Gastrointestinal mucus, the viscous gel that covers most of the mucosal surfaces of the gastrointestinal tract, derives its ability to protect epithelial surfaces from its major gelforming glycoprotein components called mucins. Mucin secretion is a unique function of columnar epithelial cells, and is not present in other cell types such as squamous or transitional epithelia. Columnar epithelial cells, including specialized goblet cells, respond to multiple stimuli and possess a variety of receptors coupled to an assortment of intracellular transduction pathways. Most of the information available today on mucin secretion by the intestine has come from studies using immortalized mucin-secreting cell lines derived from colonic adenocarcinomas. A rise in intracellular Ca2+ has been reported to trigger mucin secretion. This has been demonstrated by stimulation with Ca2+ ionophores in the colonic adenocarcinoma cell lines T84 (Marcon et al., 1990; McCool et al., 1990) and the HT29-18N2 clone (Lencer et al., 1990). Also protein kinase C (PKC) appears to function in mucin secretion. Diacylglycerol (DAG) and the phorbol ester PMA stimulate mucin secretion by T84 (McCool et al., 1990) and HT29-18N2 cells (Philips, 1992). When T84 cells are stimulated by maximally effective concentrations of Ca2+ ionophore, increased mucin secretion occurs when PMA is added, which suggests that Ca2+ and PKC activate complementary but separate portions of the exocytotic response (McCool et al., 1990).
Recently, cultured dog gallbladder epithelial cells were used to study the cellular response in mucin secretion to a number of putative secretagogues, as well as to determine the effective signal transduction pathways mediated by these secretagogues (Oda et al., 1991; Kuver et al., 1994). Intracellular cAMP increased in response to Prostaglandin E1, Prostaglandin E2, vasoactive intestinal peptide, epinephrine, and isoproterenol. Assay of mucin secretion showed that the same secretagogues led to an increase in mucin secretion (Kuver et al., 1994).
More recently, we demonstrated that bile salts play an important role in the regulation of mucin secretion by these cultured dog gallbladder epithelial cells (Klinkspoor et al., 1995). Bile salts were shown to have a dose-dependent stimulatory effect on mucin secretion. Hydrophobic bile salts were found to be more potent stimulators of mucin secretion than hydrophilic bile salts. Moreover, the stimulatory effect of bile salts on mucin secretion could not be ascribed to a cytolytic effect of the bile salts. Bile salts were shown to stimulate mucin secretion, without causing a concomitant increase in intracellular cAMP or Ca2+ levels in these cells. Phorbol esters, such as PMA, were found to be as effective as bile salts in causing mucin hypersecretion by the gallbladder cells. However, our studies provided no evidence that bile salts act on mucin secretion by means of a direct activation of protein kinase C (Klinkspoor et al., 1996).
Figure 1. Effect of tauroursodeoxycholic acid on mucin secretion by LS174T cells. LS174T cells were grown to confluence and incubated either with serum-free media (control) or solutions of the bile salts tauroursodeoxycholic acid (TUDC) in media for 4 h. TUDC concentrations ranged from 0.625 to 10 mM. Mucin secretion was then measured as described in the Materials and methods section. Values are mean of duplicate wells.
We suggest that stimulation of mucin secretion by bile salts might be a common mechanism by which gastrointestinal epithelia protect themselves against the detergent action of bile salts. Therefore, in this study we have systematically investigated whether mucin secretion is also regulated by bile salts in human colonic epithelium, using the previously described human colon adenocarcinoma cell line LS174T. The colonic epithelial cell line LS180 was first described by Tom et al. (Tom et al., 1976) and originated from an adenocarcinoma of the colon. The LS174T cell line was produced by trypsinization of the parent cell line LS180. Mucin synthesis by the LS174T cell line has been well established (Hand et al., 1985; Johnson et al., 1986; Kuan et al., 1987; Byrd et al., 1988). Here we have studied the effect of both unconjugated and conjugated bile salts on mucin secretion by the cultured LS174T colon cells. Also the possible involvement of protein kinase C in the regulation of mucin secretion was examined.
