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Vibrio parahaemolyticus is an important cause of gastroenteritis, with outbreaks now occurring all over the world (2, 17). However, the mechanisms underlying the diarrheic action of this pathogen are not completely understood. V. parahaemolyticus can be categorized according to the presence or absence of the Kanagawa phenomenon (KP), in which there is hemolysis due to thermostable direct hemolysin (TDH) produced by the organism on Wagatsuma agar medium (14). TDH has been considered a major virulence factor for this disease (8, 23); however, some cases of V. parahaemolyticus gastroenteritis are due to KP-negative strains (10, 15, 16). From clinically isolated KP-negative V. parahaemolyticus, we identified a new hemolysin, named TDH-related hemolysin (TRH), and demonstrated that this TRH was also an important virulence factor (10, 24). For example, TRH stimulated fluid secretion in the rabbit loop test (8, 10). Thus, there is a possibility that TRH induces diarrhea. TRH is immunologically related, but not identical, to TDH ordinary human virulence factor. Unlike that of TDH, the activity of TRH is labile to heat treatment at 60°C for 10 min (10). Although we recently demonstrated that TDH induced Cl secretion in human colonic epithelial cells (22), there is no evidence how TRH induces fluid secretion.

The present study was designed to investigate how TRH affects short-circuit current (Isc) in human colonic epithelial cell (Caco-2 cell) monolayers and the intracellular Ca²⁺ concentration ([Ca²⁺]_in) in Caco-2 cells after exposure to TRH. The measurements of Isc and [Ca²⁺]_in in Caco-2 cells were taken by following a previously described method (22). Briefly, stock cells were trypsinized, suspended at 20 × 10⁴ cells/ml in medium, and seeded at confluent density onto 1.0-cm² transwell inserts (Costar, Cambridge, Mass.). After 3 days, 2 mM sodium butyrate (Sigma) was added. Sodium butyrate is known to induce differentiation in many cells, including Caco-2 cells (1, 12, 13). It occurs naturally in normal human colon and is used as an energy source by colonocytes (18). Fecal levels of butyrate in healthy humans may be as high as 20 mM (4). Caco-2 cells treated with butyrate have higher transepithelial resistances and greater sensitivity to transepithelial Isc increases by TRH compared with those of nontreated cells (A. Takahashi and T. Honda, unpublished observations). Thus, butyrate-treated Caco-2 cells were used in this study to investigate the effects of TRH. The cells were cultured on the transwell inserts for 6 days and were mounted into a modified Ussing chamber and maintained at 37°C in modified Ringer solution (pH 7.4; gassed with 5% CO₂-95% O₂). Transepithelial resistance was measured by applying a 5-mV pulse at intervals of 40 to 50 s, and the resistance was calculated according to Ohm's law. [Ca²⁺]_in was determined by microfluorometry using a fluorescent dye, 1-(2-(5'-carboxyoxazol-2'-yl)- 6-aminobenzofuran-5-oxy)-2(2'amino-5'-methylphenoxy) ethane-N,N,N',N'-tetraacetic acid, pentaacetoxy methyl ester (Fura-2/AM; Molecular Probes, Eugene, Ore.). Using a previously described method (22, 23), TRH was purified from a clinically isolated strain of V. parahaemolyticus (RIMD 2210531), which was KP negative (TDH negative) and yielded a single band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Fig. 1A), suggesting that the toxins were purified to homogeneity.

View larger version (20K): [in this window] [in a new window]   FIG. 1.   (A) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel of purified preparation of TRH. Lane 1, molecular mass markers (New England Biolabs Inc.) (175, 83, 62, 47.5, 32.5, 25, 16.5, and 6.5 kDa); lane 2, TRH. (B) Effect of gluconate ion and Ca2+ depletion on Isc in Caco-2 cell monolayers. , Control cells (simple exposure to TRH); , Cl replaced by gluconate ion in bath solution of Ussing chamber; , CaCl2 was omitted from bath solution and EGTA (1 mM) was added to bath solution of Ussing chamber. TRH was added on the apical side of the Caco-2 cells at the time indicated. , negative control (no exposure to TRH). Values are expressed as means ± standard deviations (n = 5). *, Significant difference (P < 0.05) versus negative control.

