TNF-alpha potentiates the ion secretion induced by muscarinic receptor activation in HT29cl.19A cells

Judith C. J. Oprins, Helen P. Meijer, and Jack A. Groot

Institute for Neurobiology, Biological Faculty, University of Amsterdam, 1098 SM Amsterdam, The Netherlands


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Chronic gastrointestinal diseases such as ulcerative colitis and Crohn's disease are characterized by severe diarrhea. Mucosal biopsies of these patients show enhanced levels of cytokines, secreted by infiltrated inflammatory cells. In this study, we investigated the effect of the cytokine tumor necrosis factor-alpha (TNF-alpha ) on ion secretion in human intestinal epithelial cells. The conventional microelectrode technique in the cell line HT29cl.19A was used, which allows for simultaneous measurements of transepithelial potential difference and intracellular potential difference across the apical membrane. Preincubation (2-78 h) with 10 ng/ml TNF-alpha did not change basal secretory activity. However, the secretory response to the muscarinic receptor agonist carbachol was strongly increased after exposure to TNF-alpha . Application of the protein kinase C (PKC) inhibitor GF 109203X (bisindolylmaleimide I) inhibited the response to carbachol as well as the TNF-alpha -potentiated response, indicating that PKC mediates the effect of carbachol in this cell line. Propranolol, a substance that inhibits the phospholipase D (PLD) pathway, strongly reduced the response to muscarinic stimulation and its potentiation by TNF-alpha . The results indicate that activation of PLD is involved in ion secretion induced by muscarinic receptor activation and that TNF-alpha can potentiate this pathway.

phospholipase D; protein kinase C; intestinal epithelia; carbachol; cytokine; tumor necrosis factor-alpha


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

CHLORIDE SECRETION ACROSS intestinal epithelium plays a key role in regulating water secretion into intestinal lumen. The Cl- secretion is under close regulation by hormonal, neural, and paracrine mediators. An increased Cl- secretion can result in severe diarrhea, due to excessive water transport from blood to lumen.

Inflammatory bowel diseases (IBD) like ulcerative colitis and Crohn's disease are characterized by diarrhea. The underlying pathophysiological mechanisms for the diarrhea remain unknown. IBD patients show an increase in cytokines in the intestinal wall, which are secreted by infiltrated inflammatory cells (8, 23).

Antibodies against tumor necrosis factor-alpha (TNF-alpha ), one of the elevated cytokines, have been applied in animal models of experimental colitis. These studies suggested a role for antibodies against TNF-alpha in the treatment of IBD (37). In a multicenter, double-blind, placebo-controlled trial, a single infusion of a monoclonal antibody (cA2) against this cytokine appeared to be an effective treatment in patients with Crohn's disease (30). This indicates the importance of TNF-alpha in the disease. Several studies have shown that cytokines are able to alter ion transport and barrier properties of intestinal epithelium (19, 21), which could contribute to the diarrhea.

TNF-alpha may mediate activation of ion secretion in intestinal epithelium, but data on this matter are quite scarce. In human distal colon, TNF-alpha was shown to increase ion secretion via an increased release of prostaglandins by subepithelial cells (27). A similar study performed in porcine ileum also showed an indirect effect of TNF-alpha on ion secretion (15).

Several epithelial cells are known to express TNF-alpha receptors, and, especially when the receptor density is increased by interferon-gamma (IFN-gamma ), they respond to TNF-alpha directly by modulating the permeability of the tight junctions (24). However, direct effects of TNF-alpha on ion secretion are not widely studied. This study aims to gain more insight into the possible direct effect of TNF-alpha on human intestinal epithelium. With intracellular electrophysiological techniques, the effect of human recombinant TNF-alpha on the basal as well as the secretagogue-induced ion secretion in human colonic epithelial cells HT29cl.19A was determined.

The data show that TNF-alpha is able to potentiate the secretion induced by the secretagogue carbachol, an activator of the Ca2+/protein kinase C (PKC)-mediated pathway, but not the secretion induced by forskolin, an activator of the cAMP pathway. The results indicate that carbachol-induced secretion involves activation of the phospholipase D (PLD) pathway and that the potentiation by TNF-alpha occurred via that pathway. As far as we know, this is the first study in intestinal epithelial cells that shows a direct effect of TNF-alpha on carbachol-induced ion secretion and the involvement of PLD in the intestinal secretory response.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture. HT29cl.19A cells were cultured as described previously (2). Briefly, the human intestinal epithelial cell line HT29cl.19A, passages 12-28, was grown in DMEM supplemented with 10% fetal bovine serum, 8 mg/l ampicillin, and 10 mg/l streptomycin. Cells were seeded in 25-cm2 flasks at 37°C in 5% CO2-95% O2 and passaged weekly. For electrophysiological experiments, cells were subcultured on Falcon filters (25 mm in diameter), and medium was replaced every other day. Confluency was reached 7 days after seeding, and the cells were used between 13 and 26 days after seeding. TNF-alpha incubations were performed in culture media for the indicated time.

