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CELLULAR AND MOLECULAR

Role of Phospholipase C- in the Modulation of Epithelial Tight Junction Permeability

Department of Pharmacology, School of Medicine (P.D.W.), Division of Medicinal Chemistry and Natural Products, School of Pharmacy (H.O.), and Division of Drug Delivery and Disposition, School of Pharmacy (D.R.T.), University of North Carolina at Chapel Hill, Chapel Hill, North Carolina

Received August 27, 2002; accepted October 8, 2002.

	Abstract

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The results presented in this study establish an association between phospholipase C- (PLC-) and tight junction permeability across Madin-Darby canine kidney (MDCK) cell monolayers, an in vitro model for epithelial tissue. These results further show that PLC- modulates tight junction permeability by affecting actin filament organization. Hexadecylphosphocholine (HPC) inhibited PLC- and increased tight junction permeability in MDCK cells. Interestingly, the analogs of HPC, a series of alkylphosphocholines containing various lengths of linear alkyl chains, inhibited PLC- and increased tight junction permeability with a wide range of potency. The potency of alkylphosphocholines as enhancers of tight junction permeability significantly correlated (p < 0.05) with their potency as PLC- inhibitors. U73122, a steroid derivative that is structurally unrelated to alkylphosphocholines, inhibited PLC- and increased tight junction permeability with potencies that fit into the correlation observed for the alkylphosphocholine series. U73122 and HPC induced disorganization of actin filaments in MDCK cell monolayers. The potencies to cause disorganization of actin filaments were consistent with the potencies of these agents as inhibitors of PLC- and enhancers of tight junction permeability. Furthermore, ATP, an activator of PLC-, attenuated U73122-induced increase in tight junction permeability as well as disorganization of actin filaments. These results provide strong evidence that PLC- inhibition leads to increased tight junction permeability across MDCK cell monolayers through disorganization of actin filaments.

The tight junction is a complex structure composed of both transmembrane and cytosolic proteins (Denker and Nigam, 1998; Fanning et al., 1999). Many of these proteins (e.g., occludin, ZO-1, ZO-2, and ZO-3) have phosphorylation sites, suggesting that these proteins are possible endpoints of various signal cascades (Anderson et al., 1988; Sakakibara et al., 1997). Currently, many signaling pathways involving tyrosine kinases, calcium, and protein kinase C (PKC) have been implicated in the regulation of tight junction permeability (Anderson and Van Itallie, 1995; Tai et al., 1996; Collares-Buzato et al., 1998; Mullin et al., 1998). The molecular mechanisms associated with this regulation, however, have not been elucidated.

Phospholipase C (PLC), an important regulatory enzyme, catalyzes hydrolysis of phosphatidylinositol-4,5-bisphosphate into inositol triphosphate and diacylglycerol in response to the stimulation of a variety of receptors, e.g., stimulation of P2Y₂ receptors by ATP (Nicholas et al., 1996). Activity of different families of PLC is regulated through different receptor-mediated pathways. For example, the activities of PLC- and PLC- are regulated through G-protein-coupled receptors (e.g., purinergic receptor) and receptor tyrosine kinases (e.g., epidermal growth factor receptor), respectively (Katan, 1998). A PLC-dependent pathway has been implicated in the assembly of the tight junction (Balda et al., 1991; Cereijido et al., 1993a,b; Emori et al., 1994; Thackeray et al., 1998; Wang et al., 1998; Fleming et al., 2000), particularly during development, although evidence for its role in the function of mature tight junctions is ambiguous at best. For example, it has been reported that a medium-chain fatty acid causes increased tight junction permeability via activation of PLC, whereas its close structural analog does not act via PLC (Lindmark et al., 1998). Hexadecylphosphocholine (HPC), an inhibitor of PLC (Pawelczyk and Lowenstein, 1993; Berkovic et al., 1996), has structural features (i.e., a long alkyl chain, and a zwitterionic functionality) that have been previously identified to cause an increase in tight junction permeability (Liu et al., 1999a). HPC was found to be a potent enhancer of tight junction permeability across Madin-Darby canine kidney (MDCK) cell monolayers and a potent inhibitor of PLC in these cells. The studies described here expand on this initial finding and demonstrate that analogs of HPC, containing alkyl chains of different lengths, exhibit a wide range of potencies as 1) inhibitors of the PLC isozyme and 2) enhancers of tight junction permeability. Thus alkylphosphocholines serve as useful mechanistic tools in our efforts to elucidate the relationship between inhibition of PLC- and increase in tight junction permeability across MDCK cell monolayers. Additionally, our results provide evidence that changes in PLC- activity modulates tight junction permeability by affecting changes in organization of actin filament network.

	Materials and Methods

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Reagents. HEPES was obtained from Lineberger Comprehensive Cancer Center (University of North Carolina at Chapel Hill). Hanks' balanced salt solution was obtained from Mediatech (Hernford, VA). Cell culture reagents were obtained from Invitrogen (Carlsbad, CA). Transwell inserts (12 wells/plate, 3-µm pore, and 1.0 cm² area with polycarbonate or polyester membranes) and plates (12-well) were obtained from Costar (Cambridge, MA). [¹⁴C]Mannitol and [³H]myoinositol were obtained from American Radiolabeled Chemicals (St. Louis, MO). Dodecylphosphocholine was obtained from Avanti Polar Lipids (Alabaster, AL). AG1-X8 formate columns were obtained from Bio-Rad (Hercules, CA). Phalloidin conjugated with Texas Red was obtained from Molecular Probes (Eugene, OR). All other compounds and reagents were obtained from Sigma-Aldrich (St. Louis, MO).