Table I.
TUDC (mM) | Mucin in media (% control) | Mucin in cells (% control) | LDH in media (U/mg protein) |
0 | 100 (100-100) (13,000 d.p.m./mg protein) |
100 (97-103) (145,000 d.p.m./mg protein) |
1.43 (1.41-1.45) |
10 | 250 (233-267) | 81 (77-85) | 1.47 (1.32-1.62) |
5 | 212 (210-215) | 76 (74-78) | 1.25 (1.11-1.38) |
2.5 | 174 (169-179) | 80 (79-80) | 0.83 (0.81-0.84) |
1.25 | 120 (120-121) | 85 (85-85) | 0.78 (0.59-0.96) |
0.625 | 114 (105-122) | 90 (81-100) | 0.65 (0.61-0.69) |
Effect of tauroursodeoxycholic acid on mucin secretion by LS174T cells
In our previous studies with cultured dog gallbladder epithelial cells we found that, due to its low toxicity, tauroursodeoxycholic acid (TUDC) was the bile salt of choice to characterize the effects of bile salts on mucin secretion (Klinkspoor et al., 1995). Although unconjugated bile salts are more prevalent in the colon than taurine conjugated bile salts (Ostrow, 1993), we therefore decided to first study the effect of the addition of tauroursodeoxycholic acid to the culture medium of LS174T cells. After labeling with [3H]GlcNAc the cells were incubated for 4 h with TUDC in concentrations ranging from 0.625 to 10 mM. Incubation with tauroursodeoxycholic acid caused a dose-dependent increase in mucin secretion by the LS174T cells (Table I). At a concentration of 10 mM of tauroursodeoxycholate, a maximum increase in mucin secretion to 250% of control (100%) was obtained; at higher concentrations of TUDC no further increase in mucin secretion was observed (Figure
Figure 2. Effect of tauroursodeoxycholic acid on mucin secretion by LS174T cells: Sepharose CL-4B column chromatography. LS174T cells were grown to confluence and labeled with [3H]GlcNAc. The cells were incubated either in serum-free media (control) or with 5 mM tauroursodeoxycholic acid (TUDC) in media for 4 h. Culture media and cells were harvested and treated with TCA/PTA as described in the methods section. The precipitates were subjected to gel filtration on a Sepharose CL-4B column. Collected fractions were subjected to scintillation counting and results were expressed as d.p.m./mg cell protein per fraction. To calculate the amount of radioactive glycoprotein secreted by the cells, the radioactivity in the media fractions was expressed as a percentage of the radioactivity in the corresponding cell fractions. Values are mean and standard deviation of triplicate wells. The experiment was performed twice with similar results.
Secretion of mucin, a high molecular weight glycoprotein, was confirmed by measuring the amount of [3H]GlcNAc labeled glycoproteins excluded on Sepharose CL-4B. Cells were incubated for 4 h with serum-free media or media containing 5 mM of TUDC. After the incubations the media and cells were harvested and precipitated with TCA/PTA. Media and cell precipitates were analyzed by gel filtration, and the amount of radioactivity in the fractions was expressed as d.p.m./mg cell protein. The radioactivity in the media fractions was then expressed as a percentage of the radioactivity measured in the corresponding cell fractions, to calculate the relative amounts of glycoprotein secreted by the cells treated with control media and cells treated with tauroursodeoxycholate (Figure
Next, we investigated the effect of TUDC on mucin secretion in time. LS174T cells were incubated for 24 h either in serum free media as a control or in media containing 5 mM of TUDC. After several time intervals the amount of mucin secreted into the culture medium was measured (Figure
Figure 3. Effect of tauroursodeoxycholic acid on mucin secretion by LS174T cells. LS174T cells were grown to confluence and incubated either with serum-free media (control) or a solution of 5 mM of tauroursodeoxycholic acid (TUDC) in media for 24 h. Mucin secretion was then measured at different time intervals as described in the methods section. Values are mean of duplicate wells.