T84 cells were kindly provided by Dr. H. R. de Jonge (Dept. of Biochemistry, University of Rotterdam). The cells, passages 21-29, were grown in DMEM and F-12 medium in a 1:1 mixture. The medium was supplemented with 10% fetal bovine serum, 8 mg/ml ampicillin, and 10 mg/ml streptomycin. The cells were subcultured as described above.

Electrophysiological experiments. The filter was cut from the ring, divided into four pieces, and rinsed with mannitol-Ringer. One piece was mounted in a small horizontal Ussing chamber, leaving an oblong area of 0.35 cm2. The apical and basolateral compartments were continuously perfused with mannitol-Ringer buffer at a temperature of 37°C and gassed with 5% CO2-95% O2. The composition of the Ringer solution was (in mM) 117.5 NaCl, 5.7 KCl, 25.0 NaHCO3, 1.2 NaH2PO4, 2.5 CaCl2, 1.2 MgSO4, and 28 mannitol. To increase the driving force for Cl- efflux, we used a low-Cl- (0.1 mM) solution containing (in mM) 0.1 NaCl, 117.2 sodium gluconate, 5.7 potassium gluconate, 2.5 CaSO4, 1.2 MgSO4, 1.2 NaH2PO4, 25 NaHCO3, and 28 mannitol. This solution was applied to the apical side of the monolayer 15 min before addition of carbachol. Therefore, the electrophysiological response to carbachol was not affected by junction potentials.

Agar bridges were placed in apical and basolateral compartments and were connected to Ag-AgCl electrodes for monitoring of the transepithelial potential difference (Vt). An extra Ag-AgCl electrode was placed in the apical bath, serving as a common ground. Apical membrane potential (Va) was measured by impalement with a glass microelectrode pulled from capillaries (1 mm in outer diameter; Clark Electromedical, Reading, UK) with a Flaming Brown P-87 micropipette puller. The microelectrode was filled with 0.5 M KCl solution. The tip resistance was 100-200 MOmega , and the tip potential was 2-5 mV. Current electrodes (Ag-AgCl) were placed in the walls of both compartments; these were used to apply bipolar current pulses from a floating current source of 10 and 50 µA, at 30-s intervals, in order to calculate the transepithelial resistance (Rt) and the fractional resistance (fRa). fRa = Ra/(Ra + Rb), where Ra and Rb are the resistances of the apical and basolateral membranes, respectively. The equivalent short-circuit current (Isc) was calculated from Vt and Rt.

The potentials were measured differentially with M-4A electrometer probes (W-P-Instruments, New Haven, CT). The potential differences were continuously recorded on a multipen recorder and on a computer using custom-made software.

The measurements were corrected for the offset of the electrodes and for the resistances of the fluid and filter without cells. Values are means ± SE. Statistical significance was evaluated using Student's t-test.

Histology. Filters containing cells were exposed to 10 ng/ml TNF-alpha for 24 h, after which they were cut into small pieces. The filters were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for 2 h at room temperature and rinsed three times for 20 min each in 0.1 M sodium cacodylate buffer (pH 7.4). The filters were rinsed overnight (4°C) in 0.1 M sodium cacodylate buffer (pH 7.4) and then processed for routine electron microscopy.

Chemicals. TNF-alpha , GF 109203X (bisindolylmaleimide I), 4-beta -phorbol-12,13-dibutyrate (PDBu), U-73122, and U-73143 were obtained from Calbiochem. Cycloheximide, propranolol, and forskolin were purchased from Sigma, and carbachol was from Brunschwig. TNF-alpha , propranolol, and carbachol were dissolved in water. Forskolin and cycloheximide were dissolved in ethanol (maximal concentration in the Ussing chamber was 0.1%). PDBu and GF 109203X were dissolved in DMSO. U-73122 and U-73143 were dissolved in chloroform. The maximal concentration of the latter two solvents was 0.01%. Maximal concentrations of carrier solvents were without electrophysiological effect on the cells.