Cell Culture. MDCK epithelial cell line strain II was obtained from the American Type Culture Collection (Manassas, VA) through the Lineberger Comprehensive Cancer Center. MDCK cells derived from normal proximal kidney epithelial cells of a cocker spaniel, which serve as a model for epithelial transport experiments (Cho et al., 1989), were grown to confluence on Transwell inserts with polycarbonate membranes to determine the effect of absorption enhancers on TEER and mannitol transport. The cells were grown on Transwell inserts with transparent polyester membranes for confocal studies. Each well was seeded with cells at a density of 100,000 cells/cm². Cells were then grown in cell medium (minimum essential medium containing 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B, and supplemented with 10% fetal bovine serum and 0.1 mM nonessential amino acids) and were maintained at 37°C under 5% CO₂. Cells were grown for 4 days during which they differentiated into epithelial cell monolayers, as evidenced by the establishment of a stable TEER between 150 and 250 •cm² [EVOM Epithelial Tissue Voltohmeter (World Precision Instruments, Sarasota, FL) and an Endohm-12 electrode].

Determination of Tight Junction Permeability. TEER and mannitol flux across MDCK cell monolayers are indicators of tight junction permeability of the cell monolayer (Liu et al., 1999a,b). A decrease in TEER and/or an increase in mannitol flux across MDCK cell monolayers were used as parameters to measure the efficacy of tight junction permeability enhancers.

Determination of the Effect of Alkylphosphocholines and U73122 on TEER. Cell media were aspirated from both apical and basolateral compartments of Transwell inserts and replaced with 0.5 and 1.5 ml, respectively, of Hanks' balanced salt solution, supplemented with 10 mM HEPES, pH 7.4 (transport buffer). MDCK cell monolayers were then incubated at 37°C for 30 min, and the TEER of each cell monolayer was then measured. Experiments were initiated by replacement of transport buffer from the apical compartment of Transwell with transport buffer containing an alkylphosphocholine, U73122, or vehicle (0.5 ml). MDCK cell monolayers were incubated at 37°C, and TEER values were measured after 30 min. Data from each experiment were normalized to the response from the vehicle and were reported as the mean ± S.D. (n = 3). The effect of alkylphosphocholines and U73122 on TEER was evaluated at several concentrations, and their EC₅₀ values, defined as the concentration that caused a 50% decrease in TEER with respect to the untreated control, were determined (Liu et al., 1999a,b). EC₅₀ values were reported as the mean ± S.D. of three experiments, each performed in triplicate.

Determination of the Effect of Alkylphosphocholines and U73122 on Mannitol Transport. Cell media were aspirated from apical and basolateral compartments of Transwell and replaced with transport buffer. MDCK cell monolayers were then incubated at 37°C for 30 min. The integrity of tight junctions of the cell monolayer was monitored by measurement of TEER prior to the experiment (150–250 •cm²). Transport experiments were initiated by replacing the transport buffer in the apical compartment with 0.5 ml of transport buffer containing an alkylphosphocholine, U73122, or vehicle, and [¹⁴C]mannitol (25 µM, 55 mCi/mmol). Transport rates were monitored by quantifying the amount of [¹⁴C]mannitol accumulated (Packard Tri Carb 4000 Series liquid scintillation spectrophotometer) in the basolateral side (1.5 ml) between 30- and 60-min intervals after initiating the treatment. All transport experiments were conducted under sink conditions (less than 10% of the total amount of [¹⁴C]mannitol was present on the basolateral side at any given time). The flux of [¹⁴C]mannitol in the presence of an alkylphosphocholine or U73122 was normalized to that in the vehicle-treated cells and was reported as the mean ± S.D. (n = 3). EC_10x, defined as the concentration of an alkylphosphocholine or U73122 that causes a 10-fold increase in mannitol flux with respect to the vehicle-treated control (Liu et al., 1999a,b), was determined for each enhancer of tight junction permeability, and was reported as the mean ± S.D. of three experiments, each performed in triplicate.

Determination of PLC- Activity by Cellular Assay. The activity of PLC- in MDCK cells, transfected with P2Y₂ receptors, was determined by an adaptation of a previously published method (Schachter et al., 1997). MDCK cells, transfected with P2Y₂ receptors, were seeded at 400,000 cells/cm² and subsequently cultured for 4 days. The cell monolayers were then labeled with [³H]myo-inositol (1.6 µCi/well in 0.4 ml of inositol-free medium) for 24 h at 37°C. An alkylphosphocholine or U73122 was added at different concentrations to the medium, and the cells were incubated for 30 min at 37°C. Assays were initiated by immediately supplementing the cells with 100 µl of 250 mM HEPES (pH 7.3), containing 100 mM LiCl, with ATP (final concentration, 100 µM). The cells were then incubated at 37°C for 15 min to allow accumulation of [³H]inositol phosphates. Incubations were terminated by aspiration of the medium and addition of 1 ml of boiling 10 mM EDTA (pH 8.0). The supernatant was applied to AG1-X8 formate columns for chromatographic isolation of [³H]inositol phosphates (Berridge et al., 1983). The amount of [³H]inositol phosphates was measured by liquid scintillation counting in a Packard Tri Carb 4000 series spectrophotometer. Data from each experiment were normalized to the response observed with 100 µM ATP alone and were reported as the mean ± S.D. (n = 3). IC50(PLC), defined as the concentration of an alkylphosphocholine or U73122 that causes a 50% decrease in ATP-stimulated PLC- activity (accumulation of [³H]inositol phosphates), was determined and reported as the mean ± S.D. of three experiments, each performed in triplicate. The cell viability upon the inhibition treatment was determined by the MTT assay (Mosmann, 1983).

