PLD pathway involved in carbachol-induced Clminus secretion: possible role of TNF-alpha

Judith C. J. Oprins, Claudia van der Burg, Helen P. Meijer, Teun Munnik, and Jack A. Groot

Swammerdam Institute for Life Sciences, University of Amsterdam, 1090 GB Amsterdam, The Netherlands


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In a previous study, it was found that exposure to tumor necrosis factor-alpha (TNF-alpha ) potentiated the electrophysiological response to carbachol in a time-dependent and cycloheximide-sensitive manner. It was deduced that the potentiation could be due to protein kinase C activity because of increased 1,2-diacylglycerol. It was also observed that propranolol could decrease the electrophysiological response to carbachol (Oprins JC, Meijer HP, and Groot JA. Am J Physiol Cell Physiol 278: C463-C472, 2000). The aim of the present study was to investigate whether the phospholipase D (PLD) pathway plays a role in the carbachol response and the potentiating effect of TNF-alpha . The transphosphatidylation reaction in the presence of the primary alcohol 1-butanol [leading to stable phosphatidylbutanol (Pbut) formation] was used to measure activity of PLD. The phosphatidic acid (PA) levels were also measured. Muscarinic stimulation resulted in an increased formation of Pbut and PA. TNF-alpha decreased levels of PA.

intestinal epithelia; protein kinase C; phosphatidic acid; thin-layer chromatography


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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CL- secretion across intestinal epithelium plays an important role in regulating water secretion into intestinal lumen and is under close regulation of hormonal, neuronal, and paracrine mediators. A disturbed regulation of Cl- secretion can result in a change in the water balance and cause pathophysiological situations like diarrhea, in which excessive water secretion occurs. In patients suffering inflammatory bowel diseases (IBD) like Crohn's disease (CD) and ulcerative colitis, severe diarrhea occurs. Little is known about the pathogenesis of this diarrhea. The role of the cytokine tumor necrosis factor-alpha (TNF-alpha ) has drawn increased attention. In patients with CD, enhanced numbers of TNF-alpha -expressing cells and increased concentrations of TNF-alpha have been found (6, 32). Moreover, therapy using antibodies against TNF-alpha reduced the symptoms of diarrhea, suggesting a crucial role for TNF-alpha in the pathogenesis of diarrhea in IBD (38, 45). However, the precise mechanism of action of TNF-alpha on intestinal epithelium is still unknown. Several studies have shown that cytokines are able to alter ion transport and barrier properties of intestinal epithelium, which could contribute to the diarrhea (23, 26, 36). We recently demonstrated that TNF-alpha did not affect the basal Cl- secretion in the intestinal epithelial cell line HT29cl.19A. Also, neither the cAMP-dependent, the Ca2+-dependent, nor the protein kinase C (PKC)-dependent Cl- secretion was affected by TNF-alpha . However, Cl- secretion induced by muscarinic receptor activation by carbachol was potentiated after exposure to TNF-alpha (30). This phenomenon had not been previously described and deserves further investigation. This effect occurred only after at least 2 h of incubation with TNF-alpha and was cycloheximide sensitive.

It has been widely documented that activating the muscarinic receptor with carbachol results in hydrolysis of the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) by the phospholipase C pathway (PLC), generating two signaling molecules: 1,2-diacylglycerol (DAG) and D-myo-inositol 1,4,5-trisphosphate (IP3) (15). IP3 liberates Ca2+ from the stores in the endoplasmic reticulum, thereby increasing the intracellular Ca2+ concentration levels ([Ca2+]i). DAG, with or without Ca2+, can stimulate various members of the PKC family. In the human intestinal epithelial cell line HT29cl.19A, it has been described that carbachol activates the Cl- secretion in these cells via increases of [Ca2+]i and DAG and activation of isotype PKC-alpha (3). We have recently provided additional evidence for the involvement of PKC in the carbachol response in these cells (30).

Increases in DAG, and therefore subsequent activation of PKC, are not solely controlled by the PI-PLC (phosphatidylinositol-specific PLC) pathway. DAG can also be derived from PLC acting on phosphatidylcholine (PC) (13). Additionally, DAG can be generated by phospholipase D (PLD) that hydrolyzes structural lipids like PC to phosphatidic acid (PA). The latter is subsequently broken down by the enzyme PA phosphatase (PAP) to DAG. On the contrary, DAG can be phosphorylated by DAG kinase to return PA. A schematic presentation of the cell signaling pathways involved in muscarinic receptor activation is shown in Fig. 1.


