Theodore Cooper Surgical Research Institute, Department of Surgery, Saint Louis University Health Sciences Center, St. Louis, Missouri 63104
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ABSTRACT |
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We have developed an in vitro model of adaptive cytoprotection induced by deoxycholate (DC) in human gastric cells and have shown that pretreatment with a low concentration of DC (mild irritant, 50 µM) significantly attenuates injury induced by a damaging concentration of DC (250 µM). This study was undertaken to assess the effect of the mild irritant on changes in intracellular Ca2+ and to determine if these perturbations account for its protective action. Protection conferred by the mild irritant was lost when any of its effects on intracellular Ca2+ were prevented: internal Ca2+ store release via phospholipase C and inositol 1,4,5-trisphosphate sustained Ca2+ influx through store-operated Ca2+ channels or eventual Ca2+ efflux. We also investigated the relationship between Ca2+ accumulation and cellular injury induced by damaging concentrations of DC. In cells exposed to high concentrations of DC, sustained Ca2+ accumulation as a result of extracellular Ca2+ influx, but not transient changes in intracellular Ca2+ content, appeared to precede and induce cellular injury. We propose that the mild irritant disrupts normal Ca2+ homeostasis and that this perturbation elicits a cellular response (involving active Ca2+ efflux) that subsequently provides a protective action by limiting the magnitude of intracellular Ca2+ accumulation.
bile acids; AGS cells; store-operated calcium influx; phospholipase C; inositol trisphosphate; prostaglandins
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INTRODUCTION |
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THE CONCEPT OF cytoprotection was first described by Robert (44) in the late 1970s. Briefly, direct cytoprotection was defined as the ability of exogenous PGs, independent of effects on acid secretion, to protect mucosae of the gastrointestinal (GI) tract against damage induced by various injurious agents and noxious insults (46). Adaptive cytoprotection, in contrast, is that process whereby administration of a low concentration of a damaging agent (not damaging by itself), termed a "mild irritant," is able to attenuate injury to GI mucosae on subsequent exposure to higher concentrations of the same or even differing damaging agents (45).
The majority of early work investigating adaptive cytoprotection utilized in vivo models. Chandhury and Robert (11) and others (2, 25, 38) demonstrated that orally administered pretreatment with mild irritants (ethanol, bile salts, or hydrochloric acid) protected gastroduodenal mucosae against injury induced by a variety of dissimilar necrotizing agents and that a certain dose (or concentration) of the mild irritant was required to elicit this protective response. In addition, because indomethacin blocked this protective response and some mild irritants were purported to stimulate PG synthesis, it was argued that adaptive cytoprotection was in large part mediated by endogenous PGs (2, 11, 45). This view, however, has been challenged by a number of investigators (15, 31, 49), and recent studies have suggested that other mediators may play contributory roles in adaptive cytoprotection, independent of PGs, including nitric oxide, glutathione, dopamine, the internal enteric reflex, mucus and/or bicarbonate secretion, salivary secretions, and the formation of a physical protective covering of surface debris (10, 24, 31-33, 37).
We have recently investigated adaptive cytoprotection under in vitro conditions in gastric cells derived from a human carcinoma cell line. We determined that pretreatment of these cells with a low concentration of deoxycholate (DC) significantly attenuated both cell injury and permeability changes induced by subsequent exposure to damaging concentrations of DC (34). This protection was dependent on both the concentration and duration of mild irritant exposure. In addition, whereas DC exposure increased PG synthesis, the concentrations required were much higher than those required to initiate protection. We did not observe enhanced PG synthesis in response to a mild irritant concentration. This work suggested that adaptive cytoprotection exists in AGS cells under in vitro conditions independent of intact blood flow, neural innervation, or circulating humoral mediators. Furthermore, our findings indicated that stimulation of endogenous PG synthesis was not a prerequisite in mediating this protective response.
It has been proposed that Ca2+ homeostasis is critical in maintaining mucosal integrity (52) and that this cation plays a major role in promoting mucosal injury induced by various noxious agents, such as indomethacin, ethanol, and excessive nitric oxide (22, 51, 53). The relationship between Ca2+ and cellular injury is by no means specific to GI mucosae and has been described in many other cell types in which injury was initiated by diverse causes including ischemia and/or reperfusion, chemical exposure, radiation, and infection (20). Schanne and associates (47), in the late 1970s, suggested that the influx of Ca2+ across damaged cell membranes may be the final common pathway to cell death.
