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
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ABSTRACT |
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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
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INTRODUCTION |
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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.
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MATERIALS AND METHODS |
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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.
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RESULTS |
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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.
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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.
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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).
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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.
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DISCUSSION |
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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.
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.
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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
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