Theodore Cooper Surgical Research Institute, Department of Surgery, Saint Louis University Health Sciences Center, St. Louis, Missouri 63104
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Indomethacin and other nonsteroidal anti-inflammatory drugs are commonly used to indirectly deduce the possible role of PGs in a process being studied. The objective of this study was to determine if indomethacin, at concentrations comparable to plasma and tissue levels obtained in humans taking therapeutic doses, predisposes human gastric cells to injury through inhibition of PGs or acts through an alternate mechanism. The role of intracellular Ca2+ in this damaging process was also assessed. Indomethacin pretreatment, although by itself nondamaging, was associated with elevated intracellular Ca2+ concentrations and an increased cellular permeability, an effect that was dependent on extracellular Ca2+. Furthermore, indomethacin pretreatment significantly predisposed AGS cells to injury induced by two dissimilar agents (deoxycholate and A-23187), both of which are associated with intracellular Ca2+ accumulation. The addition of exogenous PGs did not reverse the predisposition to injury induced by indomethacin. The observed effects of indomethacin were dependent on concentration and not on ability to inhibit PG synthesis. Similar effects were not observed with equipotent concentrations of ibuprofen or aspirin. Finally, the exacerbation of deoxycholate-induced injury induced by indomethacin was not observed when extracellular Ca2+ was removed. Indomethacin, by disturbing intracellular Ca2+ homeostasis, predisposes human gastric cells to injury through mechanisms independent of PG synthesis. The current study suggests that data resulting from studies employing only indomethacin as a PG synthesis inhibitor should be interpreted with caution.
nonsteroidal anti-inflammatory drugs; bile salts; A-23187; adaptive cytoprotection; calcium; AGS cells
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
NONSTEROIDAL ANTI-INFLAMMATORY drugs (NSAIDs), because of their analgesic and anti-inflammatory actions, are the most frequently used medicines in the world and account for nearly 5% of all prescribed medications (34). However, the clinical efficacy of NSAIDs is not without adverse side effects. It is well accepted that NSAID administration is associated with gastrointestinal (GI) complications ranging from dyspepsia and abdominal pain to bleeding and perforated ulcers. The incidence of serious or life-threatening complications associated with NSAID use is 1-2% per NSAID user per year (10).
In the early 1970s, Sir John Vane proposed that aspirin (acetylsalicylic acid; ASA) and other NSAIDs inhibit PG synthesis and that this intrinsic property may account for both their anti-inflammatory properties and their ability to induce GI injury (41). After that original proposal and as a result of numerous studies, it has become widely accepted that the GI complications induced by NSAIDs are caused in large part by the systemic effects of endogenous PG inhibition (15, 25, 30, 44). Suggested mechanisms whereby PG inhibition results in GI injury include the reduction of mucus and/or bicarbonate release and decreased mucosal blood flow (26). However, several studies indicate that both the therapeutic actions and the ability of NSAIDs to promote injury may not only be related to their capacity to inhibit PG synthesis but may also involve additional mechanisms (1, 24, 42, 43).
We have recently investigated adaptive cytoprotection under in vitro conditions in a human gastric cell line (22). Adaptive cytoprotection can be defined as that process whereby administration of a low concentration of a damaging agent (not damaging by itself) is able to attenuate injury to GI mucosae on subsequent exposure to higher concentrations of the same or differing damaging agents. Our previous data suggested that adaptive cytoprotection exists under in vitro conditions independent of intact blood flow, neural innervation, or circulating humoral mediators. Furthermore, this protective response did not appear to be dependent on endogenous PG synthesis. However, our data also suggested that pretreatment of human gastric cells with indomethacin reversed protection conferred by the mild irritant and also appeared to increase cellular susceptibility to injury. These findings suggested one of two possibilities: that either the inhibition of basal PGs significantly disrupts cellular integrity (as has been suggested in the literature for the past several decades) or indomethacin may have other actions in addition to cyclooxygenase inhibition which may account for an increased susceptibility to gastric mucosal injury.