We also studied whether bile salts need to be continuously present to exert their stimulatory effect on mucin secretion. After labeling with [3H]GlcNAc and washing, the LS174T cells were first preincubated for 15 min with 5 mM of TUDC, then the cells were washed with serum free culture media and finally incubated for another 4 h in the absence of bile salt in serum free media. Control cells were either incubated for 4 h in normal culture media without FCS or in media containing 5 mM of TUDC, without preincubation. After 4 h of incubation no stimulation of mucin secretion could be demonstrated in the cells preincubated with the bile salt (85% versus control 100%), in contrast to the cells that were incubated in the presence of the bile salt (238%). On the contrary, cells preincubated with the bile salt showed a small decrease in mucin secretion when compared to the control cells, this is probably due to loss of labeled glycoproteins during the 15 min of preincubation with the bile salt. We therefore conclude that bile salts need to be in constant contact with the cells to have a stimulatory effect on mucin secretion.
Effect of taurochenodeoxycholic acid on mucin secretion by LS174T cells
To test whether a more hydrophobic bile salt is more effective in stimulating mucin secretion by the LS174T cells, we examined the effect of the bile salt taurochenodeoxycholic acid (TCDC). TCDC, because of its toxicity, was used in much lower concentrations of 0.1-2.0 mM. The results of this experiment are shown in Table II. Again the bile salt caused a dose-dependent increase in mucin secretion. Compared with tauroursodeoxycholic acid, taurochenodeoxycholic acid was a much more potent stimulator of mucin secretion. At the concentrations used, no cytotoxic effect was observed. Concentrations of TCDC above 2 mM did cause cell damage (results not shown).
Table II.
TCDC (mM) | Mucin in media (% control) | Mucin in cells (% control) | LDH in media (U/mg protein) |
0 | 100 (98-102) (12,000 d.p.m./mg protein) |
100 (99-101) (129,000 d.p.m./mg protein) |
1.69 (1.67-1.70) |
2 | 471 (468-474) | 87 (87-88) | 1.06 (1.03-1.08) |
1.5 | 266 (246-287) | 93 (82-103) | 0.65 (0.58-0.73) |
1 | 174 (150-198) | 87 (85-90) | 0.31 (0.30-0.31) |
0.5 | 150 (149-151) | 96 (94-98) | 0.35 (0.26-0.44) |
0.1 | 131 (121-142) | 107 (102-112) | 0.42 (0.41-0.43) |
Figure 4. Effect of unconjugated bile salts on mucin secretion by LS174T cells. LS174T cells were grown to confluence and incubated either with serum-free media or solutions of the unconjugated bile salts ursodeoxycholic acid (UDCA), cholic acid (CA) and deoxycholic acid (DCA) in media for 4 h. Mucin secretion was then measured as described in the methods section. Values are mean of duplicate wells.
Effect of unconjugated bile salts on mucin secretion by LS174T cells
Since the bile salts found in the colon are mostly unconjugated bile salts (Ostrow, 1993), we also examined the effect of unconjugated bile salts on mucin secretion by the LS174T cells. After labeling with [3H]GlcNAc and washing with PBS and serum free media, as described in the Materials and methods section, the cells were incubated for 4 h with three different unconjugated bile salts. The most hydrophilic bile salt used, ursodeoxycholic acid (UDCA), was added to the culture media in concentrations ranging from 0.313 to 2.5 mM. Cholic acid (CA) concentrations ranged from 0.125 to 2 mM. Deoxycholic acid (DCA) was added to the culture media in concentrations ranging from 0.063 to 0.5 mM. After the incubations the culture media was harvested and mucin secretion measured as described above. The results of the experiment are shown in Figure
Table III.
