Vol. 279, Issue 5, C1385-C1392, November 2000
Effect of HCO3
on TPA- and IBMX-induced
anion conductances in Necturus gallbladder epithelial
cells
Pamela
Lyall1,
William
McD.
Armstrong
,1, and
Vijay
Lyall2
1 Department of Physiology and Biophysics, Indiana
University School of Medicine, Indianapolis, Indiana 46202-5102;
and 2 Department of Physiology, Virginia Commonwealth
University, Richmond, Virginia 23298-0551
 |
ABSTRACT |
Effects of HCO3
on protein kinase C (PKC)-
and protein kinase A (PKA)-induced anion conductances were investigated
in Necturus gallbladder epithelial cells. In
HCO3-free media, activation of PKC via
12-O-tetradecanoylphorbol 13-acetate (TPA) depolarized
apical membrane potential (Va) and decreased fractional apical voltage ratio (FR). These effects were
blocked by mucosal 5-nitro-2-(3-phenylpropylamino) benzoic acid
(NPPB), a Cl channel blocker. In HCO3
media, TPA induced significantly greater changes in
Va and FR. These effects were
blocked only when NPPB was present in both mucosal and basolateral
compartments. The data suggest that TPA activates NPPB-sensitive apical
Cl conductance (gCla) in the
absence of HCO3; in its presence, TPA stimulated both
NPPB-sensitive gCla and basolateral
Cl conductance (gClb).
Activation of PKA via 3-isobutyl-1-methylxanthine (IBMX) also decreased Va and FR; however, these
changes were not affected by external HCO3. We
conclude that HCO3 modulates the effects of PKC on
gClb. In HCO3 medium, TPA
and IBMX also induced an initial transient hyperpolarization and
increase in intracellular pH. Because these changes were independent of
mucosal Na+ and Cl, it is suggested that TPA
and IBMX induce a transient increase in apical HCO3 conductance.
protein kinase A; protein kinase C; 5-nitro-2-(3-phenylpropylamino)benzoic acid; membrane potential; intracellular pH; 12-O-tetradeconoylphorbol
13-acetate; 3-isobutyl-1-methylxanthine
| |
INTRODUCTION |
THE
GALLBLADDER OF Necturus absorbs NaCl and
H2O in near-isosmotic proportions. A reciprocal regulation
of NaCl and fluid transport via protein kinase A (PKA) and protein
kinase C (PKC) suggests that these are important physiological
regulators of transport and cell volume in gallbladder cells (8,
13). Activation of PKA decreases net transepithelial salt and
fluid transport (28), and its inhibition increases the
rate of NaCl entry across the apical membrane of gallbladder cells
(8). In contrast, activation of PKC by phorbol esters
increases apical NaCl entry (13). Several studies suggest
that PKA and PKC regulate salt and fluid transport via integrated
changes in Na+/H+ exchange
(27), Cl/HCO3 exchange
(25), the mode of NaCl entry (8, 13), and
apical Cl conductance (gCla)
(6, 13, 14, 21, 28). Although the effects of cAMP (an
activator of PKA) on gCla and basolateral
Cl conductance (gClb) have
been investigated (6, 8, 10, 14, 21, 28), little
information is available on the effects of PKC activation on anion
conductances in gallbladder cells under physiological conditions.
Phorbol 12-myristate 13-acetate (PMA), an activator of PKC, stimulates
gCla in gallbladder cells (14),
but it is not known if this conductance is different from
cAMP-activated gCla (6, 14,
21). In contrast to gCla,
gClb appears to be cAMP independent
(6). It is not known whether PKC activates
gClb in gallbladder cells.
In this study, we investigated the effect of the phorbol ester
12-O-tetradecanoylphorbol 13-acetate (TPA; an activator of PKC) on the anion conductances of gallbladder cell membranes. In some
experiments, the TPA effects on membrane conductances were compared
with those of the phosphodiesterase inhibitor
3-isobutyl-1-methylxanthine (IBMX), an agent that increases
intracellular cAMP and activates PKA (34). To investigate
the regulation of anion conductances under physiological conditions
(i.e., in the presence of external HCO3), the effects
of TPA and IBMX were studied in media buffered with
HCO3 and in nominally HCO3-free
media buffered with imidazole. Our data demonstrate that, in the
presence and absence of HCO3, both TPA and IBMX
increase gCla. However, in
HCO3-buffered media, only TPA activated
gClb, suggesting that under physiological
conditions PKC regulates gClb in gallbladder cells.
| |
MATERIALS AND METHODS |
Necturus maculosus, obtained from Nasco Biological
(Ft. Atkinson, WI) or Kon's Scientific (Germantown, WI) were kept in
an aquarium at 4°C and fed live minnows. Animals were anesthetized with a 1% solution of tricaine methane-sulfonate and immediately killed after surgery. The gallbladders were removed, cut
longitudinally, and rinsed in one of the control Ringer solution
described below. The gallbladders were mounted (apical surface upward)
as flat sheets in a divided Lucite chamber with an exposed area of 0.38 cm2 as described previously (7, 19, 34).
During the experiment, the mucosal and serosal surfaces of the tissue
were independently and continuously perfused by gravity. All
experiments were conducted at room temperature (23 ± 1°C).