The concentration of GF 109203X (1 µM) was chosen because it was without effect on protein kinase A (PKA)-mediated response (see PKC is involved in electrical response to carbachol). Concentrations were taken from the literature for PDBu (6), U-73122 and U-73143 (39), and cycloheximide (41). Propranolol concentrations were tested in a range from 100 to 500 µM. At the concentration used in this study, the potentiation of the intracellular response to carbachol after exposure to TNF-alpha was fully reversed (see PLD activation is involved in carbachol-mediated secretory response).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

TNF-alpha potentiates carbachol-induced secretion. The electrophysiological response to muscarinic receptor activation with carbachol and its relation to Cl- secretion in HT29cl.19A cells have been previously reported (4). A typical electrical response to 100 µM carbachol is presented in Fig. 1. The mean values of the electrical parameters and their changes induced by carbachol are shown in Table 1. For the present description, the response was divided into two phases, which are marked by the two thin lines in Fig. 1. The data in Table 1 were taken at the time points indicated by these lines. Phase 1 was a fast depolarization of the Va together with an increase in fRa while the Vt decreased. Because the Rt was not affected, the decrease of Vt must be caused by a small but significant decrease of the Isc. On the basis of previous experiments (4), phase 1 is attributed to a sharp increase of the intracellular Ca2+ activity, primarily from the inositol 1,4,5-trisphosphate (IP3)-sensitive intracellular pool, which activates Cl- conductances located in the apical membrane and, more prominently, in the basolateral membrane. Phase 2 was characterized by repolarization leading to a hyperpolarization of the apical membrane, concomitant with an increase in Vt and a return of the fRa and Rt to control values. The increase of the Isc during phase 2 of the carbachol response is ascribed to the opening of basolateral Ca2+-sensitive K+ channels (4), which increase the driving force for Cl- efflux through apical Cl- channels. Previous work in HT29cl.19A cells suggests that these channels are different from the Ca2+-sensitive Cl- channels and that PKCalpha is involved in their activation (4, 35).


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Fig. 1.   Changes in electrophysiological parameters of HT29cl.19A cells after addition of 100 µM carbachol in absence or presence for 48 h of tumor necrosis factor-alpha (TNF-alpha ; 10 ng/ml). Two tracings (control and TNF-alpha -treated group) represent experiments in which intracellular recordings could be obtained. Two thin lines divide responses into phases 1 and 2 as described in RESULTS. Vt, transepithelial potential; Va, intracellular potential; fRa, fractional apical resistance; Rt, transepithelial resistance; Isc, short-circuit current. For statistics, see Table 1.


                              
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Table 1.   Effect of 48-h incubation with 10 ng/ml TNF-alpha on changes in electrical parameters induced by 100 µM carbachol

Incubation for 48 h with TNF-alpha (10 ng/ml bilaterally) significantly increased Rt, whereas the other basal electrical parameters were not changed. However, it appeared to be much more difficult to obtain stable intracellular recordings.

Figure 1 shows the change in electrophysiological parameters induced by carbachol after 48 h of exposure to TNF-alpha . The mean values of nine experiments are given in Table 1.

The sharp depolarization in phase 1 and the increase of fRa were not affected by the exposure to TNF-alpha ; however, the first extracellular potential change (basolateral side negative) and the change in Isc were smaller. Phase 2 showed large differences due to TNF-alpha exposure; the depolarization of Va was prolonged, concurrent with a strong decrease in the fRa (0.35 vs. 0.08 in control) and Rt (28 vs. 2% in control). The carbachol-induced increase of Isc, with respect to basal values, was much larger after treatment with TNF-alpha .

To examine whether the increased current was due to a Cl- efflux or a Na+ influx, we replaced Cl- in the apical bath with gluconate, thereby increasing the Cl- gradient across the apical membrane. Monolayers exposed to TNF-alpha under this condition responded to carbachol with changes in Vt (175 ± 29%) and Isc (150 ± 19%) larger than those of the same monolayers exposed to TNF-alpha in normal Ringer (100%; n = 4). This indicates that the cytokine potentiated the carbachol-induced Cl- efflux across the apical membrane.

Dose-response curve of TNF-alpha . To define the dose dependence of the action of TNF-alpha , filters were incubated for 24 h with different concentrations of TNF-alpha varying between 1 and 100 ng/ml. Figure 2 shows the effects of the different concentrations of TNF-alpha on the carbachol-induced Isc. The optimal potentiation takes place at a concentration of 10 ng/ml TNF-alpha . Therefore, this concentration was used in the following experiments.


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Fig. 2.   Dose-response curve of potentiating effect of TNF-alpha on carbachol-induced Isc. Monolayers were exposed for 24 h to different concentrations of TNF-alpha , after which change in Isc after carbachol was measured. Maximal potentiation took place at a concentration of 10 ng/ml TNF-alpha . * P < 0.05 compared with control carbachol, n = 4.

Time dependence of TNF-alpha action. To define the time dependence of the potentiating effect of TNF-alpha , cells were incubated with 10 ng/ml TNF-alpha for different times, varying between 30 min and 78 h. Figure 3 shows the relation between the carbachol-induced Isc and the time of exposure to TNF-alpha . The maximal change in Isc is presented as the percentage of the carbachol response of monolayers not exposed to TNF-alpha (= 100%). Until 2 h of incubation, TNF-alpha did not show a significant potentiation. However, incubation for 2.5 h with TNF-alpha enhanced the carbachol response significantly (206 ± 7%), and, with increasing incubation times, the potentiating effect of TNF-alpha on the carbachol response became larger (r = 0.70). After 78 h of incubation, the response was increased to >900%. We decided to use a shorter exposure time for TNF-alpha in some of the experiments, since this permitted more stable intracellular measurements.