UDCA (mM) (Table IIIA) |
Mucin in media (% control) | Mucin in cells (% control) | LDH in media (U/mg protein) |
0 | 100 (94-106) (10,000 d.p.m./mg protein) |
100 (92-108) (123,000 d.p.m./mg protein) |
1.43 (1.41-1.45) |
2.5 | 223 (192-225) | 50 (48-51) | 5.80 (5.37-6.22) |
1.25 | 142 (132-152) | 86 (81-90) | 1.16 (0.98-1.33) |
0.625 | 123 (118-129) | 85 (79-91) | 0.91 (0.77-1.04) |
0.3125 | 120 (120-120) | 91 (87-94) | 0.94 (0.94-0.94) |
CA (mM) (Table IIIB) |
|||
0 | 100 (99-101) (4700 d.p.m/mg protein) |
100 (99-101) (76,000 d.p.m/mg protein) |
1.30 (1.23-1.37) |
2 | 289 (280-297) | 72 (71-74) | 0.17 (0.17-0.18) |
1 | 188 (173-202) | 81 (77-86) | 0.14 (0.14-0.15) |
0.5 | 139 (128-151) | 88 (81-96) | 0.18 (0.17-0.20) |
0.25 | 124 (122-126) | 92 (90-94) | 0.24 (0.20-0.28) |
0.125 | 114 (107-120) | 91 (88-95) | 0.35 (0.33-0.37) |
DCA (mM) (Table IIIC) |
|||
0 | 100 (88-112) (3900 d.p.m./mg protein) |
100 (99-101) (76,000 d.p.m./mg protein) |
1.24 (1.18-1.30) |
0.5 | 428 (401-455) | 72 (61-84) | 2.15 (2.12-2.18) |
0.25 | 339 (333-345) | 78 (77-79) | 1.18 (1.17-1.20) |
0.125 | 186 (168-203) | 89 (86-92) | 0.57 (0.55-0.58) |
0.0625 | 101 (80-123) | 80 (66-94) | 0.58 (0.54-0.61) |
It is also interesting to note that taurine conjugated bile salts were inferior stimulants of mucin secretion compared to unconjugated bile salts, taurine conjugated bile salts being more hydrophilic than the corresponding unconjugated bile salts. Whereas 1.25 mM of ursodeoxycholic acid caused an increase in mucin secretion to 142% of control (Table IIIA), 1.25 mM of tauroursodeoxycholic acid only increases mucin secretion to 120% of control (Table I).
Effect of protein kinase C activators on mucin secretion by LS174T cells
Bile salts have been reported to affect protein kinase C (PKC) activity. We investigated the effect of activators of PKC on mucin secretion by the LS174T cells. The phorbol ester PMA has been reported to specifically activate calcium dependent protein kinase C isoenzymes. The phorbol ester was dissolved in serum-free media in concentrations varying from 10 ng/ml to 1 µg/ml and added to the culture plates containing the LS174T cells. After an incubation for 4 h with either control media or the PKC activator, only a very small stimulatory effect of PMA on mucin secretion could be demonstrated. No increase in LDH leakage from the cells was observed (Table IV). Another phorbol ester PDB, when added to the cells in the same concentrations as PMA, also had only a minor effect on mucin secretion by the LS174T cells (Table IV).
Table IV.
