Inhibition of phosphatidylinositol 3-kinase does not alter forskolin-stimulated Clminus secretion by T84 cells

Jeffrey L. Dickson, Tracy D. Conner, and Tom W. Ecay

Department of Physiology, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, Tennessee 37614


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Wortmannin is a potent inhibitor of phosphatidylinositol 3-kinase (PI3K) and membrane trafficking in many cells. To test the hypothesis that cystic fibrosis transmembrane conductance regulator (CFTR) traffics into and out of the plasma membrane during cAMP-stimulated epithelial Cl- secretion, we have studied the effects of wortmannin on forskolin-stimulated Cl- secretion by the human colonic cell line T84. At the PI3K inhibitory concentration of 100 nM, wortmannin did not affect significantly forskolin-stimulated Cl- secretion measured as short-circuit current (ISC). However, 500 nM wortmannin significantly inhibited forskolin-stimulated ISC. cAMP activation of apical membrane CFTR Cl- channels in alpha -toxin-permeabilized monolayers was not reduced by 500 nM wortmannin, suggesting that inhibition of other transporters accounts for the observed reduction in T84 Cl- secretion. Forskolin inhibits apical endocytosis of horseradish peroxidase (HRP), but wortmannin did not alter forskolin inhibition of apical HRP endocytosis. In the absence of forskolin, wortmannin stimulated HRP endocytosis significantly. We conclude that, in T84 cells, apical fluid phase endocytosis is not dependent on PI3K activity and that CFTR does not recycle through a PI3K-dependent and wortmannin-sensitive membrane compartment.

wortmannin; epithelia; short-circuit current; cystic fibrosis transmembrane conductance regulator; endocytosis


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ABSTRACT
INTRODUCTION
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CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR (CFTR) is a cAMP-activated Cl- channel that mediates the Cl- secretory activity of many different epithelia (19, 25). The rate of epithelial Cl- secretion is determined in part by the gating of apical membrane resident CFTR Cl- channels and may be regulated additionally by insertion and retrieval of CFTR or other Cl- channels at the apical membrane. This type of mechanism is analogous to regulation of gastric acid secretion and renal water transport by membrane insertion and retrieval of proton pumps and water channels, respectively (reviewed in Ref. 7). In many epithelial tissues and cell lines, cAMP stimulation of Cl- secretion is accompanied by changes in endocytosis and exocytosis rates. A model of CFTR insertion and retrieval at the plasma membrane has been proposed to explain the link between cAMP-stimulated Cl- secretion and altered plasma membrane turnover (7, 25). This model may not be universal and its applicability may depend on the precise transport functions of specific epithelia (20).

Evidence supporting CFTR insertion and retrieval at the apical membrane comes from studies in human bronchial and shark rectal gland epithelia, CFTR mRNA-injected Xenopus oocytes, and the cell lines T84, Madin-Darby canine kidney (MDCK), and A6 (6). The human adenocarcinoma cell line T84 is often used as a model for studies of colonic fluid secretion and CFTR function. These cells maintain a high degree of phenotypic differentiation characteristic of colonic crypt cells, express high levels of CFTR, and have a large capacity for cAMP-stimulated epithelial Cl- secretion (2, 14). In quiescent T84 cells, plasma membrane CFTR undergoes continuous endocytosis and recycles back to the plasma membrane with rapid kinetics (33). The presence of functional CFTR in clathrin-coated vesicles and endosomal membranes isolated from T84 cells has been demonstrated (5, 9). cAMP stimulation of CFTR-catalyzed Cl- secretion causes a concomitant decrease in fluid phase and CFTR endocytosis (11, 33) and a doubling of the CFTR content of T84 apical membranes (39). How CFTR recycles to the plasma membrane following clathrin-mediated endocytosis and the effects of cAMP on CFTR recycling rates have not been defined. Thus the relative contributions of decreased endocytosis and potentially increased exocytosis to CFTR abundance in the apical membrane following cAMP are unknown. However, it has been shown in T84 cells that cAMP stimulates mucin secretion and apical secretion of glucosaminoglycans (18, 22). The membrane vesicles that carry these products may provide a vehicle for CFTR return to the plasma membrane, or CFTR may recycle in other, yet to be defined, transport vesicles.