Two control Ringer solutions were used. One contained (in mM) 100 NaCl,
2.5 KCl, 1.8 CaCl2, and 5 imidazole (pH 7.4). This solution
was bubbled with 100% O2. The second solution contained (in mM) 75 NaCl, 25 NaHCO3, 2.5 KCl, and 1.8 CaCl2 and was buffered at pH 7.4 with 95%
O2-5% CO2. A Na+-free medium
without HCO3 was prepared in which NaCl was replaced
with Tris. When HCO3 was present, NaCl was replaced
with Tris · HCl, and choline bicarbonate was substituted for
NaHCO3. Cl-free media were also prepared in
which Cl was completely replaced with gluconate. To keep
the external Ca2+ activity in these solutions at the level
present in control solutions, the Ca2+ concentration was
increased from 1.8 to 8.0 mM (19).
TPA was dissolved in ethanol and used at a final concentration of 50 µM. The final concentration of ethanol in Ringer solution was
0.025%. It was established in separate experiments that this concentration did not affect the electrophysiological characteristics of Necturus gallbladder.
5-Nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) was dissolved in
DMSO and used at a final concentration of 10 µM. 3,4,5- Trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8)
hydrochloride was dissolved in DMSO and used at a final concentration
of 6 µM. Corresponding amounts of DMSO were added to the control
solutions, which did not affect the tissue. 1-(5-Isoquinolinylsulfonyl)-2-methylpiperazine (H-7) dihydrochloride was dissolved directly in the Ringer solution at a final concentration of 100 µM. In some experiments, gallbladders were exposed to 100 µM
mucosal IBMX (34). TPA, H-7, TMB-8, and IBMX were obtained from Sigma Chemical (St. Louis, MO). NPPB was a generous gift from Dr.
H. J. Lang (Hoechst Aktiengesellschaft, Pharma-Synthese, Frankfurt, Germany).
Fabrication of microelectrodes.
Single-barrel, open-tip microelectrodes with a tip diameter of <1 µm
were fabricated as described previously (11) and filled with 0.5 M KCl. Tip resistances ranged from 10 to 40 M
when immersed in control Ringer solution. Double-barreled, pH-sensitive
microelectrodes with an overall tip diameter of 1 µm or less were
prepared from double-barreled borosilicate glass capillary tubing as
described previously (17). The electrodes were calibrated
in Tris-buffered solutions of pH 6.0, 7.0, 7.4, and 8.0 containing (in
mM) 10 NaCl, 100 KCl, and 0.01 CaCl2 (slope = 55.0 ± 0.6 mV/pH unit; n = 23).
Electrical measurements.
Apical membrane potential (Va), transepithelial
potential (Vt), transepithelial resistance
(Rt), and the fractional apical voltage ratio
(FR) were measured as described previously
(12), all with reference to the mucosal solution. Measured
changes in potential after alterations in the composition of the
mucosal solutions were corrected for liquid junction potentials arising at the tips of the agar bridges. Liquid junction potentials were measured by using the method of Garcia-Diaz et al. (12).
Statistical analysis.
Results are presented as means ± SE. Student's t-test
was employed to analyze the differences between sets of data.
| |
RESULTS |
Studies with TPA.
The effects of TPA on the electrophysiological properties of
Necturus gallbladder were examined in the absence and
presence of external HCO3. In preliminary experiments
in HCO3-free media, TPA was added to the mucosal
medium at 50 µM. The principal responses of gallbladder epithelial
cells to TPA at these concentrations were a depolarization of
Va and a reduction in FR. At TPA
concentrations of 10 and 20 µM, these effects were inconsistent.
Experiments in HCO3-free media.
Figure 1 shows the effect of TPA on a
gallbladder epithelium perfused on both sides with
HCO3-free Ringer solution. About 2 min after exposure
to 50 µM TPA, Va began a decline (which lasted
~4 min) from its initial value of 
52 to 38 mV. After this,
Va slowly recovered to 48 mV. The initial
reduction in FR (from 0.42 to 0.18) coincided with the depolarization of 14 mV in Va. However, during
the time that Va was recovering, there was no
change in FR. The effects of TPA on
Vt, Va, FR,
and Rt in six tissue samples are summarized in Table 1, from which it is
apparent that TPA had no effect on Vt
or Rt.
View larger version (29K):
[in this window]
[in a new window]
|
Fig. 1.
Effects of 12-O-tetradecanoylphorbol
13-acetate (TPA) on apical membrane potential
(Va; top), fractional apical voltage
ratio (FR; middle), and transepithelial
potential (Vt; bottom) in
Necturus gallbladder bathed in HCO3-free
media. Tissue was exposed to mucosal 50 µM TPA at the arrow. The
apparent delay in response may be due to bath exchange time. Voltage
spikes were elicited by transepithelial current pulses and were used to
calculate transepithelial resistance (Rt) and
FR.
|
|
View this table:
[in this window]
[in a new window]
|
Table 1.
Effect of mucosal TPA on electrophysiological parameters of Necturus
gallbladder in the absence and presence of external
HCO3
|
|
In three experiments, TPA was washed with HCO3-free
Ringer solution for 30-60 min and reapplied without affecting
Va or FR (data not shown). These
results suggest that the effects of TPA are irreversible, at least
within the time frame of our experiments, and are consistent with
previous findings (14).
Phorbol esters can affect Na+/H+
antiport, Na+-H+-2Cl symport, and
Na+ channels (1, 3, 5). To investigate whether
the TPA effects depend on luminal Na+, gallbladders were
perfused with Na+-free Ringer solution on the apical
side, whereas the basolateral side was perfused with the control Ringer
solution. In the absence of apical Na+, the effects of TPA
(Table 2) were qualitatively similar to those shown in Fig. 1 and Table 1.