In view of these considerations and the results of our aforementioned studies (34) validating the existence of adaptive cytoprotection under in vitro conditions, we initiated the present study to explore the potential role that maintenance of intracellular Ca2+ homeostasis might play in this protective response. The major objective of this study was to assess the effect of a mild irritant concentration of DC on changes in intracellular Ca2+ and to determine if these effects account for its protective action. We also investigated the relationship between Ca2+ accumulation and cellular injury induced by damaging concentrations of DC. Our prior work enabled us to use a well-defined, in vitro model of adaptive cytoprotection in human gastric cells.
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MATERIALS AND METHODS |
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Cells. A human gastric cell line, known as AGS and derived from carcinoma cells, was obtained from American Type Culture Collection (Rockville, MD) at passage 49. We have previously characterized this cell line and have determined AGS cells to be morphologically similar to gastric mucous cells (PAS+, alcian blue+), with an ability to differentiate when postconfluent (34). Cells were maintained at 37°C in an atmosphere of 5% CO2 and 100% relative humidity. Cells were split on a weekly basis at a ratio of 1:6 on reaching confluency. Cells were detached using 0.5 g porcine trypsin and 0.2 g EDTA tetrasodium combined with Hanks' balanced salt solution (HBSS) and then plated into either 24- or 48-well plates (Costar, Cambridge, MA) for experiments, four-well cover-glass chambers (Nunc, Naperville, IL) for confocal microscopic imaging, or into 150-cm2 flasks for propagation. All experiments were performed at 80-90% confluence. Cell passage was maintained between 50 and 65, and medium was changed every 2-3 days. AGS media consisted of Ham's F-12 supplemented with 10% fetal bovine serum, 100 µg/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B.
Solutions. Before all experiments, medium was aspirated and replaced with HBSS plus 10 mM HEPES (Sigma Chemical, St. Louis, MO), consisting of (in mM) 137 NaCl, 5.7 NaHCO3, 5.3 KCl, 1.26 CaCl2, and 0.8 MgSO4. Experiments involving Ca2+-free buffer utilized HBSS plus 10 mM HEPES and 2 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (Sigma), consisting of (in mM) 137 NaCl, 5.7 NaHCO3, and 5.3 KCl. All test compounds were dissolved in either HBSS or HBSS minus Ca2+. Sodium DC, neomycin sulfate (neomycin), lanthanum chloride (La3+), verapamil, nifedipine, quercetin, and sodium orthovanadate (vanadate) were obtained from Sigma. The aminosteroids U-73122 and U-73343 were purchased from Calbiochem (La Jolla, CA), and 45CaCl2 was obtained from Amersham (Arlington Heights, IL). Treatment with the above antagonists and/or inhibitors involved a variable preincubation time followed by the addition of the respective inhibitor to all subsequent solutions within treatment groups. Thapsigargin (TG) was purchased from Molecular Probes (Eugene, OR) in 50-µg aliquots. Experiments were performed at 37°C in a humidified incubator.
Measurement of [Ca2+]i and extracellular Ca2+ influx. Changes in intracellular Ca2+ concentration ([Ca2+]i) were quantitated using the single-wavelength Ca2+ indicator fluo 3 (fluo 3-AM; Molecular Probes). Fluo 3 was chosen as a Ca2+ indicator because it exhibits a large fluorescent enhancement on Ca2+ binding (40-fold), enables imaging of cells using an argon lamp with confocal microscopy, and exhibits an enhanced resistance to autobleaching (48).
Before fluo 3 loading cells were washed twice with HBSS. Fluo 3 was initially dissolved in pluronic F-127 (20% solution in DMSO; Molecular Probes) to make a 1 mM working solution and subsequently added to HBSS plus 1% fetal bovine serum for a final loading concentration of 4 µM (30). Cells were then loaded with fluo 3 for 50 min at 25°C in an atmosphere of 5% CO2 and 100% relative humidity. Loading at a lower temperature significantly decreases indicator compartmentalization into the endoplasmic reticulum or mitochondria (30). AGS cells were then washed three times to ensure removal of all unloaded fluo 3, and control and test solutions were added to the respective wells. At each time point [Ca2+]i was calculated using the equation
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Measurement of Ca2+ efflux. AGS cells were incubated overnight with 45CaCl2 (10 µCi/ml), washed three times with HBSS, and subsequently treated with either control or test solutions. The supernatant was then removed at the designated time points, and cells were lysed with 1% Triton X-100. Radioactivity in the supernatant and within the lysed cells was determined by scintillation counting (Beckman LS 5000CE; Irvine, CA). Results are expressed as percent Ca2+ efflux based on the equation
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Measurement of cell injury. Cellular injury was quantitated using two different assays, one measuring plasma membrane integrity and the other measuring cytoplasmic enzyme leakage. We employed the fluorescent agent ethidium homodimer-1 (Et, 4 µM; Molecular Probes) to monitor plasma membrane integrity. Et enters cells through damaged membranes and exhibits enhanced fluorescence on binding to nucleic acids. This fluorescent probe produces a bright red fluorescence in dead cells, which was measured with a Fluorescent Multi-well Plate Reader at 485 nm excitation and 620 nm emission wavelengths (26, 40). Injury is reported as relative fluorescence.