Many membrane proteins cycle between the plasma membrane and intracellular organelles. Examples include receptor proteins such as the transferrin, low-density lipoprotein (LDL), and mannose 6-phosphate (Man-6-P) receptors and transporting proteins such as the insulin-stimulated glucose transporter GLUT-4, the Na+-pumping Na+-K+-ATPase, the Na+/H+ exchanger NHE3, and, in some cells, CFTR. Extensive studies have shown that, for many of the recycling membrane proteins, one or more steps in the recycling pathway is dependent on phosphatidylinositol 3-kinase (PI3K) activity. Wortmannin, a fungal metabolite, is a potent inhibitor of PI3K activity [inhibitor constant (Ki<=  5 nM], and it blocks the movement of many membrane proteins at some point in the endosome-to-plasma membrane recycling pathway (31, 36, 38). One effect of wortmannin is to reduce the plasma membrane content of recycling proteins by trapping them in an endosomal compartment. For instance, wortmannin blocks exocytosis of GLUT-4 without affecting its endocytosis (13). Similarly, wortmannin inhibits exocytic recycling of transferrin receptors while at the same time increasing the rate of endocytosis (37). The net effect is a reduction in the steady-state level of plasma membrane transferrin receptors. Additionally, wortmannin causes accumulation of NHE3 in an endosomal compartment in NHE3-transfected Chinese hamster ovary (CHO) cells (27). Reports from a number of cell systems suggest that wortmannin inhibits trafficking within the endosomal system at multiple steps. In mouse L cells, wortmannin has no effect on the internalization of the Man-6-P receptor or its ligand, beta -glucuronidase. However, wortmannin inhibits recycling of the Man-6- P receptor back to the trans-Golgi network as well as beta -glucuronidase delivery to lysosomes (25). Thus, while there may be multiple steps in vesicular trafficking sensitive to wortmannin, a common observation is wortmannin inhibition of trafficking out of the endosomal compartment (34). Finally, it is important to keep in mind that wortmannin has additional affects on vesicular trafficking beyond inhibition of postendosomal sorting and recycling. Wortmannin is reported to inhibit the endocytosis of plasma membrane proteins such as Na+-K+-ATPase alpha -subunits in dopamine-stimulated renal proximal tubules (12).

We have tested the hypothesis that CFTR cycles into and out of the apical plasma membrane through a PI3K-dependent compartment by studying the effects of wortmannin on cAMP-stimulated Cl- secretion in monolayers of T84 cells. We reasoned that wortmannin might block plasma membrane insertion of CFTR, analogous to its effects on insulin-stimulated GLUT-4 exocytosis in adipose and skeletal muscle, or wortmannin might trap CFTR in endosomes, analogous to its effects on NHE3 recycling in NHE3-transfected CHO cells. It has already been shown that T84 cells express a wortmannin-inhibitable PI3K activity, which can be stimulated by epidermal growth factor (EGF) (40). Wortmannin inhibits EGF activation of PI3K and blocks EGF regulation of Ca2+-activated Cl- secretion in T84 cells (40). However, there have been no reports of wortmannin effects on cAMP-stimulated Cl- secretion in these cells.


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Materials. Cell culture medium, antibiotics, and newborn calf serum (NBCS) were from Sigma Chemical (St. Louis, MO). The permeable supports used to culture T84 cells as monolayers for electrophysiology were Millicell-HA from Millipore (Bedford, MA). Nystatin, forskolin, cAMP, horseradish peroxidase (HRP; type II, 200 U/mg protein), and 3,3',5,5'-tetramethylbenzidine (TMB) were from Sigma (St. Louis, MO). Wortmannin, LY-294002, thapsigargin, and EGF were from Calbiochem (La Jolla, CA). alpha -Toxin was from GIBCO BRL (Grand Island, NY). All other chemicals and reagents were from Sigma.

Cell culture. The nutrient medium used in this study was DMEM/F-12 supplemented with 15 mM HEPES, 2 mM glutamine, 5% NBCS, 100 U/ml penicillin, and 100 µg/ml streptomycin. For electrophysiology, 5 × 105 T84 cells were seeded into Millicell-HA culture plate inserts (12 mm diameter; 0.6 cm2 surface area). For HRP endocytosis studies, T84 cells were grown in 12-well plates at an initial plating density of 2.5 × 105 cells/well, and cultures were used at confluence. All cultures were fed fresh medium every other day and the day before use.