View this table:
[in this window]
[in a new window]
|
Table 2.
Effect of TPA on electrophysiological parameters of Necturus
gallbladder in mucosal Na+-free or
mucosal Cl-free Ringer solution
|
|
In gallbladders bathed on both sides with Na+-free
Ringer solution, mucosal TPA decreased Va
from 40.0 ± 2.4 to 33.0 ± 1.0 mV
(P < 0.025, n = 7) and decreased
FR from 0.37 ± 0.08 to 0.15 ± 0.03 (P < 0.01). These data indicate that the TPA-induced
changes in Va and FR are
Na+ independent.
We further tested the possibility that the TPA-induced changes in
Va and FR depend on external
Cl. In the absence of mucosal Cl, TPA
induced only minimal changes in Va and
FR (Table 2). These data suggest that in the absence of
HCO3 the TPA-induced changes in
Va and FR depend on mucosal
Cl.
To determine whether the TPA-induced effects involved changes in
Cl conductance, tissues were perfused on the apical side
with Ringer solution containing 10 µM NPPB, a Cl
channel blocker (33), for 1 h. In gallbladders
treated with apical NPPB, TPA decreased Va from
48.0 ± 2.0 to 44.0 ± 2.0 mV and FR from
0.30 ± 0.03 to 0.24 ± 0.03 (P < 0.025, n = 9). These changes were significantly less than
those shown in the absence of NPPB (Table 1). In contrast, when
administered on the basolateral side, 10 µM NPPB had no effect on
TPA-induced changes in Va or FR
(n = 6; data not shown).
In gallbladders treated with 100 µM H-7 (a PKC inhibitor) on the
mucosal side alone, TPA had no effect on Va and
FR (n = 5; data not shown). Aside from its
inhibitory effects on PKC, H-7 has been reported to impair the activity
of other enzymes, such as ATPases (15). For example, if
H-7 inhibited the Na+-K+-ATPase activity, one
would expect changes in the electrophysiological parameters of
gallbladder cells. However, in our studies, the steady-state values of
Va, FR, Rt,
and Vt were not affected by H-7 treatment alone.
Phorbol ester effects involve mobilization of Ca2+ from
intracellular stores (20). We tested the ability of TMB-8
(6 µM), an intracellular Ca2+ antagonist
(9), which was applied for 30 min, to block TPA changes.
This concentration of TMB-8 is lower than that required for a direct
inhibitory effect on PKC (4). TPA had no detectable effect
after mucosal TMB-8 treatment (n = 6; data not shown).
Experiments in HCO3-containing media.
In Ringer solution containing HCO3 (Fig.
2), within minutes of apical TPA
exposure, there was a significant hyperpolarization of
Va (from 67 to 72 mV) without a change in
FR. The hyperpolarization was transient, after which both
Va and FR started to decline and achieved a new steady state in the next 5 min. Unlike that shown in
HCO3-free media (see Fig. 1), the changes in
Va did not spontaneously recover in the
continuous presence of TPA. Data summarized in Table 1 indicate that
the resting values of Va and FR in
HCO3-buffered media were significantly higher than
those in tissues bathed in imidazole-buffered solutions (22,
30). After TPA exposure, the initial mean peak hyperpolarization
of Va was ~4 mV. This transient increase in
Va was followed by a mean depolarization of 24 mV and a mean decrease in FR of 0.3. These values are
significantly greater than those observed in imidazole-buffered Ringer
solutions. In three experiments, the control Ringer solution was
reintroduced into the mucosal bath for 30-60 min after TPA
treatment. During this time, neither Va nor
FR recovered toward their initial control values.
View larger version (31K):
[in this window]
[in a new window]
|
Fig. 2.
Effects of TPA on Va
(top), FR (middle), and
Vt (bottom) in Necturus
gallbladder bathed in medium containing 25 mM HCO3.
Tissue was exposed to mucosal 50 µM TPA at the arrow.
|
|
In the absence of apical Na+, TPA initially induced a small
hyperpolarization in Va
(
Va = 3.0 ± 1.0 mV) followed by
depolarization of Va from 68.0 ± 5.0 to
37.0 ± 5.0 mV (P < 0.001, n = 6). During the same period, FR decreased from 0.73 ± 0.08 to 0.28 ± 0.06 (P < 0.005).
In the absence of apical Cl (Fig.
3), TPA initially induced a small
hyperpolarization (Va = 6.0 ± 2.0 mV) followed by depolarization of Va from
73.0 ± 7.0 to 54.0 ± 4.0 mV (P < 0.025, n = 6). During the same period, FR
decreased from 0.72 ± 0.05 to 0.50 ± 0.03 (P < 0.025). It is interesting to note that, in
HCO3-free media (Table 2), removal of
Cl from the apical solution completely blocked
TPA-induced changes in Va and FR. In
contrast, a similar maneuver in the presence of
HCO3-containing media only partially attenuated the
TPA-induced changes in Va, from 24.0 ± 3.5 mV (see Table 1) to 19.0 ± 6.0 mV.
View larger version (20K):
[in this window]
[in a new window]
|
Fig. 3.