Cell injury was also assessed by release of lactate dehydrogenase (LDH) into the buffer. Measurement of LDH release in cultured cells has been deemed a reliable and reproducible indicator of cellular injury (5, 41). LDH content was determined using the CytoTox 96 assay (Promega, Madison, WI), which is based on a coupled enzymatic reaction that results in the conversion of a tetrazolium salt into a red formazan product. The amount of red formazan product is directly proportional to the amount of LDH in the buffer. After the reaction, the formazan product was quantified spectrophotometrically by measuring its absorbance at 490 nm (Bio-Rad model 3550 microplate reader, Hercules, CA) in 96-well plates (Costar). Experimental values are reported as the percentage of LDH released from control cells.Confocal imaging. Confocal images were acquired with a Zeiss LSM 410 laser scanning confocal microscope system utilizing a Zeiss Axiovert 135 optical microscope. The samples were viewed with a Zeiss Plan-Neofluar 63×, 1.25 NA oil objective. The samples were then illuminated with an argon-krypton multiline laser, and confocal images were recorded with dual, simultaneous fluorescence detectors. The data system was operated with Zeiss software LSM (Rev. 3.92). Time-lapse images were acquired using the time series macros, which are part of this software package.
Experimental design. Previous work suggested that protection conferred by the mild irritant was dependent on both concentration and duration of exposure. We determined that pretreating cells with 50 µM DC for 20 min before subsequent exposure to a damaging concentration (250 µM) of DC provided optimal protection compared with other mild irritant concentrations or pretreatment durations (34). Therefore, we initially investigated the mechanism(s) whereby 50 µM DC elicits changes in intracellular Ca2+. The roles of phosphoinositide-specific phospholipase C (PLC) and subsequent inositol 1,4,5-trisphosphate (IP3) generation were determined using the aminosteroid U-73122, an inhibitor of PLC catalyzed hydrolysis of phosphatidylinosotol 4,5-bisphosphate, its inactive analog U-73343, and neomycin (an inhibitor of IP3 generation) (9, 18). The mechanism of sustained influx of extracellular Ca2+ was investigated with Ca2+-free buffer, the store-operated Ca2+ channel (SOCC) blocker lanthanum (3), and the voltage-operated Ca2+ channel (VOCC) antagonists verapamil and nifedepine. Ca2+ efflux after 50 µM DC treatment was then measured, and the effect of the plasma membrane Ca2+-ATPase inhibitors quercetin and vanadate (36) was quantitated. The above studies were then repeated, and cells were subsequently exposed to 250 µM DC to determine what effect mild irritant Ca2+ mobilization had on protection against injury induced by a damaging concentration of DC.
The second series of experiments investigated the role of Ca2+ on cellular injury induced by 250 µM DC. We initially assessed the temporal relationship between intracellular Ca2+ elevation and cell injury. This was achieved, in separate studies, with the simultaneous measurement of changes in intracellular Ca2+ content and Et uptake or LDH release. We then examined the effect of Ca2+ removal on injury induced by DC. Ca2+-free buffer was utilized to determine the role of extracellular Ca2+. To investigate the role of intracellular Ca2+, separate cells were pretreated with TG (10 min) and subsequently exposed to DC in Ca2+-free buffer. TG, a microsomal Ca2+-ATPase inhibitor, rapidly depletes intracellular Ca2+ stores (8).Statistics. Statistical evaluation was performed by ANOVA with a Scheffé post hoc test. Data (n = 6-12 per group) are reported as means ± SE. P < 0.05 was taken to represent statistical significance.