Monolayer development and integrity in Millicell-HA cultures were determined by measuring transepithelial electrical resistance (Rte) with the epithelial volt-ohm meter (EVOM) apparatus (World Precision Instruments, Sarasota, FL). To maintain sterility before use, EVOM electrodes were soaked in 70% ethanol and equilibrated in sterile PBS. Maximal Rte (1,200-2,000 Omega  · cm2) developed between days 7 and 12 of culture.

Transepithelial electrophysiology. In initial experiments, the EVOM apparatus was used to measure the effect of wortmannin on basal Rte. Measurement of short-circuit current (ISC) was conducted as previously described (16). Briefly, Millicells were mounted in water-jacketed Ussing chambers (Jim's Instrument Manufacturing, Iowa City, IA) and bathed in bicarbonate-buffered T84 Ringer solution (in mM: 115 NaCl, 5 KCl, 25 NaHCO3, 1.5 CaCl2, 1.5 MgCl2, and 5 glucose, as well as 0.005% phenol red) with continuous bubbling of 95% O2-5% CO2. The potential difference (PD) across the cell monolayer was measured with calomel electrodes in 3 M KCl and monitored with a Physiologic Instruments (San Diego, CA) current-voltage clamp. Monolayer PD was clamped to 0 mV with Ag-AgCl electrodes connected to the Ussing chamber by agar bridges. At intervals of 20-30 s, a biphasic pulse of 5 mV was applied, and the resulting current deflection was used to calculate monolayer conductance (G) by Ohm's law. All values for ISC, PD, and G were recorded on a Pentium-based microcomputer equipped with a DATAQ data acquisition board and running Acquire and Analyze software (Physiologic Instruments).

Apical plasma membrane Cl- channel activity was measured in alpha -toxin-permeabilized T84 monolayers. For this, the basolateral side of Millicell cultures was treated with 500 U/ml of alpha -toxin for 30 min before being mounted in Ussing chambers. When mounted in Ussing chambers, monolayers were bathed in Ringer solutions formulated to produce an apical-to-basolateral Cl- gradient [apical Ringer composition (in mM): 135 NaCl, 2.4 K2HPO4, 0.6 KH2PO4, 1.2 CaCl2, 1.2 MgCl2, 10 glucose, and 10 HEPES, as well as 0.005% phenol red; basolateral Ringer composition (in mM): 30 NaCl, 100 sodium gluconate, 2.4 K2HPO4, 0.6 KH2PO4, 0.1 CaCl2, 3 MgCl2, 10 glucose, and 10 HEPES, as well as 0.005% phenol red]. Ringer solution was bubbled continuously with 100% O2. Preliminary experiments demonstrated that 40 µM cAMP added to the basolateral hemichamber produced maximal Cl- current (ICl), which was insensitive to bumetanide but was blocked by apical diphenylamine carboxylic acid (DPC).

Basolateral membrane Na+ pump activity was measured in T84 monolayers permeabilized with apical nystatin by methods similar to those described previously (16). Briefly, monolayers were mounted in Ussing chambers and bathed in symmetrical HEPES-buffered T84 Ringer (in mM: 125 NaCl, 5 KCl, 2.4 K2HPO4, 0.6 KH2PO4, 1.5 CaCl2, 1.5 MgCl2, 5 glucose, and 10 HEPES, as well as 0.005% phenol red) and bubbled with 100% O2. Nystatin was diluted in Ringer and added to the apical hemichamber by solution exchange at a final concentration of 350 µg/ml (0.7% DMSO). Gassing of the apical hemichamber was reduced to minimize frothing. Addition of nystatin produced a rapid increase in current (INa). This current was completely (>=  95%) inhibited by 10-4 M ouabain added to the basolateral hemichamber, indicating that it resulted from Na+-K+-ATPase activity.