Synthesis of Alkylphosphocholines. Arachidyl 2-(N,N,N-trimethylamino)ethyl phosphate (C20), yield 7.8%; octadecyl 2-(N,N,N-trimethylamino)ethyl phosphate (C18), yield 34.1%; tetradecyl 2-(N,N,N-trimethylamino)ethyl phosphate (C14), yield 54.0%; and decyl 2-(N,N,N-trimethylamino)ethyl phosphate (C10), yield 81.8% (Fig. 1) were synthesized through a two-step reaction from corresponding alcohols by previously published procedures (Hanson et al., 1982; Surles et al., 1993). The final products were characterized by mass spectrometry and nuclear magnetic resonance spectrometry.

View larger version (11K): [in this window] [in a new window]   Fig. 1. Structures of alkylphosphocholines.

Determination of Actin Organization with Phalloidin. Cell media were aspirated from both apical and basolateral compartments of Transwell inserts with transparent polyester membranes and replaced with transport buffer. MDCK cell monolayers were then incubated at 37°C for 30 min, and TEER of each cell monolayer was measured. Transport buffer containing different concentrations of an alkylphosphocholine, U73122, or vehicle was added to the apical side of these cells. MDCK cell monolayers were incubated at 37°C, and TEER values were measured after 30 min. Transport buffer was replaced with phalloidin conjugated with Texas Red (final concentration, 200 nM) in 3.7% formaldehyde (0.2 ml), incubated for 30 min at 4°C, and subsequently washed with transport buffer. Actin filament organization was viewed with Zeiss LSM-410 inverted laser scanning microscope (Carl Zeiss, Oberkochen, Germany), fitted with a 100x objective. Several vertical sections (x–z) of the cell monolayers were taken to define the top and bottom of this cell monolayer. En face sections (x–y) were then selected from the cortical (mid-cell) sections of the cell monolayers.

Data Analysis. Student's t test for unpaired data was used to determine significant differences (p < 0.05) between the mean ± S.D. from untreated and treated MDCK cell monolayers. The relation between PLC- activity and increase in tight junction permeability was examined by linear regression analysis, and the correlation was expressed by the Pearson correlation coefficient (r).

	Results

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Effect of HPC on TEER. When MDCK cell monolayers were treated with HPC (see Fig. 1 for structure) on the apical side, TEER across cell monolayers decreased as a function of time over a 60-min exposure, indicative of increased tight junction permeability. The time course of decrease in TEER was very similar to that observed with dodecylphosphocholine (C12) (Liu et al., 1999a,b). The drop in TEER when measured at a fixed time (i.e., 30 min after initial treatment) was concentration-dependent (Fig. 2). The concentration that decreased TEER by 50% (EC₅₀) was estimated to be 29 ± 5 µM (Table 1). Thus HPC appeared to be a significantly more potent enhancer of paracellular permeability than its homolog C12, which was previously tested as an enhancer of paracellular permeability in a related cell culture model of intestinal epithelium, Caco-2 cell monolayers (EC₅₀ = 650 ± 50 µM) (Liu et al., 1999a).

View larger version (26K): [in this window] [in a new window]   Fig. 2. The effect of HPC on TEER and ATP-stimulated PLC- activity in MDCK cells. HPC was administered apically, and TEER was measured after 30-min incubation with HPC at 37°C. For measurement of PLC- activity, MDCK cells transfected with P2Y2 receptors were labeled with [3H]myo-inositol for 24 h at 37°C. The cells were then treated with HPC for 30 min at 37°C. [3H]Inositol phosphates were isolated by chromatography following treatment of the cells with ATP (100 µM) and quantified by liquid scintillation spectrometry. Data points represent mean ± S.D. (n = 3).

View this table: [in this window] [in a new window]   TABLE 1 Effect of alkylphosphocholines and U73122 on TEER (EC50), mannitol flux (EC10x) and PLC- activity (IC50(PLC)) in MDCK cells

Effect of HPC on PLC- Activity. HPC was reported as an inhibitor of PLC in human leukemia cells (Berkovic et al., 1996). To determine whether the PLC inhibitory activity was related to its effect on TEER, it was necessary to determine whether HPC inhibited PLC activity in MDCK cell monolayers. As shown in Fig. 2, HPC decreased ATP-stimulated PLC activity (i.e., PLC- activity; Nicholas et al., 1996) in P2Y₂-transfected MDCK cells. The concentration of HPC that decreased PLC- activity by 50% [IC_50(PLC)] was estimated to be 18 ± 1 µM (Table 1). It is important to note that HPC, at concentrations that markedly inhibited ATP-stimulated PLC- activity, had no effect on the activity of epidermal growth factor (EGF)-stimulated PLC- activity (Ward et al., 2002).

Effect of Alkylphosphocholines on Tight Junction Permeability across MDCK Cell Monolayers. Previous studies have shown that changes in length of the alkyl chain of glycerophosphocholines markedly affect their potencies to cause an increase in tight junction permeability (Liu et al., 1999a,b; Ouyang et al., 2002). Therefore, analogs of HPC were synthesized with systematic changes (two methylene units at a time) in the alkyl chain (Fig. 1), and their potencies to cause increase in tight junction permeability and inhibition of PLC- activity were assessed. All compounds decreased TEER in a concentration-dependent manner, such that, at maximum concentrations, TEER was decreased to less than 20% of control values (Fig. 3A), and the EC₅₀ values varied markedly (Table 1). Interestingly, small variations in the alkyl chain length of alkylphosphocholines produced significant (p < 0.05) changes in the EC₅₀ values of these compounds. For example, HPC and C12 differed by only four methylene units in their alkyl chain, and yet their respective EC₅₀ values differed by approximately 25-fold. The potency of alkylphosphocholines (EC₅₀) in this series varied over a 100-fold range, and a large drop in potency was observed between C14 and C12 (Table 1).