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RESULTS |
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Intracellular Ca2+ changes induced by mild irritant: role of PLC-IP3. AGS cells exposed to 50 µM DC demonstrated an initial increase in intracellular Ca2+ present within 2 min, followed by a sustained elevation lasting up to 10 min, and then a gradual decrease to baseline. Cells pretreated with neomycin (100 µM, 10 min) or U-73122 (1 µM, 15 min) and subsequently treated with 50 µM DC displayed no such elevation in intracellular Ca2+, and their levels were noted to be quite similar to control cells throughout the period of study. In contrast, cells pretreated with the inactive analog U-73343 (1 µM, 15 min) and subsequently exposed to 50 µM DC demonstrated no difference with regard to changes in intracellular Ca2+ compared with cells treated with only 50 µM DC. Data from these studies are depicted in Fig. 1. These data suggest that the initial increase in intracellular Ca2+ content induced by a low concentration of DC involves the release of intracellular Ca2+ stores via a PLC- and IP3-related mechanism.
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Intracellular Ca2+ changes induced by mild irritant: role of SOCI. Sustained intracellular Ca2+ elevations (2-10 min) were not observed in cells treated with 50 µM DC in the absence of extracellular Ca2+. Quenching of fluo fluorescence by Mn2+ was first evident 6 min after 50 µM DC exposure, which suggests the influx of extracellular Ca2+ over this time period. Pretreatment of AGS cells with either La3+ (25 µM, 15 min), verapamil (20 µM, 15 min), or nifedipine (10 µM, 15 min) alone did not significantly affect intracellular Ca2+ levels (data not shown). The SOCC blocker La3+, but not the VOCC antagonists verapamil or nifedipine, inhibited both the sustained intracellular Ca2+ plateau and Mn2+ uptake after 50 µM exposure. Data from these studies are shown in Fig. 2, A and B. These data suggest that the sustained Ca2+ elevation, as induced by 50 µM DC, is mediated by the influx of extracellular Ca2+ through SOCCs, observations that are consistent with store-operated Ca2+ influx (SOCI).
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Ca2+ efflux induced by mild irritant. Compared with controls, AGS cells exposed to 50 µM DC demonstrated initial signs of net Ca2+ efflux at 10 min postexposure followed by significant efflux at 20 min (Fig. 3A). In separate studies, Ca2+ efflux at 20 min after 50 µM DC exposure was not observed in cells pretreated with the plasma membrane Ca2+-ATPase inhibitors quercetin (10 µM, 10 min) or vanadate (10 µM, 10 min) (Fig. 3B). Furthermore, in cells pretreated with quercetin or vanadate and subsequently exposed to 50 µM DC, intracellular Ca2+ content did not return to baseline but remained elevated during the entire 20-min time period (Fig. 4). These data suggest that under normal conditions 50 µM DC elicits active Ca2+ efflux at later time points (10-20 min), causing a return to resting intracellular Ca2+ values.
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Mild irritant Ca2+ mobilization and adaptive cytoprotection. AGS cells, with or without pretreatment by the aforementioned inhibitors, were then exposed to 50 µM DC for 20 min, washed three times to remove any remaining inhibitor, and subsequently exposed to 250 µM DC for 20 min. Compared with controls, cells pretreated with buffer and subsequently exposed to 250 µM DC demonstrated sustained intracellular Ca2+ accumulation and significant injury as measured by both Et uptake and LDH release. In contrast, the elevation in intracellular Ca2+ content and cellular injury induced by 250 µM DC were significantly attenuated when cells were pretreated with 50 µM DC, with or without the inactive PLC inhibitor U-73343. Furthermore, protection conferred by the mild irritant was lost when any of its effects on changes in intracellular Ca2+ were prevented: internal store release via PLC and IP3 (neomycin or U-73122 pretreatment), sustained extracellular Ca2+ influx through SOCCs (Ca2+-free buffer or La3+ pretreatment), or eventual Ca2+ efflux (quercetin or vanadate pretreatment). These data are depicted in Table 1.