HRP endocytosis. T84 cells were grown in 12-well plates and used on the day they reached full confluence. Cultures were washed twice with warm (37°C) DMEM/F-12 and cultured for an additional 2 h in DMEM/F-12 that contained inhibitors as indicated. Cells were then cultured with 0.5 mg/ml HRP in DMEM/F-12 + 1% BSA to minimize nonspecific HRP binding. Uptake was terminated by cooling to 4°C and washing three times for 5 min each with DMEM/F-12 + 1% BSA followed by three 5-min washes in DMEM/F-12. Cells were extracted in 1 ml PBS + 0.2% Triton X-100 for 30 min at 4°C with constant gentle agitation on an orbital shaker. HRP activity in 2-µl samples of cell lysate was determined in a microtiter plate assay (3-5 wells/lysate) using TMB as substrate. The reaction was terminated at 5 min by addition of 1 N H2SO4 and the absorbance determined at 450 nM using a Bio-Tek Instruments (Winooski, VT) 312e plate reader controlled by a microcomputer running KinetiCalc software. In preliminary experiments, HRP uptake was linear for at least 2 h, and nonspecific HRP binding (defined as cell-associated HRP activity in cultures maintained at 4°C) was <20% of the activity in vehicle control (0.1% DMSO) cultures maintained at 37°C. The protein concentration of cell lysates was determined by the bichinchoninic assay (Pierce, Rockford, IL) using BSA as standard.

Statistical analysis. Unless noted otherwise, all values are reported as means ± SE. Statistical significance (P <=  0.05) was determined using Student's t-test.


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Wortmannin blocks EGF inhibition of thapsigargin-stimulated Cl- secretion. EGF inhibits thapsigargin-stimulated Cl- secretion by T84 cells, an effect shown to be due to EGF activation of PI3K (40). Wortmannin (50 nM) blocked the effects of EGF on thapsigargin-stimulated Cl- secretion (40). We have replicated these experiments to verify that our T84 cells are sensitive to wortmannin inhibition of PI3K activity and to verify the concentration of our wortmannin stock solutions. Figure 1 illustrates our results. Thapsigargin stimulated ISC (peak ISC = 8.5 ± 1.4 µA/cm2, n = 6). Within 15 min of administration, 20 nM EGF inhibited the thapsigargin-stimulated ISC (peak ISC = 4.2 ± 0.8 µA/cm2, n = 11, P < 0.05). At 50 nM, wortmannin rapidly (within 30 min) and completely blocked the inhibitory effects of EGF on thapsigargin-stimulated ISC (Fig. 1). In our experiments, as in those reported by Uribe et al. (40), wortmannin enhanced the ISC response to thapsigargin even in the presence of EGF (Fig. 1).


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Fig. 1.   Effects of 50 nM wortmannin (Wort) on epidermal growth factor (EGF) inhibition of thapsigargin (Thap)-stimulated short-circuit current (ISC). T84 monolayers were mounted in Ussing chambers and treated sequentially with 50 nM wortmannin, 20 nM EGF, and 1 µM thapsigargin as indicated. A: a set of recordings from 4 separate monolayers in a typical experiment. B: means ± SE for peak ISC values; n, no. of monolayers in each group.

For one set of monolayer cultures treated as described above, 10 µM forskolin was added to the basolateral hemichamber immediately following the peak of the thapsigargin response (~45 min after wortmannin addition). Forskolin-stimulated ISC was reduced in wortmannin-treated monolayers (162.3 ± 13.3 vs. 144.3 ± 8.8 µA/cm2, n = 3), but the reduction was not significant (P = 0.32). Thus, in cultures in which 50 nM wortmannin completely blocked the effects of EGF on thapsigargin-stimulated ISC, we observed a small but insignificant reduction in forskolin-stimulated ISC.

In the low nanomolar range, wortmannin is highly selective but not absolutely specific for PI3K inhibition (36, 38). Therefore, we have studied the effects of LY-294002, a less potent (IC50 = 1.4 µM) but reportedly more specific inhibitor of PI3K (41). We performed a series of experiments identical to those described in Fig. 1, except that LY-294002 was substituted for wortmannin. Results of these experiments are shown in Table 1. LY-294002 at 15 µM completely reversed EGF inhibition of thapsigargin-stimulated ISC. As with wortmannin, forskolin-stimulated ISC in these same monolayers was reduced, but the reduction was not statistically significant (P = 0.099).

                              
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Table 1.   Effects of LY-294002 and EGF on thapsigargin- and forskolin-stimulated ISC

Low concentrations of wortmannin do not inhibit forskolin-stimulated Cl- secretion. Addition of up to 500 nM wortmannin to forskolin-stimulated monolayers did not reduce stimulated ISC (not shown). Acute addition of up to 500 nM wortmannin to T84 monolayers mounted in Ussing chambers did not have an immediate effect on basal transepithelial resistance or basal ISC values (not shown). However, beginning within a few minutes of addition, 100 and 500 nM wortmannin caused a gradual, small, and sustained increase in basal ISC (Fig. 2). After 30 min in the Ussing chambers, the basal ISC of 0.1% DMSO control monolayers rose by 0.5 ± 0.1 µA/cm2 (n = 14), while at the same time the ISC in 100 nM wortmannin-treated monolayers rose by 2.6 ± 0.2 µA/cm2 (n = 9, P < 0.01). Currents in the 500 nM wortmannin-treated monolayers rose to 1.5 ± 0.1 µA/cm2 (n = 10), which was significantly greater than the DMSO controls (P < 0.01) but significantly less than those with 100 nM wortmannin (P < 0.01).