PMA (µg/ml) | Mucin in media (% control) | Mucin in cells (% control) | LDH in media (U/mg protein) |
0 | 100 (97-103) (6500 d.p.m./mg protein) |
100 (98-102) (97,000 d.p.m/mg protein) |
1.53 (1.44-1.62) |
1 | 125 (113-136) | 98 (94-102) | 1.71 (1.52-1.89) |
0.1 | 97 (88-107) | 91 (81-101) | 1.46 (1.35-1.89) |
0.01 | 92 (91-94) | 89 (87-91) | 1.61 (1.52-1.71) |
PDB (µg/ml) | |||
0 | 100 (79-121) (6500 d.p.m./mg protein) |
100 (75-125) (97,000 d.p.m./mg protein) |
1.77 (1.54-2.00) |
1 | 176 (159-193) | 98 (92-105) | 1.54 (1.46-1.62) |
0.1 | 146 (140-151) | 103 (95-111) | 1.50 (1.39-1.61) |
0.01 | 104 (81-151) | 96 (75-118) | 1.41 (1.21-1.62) |
These studies show that bile salts are stimulators of mucin secretion in cultured human colon epithelial cells. Mucin secretion by the LS174T cells was demonstrated to be increased by bile salts in a dose-dependent fashion. Bile salts were found to stimulate mucin secretion at concentrations below their critical micellar concentration, suggesting that it is not the detergent action of bile salt micelles that is responsible for the increased mucin secretion. The stimulatory effect of the bile salts depended on their hydrophobicity. Hydrophobic bile salts were found to be more potent than hydrophilic bile salts. Therefore, unconjugated bile salts were more effective than the corresponding taurine conjugated bile salts in stimulating mucin secretion. These results are compatible with our findings reported previously, in cultured dog gallbladder epithelial cells similar phenomena were observed (Klinkspoor et al., 1995). Recently, Hata et al. (Hata et al., 1994) reported bile salt induced stimulation of mucus glycoprotein secretion by cultured rabbit gastric mucosal cells. Low doses of dihydroxy bile salts were found to stimulate mucus glycoprotein release from these cells, the stimulatory effect was not found to be attributable to cell damage. In another study it was demonstrated that chenodeoxycholate produces a dose-related output of mucin, water and sodium, and DNA in the rabbit colon. However, the increase in mucin secretion was ascribed to mucosal damage resulting from the detergent effect of the bile salts on the mucosa (Camilleri et al., 1980). More recently, Shekels et al. investigated the effect of bile salts on two different human colon cancer cell lines, Caco-2 and HT29 cells. They reported that short term incubations (30 min) with either unconjugated bile acids or taurodeoxycholic acid resulted in increased mucin release relative to bile acid hydrophobicity. Longer incubations were found to be cytotoxic. Tauroursodeoxycholic acid and ursodeoxycholic acid did not alter mucin levels. Therefore, they concluded that the induced mucin release was primarily due to cytotoxicity. (Shekels et al., 1996). However, somewhat contradictory to our results, together these studies suggest that regulation of mucin secretion by bile salts might be a common mechanism, by which the different types of columnar epithelia protect themselves against the toxic action of bile salts to which they are exposed throughout the gastrointestinal tract.
Interestingly, stimulation of mucin secretion by the LS174T cells occurred at much lower bile salt concentrations than in the experiments with the cultured gallbladder epithelial cells. For example, whereas in the colon cells a maximum stimulation of mucin secretion was obtained at a concentration of 10 mM of tauroursodeoxycholic acid, maximum stimulation of the gallbladder cells was reached at 40 mM of TUDC. In the colon cells cytotoxicity also occurred at lower bile salt concentrations. Such an attenuated response makes sense, since in vivo the colon is exposed to much lower bile salt concentrations (0.1-2 mM) than the gallbladder (Ostrow, 1993). This suggests that the extent to which the different epithelia respond to bile salts seems to be adapted to the actual bile salt concentrations to which they are normally exposed. How this differential response to bile salts by these two cell types is achieved has not been investigated in this study.
Another surprising finding was that all the bile salts used, when added to the cells in low concentrations, caused a decrease in LDH release from the cells, when compared to control, whereas when added to the cells in high concentrations they caused an increase in LDH release, due to their cytotoxicity. We have tested the effect of bile salts on the LDH assay and no inhibitory effect of bile salts on LDH activity could be measured. In our experiments with the dog gallbladder epithelial cells, using the same assay under similar circumstances, no decrease in the release of LDH from the cells was observed after the addition of bile salts. Perhaps bile salts, like other toxic compounds, exert a proliferative or cytoprotective effect on the cells, when added in low concentrations, resulting in increased viability of the cells.