View larger version (28K): [in this window] [in a new window]   Fig. 3. The effect of alkylphosphocholines and U73122 on TEER (A), mannitol flux (B), and ATP-stimulated PLC- activity (C) in MDCK cells. For TEER measurement the test compound was added to the apical compartment, and TEER was measured after 30-min incubation at 37°C. For measurement of mannitol flux, the test compound and [14C]mannitol were added to the apical compartment, and the amount of [14C]mannitol accumulated in the basolateral side (1.5 ml) during the 30- to 60-min interval after treatment with the test compound or vehicle was measured. PLC- activity was measured in P2Y2 receptor-transfected MDCK cells as described in Fig. 2. Data points represent mean ± S.D. (n = 3). Symbols that represent the compounds are: , C10; , C12; •, C14; +, HPC; -, C18; , C20; and x, U73122.

Alkylphosphocholines increased mannitol flux in a concentration-dependent manner (Fig. 3B), further confirming their ability to increase tight junction permeability. The potency of these compounds to increase tight junction permeability was expressed as EC_10x, defined as the concentration of compound that increased mannitol flux by 10-fold (cf. Liu et al., 1999a,b). As expected, the EC_10x of alkylphosphocholines (Table 1) had a good correspondence with their respective EC₅₀ values. As with EC₅₀, EC_10x values varied over a 100-fold range with a large drop in potency between C14 and C12.

Effect of Alkylphosphocholines on PLC- Activity in MDCK cells and Its Relationship to the Effect on Tight Junction Permeability. To determine whether PLC- inhibition is associated with the ability of alkylphosphocholines to increase tight junction permeability in MDCK cells, the effect of these compounds on ATP-stimulated PLC- activity (increase in inositol phosphate production) was measured as a function of concentration. As shown in Fig. 3C, many of the alkylphosphocholines tested caused inhibition of PLC- activity. The cell viability (>95%) was not compromised, as determined by the MTT assay (Mosmann, 1983), by any of the alkylphosphocholines used in the experiments performed to determine the effect on the enzyme activity as well as on TEER and mannitol transport. The potency of alkylphosphocholines as inhibitors of PLC-, expressed as the concentration that inhibited PLC- activity by 50% (IC50(PLC)), varied markedly (Fig. 3C, Table 1). For example, HPC, a potent enhancer of tight junction permeability, inhibited PLC- with an IC50(PLC) of 18 ± 1 µM; whereas, the IC50(PLC) of C12, a weak enhancer of tight junction permeability, was 275 ± 135 µM (Table 1). The full extent of PLC- inhibition by these compounds also varied. For example, C10, the least potent enhancer of tight junction permeability in this series, is not included in Table 1 because C10 could not reduce ATP-stimulated PLC- activity (increase in inositol phosphate production) by 50% even at the highest concentration tested (5 mM). The full extent of PLC- inhibition from treatment with C12 was 50% at 1 mM. This result is not surprising since C10 and C12 are the least potent enhancers of tight junction permeability in this series (Table 1). The changes in PLC inhibitory activity with changes in the chain length of alkylphosphocholines appears to be due to differences in the binding affinities of these compounds to PLC- and not due to their different membrane permeability. This is because the precipitous drop in the PLC inhibitory activity observed between C14 and C12, and even more so between C12 and C10 (C10 is inactive), and conversely, relatively small drop of PLC inhibitory activity upon changing the chain length by six methylene groups (C14 and C20) (Table 1), is not consistent with the cell membrane permeability being a key determinant of the PLC inhibitory activity of alkylphosphocholines. The relationship between PLC- inhibitory activity [IC_50(PLC)] of alkylphosphocholines and their potency as enhancers of tight junction permeability (EC₅₀ and EC_10x) is depicted in Fig. 4 (also see Table 1). It appears that the potency of alkylphosphocholines is related to their potency to cause a drop in TEER and an increase in mannitol flux.

View larger version (18K): [in this window] [in a new window]   Fig. 4. Correlation between the IC50(PLC) and EC10x values (A) and IC50(PLC) and EC50 values (B) for alkylphosphocholines and U73122. The initial correlation was observed with alkylphosphocholines only [r = 0.901 (EC50), r = 0.969 (EC10x)], and the IC50(PLC), EC10x, and IC50(PLC) values for U73122 (data enclosed in a box) were plugged in subsequently. The correlation coefficients shown in the figure include data for alkylphosphocholines and U73122. Data points represent mean ± S.D. of three experiments. The data in each experiment were obtained in triplicate.

Effect of U73122 (a PLC- Inhibitor) on Tight Junction Permeability across MDCK Cell Monolayers. U73122, a PLC- inhibitor (Bleasdale et al., 1989) that is structurally unrelated to alkylphosphocholines (Fig. 5A), decreased ATP-stimulated PLC- activity and TEER in a concentration-dependent manner: IC50(PLC) and EC₅₀ were estimated to be 5 ± 3 µM and 6 ± 2 µM, respectively (Fig. 5B; Table 1). Thus U73122 appears to be one of the most potent inhibitors of PLC- and enhancers of tight junction permeability identified thus far (Table 1). Interestingly, the relationship between the potencies of U73122 to increase tight junction permeability and inhibit PLC- was the same as that observed for alkylphosphocholines; this was evident by the fact that the IC50(PLC), EC_10x, and EC₅₀ values for U73122 fitted well in the plot of IC50(PLC) versus EC_10x or EC₅₀ for alkylphosphocholines—r values improved from 0.969 to 0.982 (EC_10x) and from 0.901 to 0.947 (EC₅₀) by inclusion of U73122 in the correlation (Fig. 4). U73343, the structural analog of U73122 (Fig. 5A), shown to be inactive as an inhibitor of PLC (Bleasdale et al., 1989), showed no effect on tight junction permeability (Fig. 5B). These results showed that even when PLC- inhibitors are structurally different, the relationship between inhibition of the enzyme and enhancement of the tight junction permeability remains intact. This represents more definitive evidence for a relationship between inhibition of PLC- and enhancement of tight junction permeability. Further confirmation of this hypothesis was obtained from the observation that reversing the inhibition of PLC- could significantly attenuate the enhancement of tight junction permeability by U73122. The reversal of PLC- inhibition was achieved by treatment of the cells with excess ATP (Fig. 6), which can activate PLC- via G protein-coupled receptors (e.g., P2Y₂ receptors) (Katan, 1998).