It has been proposed that Ca2+ homeostasis is critical in maintaining mucosal integrity (37) and that this cation plays a major role in promoting mucosal injury induced by a variety of noxious agents (38, 39). This relationship between Ca2+ and cellular injury is by no means specific to GI mucosae and has been described in many other cell types initiated by diverse causes of injury including ischemia-reperfusion, chemical exposure, radiation, and infection (9). The objective of this study was to determine if indomethacin increases cellular susceptibility to injury through the inhibition of PGs or acts through an alternate mechanism and what role Ca2+ may play in this process. Although indomethacin has limited use in the clinical arena and its popularity has decreased recently with the advent of newer and safer NSAIDs, this cyclooxygenase inhibitor has been and will continue to be extensively employed in the research setting to indirectly deduce the physiological role of PGs. For this reason the current study is highly relevant.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cells. The human gastric cell line, known as AGS (CRL 1739), was obtained from American Type Culture Collection (Rockville, MD) at passage 49. We have previously characterized this cell line morphologically and have determined AGS cells to be quite similar to gastric mucous cells (PAS+, alcian blue+) with an ability to differentiate when postconfluent (22). 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 and were detached using 0.5 g porcine trypsin and 0.2 g EDTA tetrasodium per liter Hanks' balanced salt solution (HBSS), and then plated into either 24- or 48-well plates (Costar, Cambridge, MA) for experiments or into 150-cm2 flasks for propagation. Cells grown for permeability work were split at a ratio of 1:2 into 3-µM Biocoat Collagen I Cells Culture Inserts (0.3-cm2 growth area; Becton Dickinson Labware, Bedford, MA), and experiments were performed at 7 days postconfluence. All other experiments were performed at 1 day postconfluence. Cell passage was maintained between 50 and 65, and medium was changed every 2-3 days. AGS medium 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, the medium was aspirated and replaced with HBSS
plus 10 mM HEPES (H-8264, Sigma Chemical, St. Louis, MO). 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 (H-6648, Sigma, 137 mM NaCl, 5.7 mM NaHCO3, and 5.3 mM
KCl). All test compounds were dissolved in either HBSS or
HBSS (Ca2+ free). A-23187
(4-bromo A-23187; Molecular Probes, Eugene, OR), a
Ca2+ ionophore, was stored at
80°C as a stock solution of 1 mg/500 µl DMSO. Experiments
with indomethacin, ASA, and ibuprofen (Sigma) involved a 60-min
preincubation followed by the addition of the respective cyclooxygenase
inhibitor to all subsequent solutions within treatment groups.
16,16-Dimethylprostaglandin E2
(Sigma) was maintained at
20°C as a stock solution of 1 mg/1
ml in ethanol.
Measurement of [Ca2+]i. 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) and an enhanced resistance to autobleaching (33).
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 (19). 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 (19). 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. Using a Cytofluor II fluorescent multiwell plate reader (PerSeptive Biosystems), fluorescent signals were obtained using the appropriate spectra for fluo 3 (485 nm excitation and 530 emission wavelengths). At each time point [Ca2+]i was calculated using the following equation
![]() |
Measurement of cellular 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; 8 µM, Molecular Probes) to monitor plasma membrane integrity. Et enters cells through damaged membranes and exhibits enhanced fluorescence on binding to nucleic acids (18). This fluorescent probe produces a bright red fluorescence in dead cells which was measured with a fluorescent multiwell plate reader at 485 nm excitation and 620 nm emission wavelengths. Injury is reported as relative fluorescence.
Cellular injury was also assessed by release of lactate dehydrogenase (LDH) into the buffer. 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). Total protein concentration was also quantitated with a bicinchoninic acid (BCA) protein assay kit (Pierce Chemicals, Rockford, IL), and data are presented as mIU/mg protein.PG synthesis.
Newly synthesized PGs are not stored intracellularly but released into
the extracellular space. For this reason, we used the following
protocol to quantitate PG synthesis in AGS cells. After the respective
experiment buffer was immediately transferred to microcentrifuge tubes
and stored at 80°C, and plates containing cells were
immediately frozen for protein determinations. Samples were then
thawed, and PGE2 content was
assayed using a commercial enzyme immunoassay kit (Cayman Chemical, Ann
Arbor, MI). This assay utilizes an acetylcholinesterase
tracer and a specific PGE2 monoclonal antibody. PGE2
concentrations were determined by spectrophotometric analysis of all
samples after addition of Ellman's reagent and compared with standard
curves generated under identical conditions. Levels of other
eicosanoids [PGI2,
leukotriene B4
(LTB4), and thromboxane
B2
(TxB2)] were measured in a
similar fashion. Total protein concentration per well was measured
colorimetrically with BCA protein assay kits (Pierce Chemicals) to
determine picogram per milligram protein. To optimally evaluate the
efficacy of the various cyclooxygenase inhibitors,
PGE2 release in response to a
positive agonist (20 µM digitonin) was quantitated.