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Fig. 3.   Time dependence of potentiating effect of TNF-alpha on carbachol response. Monolayers were exposed to 10 ng/ml TNF-alpha for between 0 and 78 h, and maximal change in Isc after addition of carbachol was measured. Data are expressed as percentage change in Isc compared with change in Isc after carbachol alone (100%). Longer incubation times with TNF-alpha resulted in larger changes in Isc with a correlation coefficient of r = 0.70.

Histology. Figure 4, A and B, shows typical electron micrographs (×3,100) of HT29cl.19A monolayers with (Fig. 4B) or without (Fig. 4A) exposure to 10 ng/ml TNF-alpha for 48 h. No differences are seen compared with time-matched control monolayers. Propidium iodide staining combined with Hoechst 33258 was used to check for apoptosis. Even after 48 h of exposure to TNF-alpha , no indication was found for TNF-alpha -induced apoptosis (not shown).


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Fig. 4.   Electron micrographs of monolayers with and without exposure to TNF-alpha for 48 h. A: representative control monolayer. B: representative monolayer exposed to 10 ng/ml TNF-alpha for 48 h. No morphological differences are seen between monolayers. Photographs are oriented with filter side down. Magnification: ×3,100.

TNF-alpha action is dependent on protein synthesis. We examined whether the action of TNF-alpha was dependent on protein synthesis using the protein synthesis inhibitor cycloheximide. We incubated the cells with 10 µg/ml cycloheximide (bilaterally) for 1 h before TNF-alpha exposure (41). The cells were then exposed to TNF-alpha in the presence of the inhibitor for 4 h, and the maximal change in Isc induced by 100 µM carbachol was measured. Figure 5 shows the effects of cycloheximide on the carbachol response with or without exposure to TNF-alpha . The increase in Isc induced by carbachol in control monolayers was not affected by incubation with cycloheximide (7.1 ± 1.2 µA/cm2 with cycloheximide vs. 5.8 ± 1.5 µA/cm2 for control, n = 4, P > 0.1). However, in the presence of cycloheximide, the potentiating effect of TNF-alpha was completely abolished (31.8 ± 11.2 µA/cm2 for TNF-alpha vs. 6.5 ± 1.1 µA/cm2 for cycloheximide + TNF-alpha , n = 4, P < 0.05). This, together with the time lag, suggests that TNF-alpha potentiates the carbachol response via a mechanism that requires protein synthesis.


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Fig. 5.   Effect of cycloheximide on maximal change in Isc induced by carbachol (carb) with or without exposure to TNF-alpha . Cells were incubated with 10 µg/ml cycloheximide (cyclo) bilaterally for 1 h before TNF-alpha exposure (4 h). Means ± SE from 4 monolayers. * P < 0.05 compared with carbachol.

PKC is involved in electrical response to carbachol. We examined the involvement of PKC in the response to carbachol with and without exposure to TNF-alpha by using an inhibitor of PKC, GF 109203X, that is specific for PKC but can also inhibit PKA at high concentrations (32). We tested whether GF 109203X could inhibit PKC specifically, but not PKA, in HT29cl.19A cells. Figure 6 shows the effects of bilateral application of consecutive 0.1 and 1 µM GF 109203X on the changes in Va induced by PKA and PKC activation. Forskolin stimulates PKA-activated Cl- channels, via direct activation of adenylyl cyclase. Application of 1 µM PDBu results in activation of PKC (6). Addition of forskolin or PDBu, although with different time constants, resulted in a depolarization of Va, due to the increase in apical Cl- conductance (3). Application of GF 109203X did not affect the forskolin-induced depolarization. However, 1 µM GF 109203X reduced the depolarization of Va induced by PDBu. Because the full effect of GF 109203X required ~20 min, in the following experiments preincubation was performed for 30 min. As shown in Table 2, 1 µM GF 109203X did not show an effect on the basal electrical parameters. The increase of Isc induced by forskolin was not different with or without 30 min of preincubation with the inhibitor (43 ± 5 and 47 ± 11 µA/cm2, respectively; n = 9 and 4, respectively). However, the Isc induced by application of 1 µM PDBu in the presence of the inhibitor was significantly reduced by 64% from 12 ± 2 to 5 ± 1 µA/cm2 (n = 6, P < 0.05). Thus 30 min of exposure to 1 µM GF 109203X specifically inhibits PKC in HT29cl.19A. GF 109203X was therefore used to test whether PKC was involved in the response to carbachol and its potentiation by TNF-alpha . The cells were preincubated for 30 min with 1 µM GF 109203X, after which the effects of carbachol and of exposure to TNF-alpha were measured. After TNF-alpha exposure, one part of the filter was used to measure the potentiated carbachol response. The second part of the filter was preincubated for 30 min with 1 µM GF 109203X, after which the effect of carbachol was measured. Figure 7 shows the relative decrease of the carbachol-induced increase in Isc after preincubation with GF 109203X with or without exposure to TNF-alpha . In the presence of the inhibitor, the maximal change in Isc after carbachol alone was significantly reduced by 71 ± 14%. After exposure to TNF-alpha , the potentiated carbachol response was inhibited by 64 ± 6%. We conclude that PKC activation is involved in the normal carbachol-induced secretion as well as in the TNF-alpha -potentiated response in this cell line.