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Intracellular Ca2+ accumulation and cellular injury induced by DC. DC exposure elicited a dose-dependent rise in Ca2+ accumulation in AGS cells. A large, initial surge of intracellular Ca2+ was observed within the first 2 min after DC exposure (100-500 µM) followed by a lower, sustained elevation, which persisted for at least 20 min. However, cells exposed to the lowest DC concentration, 50 µM, demonstrated an initial intracellular Ca2+ elevation followed by a return toward resting Ca2+ levels. Injury, as quantitated by Et uptake and LDH release, was subsequently measured in cells exposed to graded concentrations of DC for 20 min. The higher concentrations of DC that elicited sustained elevations in intracellular Ca2+ (100-500 µM) were associated with significant cellular damage, whereas 50 µM DC did not appear to induce cell injury compared with control cells. These data are depicted in Table 2. There was a strong correlation between [Ca2+]i and both Et uptake (r2 = 0.949, P < 0.001) and LDH release (r2 = 0.746, P < 0.001). Whereas these data suggest that there was a significant association between intracellular Ca2+ accumulation and cell injury in response to damaging concentrations of DC, cause and effect remain unclear.
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Temporal relationship of Ca2+ accumulation and cell injury. Figure 5A demonstrates the simultaneous measurement of intracellular Ca2+ accumulation and cell injury (Et uptake). Although not shown, intracellular Ca2+ levels and Et uptake remained constant over the 20-min time period in control cells. Treatment with 250 µM DC elicited a large, initial intracellular Ca2+ surge followed by a decline toward basal levels. However, at 20 min the intracellular Ca2+ content remained elevated. AGS cells showed initial signs of significant Et uptake at 10 min postexposure followed by significant Et uptake at 20 min. In a separate experiment, similar patterns were observed with the simultaneous measurement of intracellular Ca2+ accumulation and LDH release (Fig. 5B). However, a significant increase in LDH release was first evident at an earlier time point (6 min postexposure).
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Confocal imaging of Ca2+ changes and Et uptake. Living AGS cells previously loaded with fluo 3 were subsequently exposed to 250 µM DC plus Et for time-lapse confocal imaging. Before DC exposure, images demonstrated a low background intracellular Ca2+ signal (green) with minimal fluo 3 compartmentalization. A minority of injured cells demonstrating Et uptake (red) could be detected at this early time point. Two minutes after DC exposure, the majority of AGS cells demonstrated a large increase in green fluorescence (or [Ca2+]i). At this time point, no increase in the number of injured cells was apparent. Ten to twenty minutes after exposure to 250 µM DC, AGS cells demonstrated slight increases in green fluorescence compared with the same cells at baseline, and an increasing number of cells demonstrated Et uptake (red cytosolic staining).
Removal of extracellular and intracellular Ca2+. Preliminary data indicated that the effect of TG on changes in intracellular Ca2+ was not concentration dependent within the range of 500 nM to 5 µM. We therefore employed the lowest concentration (500 nM). Pilot studies in AGS cells indicated that TG, in Ca2+-containing buffer, elicited SOCI (or capacitive Ca2+ entry). Cells treated with TG in Ca2+-free buffer elicited an initial increase in [Ca2+]i followed by a steady decline (at 5 min postexposure) to baseline values. These experiments suggested that intracellular Ca2+ stores were effectively depleted (data not shown).
AGS cells were exposed to 250 µM DC in 1) Ca2+-containing buffer, 2) Ca2+-free buffer, and 3) Ca2+-free buffer after pretreatment with TG. In the presence of extracellular Ca2+, 250 µM DC exposure caused a large, initial increase in intracellular Ca2+ content followed by a lower, sustained elevation. Interestingly, in the absence of extracellular Ca2+, 250 µM DC elicited an initial intracellular Ca2+ surge followed by a rapid return to baseline values. In AGS cells pretreated with TG, subsequent exposure to 250 µM DC in Ca2+-free buffer did not appear to evoke any changes in intracellular Ca2+ content (data not shown). Cellular injury was quantitated, with both Et uptake and LDH release, 20 min after the aforementioned experiment. In control cells, the absence of extracellular Ca2+, with or without TG pretreatment, did not appear to induce significant cellular injury. Removal of extracellular Ca2+ reversed injury induced by 100 µM DC and significantly attenuated injury elicited by 250 µM DC. In AGS cells exposed to 500 µM DC, elimination of extracellular Ca2+ only marginally reduced cellular injury. Further depletion of intracellular Ca2+ stores with TG, compared with only extracellular Ca2+ removal, did not appear to further decrease cellular injury with any of the concentrations of DC employed. These data are shown in Fig. 6, A and B.