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Fig. 1.   Schematic representation of signaling mechanism involved in muscarinic receptor activation. PC, phosphatidylcholine; PIP2, phosphatidylinositol 4,5-bisphosphate; PI-PLC, phosphatidylinositol-specific phospholipase C; PLD, phospholipase D; DAG, 1,2-diacylglycerol; PA, phosphatidic acid; IP3, D-myo-inositiol 1,4,5-trisphosphate; DGK, DAG kinase; PAP, PA phosphatase; PKC, protein kinase C.

The involvement of PLD in muscarinic receptor-related signaling has been widely studied. Muscarinic receptor-stimulated PLD activity is reported in several cell types, including lacrimal gland acini, neuroblastoma cells, and tracheal smooth muscle cells (24, 33, 47). Recently, we showed that the carbachol-induced Cl- secretion in human intestinal epithelium is diminished by propranolol, a putative inhibitor of PAP (30). Hence, these results suggest a role for the PLD pathway in the carbachol-induced ion secretion in HT29cl.19A cells. Moreover, the potentiating action of TNF-alpha was completely abolished by propranolol. This strongly suggests that TNF-alpha exerts its effect via the PLD pathway, so that more DAG is generated and more PKC is activated, which then generates an increased Cl- conductance and consequent Cl- secretion.

The aim of the present study was to investigate the possible involvement of the PLD pathway in the carbachol response in the colonic epithelial cell line HT29cl.19A and the possible upregulation of this pathway by TNF-alpha . The activity of PLD can be measured by using the unique feature of the enzyme to transphosphatidylate its substrate (46). In the presence of a primary alcohol like 1-butanol, PLD is able to form the stable product phosphatidylbutanol (Pbut) in addition to its physiological product PA. The level of Pbut can be used as a relative measure for the activity of PLD.

Here, we show that muscarinic receptor activation in HT29cl.19A cells results in activation of PLD. PLD could also be activated by phorbol ester, which shows that PLD activation could be downstream of PLC activation. However, in contrast to the phorbol ester-induced PLD activation, the muscarinic receptor-activated PLD could not be blocked by the PKC inhibitor GF-109203X. Furthermore, we show that TNF-alpha affects the PLD pathway by decreasing the level of PA. This could result in an increased formation of DAG and subsequent PKC activation, which may explain the potentiating effect of TNF-alpha on the secretory response to carbachol in these cells.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Cell culture. HT29cl.19A cells were cultured as described previously (2). Briefly, cells (passages 16-32) were grown in DMEM, supplemented with 10% fetal bovine serum, 8 mg/l ampicillin, and 10 mg/l penicillin/streptomycin. They were seeded in 25-cm2 flasks and passaged every week. For experiments, cells were subcultured on 25-mm Falcon filters for 14 days, and medium was replaced every other day. Labeling of the cells with 32Pi and TNF-alpha incubations were performed in culture media in the incubator for the indicated time.

Labeling, stimulation, and lipid extraction. Cells were labeled in culture medium for 24 h with 1.85 × 106 - 3.7 × 106 Bq of carrier-free 32Pi (Amersham International) added to the mucosal side of the cells. Cells were then washed with mannitol Ringer at 37°C to remove the label that was not incorporated. The composition of the Ringer was (in mM) 117.5 NaCl, 5.7 KCl, 25.0 NaHCO3, 1.2 NaH2PO4, 2.5 CaCl2, 1.2 MgSO4, and 28.0 mannitol. Cells were stimulated with carbachol in the presence of 0.05% 1-butanol for 30 min with or without preexposure to different blockers. The stimulation procedure was optimized as described in Time and dose-response curves of 1-butanol stimulation. The reaction was stopped by adding 0.1 M HCl, and lipids were extracted as previously described by Munnik et al. (27). Extracted lipids were separated on heat-activated TLC plates (Merck; 20 × 20 × 0.1 cm) using a modified ethyl acetate solvent system [ethyl acetate/isooctane/formic acid/water (13:2:3:10, by vol)] (28). The levels of [32P]PA and [32P]Pbut were quantified by phosphoimaging (Storm; Molecular Dynamics).