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Fig. 6.   Effect of GF 109203X (bisindolylmaleimide I) on changes in Va after addition of forskolin (10 µM, basolateral) and 4-beta -phorbol-12,13-dibutyrate (PDBu; 1 µM, apical). GF 109203X was added to both sides of cells in concentrations of 0.1 and 1 µM. See RESULTS for effects of preincubation of cells with GF 109203X on forskolin- and PDBu-induced changes in Isc.


                              
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Table 2.   Effects of 30-min incubation with 1 µM GF 109203X on basal electrical parameters



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Fig. 7.   Effect of 30 min of preincubation with 1 µM GF 109203X (GF; bilateral) on Isc induced by carbachol with or without exposure to TNF-alpha . Data are presented as percentage changes compared with pairwise control experiments without inhibitor. Means ± SE from 4 or 5 monolayers. Cells were exposed to TNF-alpha between 4 and 48 h. * P < 0.05 compared with carbachol; # P < 0.05 compared with carbachol + TNF-alpha .

TNF-alpha does not affect PDBu- and forskolin-induced secretion. One mechanism by which TNF-alpha could potentiate the effect of carbachol is a direct upregulation of PKC. To investigate this possibility, we preincubated the cells with TNF-alpha for between 6 and 24 h and measured the Isc induced by the PKC activator PDBu. The change in Isc induced by 1 µM PDBu alone was 9.9 ± 1.2 µA/cm2 (n = 5), and the change induced by PDBu after TNF-alpha was 9.3 ± 0.7 µA/cm2 (n = 4). In HT29cl.19A, the secretory response activated by PDBu is mediated by the same Cl- channels as are the PKA-activated channels, namely the cystic fibrosis transmembrane conductance regulator (5). To test whether TNF-alpha had an effect on the PKA-mediated activation of the apical Cl- channels, we applied forskolin. The Isc induced by 10 µM forskolin alone was 43 ± 5 µA/cm2 (n = 9). After exposure to TNF-alpha for 48 h, the Isc induced by forskolin was 51 ± 3 µA/cm2 (n = 4), which is not significantly different.

PLD activation is involved in carbachol-mediated secretory response. The foregoing results indicate that the potentiation by TNF-alpha must be sought upstream from PKC. One possibility is that the effect is based on a stronger activation of PKC by an increased production of diacylglycerol (DAG). DAG can be produced by several reactions: first, the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) by phosphatidylinositol-specific phospholipase C (PI-PLC; this pathway has been demonstrated for muscarinic and histaminic receptor activation in intestinal epithelial cells; Ref. 13); second, hydrolysis of phosphatidylcholine by phosphatidylcholine-specific PLC (PC-PLC); or third, hydrolysis of phospholipids to phosphatidic acid (PA) by PLD and further to DAG by phosphatidate phosphohydrolase (PAP; reviewed in Ref. 11). However, as far as we know, the two last mechanisms are not documented in intestinal epithelial cells. To test the possible involvement of the PI-PLC pathway, we attempted to block PLC with U-73122, a putative PLC-PIP2-specific antagonist (39). However, addition of this compound (10 µM) did not show inhibitory effects, and the negative control U-73343 did show the same quantitative effects on Va. Thus this compound could not be used as a tool to investigate the role of PI-PLC.

