact that Cd2+ does not leave a residual, apparently toxic, effect when its contact with the tissue takes
place in the presence of Ca2+ may result from a competitive interaction with Ca2+ for a common binding site,
which most probably is E-cadherin. In support of this interpretation are the observations in LLC-PK1 cell monolayers that Cd2+ shows a higher binding affinity at low
(0.1 mM) than at high (10 mM) Ca2+ concentrations
(Prozialeck and Lamar, 1993), and also the experiments of Cd2+ binding to E-CAD1, a Ca2+ binding polypeptide
analog of E-cadherin (Prozialeck et al., 1996
).
Previous studies in frog skin (Castro et al., 1993) and
urinary bladder (Lacaz-Vieira and Kachar, 1996
) have
shown that apical Ca2+ may reach the sites that control
the TJs when the permeability of the TJs was increased.
As Ca2+ channel blockers in the apical solution did not
block the effect of apical Ca2+ (Lacaz-Vieira and Kachar, 1996
), it can be inferred that apical Ca2+, passing
through partially opened TJs, may reach the sites that affect the TJs located at the zonula adhaerens. In the
present study, we explored in more detail this subject
and showed that at concentrations higher than those of
the Ringer's solution, apical Ca2+ is able to effectively
fulfill the role of basolateral Ca2+, maintaining the TJs
closed or even causing the resealing of open TJs in the
absence of basolateral Ca2+. The fact that to be effective apical Ca2+ concentrations must be higher than
that needed in the basolateral solution is reasonable
if we take into consideration the diffusion barrier imposed by the TJs before Ca2+ reaches the zonula adhaerens.
The fact of Mg2+ competitively inhibiting the Ca2+-induced recovery of open TJs means that Mg2+ interacts with the Ca2+ binding sites, despite the fact that
this interaction, in the absence of Ca2+ has no effect
whatsoever on the stability of the TJs, the halting of the
opening process, or recovery of the TJ seal. This competitive inhibition is in agreement with findings in A6
cell monolayers (Jovov et al., 1994).
Another aspect of the interaction of Mg2+ with the TJ
regulatory system is the dissociation between the ability
of basolateral Ca2+ to stop almost instantly the process
of TJ opening, which is preserved, and the ability to induce the recovery of TER, which is abolished by Mg2+.
This dualistic behavior may be regarded as an argument
in favor of a dual effect of Ca2+. The preserved effect
could be due to the formation of ionic bridges between
components in adjacent cells or, most likely, Ca2+-
induced changes in E-cadherin adhesiveness (Ringwald
et al., 1987). The other, slower, abolished by Mg2+, is
the recovery of the TJ seal, presumably resulting from a rearrangement of the TJ molecular organization mediated by cell signaling triggered by the interaction of
Ca2+ with E-cadherin.
The fact that 200 µM La3+ causes a reversible TJ
opening in the presence of 1 mM Ca2+ characterizes an
antagonistic effect for Ca2+ in an apparent competitive
interaction. Interaction of La3+ with Ca2+ binding sites
has been studied in different structures (Weiss, 1974).
La3+ is a modulator of gating activity of ionic channels
(Takata et al., 1966
; Vogel, 1974
; Hille et al., 1975
;
Armstrong and Cota, 1990
; Watkins and Mathie, 1994
),
a potent Ca2+ channel blocker (Nelson, 1987
; Poncet
et al., 1992
; Clarke et al., 1994
), and may also act as a
Ca2+ agonist (Powis et al., 1994
). These effects may result from the fact that La3+, by virtue of an effective
ionic radius (1.10 Å) similar to that of Ca2+ (1.06 Å)
(Snyder et al., 1990
) and a valence higher than Ca2+, is
expected to bind at Ca2+ sites more tightly than does
Ca2+. The action of La3+ on the TJs is a complicated
matter since different effects have been described. Our
present finding that La3+ promotes TJ opening in the
presence of Ca2+ contrasts with those in MDCK cells,
where La3+ was used as a Ca2+ channel blocker and
found not to interfere with junction sealing (Contreras
et al., 1992
), with neurophysiological experiments (where La3+ was used as a TJ blocker) (Sostman and Simon, 1991
; Simon, 1992
; Bryant and Moore, 1995
;
Wang et al., 1993
), and with experiments in A6 cell
monolayers, where a toxic effect was reported (Jovov et
al., 1994
). In the frog skin, however, the reversible
opening of the TJs by an apical hypertonicity (Ussing,
1965
) was made irreversible by the presence of La3+ in
the apical solution (Erlij and Martinez-Palomo, 1972
),
indicating that La3+ interacted with open TJs, preventing their resealing. Transient effects of La3+ on TER
and changes of ion selectivity have been described in rabbit gallbladder and ileum (Machen et al., 1972
).