Materials. TNF-alpha , GF-109203X (bisindolylmaleimide I), 4-beta -phorbol 12,13-dibutyrate (PDB), and DAG kinase inhibitors I and II (R59022 and R59949) were from Calbiochem. Propranolol, carbachol, and 1-butanol were purchased from Sigma, Brunschwig, and Brocades, respectively. Silica 60 TLC plates and reagents for lipid extraction were from Merck. [32P]orthophosphate (32Pi; carrier free) was from Amersham International. TNF-alpha , propranolol, and carbachol were dissolved in water. PDB, GF-109203X, R59022, and R59949 were dissolved in DMSO and used at a maximal concentration of the solvent of 0.01% (vol/vol). Maximal concentrations of solvents were without electrophysiological effects on the cells.

Statistics. Data are presented as means ± SE of the means. Statististical significance was calculated, comparing two groups using a paired Student's t-test as indicated in figure legends.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Muscarinic receptor activation with carbachol results in activation of PLD. To establish whether in intestinal epithelial cells (HT29cl.19A cells) the PLD activity is increased after activating the muscarinic receptor with carbachol, we studied the formation of Pbut, which is used as a measure for PLD activity (46). The level of [32P]Pbut was increased after incubating the cells with 100 µM carbachol. Figure 2A shows that application of carbachol resulted in an increased level of [32P]Pbut to 124 ± 6%, compared with controls without carbachol (100%) (P < 0.001; n = 21). The level of [32P]PA is increased to 242 ± 11%, compared with control (P < 0.001; n = 21) (Fig. 2B). These results illustrate that in HT29cl.19A cells, application of a muscarinic receptor agonist activates PLD.


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Fig. 2.   Effect of carbachol (carb) on levels of [32P]phosphatidylbutanol ([32P]Pbut; A) and [32P]phosphatidic acid ([32P]PA; B) in HT29cl.19A cells. Cells were stimulated during 30 min with 100 µM carbachol in the presence of 0.05% 1-butanol. Data are shown as percentage of control monolayers (100%). Basal [32P]PA level is 2.6 ± 0.1% of total labeled phospholipids, and basal [32P]Pbut level is 0.51 ± 0.03% of total labeled phospholipids. Approximately 20% of the total label was incorporated into phosphatidylcholine. The other labeled phospholipids cannot be distinguished on this system. Data are means ± SE of 21 experiments. *P < 0.001.

Time and dose-response curves of 1-butanol stimulation. To determine the time dependence of [32P]Pbut formation, cells were incubated with carbachol plus 1-butanol (0.05% vol/vol) for different time intervals. Figure 3 shows that at time (t) = 30 min, the maximal levels of Pbut and PA are seen. Therefore, in all following experiments, cells were incubated during 30 min with 1-butanol. To determine the concentration of 1-butanol to be used, electrophysiological experiments were performed with concentrations of 1-butanol varying between 0.05% and 0.5%. We found that 1-butanol concentrations >0.05% induced a transient increase in transepithelial potential of 0.6 mV (for 0.15%) and 3 mV (for 0.5%). At 0.5% 1-butanol, the carbachol response was significantly inhibited with 37 ± 4%. The concentration of 0.05% did not show electrophysiological effects and was, therefore, used in these experiments.


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Fig. 3.   Time course of carbachol-induced formation of [32P]Pbut and [32P]PA. Cells were incubated with 100 µM carbachol and 0.05% 1-butanol during time periods varying between 10 and 180 min. The data of [32P]PA and [32P]Pbut induced by carbachol are presented as % of (time) controls without carbachol. Data are means ± SE of 6-8 experiments.

Propranolol increases PA levels. In electrophysiological experiments, we observed that the short-circuit current induced by carbachol could be inhibited 47% by adding 100 µM propranolol (30). This inhibitor was claimed to block PAP activity (39). Figure 4A shows that the presence of propranolol (100 µM) induced an increase in the basal [32P]PA levels (208 ± 9%) and in the carbachol-induced [32P]PA levels (136 ± 12% compared with carbachol alone) (P < 0.05; n = 5). Neither the basal nor the carbachol-induced levels of [32P]Pbut were significantly affected by incubation with propranolol (Fig. 4B). These results confirm that exposure to 100 µM propranolol in these cells inhibits PAP activity.