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Fig. 2.   Effect of wortmannin on forskolin-stimulated ISC. T84 monolayers were mounted in Ussing chambers and treated with wortmannin or 0.1% DMSO and 30 min later with 10-5 M forskolin as indicated. A: a set of recordings from a typical experiment. B: compiled results from several experiments (n = 8 monolayers in each wortmannin test group). For all experiments, forskolin-stimulated ISC was 25.0 ± 1.4 µA/cm2 in the 0.1% DMSO control monolayers (n = 10). * P < 0.02 vs. 0.1% DMSO control.

Because 50 nM wortmannin completely reversed EGF effects within 30 min, we exposed T84 monolayers to 100 nM wortmannin for 30 min before stimulation with forskolin. In these experiments, 100 nM wortmannin produced a small but insignificant inhibition of forskolin-stimulated ISC (Fig. 2). This demonstrates again that concentrations of wortmannin that effectively block PI3K-mediated EGF effects have no effect on forskolin-stimulated ISC. However, when wortmannin was raised to 500 nM, we observed a 35% reduction in forskolin-stimulated ISC (Fig. 2). Because 500 nM wortmannin inhibits myosin light chain kinase (30), mitogen-activated protein kinase (17), and possibly other enzymes (36, 38), we hypothesize that the inhibitory effect of 500 nM wortmannin on forskolin-stimulated ISC involves inhibition of one or more of these enzymes in addition to PI3K.

To define further the role of PI3K in T84 Cl- secretion, we tested LY-294002 for effects on basal and forskolin-stimulated ISC. Similar to 100 nM wortmannin, 15 µM LY-294002 caused a gradual increase in basal ISC [5.8 ± 0.8 µA/cm2 (n = 6) vs. 2.1 ± 0.3 µA/cm2 (n = 6), P < 0.01]. This increase in basal ISC was sustained for 30 min. After 30 min, 10 µM forskolin stimulated ISC in the 0.1% DMSO control monolayers by 50.7 ± 2.7 µA/cm2 (n = 6) and in the 15 µM LY-294002-treated monolayers by 45.2 ± 4.2 µA/cm2 (n = 6). The difference was not statistically significant (P = 0.29). Thus the effects of wortmannin and LY-294002 are consistent; both block the PI3K-mediated effects of EGF and neither inhibit forskolin-stimulated ISC.

Wortmannin does not inhibit apical membrane cAMP-activated Cl- channels. In a separate series of experiments we determined the time dependence for inhibition of forskolin-stimulated ISC at concentrations of wortmannin >100 nM. Using 200 nM wortmannin we found that inhibition increased to ~50% after 2 h and remained at this level for up to an additional 24 h (not shown). Also after 2 h, the Rte values of 200 nM wortmannin-treated monolayers were reduced by 30% compared with 0.1% DMSO-treated monolayers (not shown). We chose to use a 2-h exposure to wortmannin as an experimental system for investigating the mechanisms of wortmannin inhibition at concentrations >100 nM.

The rate-limiting step for epithelial Cl- secretion is the activity of apical Cl- channels. In T84 cells these are predominantly CFTR Cl- channels (1). To address the possibility that elevated wortmannin inhibits the activation and function of CFTR, we have used alpha -toxin permeabilization methods coupled to ISC measurements (4). By this technique the activity of apical membrane CFTR Cl- channels can be measured in T84 cells grown as a polarized monolayer. The advantage of this approach is that apical membrane Cl- channel activity is measured in cells with the three-dimensional morphology characteristic of epithelial tissues. We use cAMP to activate Cl- channels in this preparation because it will enter only the permeabilized cells, and, therefore, all of the resulting current originates in these cells (4). Cells not permeabilized by the alpha -toxin treatment do not generate current under these conditions.