Bile salts have been reported to affect protein kinase C (PKC) activity. For example, Huang et al. (Huang et al., 1992) showed that deoxycholic acid triggered the activation of protein kinase C under physiologic conditions. Therefore, they suggested that direct or indirect activation of PKC by bile salts may account for the tumor promoting effects of bile acids in colorectal cancer. Both primary and secondary bile salts were found to be potent and selective activators of partially purified protein kinase C isoenzymes from normal colorectal mucosa (Pongracz et al., 1995).
An activation of PKC has been demonstrated to result in a stimulation of mucin secretion in T84 (McCool et al., 1990) and HT29-18N2 (Philips, 1992) cells. Recently, it was reported by Hong et al. (Hong et al., 1996) that, in both T84 and HT29 cells, PMA increases the expression of two mucin genes on chromosome 11p15, MUC2 and MUC5AC, suggesting that both genes are coordinately regulated through a common phorbol ester (PKC) response element. In contrast, MUC6 expression was not found to be responsive to PMA. Niv et al. (Niv et al., 1992) demonstrated, using cDNA probes for two distinct human intestinal mucins, that the LS174T cells express mRNA for both MUC2 and MUC3 mucin. More recently it was demonstrated by Van Klinken et al. that LS174T cells synthesize and secrete the goblet cell specific mature MUC2 mucin, as well as human gallbladder mucin (van Klinken et al., 1996). However, activation of PKC by the phorbol ester PMA, resulted in only a minor increase in mucin secretion by the LS174T cells. Differences in mucin gene expression might also explain the observations by Shekels et al. (Shekels et al., 1996), since Caco-2 and HT29 cells were observed to contain high levels of MUC3 mRNA, but neither cell type showed detectable expression of intestinal MUC2 or MUC6 RNA.
In contrast, addition of 1 µg/ml of PMA to the culture media of the dog gallbladder cells did result in a dramatic increase in mucin secretion to 342% of control (100%; Klinkspoor et al., 1996). Perhaps the differential expression of the mucin genes, as reported by Hong et al. (Hong et al., 1996) can explain the differences in response to phorbol esters by the LS174T cells and the dog gallbladder epithelial cells. However, no evidence could be obtained linking bile salts with PKC in the dog gallbladder cells. Therefore, the results for the LS174T cells are in accordance with our findings in the gallbladder cells, apparently bile salts do not stimulate mucin secretion by means of a direct activation of protein kinase C.
In vivo, normally no drastic changes in the local bile salt concentration will occur. However, in case of obstructive jaundice, impaired gastrointestinal bile flow will lead to a decreased bile salt concentration in the intestine. It is well known that patients with obstructive jaundice are more prone to postoperative complications than non-jaundiced patients (Pain et al., 1985; Wait et al., 1989). Most frequent are septic complications and renal impairment (Wardle et al., 1970; Armstrong et al., 1984). A factor suggested to play an important role in the pathogenesis of the enhanced morbidity and mortality in obstructive jaundice is endotoxemia (Kocsar et al., 1969; Pain and Bailey, 1987). Endotoxemia in obstructive jaundice is considered to result from an increased absorption of gut-derived endotoxins, following absent intestinal bile flow (Bailey, 1976; Cahill, 1983). It is thought that the absence of bile salts in the intestinal tract allows endotoxin to be absorbed, because bile salt binding of endotoxin is lacking. Bile salts also have a direct detergent effect on the lipopolysaccharide endotoxin molecule (Shands et al., 1980). The predominant pathogenic microorganisms in sepsis in patients with obstructive jaundice are enteric gram-negative bacteria, these bacteria might cross the intestinal mucosal barrier and invade remote organs and tissues, a phenomenon called bacterial translocation (Deitch et al., 1990). Oral administration of bile acids has proved to be effective in the prevention of endotoxemia in obstructive jaundice, unconjugated bile acids seemed more efficient than conjugated ones (Cahill, 1983; Thompson et al., 1986; Pain and Bailey, 1988). Ding et al. showed that oral administration of cholic acid, deoxycholic acid or whole bile inhibited bacterial translocation and endotoxin absorption in obstructive jaundice in rats (Ding et al., 1993).