Effects of TPA on Va
(top), FR (middle), and
Vt (bottom) in Necturus
gallbladder bathed in Cl-free medium. The gallbladder was
initially perfused with a Cl-free medium containing 25 mM
HCO3 in the apical compartment; the serosal
compartment was perfused with control HCO3-containing
medium. Tissue was exposed to mucosal 50 µM TPA at the arrow.
|
|
To determine whether the TPA effects involved changes in
Cl conductance, experiments were performed with NPPB. As
shown in Fig. 4A, mucosal NPPB
only partially attenuated TPA-induced changes in
Va and FR. TPA depolarized
Va by 12.0 ± 7.0 mV (from 54.0 ± 5.0 to 42.0 ± 4.0 mV; P < 0.025, n = 5). During the same period, FR
decreased from 0.44 ± 0.06 to 0.27 ± 0.04. These data
suggest that mucosal NPPB alone inhibits TPA-induced changes in
Va by 50% (see Table 1). However, when tissues
were treated with NPPB on both sides (Fig. 4B), the
TPA-induced changes in these parameters were minimal. TPA depolarized
Va by 3.0 ± 3.0 mV
(Va decreased from 55.0 ± 4.0 to
52.0 ± 6.0 mV; P > 0.05, n = 5). During the same period, FR decreased from 0.42 ± 0.05 to 0.34 ± 0.07. Together, these data suggest that, in
HCO3-containing media, NPPB had to be present in both
compartments to completely block TPA-induced changes in
Va and FR.
View larger version (25K):
[in this window]
[in a new window]
|
Fig. 4.
Effects of TPA on Va and
FR in Necturus gallbladder in the presence of
nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), a Cl
channel blocker. A: gallbladder was initially perfused with
HCO3-buffered solution containing, in addition, 10 µM NPPB in the apical compartment, whereas the serosal compartment
was perfused with control HCO3-buffered medium.
B: gallbladder was initially perfused with
HCO3-buffered solution containing, in addition, 10 µM NPPB bilaterally. Tissue was exposed to mucosal 50 µM TPA at the
arrow.
|
|
In the presence of 100 µM mucosal H-7, TPA depolarized
Va from 77 ± 7 to 68 ± 7 mV
(Va = 9.0 ± 3.0 mV;
P > 0.05, n = 4) and decreased
FR from 0.76 ± 0.13 to 0.55 ± 0.12 (P > 0.05). These values are significantly less than
those in the absence of the drug (Table 1). Consistent with our
previous results, these data suggest that TPA effects are sensitive to
H-7.
Studies with IBMX.
Our data strongly suggest that TPA actions on gallbladder cells are
modulated by external HCO3. In our previous studies
(34), IBMX also depolarized Va and decreased FR. Therefore, we wondered if cAMP-induced
changes in electrophysiological parameters of gallbladder cells also
depend on external HCO3. Gallbladders perfused with
imidazole-buffered Ringer solutions were impaled with double-barreled,
pH-sensitive microelectrodes and then exposed to 100 µM mucosal IBMX.
Similar to the effects of TPA (compare with Fig. 1 and Table 1), IBMX
did not induce any significant hyperpolarization of
Va (within 1 min after IBMX exposure). However,
IBMX depolarized Va by 10.1 ± 3.5 mV and
decreased FR by 0.34 ± 0.03 (Table
3). These data confirm and extend our previous results (34).
View this table:
[in this window]
[in a new window]
|
Table 3.
Effect of IBMX on the electrical parameters of Necturus gallbladder
cells in the absence and presence of external
HCO3
|
|
In HCO3 media, immediately after IBMX exposure,
Va hyperpolarized from 69.1 to 81.5 mV (Fig.
5, top trace). The
hyperpolarization was transient, after which Va
slowly depolarized to 59.4 mV. During this period, FR
decreased from 0.79 to 0.61 (data not shown). Table 3 also summarizes
data from five similar experiments and compares these parameters at 1 and 11 min after IBMX exposure. The initial mean peak hyperpolarization
of Va was 11.2 mV. This transient increase in
Va was followed by a mean depolarization of 7.6 mV and a 0.14 decrease in FR. This decrease in
Va was not statistically different from the
IBMX-induced depolarization in the absence of HCO3
(10.1 ± 3.5 mV; Table 3). These data indicate that, like TPA, the
IBMX-induced initial transient hyperpolarization of
Va occurs only in the presence of
HCO3. However, unlike TPA, the IBMX-induced
depolarization of Va is not affected by external
HCO3. On removal of IBMX, both
Va and FR recovered completely.
View larger version (25K):
[in this window]
[in a new window]
|
Fig. 5.
Effect of mucosal 3-isobutyl-1-methylxanthine (IBMX) on
Va and intracellular pH (pHi) in
Necturus gallbladder bathed in medium containing 25 mM
HCO3. A cell was impaled with a double-barreled,
pH-sensitive microelectrode. Top trace:
Va recorded by the open-tip reference barrel.
Bottom trace: difference between the potentials recorded by
the ion-selective barrel and the reference electrode
(VH Va). The
scale (bottom right) represents calculated
changes in pHi. Downward arrow indicates exposure of tissue
to mucosal 0.1 mM IBMX; upward arrow indicates exposure of tissue to
the control solution.
|
|
Consistent with the effects of IBMX reported here and previously
(34), agents that elevate intracellular cAMP
[8-bromo-cAMP (8-BrcAMP), theophylline, or forskolin] also depolarize
Va and decrease FR (6, 10, 13,
14, 21, 32). Under these conditions, an increase in
gCla was confirmed by characteristic
responses to short pulses of low-Cl solutions in the
apical compartment (10, 13, 14, 21). The properties of
cAMP-activated gCla have been investigated
in detail by several workers (6, 10, 13, 14, 21, 32) and
were not investigated further (see DISCUSSION).
TPA- and IBMX-induced changes in intracellular pH.
