Effect of HCO3minus on TPA- and IBMX-induced anion conductances in Necturus gallbladder epithelial cells

Pamela Lyall1, William McD. Armstrongdagger,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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 MOmega 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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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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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.


                              
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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.

                              
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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.


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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 (Delta 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 (Delta 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.


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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.


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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 (Delta 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).

                              
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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.


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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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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

dagger 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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Cell Physiol 279(5):C1385-C1392
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