Following basolateral permeabilization with alpha -toxin and mounting in Ussing chambers, an apical-to-basolateral Cl- gradient (~4:1) was imposed across T84 monolayers. Before stimulation with cAMP, a small negative basal ICl was consistently observed, and the basal ICl was always larger for 500 nM wortmannin-treated monolayers [7.4 ± 0.9 µA/cm2 (n = 6) vs. 11.6 ± 1.2 µA/cm2 (n = 6), P < 0.05]. The basal conductance of wortmannin-treated and permeabilized monolayers was greater than for the DMSO-treated control monolayers [3.53 ± 0.68 mS/cm2 (n = 6) vs. 1.66 ± 0.09 mS/cm2 (n = 6), P < 0.05]. Thus the difference in basal ICl values likely resulted from greater Cl- movement through the paracellular pathway in wortmannin-treated monolayers. However, following addition of 40 µM cAMP to the basolateral hemichamber, a rapid increase in a negative current resulted, the magnitude of which was not affected significantly by wortmannin (Fig. 3). This result suggests that CFTR or other apical Cl- channels activated by elevated cAMP are not direct targets for wortmannin action. In addition, this result demonstrates that 500 nM wortmannin for 2 h causes no change in the cAMP-regulated Cl- conductive properties of T84 apical membranes, although it does produce a 50% reduction in forskolin-stimulated ISC.


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Fig. 3.   Wortmannin does not alter the cAMP-activated Cl- conductive properties of T84 monolayer apical membranes. Monolayers were treated with 500 nM wortmannin or 0.1% DMSO for 2 h and throughout all subsequent steps. Basolateral membranes were then permeabilized with 500 U/ml alpha -toxin for 30 min. Monolayers were mounted in Ussing chambers with an apical-to-basolateral Cl- gradient, and monolayers were short circuited. At time indicated by arrow, 40 µM cAMP was added to basolateral hemichamber. A: a set of recordings from a typical experiment. B: means ± SE for 6 monolayers in each group. ICl, Cl- current.

High wortmannin inhibits the basolateral membrane Na+ pump. We have used the monovalent ionophore antibiotic nystatin to permeabilize the apical membranes of T84 monolayer cultures and measure the resulting ouabain-inhibitable Na+ current (INa). This was done to address whether the basolateral membrane Na+-pumping Na+-K+-ATPase is a target for wortmannin. Permeabilization was carried out in symmetric Ringer solutions to eliminate current flow through tight junctions or active ion channels present in basolateral membranes. The magnitude of the ouabain-inhibitable INa following nystatin permeabilization was significantly lower in monolayers treated with 500 nM wortmannin (Fig. 4). This result suggests that inhibition of forskolin-stimulated ISC by 500 nM wortmannin results, at least in part, from inhibition of basolateral membrane Na+ pump activity.


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Fig. 4.   Wortmannin inhibits basolateral membrane Na+ pump activity in T84 monolayers. Millicell cultures were treated with 500 nM wortmannin or 0.1% DMSO (vehicle control) for 2 h before mounting in Ussing chambers. Monolayer apical membranes were permeabilized with 0.35 µg/ml nystatin in the presence of 500 nM wortmannin or 0.1% DMSO as described in MATERIALS AND METHODS. Shown are means ± SE of ouabain-inhibitable Na+ current (INa) that followed nystatin permeabilization of 5 monolayers in each group.

Wortmannin does not reduce forskolin inhibition of endocytosis. Forskolin has been reported to reduce endocytosis in T84 cells (11). To determine whether plasma membrane recycling is a target for wortmannin, we measured its effect on endocytosis. Using HRP as a marker for fluid phase endocytosis we measured a 50-75% reduction in endocytosis in T84 cells exposed to 10 µM forskolin (Fig. 5). This agrees with other reports of forskolin inhibition of HRP or fluorescent dextran endocytosis (11, 35). We tested wortmannin at concentrations up to 500 nM and found that it did not alter forskolin inhibition of HRP endocytosis (Fig. 5). Unexpectedly, we found that 50-500 nM wortmannin, in the absence of forskolin, stimulated HRP endocytosis 30-100% over 0.1% DMSO-treated cultures (Fig. 5). A similar effect has been reported in K-562 cells, where 100 nM wortmannin increased the rate constant for transferrin receptor endocytosis by 60% (37). Despite this stimulation of endocytosis, forskolin reduced HRP endocytosis to levels measured in the absence of wortmannin (Fig. 5). These results demonstrate that wortmannin has no effect on forskolin inhibition of fluid-phase (HRP) endocytosis in T84 cells.