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Fig. 4.   Effect of propranolol (prop) on basal as well as carbachol-induced levels of 32P[PA] (A) and 32P[Pbut] (B). Cells were incubated with 100 µM propranolol on the mucosal side 30 min before carbachol stimulation. Application of carbachol resulted in significantly increased levels of 32P[PA] and 32P[Pbut] (244 ± 13% and 125 ± 5%). Data are shown as percentage of control experiments (100%). Data are means ± SE of 5 experiments. *P < 0.05.

TNF-alpha affects PA levels. Since electrophysiological experiments revealed that the potentiation of the carbachol response by TNF-alpha could be prevented by propranolol, we proposed that TNF-alpha could exert its effect via the PLD pathway in HT29cl.19A cells (30). To test this, cells were exposed to 10 ng/ml TNF-alpha during the 24-h labeling period. TNF-alpha did not have an effect on the total labeling of the cells [control 5.4 ± 1.4 × 108 arbitrary units (AU) vs. 4.7 ± 1.2 × 108 AU TNF-alpha ; n =42]. After exposure to TNF-alpha , basal levels of [32P]PA were decreased to 87 ± 1% (P < 0.05; n = 6) compared with control levels (100%), and the carbachol-stimulated levels were also attenuated to 87 ± 4% (P < 0.05; n = 6), as shown in Fig. 5A. In Fig. 5B, the effect of TNF-alpha on the level of [32P]Pbut is shown. The basal levels of [32P]Pbut (100%) were not significantly affected after exposure to TNF-alpha (95 ± 5%; n = 6). However, the carbachol-induced levels were slightly but significantly decreased after exposure to TNF-alpha to 92 ± 3%, compared with control (100%) (P < 0.05; n = 6).


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Fig. 5.   Effect of tumor necrosis factor-alpha (TNF-alpha ) on basal and carbachol-induced levels of [32P]PA (A) and [32P]Pbut (B). Cells were exposed to 10 ng/ml TNF-alpha during the 24-h labeling period. Cells were stimulated with 100 µM carbachol. Application of carbachol resulted in significantly increased levels of [32P]PA and [32P]Pbut (226 ± 20% and 119 ± 3%, respectively). Data are shown as percentage changes of control experiments. Data are means ± SE of 7 experiments. *P < 0.05.

Phorbol ester-induced PKC activation increases PLD activation. PKC is known to upregulate PLD activity in many different tissues and cells (14). To establish whether PKC activated by phorbol esters can activate the PLD pathway in these cells as well, we incubated the cells during 30 min with 1 µM of the phorbol ester PDB (4) and measured the formation of [32P]Pbut. Figure 6A shows that the level of [32P]Pbut was increased to 128 ± 6% (P < 0.05; n = 8) and that the PKC inhibitor GF-109203X (1 µM) (41) could inhibit this increased [32P]Pbut formation completely. This suggests that also in these intestinal epithelial cells, stimulating PKC with phorbol ester increases PLD activity.


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Fig. 6.   Effect of the PKC inhibitor GF-109203X (GF) on the levels of [32P]Pbut induced by 4-beta -phorbol 12,13-dibutyrate (PDB; A) and carbachol (B). Cells were incubated with 1 µM PDB (mucosal) or 100 µM carbachol during 30 min with or without 30-min preexposure to GF-109203X (1 µM, bilaterally). Data are shown as percentage of control monolayers (100%). Data are means ± SE of 5-8 experiments. *P < 0.05.

Inhibition of PKC does not affect carbachol-induced PLD activation. We next asked whether in these cells, PKC, which can be activated via carbachol-induced stimulation of PI-PLC, was involved in the carbachol-stimulated PLD activity as well. Before addition of carbachol, cells were incubated for 30 min with 1 µM of the PKC-inhibitor GF-109203X bilaterally. As shown in Fig. 6B, the PKC inhibitor did not show an effect on the basal or on the carbachol-induced levels of [32P]Pbut (n = 7). These results imply that in HT29.cl19A cells, carbachol does not activate PLD downstream of PKC activation.