In our studies, we observed two distinct effects in
HCO3 media. First, IBMX and TPA each induced
transient hyperpolarization in Va. Second, the
TPA-induced depolarization of Va was greater. We
considered the possibility that one or both of the above effects may be
related to changes in apical membrane HCO3
conductance (34). We monitored changes in intracellular pH (pHi) of gallbladder cells as an indirect measure of
HCO3 conductance. Compared with the effects of IBMX
(Fig. 5 and Table 3), there was a slower time course (see Figs.
2-4) and smaller hyperpolarization of Va in
the presence of TPA (4.0 ± 1 mV; Table 1). In our preliminary
experiments (unpublished observations), these changes in
Va were associated with a small but transient increase in pHi (0.03-0.04). However, the transient
changes in Va and pHi in the
presence of IBMX were greater and are presented in more detail. Figure
5 shows that, in the presence of HCO3, the
IBMX-induced hyperpolarization of Va (top
trace) is accompanied by a temporal increase in pHi
(bottom trace). The resting pHi increased from
7.18 ± 0.03 to 7.38 ± 0.05 (P < 0.001, n = 5) 1 min after IBMX exposure. However, changes in
pHi and Va were transient and
rapidly returned to resting values. The mean resting values of
Va and pHi are within the reported
range (8, 29, 34). As further shown in Fig. 5, 11 min
after IBMX exposure, when both Va and
FR were at their lowest values, the pHi
(7.18 ± 0.06; n = 5) was not different from its
control value. These data suggest that IBMX- or TPA-induced
depolarization of Va is independent of changes
in pHi.
In nominally HCO3-free solutions, IBMX did not
hyperpolarize Va or induce an increase in
pHi. At 11 min, when both Va and FR declined to their lowest values, pHi
(7.32 ± 0.08; n = 5) was not different from its
control value (7.35 ± 0.11). However, a small decrease in
pHi (0.04-0.06) was reported in some studies after
theophylline (25) or 8-BrcAMP (27) exposure.
The physiological significance of such small changes in pHi
on gCla and gClb
is not clear.
To determine whether the IBMX-induced changes in
Va and pHi were Na+
dependent, tissues were perfused with Na+-free Ringer
solutions containing HCO3. In the absence of mucosal
Na+, the time course of IBMX-induced changes in
Va and pHi was similar to that shown
in Fig. 5. In five animals, Va reversibly
hyperpolarized from 74.5 ± 5.5 to 80.7 ± 5.3 mV
(P < 0.05) and pHi increased from
7.21 ± 0.08 to 7.31 ± 0.07 (P < 0.001) in
the first minute. These results indicate that the IBMX-induced
transient hyperpolarization of Va and the
increase in pHi were Na+ independent.
Therefore, the IBMX-induced alkalinization and its spontaneous rapid
recovery to control levels probably did not involve apical
Na+-dependent, pH-regulatory mechanisms (18).
| |
DISCUSSION |
Our results indicate that external HCO3
modulates the effects of TPA on Va and
FR in gallbladder cells. In HCO3-buffered
media, TPA induced two effects that were not observed in
imidazole-buffered media. First, it induced an initial transient hyperpolarization of Va (Fig. 2 and Table 1)
without a change in FR. Second, it induced a twofold
greater depolarization in Va and a significantly
greater decrease in FR (Table 1). We discuss the
implications of these results in relation to PKC regulation of anion
conductances in gallbladder cell membranes. To determine whether PKC
regulates specific anion conductances in gallbladder cells, we compared
the effects of TPA with IBMX, an agent that increases intracellular
cAMP and activates PKA.
Effects of TPA on Cl conductance.
TPA depolarized Va and decreased FR.
In gallbladder cells, PMA, another phorbol ester, also depolarized
Va and decreased FR (14). The characteristic responses to short pulses of
low-Cl solutions in the apical compartment before and
after PMA treatment indicated that these changes in
Va and FR are related to an increase in gCla (14). In the absence of
HCO3, the TPA-induced changes in
Va and FR were specifically blocked by mucosal Cl removal (Table 2) and by NPPB (a
Cl channel blocker) in the mucosal compartment. Together,
the data suggest that TPA activates an NPPB-sensitive
gCla in gallbladder cells in the absence of
HCO3. However, TPA effects are greater in the
presence of HCO3. With HCO3,
mucosal NPPB or unilateral removal of mucosal Cl only
partially attenuated the effects of TPA. NPPB in both apical and
basolateral compartments was necessary to block the effects of TPA. The
data suggest that TPA activates both gCla
and gClb in the presence of
HCO3. In HCO3 media, the TPA
effects on gClb are related to the presence
of Cl conductive channels in gallbladder epithelial cells
(28). There is little evidence for Cl
conductance as a major route for Cl transfer across the
apical or basolateral cell membranes in the absence of
HCO3. Without HCO3,
gCla appears to be slight or negligible
(12) and gClb amounts to no
more than 6% of the basolateral ionic conductance (7,
24). In the presence of 10 mM HCO3,
gClb increases to ~50% of the total
conductance of the basolateral cell membrane (28) and
contributes to the increase in transepithelial Cl flux
and to net fluid absorption (28-30). Our data suggest
that, in the presence of HCO3, TPA activates
basolateral Cl channels.