Intestinal mucosal integrity is crucial for the maintenance of the barrier function of the intestine, confining bacteria within the intestinal lumen. Our study suggests that an impairment of bile flow, resulting in a decreased bile salt concentration in the intestine, will lead to a reduction in mucin secretion by the intestinal epithelium. This would result in a decreased mucus layer on the epithelium, thereby reducing its barrier function. Therefore, we speculate that a depletion of bile salts in the intestinal lumen will not only lead to a decreased absorption of endotoxins by bile salts in the gut, but will also result in an increased permeability of the intestinal wall to endotoxins and microorganisms, due to a reduced mucus layer. Evidence in support of this hypothesis was recently presented (Belley et al., 1996); colonic mucins secreted by LS174T cells were demonstrated to bind to and inhibit the adherence of amoebae to Chinese hamster ovary cells. Also the killing by amoebae of LS174T cells with an intact mucus layer was found to be retarded, compared to the killing of unprotected hamster ovary cells. Oral administration of bile acids would lead to a restoration of mucin secretion, hydrophobic bile acids being more effective than hydrophilic bile acids.
In conclusion, we suggest that bile salt regulated mucin secretion represents an important mechanism, by which the different gastrointestinal epithelia protect themselves against the toxic action of bile salts.
Chemicals and reagents
Collagen type I was purchased from Sigma (St. Louis, MO). Tissue culture plates were from Costar (Cambridge, MA). Cell culture media and reagents were obtained from BioWhittaker (Walkersville, MA). Sepharose 4B column material was obtained from Pharmacia Fine Chemicals AB (Uppsala, Sweden). N-Acetyl-d-[1-3H]glucosamine ([3H]GlcNAc) was from Amersham Life Science (Arlington Heights, IL). Filters with a pore size of 0.45 µm were obtained from Millipore (Molsheim, France). Tauroursodeoxycholate was from Calbiochem (La Jolla, CA). Other bile salts, phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate (PDB), were obtained from Sigma (St. Louis, MO). All other chemicals were of analytical grade and purchased from Sigma except where noted.
Cell line and cell culture
LS174T, a previously described (Tom et al., 1976) mucin producing human colon adenocarcinoma cell line, was used for these studies. The cell line was obtained from American Type Culture Collection. Stock cultures were grown on 60 mm petri dishes coated with 2.5 µg/cm2 collagen type I in Dulbeccos Modified Eagles Medium (DMEM) with 4.5 g/l glucose, supplemented with 20% fetal calf serum, 2 mM l-glutamine, 20 mM HEPES, 100 IU/ml penicillin, and 100 µg/ml streptomycin. The cells were maintained in a 37°C incubator with 10% CO2 at a relative humidity of 95%. Media were replaced daily. The cells were passaged when confluent, using trypsine (0.5 g/l) and ethylenediaminetetraacetic acid (EDTA) (0.2 g/l) treatment. For experiments the LS174T cultures were used between passages 112-125.