Permeability. Permeability of the epithelial cell monolayer was quantified by measuring the apical-to-basolateral flux of Texas Red conjugated BSA (66 kDa, Molecular Probes). During the respective treatment fresh HBSS (750 µl) was pipetted into the basolateral chamber and experimental HBSS solutions (300 µl) containing BSA (50 µg/ml) were pipetted into the apical chamber. Duplicate samples of 100-µl aliquots were subsequently obtained from the apical and basolateral chambers at baseline and after the experiment and pipetted into 96-well plates (fluorescent clear bottom plate; Costar). Fluorescent signals were quantified using a Cytofluor II fluorescent multiwell plate reader employing 530 nm excitation and 620 nm emission spectra. Clearance (Cl) was calculated according to the following equation
![]() |
Experimental design. We have previously demonstrated that 100 µM indomethacin significantly decreases PGE2 synthesis and does not cause cellular injury (22). In addition, this concentration of indomethacin (100 µM) has been employed by other investigators to inhibit PG synthesis under in vitro conditions (5, 49). Furthermore, this concentration approximates plasma and tissue levels obtained with therapeutic doses (2, 7). Thus this concentration of indomethacin was utilized for initial experiments.
The first two experiments were designed to determine the effect of indomethacin pretreatment on changes in intracellular Ca2+ and injury in AGS cells exposed subsequently to low, nondamaging concentrations of either deoxycholate (DC) or a common Ca2+ ionophore (A-23187) or higher, damaging concentrations of both respective agents. In the third experiment we added exogenous PG during indomethacin pretreatment to ascertain whether exogenous PGs could reverse responses observed in the first experiment. The ability of varying concentrations of indomethacin, ibuprofen, and ASA to inhibit endogenous PGE2 release was then established in the fourth experiment. In the fifth experiment, the effect of varying concentrations of indomethacin on changes in intracellular Ca2+ and cellular injury in AGS cells exposed to a damaging concentration of DC was determined. The responses induced by DC exposure after indomethacin pretreatment were then compared with pretreatments with equipotent concentrations of ibuprofen and ASA in the sixth experiment. We then investigated the role of extracellular Ca2+ with regard to the predisposition to injury induced by indomethacin. Finally, we assessed permeability changes induced by exposure to the various cyclooxygenase inhibitors alone in the presence or absence of extracellular Ca2+ to determine if indomethacin alone had any effect on cellular function.Statistics. Statistical evaluation was performed by ANOVA with a Scheffé post hoc test. Data (n = 6-13/group) are reported as means ± SE. P < 0.05 was used to determine statistical significance.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Indomethacin pretreatment, changes in intracellular Ca2+, and cellular injury. In control cells intracellular Ca2+ and cellular injury remained stable over the 20-min experimental period (data not shown). Indomethacin pretreatment was associated with mild elevations in [Ca2+]i (177 ± 15 vs. 103 ± 19 nM, P < 0.05) but by itself was not damaging. Compared with control cells, 50 and 250 µM DC evoked an early increase in intracellular Ca2+ which decreased with time. This effect was more pronounced with 250 µM DC which induced cellular injury after sustained increases in intracellular Ca2+ (Figs. 1 and 2). Indomethacin pretreatment augmented the increases in intracellular Ca2+ and induced cellular injury, as measured by Et uptake and LDH release, in cells exposed to 50 µM DC and significantly accentuated both the changes in intracellular Ca2+ and injury in cells subsequently treated with 250 µM DC (see Figs. 1 and 2).
|
|
|
Effect of exogenous PGs. In view of the above findings, we next attempted to reverse the aforementioned effects of indomethacin pretreatment with exogenous PG treatment. Various concentrations of PGE2 were added concomitantly during the indomethacin pretreatment period as well as during the entire posttreatment period. Compared with control, AGS cells exposed to 250 µM DC demonstrated significant increases in both intracellular Ca2+ and cellular injury, an effect that was significantly augmented with indomethacin pretreatment. The addition of PGE2, at concentrations ranging from 2.5 to 10 µM, did not reverse the increased susceptibility to injury induced by indomethacin pretreatment (data not shown). These data suggested that indomethacin may predispose AGS cells to injury through a mechanism which is not directly related to PG metabolism.