TPA effects were blocked by H-7, a PKC blocker. It is suggested that
TPA, like PMA, induces its effect via activation of PKC (14). Regulation of the cystic fibrosis transmembrane
conductance regulator (CFTR), a putative Cl channel,
occurs via direct phosphorylation of the R domain by PKC and PKA
(23). In gallbladder cells,
gCla is activated by PKC- and PKA-dependent
phosphorylation of the channel or of a regulatory protein
(14). However, PKC activates gCla by a mechanism that does not involve
changes in intracellular cAMP (14). PKC comprises a large
family with multiple isoforms exhibiting individual characteristics and
tissue distributions (20). It has been suggested that
gallbladder cells lack a Ca2+-activated
gCla because an increase in intracellular
Ca2+ did not affect PKC- or cAMP-activated
gCla (14). However, TPA effects
were blocked in our studies by TMB-8, which presumably decreased
intracellular Ca2+ concentration
(5). However, a reduction in intracellular
Ca2+ concentration may alter the activity of a number of
other Ca2+-dependent parameters, permissive or required,
for activating gCla and
gClb by TPA (2, 5).
Effects of IBMX on Cl conductance.
Under control conditions, gallbladder cells have a sizable
gClb but no native
gCla (6, 10, 14, 21, 32). IBMX
(Table 3) and other agents that increase intracellular cAMP
(34) depolarize Va
and decrease FR. These data indicate that cAMP
stimulates gCla in gallbladder cells.
After stimulation with cAMP, gCla
becomes the predominant membrane conductance in gallbladder cells (32). Activation of gCla by
cAMP short circuits Cl influx across the apical cell
membrane (14, 21, 25, 28) and contributes to inhibition of
transepithelial fluid absorption. The cAMP-activated Cl
channel, responsible for increased gCla, is
insensitive to voltage, Ca2+, and pH (6, 14,
32). Most importantly, it is insensitive to many agents
(including NPPB) that block Cl channels in other cells
(6, 32). With the use of anti-human CFTR as the primary
antibody, it was demonstrated that the cAMP-activated Cl
channel is a CFTR homologue and is expressed in the apical but not the
basolateral membrane. However, this is not the only Cl
channel activated by cAMP. Garvin and Spring (13)
presented evidence for a gCla channel in
control gallbladders that was blocked by bumetanide and adenosine
3',5'-cyclic monophosphorothioate (Rp isomer), an inhibitor
of PKA.
In another study (10), monoclonal antibodies to
Necturus gallbladder cells bound mostly at the apical
membrane of gallbladder cells. In electrophysiological studies,
antibodies in the mucosal compartment significantly inhibited
cAMP-induced increases in gCla. The data
suggest that these antibodies recognize apical Cl
channels in gallbladder cells. The data further suggested that the
Cl channel is constitutive and that the role of cAMP is
to control the channel activation rather than its insertion into the
membrane (10).
The data shown in Table 3 indicate that, unlike the case with TPA,
external HCO3 does not affect the magnitude of
IBMX-induced gCla. It suggests that cAMP
predominantly affects gCla even in the
presence of HCO3, when
gClb represents 50% of the total
basolateral conductance (28). In gallbladders bathed on
both sides with HCO3-buffered Ringer solutions, the
most potent antibody to apical Cl channels inhibited the
cAMP response by 83% (10). These data are further
supported by the observation that cAMP-activated Cl
channel is not expressed in the basolateral membrane (6,
32). Our data suggest that, unlike cAMP, TPA activates
NPPB-sensitive apical Cl channels in the presence and
absence of HCO3 and that, in the presence of
HCO3, the basolateral membrane Cl
channels are activated by TPA that are also NPPB sensitive.
Apical membrane HCO3 conductance.
Both TPA and IBMX induced an initial transient hyperpolarization of
Va in the presence of HCO3 and
transiently increased pHi. TPA-induced hyperpolarization was slower. TPA hyperpolarized Va by 4 mV and
increased pHi by 0.04. IBMX-induced hyperpolarization was
faster. IBMX hyperpolarized Va by 11 mV and
increased pHi by 0.2. These differences may reflect different rates of PKC and PKA activation under our experimental conditions. The TPA- and IBMX-induced hyperpolarizations were independent of external Na+ and Cl
(34). Because IBMX gave greater and faster responses in
pHi, the pHi changes with IBMX are considered
in greater detail. The IBMX-induced hyperpolarization and increase in
pHi were also independent of apical Na+. The
data suggest that changes in Va and
pHi do not involve apical Na+- or
Cl-dependent pH regulatory mechanisms and that the
increase in pHi is due to a transient increase in apical
membrane HCO3 conductance (34). However,
the physiological significance of these early events is not clear.
Second, it is also unclear if this anion-selective channel is also
permeable to Cl. Because both apical and basolateral
membrane K+ conductance of gallbladder cells is increased
at alkaline pHi (26, 31), it may account for
the observed hyperpolarization of Va. Because
the depolarization phase of TPA and IBMX effects was pHi
independent, it is suggested that HCO3 conductance
changes are not involved.
Relationship between Cl and K+
conductances.
Both TPA and IBMX induce changes in Va and
FR without changes in Rt. This
suggests that the subsequent recovery of Va from initial hyperpolarization, and its slow depolarization, involve time-dependent changes in additional membrane conductances. In control
tissues, exposing the apical or basolateral membrane to high-K+ solutions depolarized Va,
indicating that both membranes are K+ conductive (6,
14, 32, 34). After an increase in intracellular cAMP,
high-K+ solutions induced smaller depolarizations. These
data suggest that the cAMP-activated increase in
gCla is accompanied by a compensatory
decrease in K+ conductance or that an increase in
Cl conductance shunts the K+ conductance.