View larger version (16K): [in this window] [in a new window]   Fig. 5. Structures of the specific PLC- inhibitor, U73122, and its inactive analog, U73343 (A), and their effect on TEER and ATP-stimulated PLC- activity in MDCK cells (B). TEER and PLC- activity were determined as described in Fig. 2. Data points represent mean ± S.D. (n = 3). The solid square symbol (f) represents the effect of 10 µM U73343 on TEER and PLC- activity.

View larger version (16K): [in this window] [in a new window]   Fig. 6. Effect of ATP on U73122-induced decrease in TEER. U73122 (5 µM) with or without ATP (100 µM) was added to the apical compartment. TEER was measured after a 30-min incubation at 37°C. The asterisk denotes a significant difference (p < 0.05) between TEER of MDCK cell monolayers treated with U73122 and with U73122 plus ATP.

Effect of HPC and U73122 on Actin Filament Organization. In searching for the cellular event that accompanied enhanced tight junction permeability caused by inhibitors of PLC- (e.g., HPC and U73122), we observed that the organization of actin was affected by these compounds. The confocal microscopic images of the untreated (control) cells revealed the presence of actin filaments (visualized with phalloidin-conjugated Texas Red) at cortical regions (i.e., mid-cell level) around the cell border (Fig. 7); this cortical actin ring has been previously observed (Hirokawa and Tilney, 1982). HPC and U73122 induced the disorganization of these filaments, as evidenced by the presence of actin aggregates in the cytoplasmic space (Fig. 7). The potency of the effect of HPC and U73122 on actin organization was consistent with their potency as enhancers of tight junction permeability. For example, HPC had no affect on tight junction permeability and actin filament organization at 10 µM but decreased TEER by 60% and induced a marked disorganization of actin filaments in some cells of the monolayer at 30 µM, as evidenced by the presence of actin in the cytoplasm near the cortical region, outlining the nucleus (Fig. 7A). A similar concentration-dependent increase in tight junction permeability and disorganization of actin filaments was caused by U73122 (Fig. 7B). At 8 µM, U73122 induced punctate actin aggregates in the cytoplasm and markedly decreased TEER (Fig. 7B), which was similar to the effect observed by cytochalasin D, a compound that directly disrupts actin cytoskeleton organization (Stevenson and Begg, 1994). It is important to note that organization of the apical actin, unlike that of cortical actin, was unaffected by treatment of cells with relevant concentrations of U73122 and HPC (data not shown). These results suggest that inhibition of PLC- induces an increase in tight junction permeability specifically via disorganization of the cortical actin filaments.

View larger version (63K): [in this window] [in a new window]   Fig. 7. Effect of HPC (A) and U73122 (B) on actin filament organization of MDCK cell monolayers. The cells were treated apically with HPC or U73122 for 30 min at 37°C. TEER was measured before and after treatment. Cell monolayers were then incubated with phalloidin conjugated with Texas Red in 3.7% formaldehyde for 30 min at 4°C and subsequently washed with transport buffer. Actin filament organization was viewed with a Zeiss confocal microscope. The bar denotes 20 µm.

Attenuation of U73122-Mediated Disorganization of Actin Filaments by ATP. Since ATP can attenuate U73122-induced increase in tight junction permeability by reversing the inhibition of PLC- (Fig. 6), its effect on U73122-induced disorganization of actin filaments was assessed. At 5 µM, U73122 decreased TEER by 40% and induced a marked disorganization of actin filaments (Fig. 8). Interestingly, the effect of U73122 on tight junction permeability and organization of actin was markedly diminished after cotreatment with ATP (100 µM) (Fig. 8). ATP alone had a negligible effect on tight junction permeability and organization of actin filaments (Fig. 8).

View larger version (83K): [in this window] [in a new window]   Fig. 8. Attenuation of U73122-mediated disorganization of actin filaments in MDCK cell monolayers by ATP. The cells were treated with U73122 (5 µM) with or without ATP (100 µM) for 30 min at 37°C. TEER was measured before and after treatment. The actin filaments were visualized as described in Fig. 7. The bar denotes 25 µm.

	Discussion

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In MDCK cell monolayers, the PLC-dependent pathway has been implicated in the assembly of the tight junction (Cereijido et al., 1993a). For example, activation of PLC by thyrotropin-1-releasing hormone stimulated tight junction assembly, and its inhibition by neomycin blocked this assembly in MDCK cells (Balda et al., 1991). Diacylglycerol is a product of the reaction catalyzed by PLC. An analog of diacylglycerol, i.e., 1,2-dioctanoylglycerol, stimulated tight junction assembly via activation of the downstream enzyme PKC (Balda et al., 1991). Interestingly, knockout of the gene coding for the PLC isozyme, PLC-3, in mice produced an embryonic lethal mutant in which the blastocoele failed to develop (Wang et al., 1998). Similarly, in Drosophila, PLC- is expressed predominantly in the blastoderm cell (Emori et al., 1994), and this PLC isozyme is involved in the Drosophila development disorder known as small wing (Thackeray et al., 1998). In light of the evidence that the tight junction formation is essential for development (i.e., blastocoele cavitation) (Fleming et al., 2000), these findings may point to the potential importance of PLC in the assembly of tight junctions during development. In contrast, the role of the PLC-dependent pathway in the regulation of the tight junction structure and function in mature epithelial tissues is not clearly understood.