The formation of DAG by PAP, and therefore the involvement of PLD, can be blocked by 100 µM propranolol (31). Using TLC of 32P-incubated monolayers, it was found that propranolol (100 µM) increased the level of PA, indicating an inhibition of the degradation of PA to DAG (Oprins et al., unpublished observations). To check whether PLD-dependent DAG formation, and therefore increased PKC activation, is involved in the (TNF-alpha -potentiated) carbachol response, we examined the effect of propranolol on the carbachol response with or without exposure to TNF-alpha . Table 3 shows that 10 min of exposure to 100 µM propranolol on the apical side of the cells slightly changed the basal electrical parameters. A small hyperpolarization occurred together with an increase in fRa, indicating an increase in basolateral K+ conductance. However, no significant effect was seen on transepithelial parameters. (At 500 µM or higher, propranolol reduced the Rt.) Propranolol at 100 µM did not have any effect on forskolin- or PDBu-induced secretion (results not shown). Figure 8 shows the effect of propranolol on the change in Va after addition of carbachol in TNF-alpha -treated cells. The prolonged depolarization induced by carbachol in the presence of TNF-alpha was completely abolished after propranolol treatment and is similar to registrations of Va after carbachol application without TNF-alpha (see Fig. 1). Figure 9 shows the relative change in Isc with respect to the carbachol-induced change of Isc (100%). After preincubation with propranolol, the carbachol-induced increase of Isc was reduced (from 14 ± 2.0 to 7.4 ± 1.7 µA/cm2, n = 6, P < 0.05). Propranolol completely abolished the potentiating effect of TNF-alpha on the carbachol response (57 ± 7 µA/cm2 for carbachol after TNF-alpha vs. 4.2 ± 0.3 µA/cm2 in the presence of propranolol, n = 6, P < 0.001).

                              
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Table 3.   Effects of 10-min incubation with 100 µM propranolol on basal electrical parameters



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Fig. 8.   Example of intracellular recording of effect of 10 min of preincubation with 100 µM propranolol (apical) on changes in Va induced by addition of carbachol after exposure to TNF-alpha , as compared with tracing without propranolol. Decreased depolarization and its shorter duration are reflected in a decrease in Isc shown in Fig. 9. Cells were exposed to TNF-alpha for 4 h.



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Fig. 9.   Effect of propranolol on Isc induced by carbachol with or without exposure to TNF-alpha . Data are presented as percentage compared with carbachol-induced response without propranolol (means ± SE from 6-9 monolayers). Cells were exposed to TNF-alpha between 4 and 48 h. Means ± SE of absolute values of changes are given in RESULTS. * P < 0.05 compared with carbachol; # P < 0.001 compared with carbachol + TNF-alpha .

T84 cells. Exposure of T84 cells to 10 ng/ml TNF-alpha for 24 h did not change basal secretory activity (Isc was 6.82 ± 1.50 µA/cm2 for control vs. 6.30 ± 1.8 µA/cm2 for TNF-alpha -exposed monolayers). Also no change in basal Rt was found after exposure to TNF-alpha (588 ± 88 Omega  · cm2 for control vs. 656 ± 99 Omega  · cm2 for TNF-alpha ). Addition of 100 µM carbachol resulted in an increase in Isc of 82 ± 25% (n = 3) compared with basal values. After 24 h of exposure to TNF-alpha , the change in Isc was 68 ± 10% (n = 3), which is not significantly different from that in control monolayers.

The carbachol-induced increase of Isc in these cells appeared not to be affected by the PKC inhibitor GF 109203X (Isc induced by carbachol in the presence of GF 109203X was 121 ± 17% compared with control values). The Va in T84 cells is -34 ± 0.6 mV (n = 3), which is lower than that in HT29cl.19A cells, suggesting a higher basal Cl- conductance. Addition of carbachol resulted in a hyperpolarization of Va by -18 ± 2 mV (n = 3), which occurred together with the increase in Isc.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In the present study, we show that exposure to TNF-alpha (10 ng/ml) potentiates the Cl- secretion induced by muscarinic receptor activation in human intestinal epithelial cells (HT29cl.19A). The potentiating effect of TNF-alpha was time dependent; a significant increase of the carbachol-induced Isc was observed after exposure to TNF-alpha for at least 2.5 h. The rather long lag time and the fact that blocking protein synthesis by cycloheximide prevented the potentiating action of TNF-alpha suggest that de novo synthesis of a link or activator in the signaling cascade occurs.

There are many potential sites where TNF-alpha could augment the effect of carbachol.

1) The basal Cl- conductance in the apical membrane may be increased, so that the carbachol-dependent increase of K+ conductance in the basolateral membrane has a larger effect. However, the observation that the basal electrophysiological parameters did not show increased secretory activity after prolonged exposure to TNF-alpha argues against this possibility.

2) TNF-alpha may increase the number of Cl- channels in the apical membrane so that activation induces a larger conductance. However, activation of the PKA route by forskolin or the PKC route by PDBu did not show potentiation by TNF-alpha . Therefore, this possibility is not likely.

3) TNF-alpha may increase the number of muscarinic receptors. Although we have not tested this possibility directly, the observation that activation by histamine after exposure to TNF-alpha also showed a potentiated secretory response (unpublished observations) indicates that potentiation is in the pathway between receptors and the Cl- channel.