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DISCUSSION |
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A considerable body of data suggests that Ca2+ is critical in maintaining GI integrity and may be a major mediator involved in mucosal injury induced by ethanol, nitric oxide, indomethacin, or stress (22, 43, 51-53). We therefore investigated the role of Ca2+ in both adaptive cytoprotection and cell injury induced by DC. Our data indicate that pretreatment with a mild irritant (50 µM DC) significantly attenuated both Ca2+ accumulation and cell injury induced by damaging concentrations of DC (250 µM). This protection was not observed when the effects of the mild irritant on changes in intracellular Ca2+ were prevented. Our findings suggest that the mild irritant initially disrupts normal Ca2+ homeostasis and that this perturbation elicits a cellular response (involving active Ca2+ efflux) that subsequently protects cells from injury induced by damaging concentrations of DC by limiting Ca2+ accumulation. In contrast, sustained Ca2+ accumulation as a result of extracellular Ca2+ influx, but not transient changes in intracellular Ca2+ content, appeared to precede and induce cellular injury by high concentrations of DC. A schematic diagram depicting these proposed events is shown in Fig. 7.
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The mechanism whereby a low, nondamaging concentration of DC elicits changes in intracellular Ca2+ appears to initially involve the release of internal Ca2+ stores through PLC activation and/or IP3 generation. This is followed by a period of sustained intracellular Ca2+ accumulation resulting from the influx of extracellular Ca2+ through SOCCs. Finally, the intracellular Ca2+ content returns to resting values as a result of active Ca2+ efflux from the cytosol into the extracellular space (see Fig. 7).
The aforementioned proposed mechanism of a mild irritant concentration of DC-induced Ca2+ mobilization is consistent with previously published observations. Although similar work has not been reported specific to gastric cells, extensive investigation has been performed in hepatocytes and colonocytes. Combettes et al. (12, 13) reported in hepatocytes that bile salts initially mobilize IP3-sensitive intracellular Ca2+ pools. This is followed by the passive entry of extracellular Ca2+ through a process that appears to be independent of bile salts themselves. DC, at concentrations ranging from 10 to 600 µM, has been shown to significantly increase PLC activity in HT-29 colon tumor cells and rat colonic mucosa (4, 6). Devor et al. (16), employing both whole cell patch-clamp techniques and fluorescent measurements in T84 colonic cells, demonstrated that taurodeoxycholate initially induced the release of Ca2+ from intracellular stores via an IP3-dependent mechanism. Craven et al. (14) reported that DC increases diacylglycerol and IP3 generation in rat colonic cells. It is currently unknown whether the effect of DC on PLC is direct or related to its detergent properties, which may alter the interaction of PLC with membrane substrates.
Several groups have also reported that low concentrations of bile salts stimulate Ca2+ efflux in hepatocytes (7, 13). Because Na+/Ca2+ exchangers are present in very low levels or absent in nonexcitable cells, Ca2+ extrusion in human gastric cells is likely Ca2+-ATPase mediated. Currently the mechanism whereby bile salts stimulate Ca2+ efflux is unclear. This effect may be indirect and regulated by the mere increase in intracellular Ca2+ content (1). Ca2+-ATPase is regulated by calmodulin and fatty acids-acidic phospholipids, and perhaps these are mobilized by low concentrations of DC. Whereas the current work suggests that DC stimulates Ca2+ efflux, other mechanisms may also partially account for a decrease in the intracellular Ca2+ elevation; these include Ca2+ buffering and sequestration.
Our observations are consistent with other reports that suggest that bile salt toxicity involving concentrations in excess of a mild irritant may evoke substantial perturbations in intracellular Ca2+ content (19, 39, 50). Dziki et al. (19) demonstrated cellular hypercalcemia in DC-induced injury in rabbit gastric cells. They reported that 200 µM DC was the minimal concentration required to elicit intracellular Ca2+ changes and observed an initial rise in intracellular Ca2+ content 2 min after exposure, followed by a continuous rise to maximal concentrations at 20 min (19, 39). We also observed that damaging concentrations of DC elicit a large increase in intracellular Ca2+ within 2 min. However, in contrast to the Dziki study, this initial increase was noted to be only transient and was followed by a lower but sustained elevation over the remaining 20 min. Our findings suggested that whereas DC elicited early, transient intracellular Ca2+ changes, sustained elevation is required to induce cell injury. In support of this contention is the observation that when cells were exposed to a high concentration of DC (250 µM) in the absence of extracellular Ca2+, injury was significantly attenuated despite a significant but transient Ca2+ elevation. Perhaps one reason our observed Ca2+ trends differed from that of Dziki and associates is that they used a primary rabbit gastric cell preparation, in which several different cell types were represented, each of which may have differing Ca2+ responses on exposure to DC than would occur with a single cell type (19, 39). The AGS cell line, on the other hand, is morphologically homogeneous. Furthermore, because AGS cells are of human origin (and therefore have human relevance) and involve cells that are commonly exposed to noxious luminal substances under in vivo conditions, this cell line is an excellent model for the study of GI damage.