In the present study, we have utilized a homologous series of alkylphosphocholines to produce systematic changes in the activity of the PLC- isozyme and in the tight junction permeability across MDCK cell monolayers. The basal PLC activity in MDCK cells was quite low, presumably comprised of multiple PLC isozymes. PLC- was selectively activated (versus PLC-) by treatment of the cells with ATP. The selective PLC- inhibitor, 3-nitrocoumarin, did not affect ATP-stimulated PLC activity, thus confirming the absence of significant PLC- activity in these cells. As reported previously (Nicholas et al., 1996), ATP activates PLC- via the G-protein-coupled receptor P2Y₂. The separation between the basal levels of [³H]inositol phosphates and those in ATP-stimulated cells was not sufficiently high (>3 fold) to allow measurement of PLC- activity and its inhibition. Thus the effect of these compounds was measured on ATP-stimulated PLC activity in MDCK cells that were transfected with a plasmid encoding the P2Y₂ receptor. The P2Y₂-transfected MDCK cells are identical to the native cells in all respects except for their greater P2Y₂ receptor activity. A relationship (r > 0.90, p < 0.05) was observed between the potency of alkylphosphocholines as inhibitors of ATP-stimulated PLC- and their potency as enhancers of tight junction permeability in MDCK cell monolayers. Such a relationship between the inhibition of PLC- activity and tight junction permeability, observed for a series of homologues, provided more unequivocal evidence for the role of PLC- in the modulation of tight junction permeability than would be possible with the use of a single inhibitor or activator. The use of a homologous series of compounds, instead of a single agent as a mechanistic tool, makes it less likely, although not impossible, to mistakenly infer that a relationship exists between two apparently unrelated events. Furthermore, U73122, a PLC- inhibitor that is structurally unrelated to alkylphosphocholines, inhibited PLC- and enhanced tight junction permeability in MDCK cell monolayers with relative potencies that were entirely consistent with those of alkylphosphocholines. In contrast, U73343, a structural analog of U73122, which is inactive as a PLC inhibitor, was also found to be inactive as an enhancer of tight junction permeability (Fig. 5B). This observation provided further confirmation for the relationship between PLC- activity and tight junction permeability, because the probability is very small that two structurally diverse compounds (or classes of compounds) would affect PLC- activity and tight junction permeability at relevant concentrations through totally unrelated mechanisms. Finally, activating this enzyme with the purinergic receptor agonist ATP significantly attenuated inhibition of PLC- by U73122, with a concurrent reversal of the enhancement of the tight junction permeability. Together, these results construct strong evidence that PLC- activity can functionally affect the tight junction permeability of an epithelial tissue. It is important to note that HPC (alkylphosphocholines) and U73122 are selective inhibitors of PLC- and do not inhibit EGF-stimulated PLC- (Ward et al., 2002), because inhibition of PLC- by 3-nitrocoumarin also leads to enhancement of tight junction permeability (Ward et al., 2002).

Our observation that these PLC- inhibitors caused disorganization of actin filaments at concentrations that enhanced the tight junction permeability suggested that the enhancement in the tight junction permeability is caused by modulation of the tight junction architecture. That ATP attenuated the effect of these agents on actin organization and on PLC- activity, is indicative of a specific mechanism-based association between the two events rather than nonspecific toxic events following inhibition of the enzyme. This is consistent with the previous observation that tight junction permeability can be regulated by actin-containing cytoskeleton (Anderson and Van Itallie, 1995). It is interesting to note that inhibition of PLC- by 3-nitrocoumarin leads to qualitatively different effect on the cortical actin filament in that a punctate staining for actin is observed at intercellular junctions, unlike the presence of actin aggregates in the cytoplasmic space observed upon inhibition of PLC- (Ward et al., 2002).

The actin cytoskeleton associates with the tight junction and plasma membrane, specifically through a network of actin filaments underneath the tight junction and through a cortical ring of actin filaments at the level of the adherens junction (i.e., perijunctional actin myosin ring) (Hirokawa and Tilney, 1982). In guinea pig ileum, disruption of the actin cytoskeleton by drugs, such as cytochalasin D, increased sodium and mannitol flux (Madara, 1986), suggesting the importance of the integrity of the cytoskeleton on the function of junctional complexes. PLC- may alter the organization of cortical actin filaments through regulation of PKC. The involvement of PKC has been implicated in zonula occludens toxin-induced disorganization of actin filaments and increased tight junction permeability (Fasano et al., 1995). Furthermore, the actin binding protein, vinculin, is a target of PKC phosphorylation during junctional assembly induced by calcium (Perez-Moreno et al., 1998). Rho may also be involved in this pathway, since inhibition of rhoA with C3 transferase induced disassembly of the cytoskeletal actin, including the perijunctional actin myosin ring, and increased tight junction permeability (Nusrat et al., 1995). Furthermore, U73122 was found to inhibit Rho activity (Nozu et al., 1999).

The present study provides early insights into how epithelial tight junctions could be regulated via receptor-initiated cellular signaling events. PLC-, a regulatory enzyme, emerges as an important player in the regulation of structure and function of epithelial tight junctions. Clearly, discrete events that mediate changes in the tight junction structure and function following changes in the PLC- activity remain to be elucidated. Such studies will lead to a better understanding of the cellular regulation underlying the barrier function of the epithelial and endothelial tissues.