4) Because the carbachol effect is mediated via PKC in HT29cl.19A cells (Ref. 4; presently illustrated by the effect of GF 109203X), it may be that TNF-alpha upregulates PKC so that a larger pool of PKC molecules is available. This possibility should be studied directly. However, because the effect of PDBu was not different in TNF-alpha -exposed cells, this possibility is less likely. Additionally, the observation that GF 109203X inhibited the PDBu effect and the potentiated carbachol effect by the same percentage suggests that the quantitative relation between the blocker and PKC is not changed by TNF-alpha . Therefore we hypothesize that the effect of TNF-alpha is between the receptor and PKC. The absence of an effect of exposure to TNF-alpha on the carbachol response in T84 cells may be in line with this assumption. In these cells, the mechanism leading to increased Cl- secretion is different from the mechanism in HT29cl.19A cells. In T84 cells, the carbachol-induced increase in the Isc is primarily due to activation of basolateral K+ channels that leads to an increased driving force for Cl- efflux through conducting apical Cl- channels (34). This view is corroborated by the present intracellular potential measurements. The resting Va is much lower than in HT29cl.19A cells, suggesting a much larger Cl- conductance. The simultaneous hyperpolarization of the Va and the increase of transepithelial potential and Isc upon carbachol addition indicate activation of basolateral K+ channels as the underlying mechanism for transepithelial current in this cell line. The refractoriness of the carbachol response to the PKC inhibitor GF 109203X confirms the finding that PKC is not involved in the carbachol response in these cells (17). Therefore, the absence of an effect of TNF-alpha on the electrophysiological effect of carbachol in these cells is an argument in favor of the hypothesis.

5) The activation of the muscarinic receptor after exposure to TNF-alpha may lead to increased activation of PI-PLC, leading to larger amounts of the intracellular messengers IP3 and the PKC activator DAG. This possibility should also be studied directly. However, because IP3 leads to an increase in Ca2+ and because Ca2+ in turn leads to the activation of phase 1 of the carbachol response (4), which was not different after TNF-alpha exposure, this possibility seems less likely. Therefore, another pathway to increase DAG may be involved. The PI-PLC inhibitor U-73122 was without effect on phase 1 of the carbachol effect, and therefore it was not possible to use this inhibitor to show the involvement of other phospholipase(s) in the generation of DAG.

6) Carbachol, after TNF-alpha exposure, may lead to increased production of DAG by activation of the PLD pathway. Although not described for epithelia, TNF-alpha can increase the PLD route in a number of cells (7, 9). Direct measurements of DAG levels and the analyses of the involvement of the PLD pathway have to be performed. However, parallel studies confirmed that, in the presence of butanol, carbachol increased the synthesis of phosphatidylbutanol, a specific product of PLD activation. This indicates that muscarinic receptor activation can increase the PLD activity (Oprins et al., unpublished observations). Participation of the PLD pathway in the carbachol response was also indicated by the electrophysiological effect of propranolol, which is considered an inhibitor of conversion of the PLD product PA to DAG by PAP (11). The inhibitory effect of propranolol on PAP in these cells was confirmed by the observation that propranolol increased the [32P]PA level in 32P-exposed cells, as analyzed with TLC (Oprins et al., unpublished observations). Other studies have shown an effect of TNF-alpha on PC-PLC (28, 36, 38). This route forms DAG directly without PA as an intermediate and does not require PAP. We cannot exclude a role for PC-PLC in the (potentiated) carbachol response. However, the large suppression of the carbachol response by propranolol suggests that DAG formation by the PLD route plays an important role in the TNF-alpha -potentiated carbachol response.

7) According to work with T84 cells, activation of PI-PLC can generate an inhibitory inositol derivative, inositol 3,4,5,6-tetrakisphosphate, that may mediate carbachol-induced inhibition of Cl- secretion (33). The presence of this mechanism in HT29cl.19A cells is unknown, and the possibility that TNF-alpha exposure alleviates a similar inhibition by reducing the synthesis of such a compound remains to be studied. However, TNF-alpha was without effect in the T84 cells, and the inhibitory effect of preexposure to carbachol on the histamine response in HT29cl.19A cells was not affected by TNF-alpha (unpublished observations).

8) In human lymphoma cells, TNF-alpha appeared to increase Na+ channels (14). If this were the case in HT29cl.19A cells and if the channels were activated by carbachol, one would not expect to observe an increased response when the driving force for Cl- was increased.

Therefore, the most plausible hypothesis for the effect of TNF-alpha in the potentiation of the effect of muscarinic receptor activation in HT29cl.19A cells is an upregulation of the PLD route leading to increased DAG formation and increased PKC-dependent Cl- conductance.

In another clone of HT-29 cells (HT-29/B6), TNF-alpha appeared to have no direct effect on the secretory status (27). However, as in HT29cl.19A cells, the effect of carbachol was potentiated (J. D. Schulzke, Freie Universitat Berlin, personal communication).