The source of PA. PA can be formed by DAG kinase from DAG generated by PI-PLC and by PC-PLC. To investigate whether DAG kinase was involved in PA formation, cells were preincubated with 10 µM DAG kinase inhibitor I (R59022) or 5 µM DAG kinase inhibitor II (R59949) 30 min before carbachol addition (9, 10). Neither DAG kinase inhibitor I (293 ± 6% carbachol vs. 285 ± 13% R59022 + carbachol; n = 4) nor DAG kinase inhibitor II (244 ± 13% carbachol vs. 243 ± 8% carbachol + R59949; n = 4) had an effect on the carbachol-induced level of [32P]PA.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In a previous study, we found that exposure to TNF-alpha potentiated the electrophysiological response to carbachol in a time-dependent and cycloheximide-sensitive manner. We deduced that the potentiation could be due to increased PKC activity because of increased DAG (30). We also observed that propranolol could decrease the electrophysiological response to carbachol. The aim of the present study was to investigate whether the PLD pathway plays a role in the carbachol response and the potentiating effect of TNF-alpha .

Therefore, we analyzed the changes in levels of 32P-labeled PA and 32P-labeled Pbut as a consequence of exposure to carbachol and/or TNF-alpha . Although the level of PA can be increased because of a large number of mechanisms, it is claimed that the transphosphatidylation of primary alcohols is a unique feature of PLD (46). The increase of the percentage of [32P]Pbut by carbachol in these cells is, therefore, evidence that muscarinic receptor activation can increase the activity of PLD in these cells. A similar observation has been made in transfected HEK-293 cells (34), submandibular acinar cells (8), thyroid cells (19), and in a number of nonepithelial cells like tracheal smooth muscle cells, neuroblastoma cells, and astrocytes (17, 24, 33), respectively. The increase of the PA level may, therefore, be partly the result of the increased formation through PLD. Another pathway for PA formation is from DAG via DAG kinase. Experiments using inhibitors of DAG kinase did not show an effect on PA levels. This could imply that DAG kinase is not involved in the generation of PA. However, we cannot exclude the possibility that the inhibitors used are not effective in our cell system. Nine isotypes of DAG kinase are now known, and it is unknown which isotypes are present in our cells and whether these are effectively inhibited by the mentioned inhibitors (40). One of the goals of this study was to establish whether propranolol reduced the electrophysiological response to carbachol via inhibition of the conversion of PA to DAG. In the presence of propranolol, PA levels were further increased, which is consistent with the postulated inhibitory role of propranolol on PAP (39). An important observation is that the potentiation of the carbachol-induced secretory response by TNF-alpha is totally prevented by propranolol (30). This indicates that the PLD pathway plays a crucial role in the potentiating effect of TNF-alpha .

We reasoned, from electrophysiological experiments, that exposure to TNF-alpha could augment the response to carbachol by increased formation of DAG. Thus TNF-alpha might have increased the activity of PLD or PAP or decreased the activity of DAG kinase. In the present study, we observed that TNF-alpha decreased basal and carbachol-induced [32P]PA levels. Because Pbut cannot be a substrate for PAP, the observed decrease of Pbut after TNF-alpha and carbachol exposure requires another explanation. It is known that PLD activity can be enhanced by PA and by PIP2, which is formed by the PA-dependent PIP kinase (16, 31). Therefore, when PA level is decreased by exposure to TNF-alpha , PLD may be activated to a lesser extent, which results in a decreased level of [32P]Pbut after TNF-alpha plus carbachol exposure. From these observations, we propose that muscarinic receptor activation and TNF-alpha exposure act synergistically in that they increase the activity of PLD and modulate the level of PA in this cell line. The decrease in PA level may be due to increased activity of PAP or decreased activity of DAG kinase. In this context, it is intriguing that recently the presence of PLD and PAP in specific membrane compartments have been described (37).