Dziki et al. (19) also reported minimal cellular injury in response to 200 µM DC exposure, and this group and others have utilized higher concentrations of DC (500 µM-1 mM) while studying injury to rabbit gastric cells and hepatocytes (19, 23, 39). In contrast, we observed a mild degree of injury with 100 µM DC, a moderate degree of cell injury induced by 250 µM DC, and extensive cell injury induced by DC concentrations exceeding 500 µM. Moreover, DC concentrations in human stomach remnants after distal gastric resections are of the order of 370 µM or lower (17). Hence, we employed concentrations that are likely to occur under physiological conditions. The differences in DC concentrations required to induce injury in our human cell line and that of the rabbit stomach cell line are most likely species specific.
The causal connection between intracellular
Ca2+ accumulation and cell death
remains moot. The cellular Ca2+
gradient between the extracellular and intracellular compartments is
among the largest in mammalian cells. Cells normally maintain (through
plasma membrane impermeability and active extrusion) a steep gradient
such that an
[Ca2+]i
of 107 M is preserved
despite being surrounded by an extracellular
Ca2+ concentration that approaches
10
3 M (21). Some
investigators believe that irreversible cellular injury is followed by
the influx of Ca2+, which then
leads to the classic morphological appearance of Ca2+ toxicity (coagulation
necrosis), whereas others contend that a reversible cellular injury
causes a loss of the maintenance of the
Ca2+ gradient, leading to
intracellular Ca2+ accumulation
and subsequent irreversible cellular injury (20, 21).
Ca2+ has been implicated in
converting reversible to irreversible cellular injury in models
investigating hepatic injury induced by ischemia-reperfusion,
phalloidin, galactosamine, and silica (20, 27, 28). Our data indicate
that intracellular Ca2+
accumulation precedes cell injury induced by DC. Our studies also
suggest that sustained rises in
[Ca2+]i
are required for cellular injury and that rapid, transient changes are
well tolerated by AGS cells. These observations are consistent with the
findings of others (20, 42).
Several mechanisms have been proposed whereby sustained increases in intracellular Ca2+ may cause cellular toxicity. Ca2+ disrupts the normal cellular cytoskeleton by dissociating of actin microfilaments from various protein structures (35). Phospholipases are also activated by Ca2+ with the subsequent hydrolysis of phospholipids and the disruption of membrane stability (35). Furthermore, Ca2+ has been implicated to activate proteases and endonucleases with resultant degradation of cytoskeletal elements (e.g., microtubules and actin), membrane proteins, and nucleic acids (42). Finally, it has been reported that excessive Ca2+ impairs mitochondrial function (42).
In summary, our data indicate that Ca2+ plays a significant role in both adaptive cytoprotection and cell injury induced by DC. Mild irritant pretreatment attenuated subsequent injury induced by damaging concentrations of DC through mechanisms that resisted intracellular Ca2+ accumulation. The mild irritant appeared to provide this protective effect through Ca2+ mobilization involving PLC and IP3 activation, sustained Ca2+ influx through SOCCs, and eventual Ca2+ extrusion through the plasma membrane. Finally, sustained Ca2+ influx preceded cell injury induced by damaging concentrations of DC and may be a significant etiologic factor in bile salt toxicity.
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ACKNOWLEDGEMENTS |
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We thank Dr. John Freeman, Saint Louis University Pediatric Research Institute, for help with confocal microscopic imaging and data collection.
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FOOTNOTES |
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The current work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-25838.
Portions of this work were presented at the annual meeting of the American Gastroenterological Association in Washington, DC, 1997, and in New Orleans, LA, 1998, and have been previously published in abstract form (Gastroenterology 112: A179, 1997 and Gastroenterology 114: A183, 1998).
Address for reprint requests: T. A. Miller, Dept. of Surgery, Saint Louis Univ. Health Sciences Center, 1402 South Grand Blvd., St. Louis, MO 63104.
Received 2 September 1997; accepted in final form 9 April 1998.
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