The effects of TNF-alpha on ion secretion in human or animal intestine have not been studied extensively. A difficulty in these studies is that in the presence of so many other cells one cannot be sure of the target for the applied TNF-alpha . For example, in human distal unstripped colon (27) and in porcine ileum (15), TNF-alpha increased the Isc. This effect could be blocked by indomethacin, indicating the release of prostaglandins. As far as we know, no experiments have been reported showing more or less acute effects of TNF-alpha on responsivity to secretagogues in isolated intestine. Pathophysiology gives no clear evidence for effects of TNF-alpha . TNF-alpha is increased in inflammatory intestinal tissue (8, 23), and isolated but unaffected tissue from inflamed intestine appeared hyporesponsive to secretagogues (1). An explanation for this finding has not been given. It may be that in some studies disruption of the tissue or altered morphology plays a role. Alternatively, the hyporesponsivity may be related to downregulation of one or more steps in the pathway of the secretagogues because the tissue is still in a secretory state or has been in this state for a prolonged period. A recent abstract claims that TNF-alpha is not responsible for the secretory dysfunction caused by inflammation (18).

From cocultures of T84 cells and activated immune cells, there is ample evidence that products from immune cells can modify the epithelial response to secretagogues (21, 22, 40). The nature of these products or the mechanism of action is not known. As shown in the present study, T84 cells lack the PKC-dependent carbachol route, and it is feasible that this cell line cannot show the potentiating effect of TNF-alpha .

An interesting question therefore is whether PKC is involved in carbachol secretory responses of small or large intestinal enterocytes. Data concerning this question are scarce, but in rabbit ileum PKC appears to be involved in the response to carbachol (10).

Transepithelial permeability appears to be modified by TNF-alpha directly. This effect occurred at high concentrations (100 ng/ml) in Caco-2 BBE cells (20) and in HT-29/B6 cells (26) and at lower concentrations (10 ng/ml) in HT29cl.19A cells when TNF-alpha exposure was performed in combination with IFN-gamma (24). The cooperative effect of the cytokines may be due to the expression of TNF-alpha receptors triggered by IFN-gamma (25). In our laboratory, coexposure of the cells to TNF-alpha and IFN-gamma made it totally impossible to obtain intracellular recordings, and also high concentrations or longer exposure to TNF-alpha alone decreased the success rate of impalements strongly. We have no explanation for this. It appeared not be due to morphological changes, since electron micrographs of monolayers exposed to TNF-alpha for even 48 h were not different from controls. From the increase of the Rt induced by exposure to TNF-alpha , we can conclude that there is no increased permeability for ions. The large, transient decrease in Rt during the potentiated carbachol response is concomitant with a decrease in fRa and therefore indicates a transcellular change in conductances. We propose that, after exposure to TNF-alpha , carbachol may induce an increased activation of PKC, and numerous other studies in various cell types have implicated a role for DAG-stimulated PKC in the effect of TNF-alpha (reviewed in Ref. 29). Massive stimulation of PKC by phorbol esters with PDBu caused a slowly increasing paracellular permeability for macromolecules in HT29cl.19A cells (12). It remains to be studied whether TNF-alpha in combination with a secretagogue like carbachol can induce a similar increase of permeability and, if so, which isotype of PKC could be involved.

This is the first study that shows a direct potentiation of receptor-activated ion secretion in intestinal epithelial cells by TNF-alpha . It remains to be studied whether the results in HT29cl.19A cells can be translated to the living animal. If so, TNF-alpha could contribute to the diarrhea in patients with IBD, especially when the cells are primed by other cytokines like IFN-gamma to express receptors for TNF-alpha . The potentiation of secretion induced by muscarinic receptor activation (and histamine H1 receptor activation; unpublished observations) suggests that TNF-alpha upregulates a common intermediate in the transduction pathway and underscores its possible role in mast cell responses. It is conceivable that this intermediate step is also involved in other PKC-dependent secretory mechanisms induced by, for example, bacterial toxins (16). Furthermore, our results suggest a place for PLD in the secretory mechanism. These aspects deserve further investigation.


    ACKNOWLEDGEMENTS

We thank Dr. H. R. de Jonge for providing the T84 cells and Greet Scholten for the electron micrographs. We thank Dr. Teun Munnik and Dr. Alan Musgrave for their help with the lipid messenger system and Dr. J.-D. Schulzke for evaluating the TNF-alpha effect in another clone of HT-29 cells.


    FOOTNOTES

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: J. C. J. Oprins, Institute for Neurobiology, Faculty of Biology, University of Amsterdam, Kruislaan 320, 1098 SM Amsterdam, The Netherlands (E-mail: oprins{at}bio.uva.nl).

Received 19 July 1999; accepted in final form 11 October 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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