TRH increased Isc in Caco-2 cell monolayers. Isc increased in Caco-2 cell monolayers when TRH was added to the apical side of the monolayers. When the Cl in the bath solution was replaced with gluconate ion in both the apical and basolateral sides of the cells, Isc did not increase by addition of TRH (Fig. 1B). This indicates that the influence of TRH on Isc is dependent on extracellular Cl. To deplete Ca²⁺ from the apical cell surface, CaCl₂ was omitted from the bath solution and EGTA (1 mM) was added; in these conditions, after exposure to TRH, there was no change in Isc (Fig. 1B). This indicates that Isc change is also dependent on extracellular Ca²⁺. From these results, we hypothesized that Isc increases after exposure to TRH flow along Ca²⁺-activated Cl channels.

To explore this hypothesis, we used four kinds of channel inhibitors. Figure 2A shows that the influence of TRH on Isc was inhibited by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) (300 µM), an inhibitor of the Ca²⁺-activated Cl channel (5, 11).

View larger version (28K): [in this window] [in a new window]   FIG. 2.   Effect of inhibitors on Isc in Caco-2 cell monolayers. TRH (10 µg/ml) was added on the apical side at the time indicated. (A) DIDS (100 µM) was added on the apical side at the time indicated. (B) Effect of Cl channel inhibitors, DIDS (100 µM), glybenclamide (300 µM), NPPB (100 µM), and gadolinium ion (Gd3+) (500 µM). Isc was measured after a 15-min exposure to TRH or 10 min after addition of Cl channel inhibitor. Values are expressed as means ± standard deviations (n = 5). *, Significant difference (P < 0.05 versus TRH.

The cystic fibrosis transmembrane conductance regulator (CFTR) is one of the major Cl channels in Caco-2 cells, and it is one of the most important Cl secretion pathways involved in human diarrhea (6). We eliminated the possibility that CFTR is a target for TRH by testing with glybenclamide (300 µM) and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) (100 µM), both of which are known to be inhibitors of CFTR (5, 19, 21); neither had any effect on the influence of TRH on Isc currents flowing through the Cl channel (Fig. 2B).

We noted cell swelling within 15 min of adding TRH to Caco-2 cells (unpublished observation), which suggested the possibility that the stretch-activated channels may have opened. We tested with gadolinium ion (Gd³⁺), an inhibitor of stretch-activated channels (3, 7), and found it had no effect on Isc (Fig. 2B). This tends to rule out the association of stretch-activated channels with the Cl secretion induced by TRH.

These observations are consistent with the conclusion that the effects of TRH on Isc are related to Ca²⁺-activated Cl channels (20).

Intracellular Ca²⁺ concentration. [Ca²⁺]_in of Caco-2 cells increased in the presence of TRH (Fig. 3A). After depletion of Ca²⁺ from the cell culture medium by withholding CaCl₂ from the bath solution and EGTA (1 mM), however, [Ca²⁺]_in did not increase. Thus, the increase in [Ca²⁺]_in was due to an influx of Ca²⁺ from the extracellular medium after exposure to TRH.

View larger version (23K): [in this window] [in a new window]   FIG. 3.   [Ca2+]in change after exposure of Caco-2 cell monolayers to TRH. (A) TRH (10 µg/ml) was added at the time indicated. , Positive control (simple exposure to Vp-TRH); , CaCl2 was depleted, and 1 mM EGTA was added to bath solution in fluorometer chamber, followed by addition of TRH and the inhibitors indicated; , negative control (no exposure to Vp-TRH). (B) DIDS (300 µM) and TRH (10 µg/ml) were added at the times indicated. Values are expressed as means ± standard deviations (n = 10).