As reported for many other cell types (14), we demonstrated that also in this cell line PLD activity can be increased by PDB-induced PKC activation. However, we revealed that carbachol does not activate PLD downstream of PKC activation in HT29cl.19A cells. It may be that this is due to lack of activation of the specific PKC isotype required for PLD activation by carbachol. Another possibility is that the activation of PLD by the muscarinic receptors occurs via another route. A detailed study of this dual activation of PLD by muscarinic receptors and by phorbol ester-activated PKC in HEK-293 cells has given insight into this phenomenon (35, 43). In these cells, phorbol ester-stimulated PLD activity appeared to be dependent on Ral GTPases, whereas muscarinic receptor-mediated PLD activation was dependent on Rho kinase-activated Rho protein and PIP2. Similarly, Mamoon et al. (24) concluded that the PLD stimulation in tracheal smooth muscle by muscarinic receptor activation is not solely due to PKC, and this was also observed in neuroblastoma cells (33). Thus our observation in the intestinal cell line seems to fit in a rather general mechanism where activation of the muscarinic receptor leads to activation of PLD. This may lead to the slow but prolonged increase of DAG, which was described for the first time in astrocytoma cells by Martinson et al. (25). Surprisingly, as far as we know, this pathway has gained no attention in studies of Cl- secretion by intestinal epithelium.

Recently, in a study of protein transport through the apical membrane using the same intestinal cell line as the present study, Auger et al. (1) concluded that carbachol did not activate PLD (and that carbachol-induced increase of PA was solely via the PC-PLC/DAG-kinase pathway). One differences between their methods and ours is that they used a very high concentration of ethanol (3%) as substrate for the transphosphatidylation instead of the 0.05% 1-butanol we used in the present study. Furthermore, they labeled lipids with [3H]myristic acid, a less sensitive method to detect phospholipids than 32Pi labeling, as used in the present study.

To our knowledge, this is the first study that suggests that TNF-alpha might affect PAP activity. Interestingly, it has been recently shown that other cytokines can affect the activity of PAP. In rat hepatocytes, transforming growth factor-beta increases the activity of PAP, probably by increased enzyme synthesis or decreased degradation (12). In human mesangial cells, interleukin-1 rapidly stimulates PAP activity (7). However, the mechanism of the regulation of PAP remains unclear.

Regulation of PAP activity/expression can influence the PA levels and consequently PA/DAG ratios following PLD activation. Although DAG is known to be an important second messenger, and lately, more attention is paid to the role of PA as a second messenger, not much is known about the mechanisms of regulation of PAP yet. Waggoner et al. (44) reviewed that PAP has putative regulation sites on COOH and NH2 termini, but the mechanisms of regulation remain poorly understood.

PLD activity is shown to be present at different sites of the intestinal system. Enterocytes express PLD activity in the mitochondria, and their activity is regulated by nitric oxide, polyamines, monoamines, and Ca2+ (20-22). Furthermore, PLC and PLD signaling have been extensively studied in intestinal smooth muscle cells (29). But as mentioned before, not much is known about the role the PLD pathway can play in the Cl- secretion in intestinal epithelium. To our knowledge, only one study showed that the primary product of the PLD pathway, i.e., PA, although exogenously applied, did modulate the Cl- secretion in another intestinal epithelial cell line, T84 (42).

A role for TNF-alpha in PLD signaling has been proposed earlier, but these studies focus on TNF-alpha as a mediator involved in apoptosis (5, 11, 18). In the HT29cl.19A cells, TNF-alpha in the concentration used (10 ng/ml) does not induce apoptosis (30).

In summary, we have shown that PLD activity is present in human intestinal epithelial cells and that this PLD activity is increased by muscarinic receptor activation and phorbol ester-induced PKC activation leading to augmented PA levels. We propose that the de novo enzyme synthesis-dependent potentiation of the electrophysiological response to carbachol after TNF-alpha exposure may be due to enhanced activity of PAP. The increased PAP activity, acting on the augmented substrate level, may lead to increased DAG-dependent PKC activity and, consequently, to increased Cl- conductivity. The testing of this hypothesis remains to be done.


    FOOTNOTES

Address for reprint requests and other correspondence: J. C. J. Oprins, Swammerdam Institute for Life Sciences, Univ. of Amsterdam, P.O. Box 94084, 1090 GB Amsterdam, The Netherlands (E-mail: oprins{at}bio.uva.nl).

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 19 July 2000; accepted in final form 25 October 2000.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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