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Fig. 5.   Wortmannin does not block forskolin inhibition of horseradish peroxidase (HRP) endocytosis. T84 cultures were treated with 0.1% DMSO, 50 nM wortmannin, or 500 nM wortmannin for 2 h. Cultures were allowed to endocytose 0.5 mg/ml HRP for 1 h in the continuous presence of DMSO or wortmannin and 10-5 M forskolin as indicated. HRP activity of cell lysates was measured as described in MATERIALS AND METHODS and normalized to lysate protein. Each value is mean ± SE for 6 cultures in each group. * P < 0.05 vs. 0.1% DMSO control without forskolin. ** P < 0.01 vs. 0.1% DMSO control without forskolin.


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ABSTRACT
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The data presented in this paper demonstrate that cAMP-stimulated Cl- secretion by T84 cells is independent of wortmannin- and LY-294002-inhibitable PI3K activity. We have confirmed a previous report that 50 nM wortmannin completely blocks EGF-stimulated and PI3K-dependent downregulation of thapsigargin-stimulated Cl- secretion in T84 cells (40). In addition, we have shown that a second PI3K inhibitor, LY-294002, blocked EGF inhibition of thapsigargin-stimulated Cl- secretion. However, under the same conditions that these PI3K inhibitors blocked EGF effects, wortmannin and LY-294002 produced only small and insignificant effects on forskolin-stimulated Cl- secretion. In a recent report, EGF was shown to inhibit carbachol-stimulated 86Rb+ efflux but not 125I- efflux from T84 cells (3). Consistent with its effects on Cl- secretion, wortmannin blocked the effect of EGF on 86Rb+ efflux without altering 125I- efflux. These results are consistent with the results presented here, both of which suggest that wortmannin has no effect on the activity of CFTR or other anion channels in T84 cells. Using clones of the HT-29 human colonic cell line, Merlin et al. (28) showed that 100 nM wortmannin had no effect on forskolin-stimulated Cl- secretion, although it did inhibit purinergic stimulation of mucin granule fusion in these cells. These results further support our conclusion that PI3K activity is not required for cAMP activation of CFTR-mediated Cl- secretion.

The observation that wortmannin blocks EGF effects on 86Rb+ efflux (40) offers an explanation for wortmannin and LY-294002 stimulation of basal ISC and potentiation of thapsigargin-stimulated ISC in T84 monolayers, as reported here and by Uribe et al. (40). We and others routinely culture T84 cells in media that contain serum, which in turn contains EGF and other growth factors that stimulate PI3K activity. By inhibition of growth factor-stimulated PI3K, wortmannin might remove a tonic inhibition of basolateral K+ channels, which would hyperpolarize the cell and increase the driving force for Cl- exit through channels open in the basal state. In addition, removal of this tonic inhibition would be expected to increase the sensitivity of basolateral K+ channels to elevated intracellular Ca2+ following thapsigargin stimulation, thereby supporting an increased rate of Cl- secretion.

At higher wortmannin concentrations (200-500 nM) and with prolonged exposure (2 h), we observed significant inhibition of forskolin-stimulated Cl- secretion in intact monolayers. However, under these conditions there was no difference in the cAMP-stimulated Cl- conductive properties of T84 monolayer apical membranes. This suggests that the inhibition observed with high wortmannin concentrations is not due to inhibition of CFTR function and that it must be due to inhibition of another transporter contributing to Cl- secretion. Here and in a preliminary report (15), we have shown that 500 nM wortmannin inhibits Na+ pump activity in T84 monolayers. Inhibition of the Na+ pump must account for some of the inhibition of forskolin-stimulated ISC observed in 500 nM wortmannin-treated monolayers. In addition, wortmannin above 100 nM is known to cause inhibition of other enzymes including myosin light chain kinase and mitogen-activated protein kinase (17, 30). It is likely that these or other wortmannin-sensitive enzymes are responsible for inhibition of Cl- secretion, alone or in combination with PI3K inhibition.