Studies of protein Ca2+ sites have indicated that La3+ is
often able to effectively replace bound Ca2+ because of
the proximity of their effective ionic radius and because many Ca2+ sites bind both divalent and trivalent
metal ions with high affinity (Brittain et al., 1976
; Horrocks, 1984
). Comparison of the dissociation constants
for the binding of spherical metal ions from groups IA,
IIA, IIIA, and lantanides indicates that both charge and
size are important parameters in determining the specificity of the protein binding sites (Snyder et al., 1990
).
The ability of La3+ to open TJs in the presence of
Ca2+ is a relevant matter to the general use of La3+ in
the evaluation of the permeability of TJs in epithelial
and endothelial membranes (Arendt, 1991; Unakar et
al., 1991
; Vu et al., 1992
; Shirai and Ikemoto, 1992
;
Caldwell and Slapnick, 1992
; Morales and Cavicchia,
1993
; Adamson and Michel, 1993
; Zhong et al., 1994
;
Pelletier, 1994
; Hochman et al., 1994
; Hara et al., 1994
;
Devalia et al., 1994
), particularly during in vivo perfusions or in the absence of a simultaneously present fixative such as glutaraldehyde or formaldehyde (Martinez-Palomo et al., 1971
; Whittembury and Rawlins, 1971
;
Machen et al., 1972
; Martinez-Palomo and Erlij, 1973
;
Tisher and Yarger, 1973
). A comparative study of the
permeability of TJs of blood barriers of the epididymis, vas deferens, and testis in the mink, using horseradish
peroxidase and lanthanum nitrate, showing that lanthanum deposits were found at the microvilli despite
the impermeability of the TJs to horseradish peroxidase, permitted the authors to suggest that the lanthanum technique yielded false positive results (Pelletier,
1994
). It is conceivable that in this case some TJs could
have been opened by the effect of La3+ before the action of fixatives could have taken place. Supporting this
interpretation are studies (with high resolution electron micrographs of TJs in different structures in which
La3+ was used during fixation) that have given no evidence that these junctions were permeable to colloidal
lanthanum (Overton, 1968
; Brightman and Reese,
1969
; Goodenough and Revel, 1970
), in contrast to experiments in which La3+ was perfused in living tissue
(Schatzki, 1969
, 1971
), where there are evidences of
lanthanum passage through the TJs.
To conclude, it is rewarding to compare (Table I) the
effects of Ca2+ and metal ions upon uvomorulin (E-cadherin) in a study of early embryogenesis (Hyafil et al.,
1981), where the authors concluded that uvomorulin
undergoes a Ca2+ (or Mn2+- or Cd2+-)-dependent transition from a trypsin-sensitive to a trypsin-resistant conformation that favors recognition of uvomorulin by a
monoclonal antibody and triggers cell compaction in
early embryogenesis, and the findings of the present
study. The close similarity of behavior observed in
those two systems in response to similar treatments is a
strong indication that the modulation Ca2+ and other
metal ions exert on the TJs is most importantly mediated through their interaction with E-cadherin molecules.
Table I.
Comparative Effects of Metal Ions on the Dynamics of TJs, as Observed in the Present Study, and on the Uvomorulin Molecule (Hyafil et al., 1981 |
Address correspondence to F. Lacaz-Vieira, Institute of Biomedical
SciencesUSP, Department of Physiology and Biophysics, 05508-900 São Paulo, SP, Brazil. Fax: 55-11-818.7285; E-mail: lacaz{at}bmb.icb1.usp.br
Received for publication 23 June 1997 and accepted in revised form 8 September 1997.
This project was supported by grants 96/3367-5 from Fundação de Amparo à Pesquisa do Estado de São Paulo, and 521869/94-3 and 303633/85-9 from Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brazil.1. | Adamson, R.H., and C.C. Michel. 1993. Pathways through the intercellular clefts of frog mesenteric capillaries. J. Physiol. (Camb.). 466: 303-327 [Abstract]. |
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