Mucin secretion assay
For the mucin assay LS174T cells were grown to confluence in collagen type I coated 6 well tissue culture plates. Mucin assays were performed as described by Kuver et al. (Kuver et al., 1994) with slight modifications. The cells were labeled overnight (16-24 h) with 2 µCi/well of [3H]GlcNAc, in media containing 20% fetal calf serum. After labeling the cells were washed with sterile phosphate buffered saline (PBS), pH 7.4, for 30 min, followed by washing for another 30 min with serum free media to remove unincorporated label. Next, 2 ml of solutions of bile salts in serum-free media were added to the wells and the plates were returned to the incubator. After the incubation 1 ml of media was harvested from each well and spun at 500 g for 10 min to pellet released cells. A half milliliter of the supernatant was then mixed with 5 ml of 10% trichloroacetic acid/1% phosphotungstic acid (TCA/PTA), vortexed and incubated overnight at 4°C. The cells were washed once with PBS and harvested with trypsin/EDTA. After collection they were again washed with PBS and spun down at 500 × g for 10 min. Aliquots of the cell pellets were sampled and used for protein determination as described by Lowry (Lowry et al., 1951). The remaining cells were treated with TCA/PTA similar to the processing of the media samples. After precipitation overnight the samples were spun at 1500 g for 15 min, the resulting protein pellet was washed twice, first with 3 ml of TCA/PTA, then with 2 ml of 90% ethanol. Finally, the pellets were dissolved in 0.5 ml of water and counted in 10 ml of scintillation fluid. Results were expressed as percentage of control in disintegrations per min (d.p.m.) per mg cell protein. All experiments were performed at least twice.
Gel filtration of labeled glycoproteins
LS174T cells were grown to confluence in collagen type I coated 6 well tissue culture plates and were labeled overnight (16-24 h) with 2 µCi/well of [3H]GlcNAc. After labeling the cells were washed with PBS and serum free media and then incubated for 4 h with serum-free media or 5 mM of tauroursodeoxycholic acid in media. Culture media and cells were harvested and treated with TCA/PTA as described above. The precipitates were dissolved in 0.5 ml of PBS and subjected to gel filtration in a Sepharose CL-4B column with dimensions of 1.0 × 25.0 cm, equilibrated with PBS, containing 0.02% sodium azide, pH 7.4. (flow rate 0.5 ml/min). After application of the samples to the column, 25 fractions of 1 ml were collected and subjected to scintillation counting. Results were then expressed as d.p.m./mg cell protein per fraction. To calculate the amount of radioactive glycoprotein secreted by the cells into the culture media, the radioactivity in the media fractions was expressed as a percentage of the radioactivity in the corresponding cell fractions. The void volume and included volume of the column were determined with dextran blue (molecular mass 2000 kDa) and [3H]GlcNAc, respectively.
Preparation of bile salt solutions
Bile salts were directly dissolved in serum-free media in concentrations ranging from 0.0625 mM to 10 mM and the pH of the solutions was adjusted to 7.4. The solutions were filtered through a 0.45 µm filter before incubation with the LS174T cells. Bile salt solutions were added to the wells, and the cells were returned to the incubator for 4 to 24 h. Mucin secretion was then measured as described above.
Measurement of cell viability
Functional preservation of the LS174T cells was assessed by measuring the leakage of an endogeneous cytoplasmic enzyme, lactate dehydrogenase (LDH) into the culture media, using the method of Amador et al. (Amador et al., 1963). Results are expressed in units of LDH activity per mg cell protein. Mean of duplicate or triplicate wells from experiments were then calculated. The effect of bile salts on the assay was tested, measurement of LDH was not affected by bile salts.
CA, cholic acid; DCA, deoxycholic acid; LDH, lactate dehydrogenase; [3H]GlcNAc, N-acetyl-d-[1-3H]glucosamine; PDB, phorbol 12,13-dibutyrate; PMA, phorbol 12-myristate 13-acetate; PTA, phosphotungstic acid; TCA, trichloroacetic acid; TCDC, taurocheno-deoxycholic acid; TUDC, tauroursodeoxycholic acid; UDCA, ursodeoxycholic acid.
4To whom correspondence should be addressed at: GMR 151L , BLD13/206, Veterans Affairs Medical Center, 1660 South Columbian Way, Seattle, WA 98108