Stimulation of T84 cell Cl- secretion is accompanied by an increase in the apical membrane content of CFTR (33, 39). This may result from increased rates of CFTR exocytosis, although the possibility that it reflects reduced CFTR endocytosis without a change in CFTR exocytosis has not been fully addressed. However, cAMP does stimulate the exocytosis of mucin-containing secretory granules, glucosaminoglycan-containing constitutive secretory vesicles, and vesicles involved in the recycling of lectin receptors in T84 cells (10, 18, 22). One or more of these may mediate delivery of CFTR to the apical membrane. It is unlikely that mucin granules or constitutive secretory vesicles mediate the return of CFTR to the apical membrane during its continuous recycling in unstimulated cells, but they may deliver additional CFTR to the apical membrane during times of stimulated Cl- secretion. Studies in mucin-secreting clones of the HT-29 cell line suggest that mucin granules contain Cl- channels that contribute to Cl- secretion under some conditions but that these are not CFTR Cl- channels (28). If cAMP does stimulate exocytosis of CFTR-containing vesicles, whatever their origin, our data suggest that this process is independent of PI3K activity.

A detailed itinerary for endocytosed CFTR is lacking. We know that internalization of CFTR in T84 cells occurs by clathrin-mediated endocytosis (8, 9). A putative tyrosine-based internalization sequence, which targets other recycling proteins to coated pits, has been identified in the carboxy-terminal tail of CFTR and has been shown to be required for efficient internalization of CFTR/transferrin receptor chimeras (32). Consistent with this is the observation that CFTR expressed by cells of the human airway line Calu-3 is present in clathrin-coated vesicles but excluded from caveolae (8). How CFTR gets from clathrin-coated endocytic vesicles back to the plasma membrane is unknown. It is unlikely that CFTR enters the sorting/recycling endosome system in T84 cells for two reasons. First, CFTR is endocytosed and recycled to the apical membrane with a half time of 1-2 min (33). Although some cells exhibit similarly rapid rates of membrane protein recycling (23), the recycling of proteins in most cell types is slower, with reported half times of 10-30 min (29). Second, the amount of CFTR in the apical membrane, as assayed by Cl- channel function, is unaltered by as much as 500 nM wortmannin for 2 h, whereas the recycling of membrane proteins in other cells is significantly reduced within minutes by 50-100 nM wortmannin (13, 26, 27, 34, 37). It may be that, in cell systems exhibiting rapid CFTR recycling, CFTR endocytic vesicles return and fuse directly with the plasma membrane and not with early endosomes.

In summary, we show that the functional expression of CFTR Cl- channels in the apical membrane of T84 cells is insensitive to inhibition of PI3K activity by wortmannin and LY-294002. The recycling of CFTR in the apical membrane of Cl--secreting epithelia is often compared with the recycling of GLUT-4 and the transferrin receptor (6, 25). Membrane recycling of these proteins is wortmannin sensitive (13, 37), and so our observations suggest that similarities between CFTR recycling and GLUT-4 or transferrin receptor recycling are limited. A factor underlying the dissimilarities may be the recycling of CFTR specifically to the apical membrane. Epithelial apical membranes are compositionally distinct from basolateral membranes and from the plasma membranes of nonpolarized cells (adipose, muscle, fibroblasts, etc.). Delivery of newly synthesized proteins to the apical membrane involves sorting and vesicular trafficking mechanisms that are biochemically distinct from those that govern delivery to the basolateral membrane (24). Similarly, recycling of apical proteins may be governed by sorting and trafficking mechanisms distinct from those operating at the basolateral membrane or in nonpolarized cells. In this regard, wortmannin inhibits transcytosis of the polymeric immunoglobulin receptor (pIgR) from basolateral to apical membrane in MDCK cells but has no effect on apical recycling of pIgR (21). We suggest that a full understanding of the molecular regulation and physiological significance of CFTR recycling necessitates development and pursuit of models of protein recycling specifically at the apical membrane and less reliance on recycling proteins expressed at the basolateral membrane or in nonpolarized cells.


    ACKNOWLEDGEMENTS

We are grateful to Thomas Wiegand for technical assistance and to Drs. R. Wondergem and B. Rowe for critical reading of the manuscript.


    FOOTNOTES

This work was supported by a grant from the American Heart Association (TN97N74) to T. W. Ecay.

A portion of this work has been published in abstract form (15).

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: T. W. Ecay, Dept. of Physiology, East Tennessee State Univ., PO Box 70576, Johnson City, TN 37614 (E-mail: ecay{at}etsu.edu).

Received 30 August 1999; accepted in final form 15 November 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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