Under control condition, gCla and
gClb are small, and K+
conductance is the major conductance that maintains the membrane potential. After TPA or IBMX treatment, as
gCla increases with time (14),
the total apical conductance becomes anion selective (6)
and basolateral K+ conductance decreases (34),
Va depolarizes, and FR decreases.
After TPA treatment, Va first depolarized and
then spontaneously recovered (Fig. 1). The spontaneous recovery of
Va was independent of external Na+
and Cl but did not occur in
HCO3-containing media (Fig. 3). Similarly, during
prolonged impalements in the presence of IBMX, there was an occasional
spontaneous repolarization of Va
(34). The mechanism(s) involved in spontaneous recovery of
Va after TPA or IBMX treatment is not known but
must also involve time-dependent changes in membrane conductances.
In summary, the data suggest that the activation of PKC by TPA
stimulates gCla in the absence of
HCO3 but in the presence of HCO3,
it stimulates both gCla and
gClb. The TPA-induced increases in
gCla and gClb
were blocked by the Cl channel blocker NPPB and by
inhibiting the mobilization of Ca2+ from intracellular
stores. In contrast, the activation of PKA by cAMP only activated
apical Cl channels that are voltage, Ca2+,
pH, NPPB, and HCO3 insensitive (6, 32).
In the presence of HCO3, both TPA and IBMX induced a
rapid initial transient hyperpolarization and increase in
pHi that may have resulted from a transient increase in
apical HCO3 conductance.
| |
ACKNOWLEDGEMENTS |
We thank Dr. Yu-Zhang Wang for pHi measurements using
double-barreled, pH-sensitive microelectrodes. We also thank Drs. Ayus Corcia, George Tanner, Judy Tanner, John A. DeSimone, Steven Price, and
George M. Feldman for many helpful discussions during the progress of
this investigation. We also thank Susan L. Brooks for technical assistance.
| |
FOOTNOTES |
Deceased 9 January 1997.
V. Lyall was supported by Veterans Affairs Merit Review. This work was
supported by National Institute of Diabetes and Digestive and Kidney
Diseases Grant DK-12715.
A preliminary report has been published (16).
Address for reprint requests and other correspondence: V. Lyall, Dept.
of Physiology, Virginia Commonwealth Univ., Sanger Hall 3002, 1101 E. Marshall St., Richmond, VA 23298-0551 (E-mail: Lyall{at}vcu.org).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 12 August 1999; accepted in final form 5 June 2000.
| |
REFERENCES |
1.
Ahn, J,
Chang EB,
and
Field M.
Phorbol ester inhibition of Na-H exchange in rabbit proximal colon.
Am J Physiol Cell Physiol
249:
C527-C530,
1985[Abstract/Free Full Text].
2.
Anderson, MP,
Sheppard DN,
Berger HA,
and
Welsh MJ.
Chloride channels in the apical membrane of normal and cystic fibrosis airway and intestinal epithelia.
Am J Physiol Lung Cell Mol Physiol
263:
L1-L14,
1992[Abstract/Free Full Text].
3.
Barthelson, RA,
Jacoby DB,
and
Widdicombe JH.
Regulation of chloride secretion in dog tracheal epithelium by protein kinase C.
Am J Physiol Cell Physiol
253:
C802-C808,
1987[Abstract/Free Full Text].
4.
Civan, MM,
Oler A,
Peterson-Yantorno K,
George K,
and
O'Brien TG.
Ca2+-independent form of protein kinase C may regulate Na+ transport across frog skin.
J Membr Biol
121:
37-50,
1991[Web of Science][Medline].
5.
Civan, MM,
Rubenstein D,
Mauro T,
and
O'Brien TG.
Effects of tumor promoters on sodium ion transport across frog skin.
Am J Physiol Cell Physiol
248:
C457-C465,
1985[Abstract/Free Full Text].
6.
Copello, J,
Heming TA,
Segal Y,
and
Reuss L.
cAMP-activated apical membrane chloride channels in Necturus gallbladder epithelium: conductance, selectivity, and block.
J Gen Physiol
102:
177-199,
1993[Abstract/Free Full Text].
7.
Corcia, A,
and
Armstrong WM.
KCl cotransport: a mechanism for basolateral chloride exit in Necturus gallbladder.
J Membr Biol
76:
173-182,
1983[Web of Science][Medline].
8.
Dausch, R,
and
Spring KR.
Regulation of NaCl entry into Necturus gallbladder epithelium by protein kinase C.
Am J Physiol Cell Physiol
266:
C531-C535,
1994[Abstract/Free Full Text].
9.
Donowitz, M,
Cusolito S,
and
Sharp GW.
Effects of calcium antagonist TMB-8 on active Na and Cl transport in rabbit ileum.
Am J Physiol Gastrointest Liver Physiol
250:
G691-G697,
1986.
10.
Finn, AL,
Tsai LM,
and
Falk RJ.
Monoclonal antibodies to the apical chloride channel in Necturus gallbladder inhibit the chloride conductance.
Proc Natl Acad Sci USA
86:
7649-7652,
1989[Abstract/Free Full Text].
11.
Garcia-Diaz, JF,
and
Armstrong WM.
The steady-state relationship between sodium and chloride transmembrane electrochemical potential difference in Necturus gallbladder.
J Membr Biol
55:
213-222,
1980[Web of Science][Medline].
12.
Garcia-Diaz, JF,
Corcia A,
and
Armstrong WM.
Intracellular chloride activity and apical membrane chloride conductance in Necturus gallbladder.
J Membr Biol
73:
145-155,
1983[Web of Science][Medline].