	Acknowledgements

 
We gratefully acknowledge Sam Wolff and Dr. Rob Nicholas (University of North Carolina Chapel Hill) for donation of transfected P2Y₂ MDCK cells, and Dr. John Lemasters (Confocal Microscopy Core) for the use of the Zeiss confocal microscope. We also thank Dr. Pieter Annaert (Janssen) for helpful discussion.

	Footnotes

 
This work was supported by Pharmaceutical Research and Manufacturers of America (PhRMA) Foundation in the form of a Predoctoral Fellowship to Peter Ward and by GlaxoWellcome (unrestricted gift).

DOI: 10.1124/jpet.102.043638.

ABBREVIATIONS: TEER, transepithelial electrical resistance; PKC, protein kinase C; PLC, phospholipase C; EGF, epidermal growth factor; MDCK, Madin-Darby canine kidney; C10, C12, C14, C18, C20, alkylphosphocholines containing indicated numbers of carbons in the alkyl chain; HPC, hexadecylphosphocholine; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium.

Address correspondence to: Dr. Dhiren R. Thakker, Division of Drug Delivery and Disposition, School of Pharmacy, CB#7360, 303B Beard Hall, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7360. E-mail: dhiren_thakker{at}unc.edu

	References

Top Abstract Materials and Methods Results Discussion References

 

Anderson JM, Stevenson BR, Jesaitis LA, Goodenough DA, and Mooseker MS (1988) Characterization of ZO-1, a protein component of the tight junction from mouse liver and Madin-Darby canine kidney cells. J Cell Biol 106: 1141–1149.[Abstract/Free Full Text]

Anderson JM and Van Itallie CM (1995) Tight junctions and the molecular basis for regulation of paracellular permeability. Am J Physiol 269: G467–G475.

Balda MS, Gonzalez-Mariscal L, Contreras RG, Macias-Silva M, Torres-Marquez ME, Garcia-Sainz JA, and Cereijido M (1991) Assembly and sealing of tight junctions: possible participation of G-proteins, phospholipase C, protein kinase C and calmodulin. J Membr Biol 122: 193–202.[CrossRef][Medline]

Ballard ST, Hunter JH, and Taylor AE (1995) Regulation of tight-junction permeability during nutrient absorption across the intestinal epithelium. Annu Rev Nutr 15: 35–55.[Medline]

Berkovic D, Goeckenjan M, Luders S, Hiddemann W, and Fleer EA (1996) Hexadecylphosphocholine inhibits phosphatidylinositol and phosphatidylcholine phospholipase C in human leukemia cells. J Exp Ther Oncol 1: 302–311.[Medline]

Berridge MJ, Dawson RM, Downes CP, Heslop JP, and Irvine RF (1983) Changes in the levels of inositol phosphates after agonist-dependent hydrolysis of membrane phosphoinositides. Biochem J 212: 473–482.[Medline]

Bleasdale JE, Bundy GL, Bunting S, Fitzpatrick FA, Huff RM, Sun FF, and Pike JE (1989) Inhibition of phospholipase C dependent processes by U-73, 122. Adv Prostaglandin Thromboxane Leukotriene Res 19: 590–593.[Medline]

Cereijido M, Gonzalez-Mariscal L, Contreras RG, Gallardo JM, Garcia-Villegas R, and Valdes J (1993a) The making of a tight junction. J Cell Sci Suppl 17: 127–132.[Medline]

Cho MJ, Thompson DP, Cramer CT, Vidmar TJ, and Scieszka JF (1989) The Madin Darby canine kidney (MDCK) epithelial cell monolayer as a model cellular transport barrier. Pharm Res (NY) 6: 71–77.[CrossRef][Medline]

Collares-Buzato CB, Jepson MA, Simmons NL, and Hirst BH (1998) Increased tyrosine phosphorylation causes redistribution of adherens junction and tight junction proteins and perturbs paracellular barrier function in MDCK epithelia. Eur J Cell Biol 76: 85–92.[Medline]

Denker B and Nigam S (1998) Molecular structure and assembly of the tight junction. Am J Physiol 274: F1–F9.

Diamond JM (1977) The epithelial junction: bridge, gate and fence. Physiologist 20: 10–18.[Medline]

Emori Y, Sugaya R, Akimaru H, Higashijima S, Shishido E, Saigo K, and Homma Y (1994) Drosophila phospholipase C-gamma expressed predominantly in blastoderm cells at cellularization and in endodermal cells during later embryonic stages. J Biol Chem 269: 19474–19479.[Abstract/Free Full Text]

Fanning A, Mitic L, and Anderson J (1999) Transmembrane proteins in the tight junction barrier. J Am Soc Nephrol 10: 1337–1345.[Abstract/Free Full Text]

Fasano A, Fiorentini C, Donelli G, Uzzau S, Kaper JB, Margaretten K, Ding X, Guandalini S, Comstock L, and Goldblum SE (1995) Zonula occludens toxin modulates tight junctions through protein kinase C-dependent actin reorganization, in vitro. J Clin Investig 96: 710–720.