13.
Garvin, JL,
and
Spring KR.
Regulation of apical membrane ion transport in Necturus gallbladder.
Am J Physiol Cell Physiol
263:
C187-C193,
1992[Abstract/Free Full Text].
14.
Heming, TA,
Copello J,
and
Reuss L.
Regulation of cAMP-activated apical membrane chloride conductance in gallbladder epithelium.
J Gen Physiol
103:
1-18,
1994[Abstract/Free Full Text].
15.
Hidaka, H,
Inagaki M,
Kawamoto S,
and
Sasaki Y.
Iso-quinolinesulfonamides, novel and potent inhibitors of cyclic nucleotide dependent protein kinase and protein kinase C.
Biochemistry
23:
5036-5041,
1984[Medline].
16.
Lyall, P,
and
Armstrong WM.
TPA stimulates Cl conductance in Necturus gallbladder (NGB) epithelial cells (Abstract).
FASEB J
6:
A1237,
1992.
17.
Lyall, V,
Belcher TS,
and
Biber TUL
Effect of changes in extracellular potassium on intracellular pH in principal cells of frog skin.
Am J Physiol Renal Fluid Electrolyte Physiol
263:
F722-F730,
1992[Abstract/Free Full Text].
18.
Lyall, V,
and
Biber TUL
Potential-induced changes in intracellular pH.
Am J Physiol Renal Fluid Electrolyte Physiol
266:
F685-F696,
1994[Abstract/Free Full Text].
19.
Lyall, V,
Corcia A,
Croxton TL,
Chao AC,
and
Armstrong WM.
A possible relationship between KCl symport and basolateral K+-conductance in Necturus gallbladder epithelial cells.
Comp. Biochem Physiol
102A:
497-505,
1992.
20.
Nishizuka, Y.
Protein kinase C and lipid signaling for sustained cellular responses.
FASEB J
9:
484-496,
1995[Abstract].
21.
Petersen, KU,
and
Reuss L.
Cyclic AMP-induced chloride permeability in the apical membrane of Necturus gallbladder epithelium.
J Gen Physiol
81:
705-709,
1983[Abstract/Free Full Text].
22.
Petersen, KU,
and
Reuss L.
Electrophysiological effects of propionate and bicarbonate on gallbladder epithelium.
Am J Physiol Cell Physiol
248:
C58-C69,
1985[Abstract/Free Full Text].
23.
Picciotto, MR,
Cohn JA,
Bertuzzi G,
Greengard P,
and
Nairn AC.
Phosphorylation of the cystic fibrosis transmembrane conductance regulator.
J Biol Chem
267:
12742-12752,
1992[Abstract/Free Full Text].
24.
Reuss, L.
Basolateral KCl cotransport in a NaCl-absorbing epithelium.
Nature
305:
723-726,
1983[Medline].
25.
Reuss, L.
Cyclic AMP inhibits Cl/HCO3 exchange at the apical membrane of Necturus gallbladder epithelium.
J Gen Physiol
90:
173-176,
1987[Abstract/Free Full Text].
26.
Reuss, L,
Cheung LY,
and
Grady TP.
Mechanisms of cation permeation across apical cell membrane of Necturus gallbladder: effect of luminal pH and divalent cations on K+ and Na+ permeabilities.
J Membr Biol
59:
211-224,
1981[Web of Science][Medline].
27.
Reuss, L,
and
Petersen KU.
Cyclic AMP inhibits Na+/H+ exchange at the apical membrane of Necturus gallbladder epithelium.
J Gen Physiol
85:
409-429,
1985[Abstract/Free Full Text].
28.
Reuss, L,
Segal Y,
and
Altenberg G.
Regulation of ion transport across gallbladder epithelium.
Annu Rev Physiol
53:
361-373,
1991[Web of Science][Medline].
29.
Reuss, L,
and
Stoddard JS.
Role of H+ and HCO3 in salt transport in gallbladder epithelium.
Annu Rev Physiol
49:
35-49,
1987[Web of Science][Medline].
30.
Stoddard, JS,
and
Reuss L.
Dependence of cell membrane conductances on bathing solution HCO3/CO2 in Necturus gallbladder.
J Membr Biol
102:
163-174,
1988[Web of Science][Medline].
31.
Stoddard, JS,
and
Reuss L.
pH effects on basolateral membrane ion conductances in gallbladder epithelium.
Am J Physiol Cell Physiol
256:
C1184-C1195,
1989[Abstract/Free Full Text].
32.
Torres, RJ,
Altenberg GA,
Conn JA,
and
Reuss L.
Polarized expression of cAMP-activated chloride channels in isolated epithelial cells.
Am J Physiol Cell Physiol
271:
C1574-C1582,
1996[Abstract/Free Full Text].
33.
Wangemann, P,
Wittner M,
Di Stefano A,
Englert HC,
Lang HJ,
Schlatter E,
and
Greger R.
Cl-channel blockers in the thick ascending limb of the loop of Henle. Structure-activity relationship.
Pflügers Arch
407:
S128-S141,
1986.
34.
Zeldin, DC,
Corcia A,
and
Armstrong WM.
Cyclic AMP-induced changes in membrane conductance of Necturus gallbladder epithelial cells.
J Membr Biol
84:
193-206,
1985[Web of Science][Medline].
Am J Physiol Cell Physiol 279(5):C1385-C1392
0363-6143/00 $5.00
Copyright © 2000 the American Physiological Society
TOP
ABSTRACT
INTRODUCTION