Fleming TP, Papenbrock T, Fesenko I, Hausen P, and Sheth B (2000) Assembly of tight junctions during early vertebrate development. Semin Cell Dev Biol 11: 291–299.[CrossRef][Medline]

Hanson W, Murari R, Wedmid Y, and Baumann W (1982) An improved procedure for the synthesis of choline phospholipids via 2-bromoethyl dichlorophosphate. Lipids 17: 453–459.[CrossRef]

Hirokawa N and Tilney LG (1982) Interactions between actin filaments and between actin filaments and membranes in quick-frozen and deeply etched hair cells of the chick ear. J Cell Biol 95: 249–261.[Abstract/Free Full Text]

Katan M (1998) Families of phosphoinositide-specific phospholipase C: structure and function. Biochim Biophys Acta 1436: 5–17.[Medline]

Lindmark T, Kimura Y, and Artursson P (1998) Absorption enhancement through intracellular regulation of tight junction permeability by medium chain fatty acids in Caco-2 cells. J Pharmacol Exp Ther 284: 362–369.[Abstract/Free Full Text]

Liu DZ, LeCluyse EL, and Thakker DR (1999a) Dodecylphosphocholine-mediated enhancement of paracellular permeability and cytotoxicity in Caco-2 cell monolayers. J Pharm Sci 88: 1161–1168.[CrossRef][Medline]

Liu DZ, Morris-Natschke SL, Kucera LS, Ishaq KS, and Thakker DR (1999b) Structure-activity relationships for enhancement of paracellular permeability by 2-alkoxy-3-alkylamidopropylphosphocholines across Caco-2 cell monolayers. J Pharm Sci 88: 1169–1174.[CrossRef][Medline]

Madara JL (1986) Effect of cytochalasin D on occluding junctions of intestinal absorptive cells: further evidence that the cytoskeleton may influence paracellular permeability and junctional charge selectivity. J Cell Biol 102: 2125–2136.[Abstract/Free Full Text]

Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65: 55–63.[CrossRef][Medline]

Mullin JM, Kampherstein JA, Laughlin KV, Clarkin CE, Miller RD, Szallasi Z, Kachar B, Soler AP, and Rosson D (1998) Overexpression of protein kinase C-delta increases tight junction permeability in LLC-PK1 epithelia. Am J Physiol 275: C544–C554.

Nicholas RA, Watt WC, Lazarowski ER, Li Q, and Harden K (1996) Uridine nucleotide selectivity of three phospholipase C-activating P2 receptors: identification of a UDP-selective, a UTP-selective and an ATP- and UTP-specific receptor. Mol Pharmacol 50: 224–229.[Abstract]

Nozu F, Tsunoda Y, Ibitayo AI, Bitar KN, and Owyang C (1999) Involvement of Rho and its interaction with protein kinase C and Src in CCK-stimulated pancreatic acini. Am J Physiol 276: G915-G923.

Nusrat A, Giry M, Turner JR, Colgan SP, Parkos CA, Carnes D, Lemichez E, Boquet P, and Madara JL (1995) Rho protein regulates tight junctions and perijunctional actin organization in polarized epithelia. Proc Natl Acad Sci USA 92: 10629–10633.[Abstract/Free Full Text]

Ouyang H, Morris-Natschke SL, Ishaq KS, Ward P, Liu D, Leonard S, and Thakker DR (2002) Structure-activity relationship for enhancement of paracellular permeability across Caco-2 cell monolayers by 3-alkylamido-2-alkoxypropylphosphocholines. J Med Chem 45: 2857–2866.[CrossRef][Medline]

Pawelczyk T and Lowenstein JM (1993) Inhibition of phospholipase C delta by hexadecylphosphorylcholine and lysophospholipids with antitumor activity. Biochem Pharmacol 45: 493–497.[CrossRef][Medline]

Perez-Moreno M, Avila A, Islas S, Sanchez S, and Gonzalez-Mariscal L (1998) Vinculin but not alpha-actinin is a target of PKC phosphorylation during junctional assembly induced by calcium. J Cell Sci 111: 3563–3571.[Abstract]

Sakakibara A, Furuse M, Saitou M, Ando-Akatsuka Y, and Tsukita S (1997) Possible involvement of phosphorylation of occludin in tight junction formation. J Cell Biol 137: 1393–1401.[Abstract/Free Full Text]

Schachter JB, Sromek SM, Nicholas RA, and Harden TKN (1997) HEK293 human embryonic kidney cells endogenously express the P2Y1 and P2Y2 receptors. Neuropharmacology 36: 1181–1187.[CrossRef][Medline]

Schneeberger EE and Lynch RD (1992) Structure, function and regulation of cellular tight junctions. Am J Physiol 262: L647–L661.

Stevenson BR and Begg DA (1994) Concentration-dependent effects of cytochalasin D on tight junctions and actin filaments in MDCK epithelial cells. J Cell Sci 107: 367–375.[Abstract]

Surles JR, Morris-Natschke S, Marx MH, and Piantadosi C (1993) Multigram synthesis of 1-alkylamido phospholipids. Lipids 28: 55–57.[CrossRef][Medline]

Tai YH, Flick J, Levine SA, Madara JL, Sharp GW, and Donowitz M (1996) Regulation of tight junction resistance in T84 monolayers by elevation in intracellular Ca2+: a protein kinase C effect. J Membr Biol 149: 71–79.[CrossRef][Medline]

Thackeray JR, Gaines PC, Ebert P, and Carlson JR (1998) small wing encodes a phospholipase C-(gamma) that acts as a negative regulator of R7 development in Drosophila. Development 125: 5033–5042.[Abstract]

Wang S, Gebre-Medhin S, Betsholtz C, Stalberg P, Zhou Y, Larsson C, Weber G, Feinstein R, Oberg K, Gobl A, and Skogseid B (1998) Targeted disruption of the mouse phospholipase C beta3 gene results in early embryonic lethality. FEBS Lett 441: 261–265.[CrossRef][Medline]

Ward PD, Klein RK, Troutman MD, Desai S, and Thakker DR (2002) Phospholipase C-gamma modulates tight junction permeability through hyperphosphorylation of tight junction proteins. J Biol Chem 277: 35760–35765.[Abstract/Free Full Text]

Ward PD, Tippin TK, and Thakker DR (2000) Enhancing paracellular permeability by modulating epithelial tight junctions. Pharm. Sci. Technol. Today 3: 346–358.[CrossRef][Medline]

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