Inhibition of PG synthesis. Under control conditions, PGE2 synthesis in AGS cells averaged 50 ± 11 pg/mg protein, levels too low to accurately measure significant decreases in response to cyclooxygenase inhibition. Thus we assessed PGE2 inhibition in cells treated with a positive agonist. Cells exposed to 20 µM digitonin for 20 min demonstrated a significant and substantial increase in PGE2 release (1,311 ± 172 pg/mg protein). Preliminary data demonstrated that indomethacin, ibuprofen, and ASA alone did not induce significant cellular injury until concentrations approached 500 µM, 500 µM, and 2 mM, respectively (data not shown). Thus we employed nondamaging concentrations of these three cyclooxygenase inhibitors for subsequent experimentation. In cells stimulated by digitonin, indomethacin (1-100 µM) inhibited PGE2 synthesis by 98-99%. Ibuprofen (100 µM) and ASA (500 µM) pretreatment significantly inhibited PGE2 release by 93 and 89%, respectively. These data are shown in Fig. 4. Although there was a trend toward increased PGE2 inhibition as induced by 100 µM indomethacin, compared with cells treated with 100 µM ibuprofen or 500 µM ASA, this did not achieve statistical significance.
|
Effect of indomethacin concentration. Because indomethacin appeared to equally inhibit PGE2 synthesis at concentrations ranging from 1 to 100 µM, the effect of varying concentrations of indomethacin on intracellular Ca2+ content and cellular injury was then investigated (Figs. 5 and 6). The augmentation by indomethacin pretreatment on changes in intracellular Ca2+ and cellular injury induced subsequently by a damaging concentration of DC was highly concentration dependent. After 20 min of exposure to 250 µM DC, there was no difference between cells pretreated with 1-10 µM indomethacin and those pretreated with buffer, whereas pretreatment with 50-100 µM indomethacin significantly augmented intracellular Ca2+ accumulation, Et uptake, and LDH release. These data further suggest that indomethacin, independent of effects on PG synthesis inhibition, increases cellular susceptibility to injury.
|
|
Effect of other cyclooxygenase inhibitors. As previously shown, there was no significant difference with regard to PGE2 synthesis inhibition among 100 µM indomethacin, 100 µM ibuprofen, and 500 µM ASA (see Fig. 4). We next investigated the effects of these equipotent concentrations of ibuprofen and ASA to determine if alternate cyclooxygenase inhibitors had similar effects to indomethacin on intracellular Ca2+ and cellular injury (Table 1). In cells exposed to DC, indomethacin (100 µM) pretreatment enhanced the increases in intracellular Ca2+, induced both Et uptake and LDH release in cells exposed to 50 µM DC, and accentuated cell injury induced by 250 µM DC treatment. This predisposition to injury was not appreciated when cells were pretreated with either 100 µM ibuprofen or 500 µM ASA.
|
Role of extracellular Ca2+. We have previously investigated, in AGS cells, the role of extracellular Ca2+ in injury induced by DC (23). Our data suggested that in the absence of extracellular Ca2+ 250 µM DC elicited an initial Ca2+ surge followed by a rapid return to baseline values. Furthermore, removal of extracellular Ca2+ significantly attenuated injury elicited by 250 µM DC. These previous observations were confirmed in the current study. We again noted that indomethacin pretreatment significantly increased intracellular Ca2+ elevations and enhanced Et uptake and LDH release induced by exposure to 250 µM DC in Ca2+-containing buffer. However, the predisposition to injury induced by indomethacin pretreatment was not observed when cells were posttreated with 250 µM DC in Ca2+-free buffer. These data are depicted in Table 2 and further suggest that indomethacin predisposes AGS cells to injury through a Ca2+-mediated mechanism.
|
Effect of NSAIDs on cellular permeability.
BSA clearance in control cells was ~1
nl · h1 · cm
2.
AGS cells treated for 1 h with 50-100 µM indomethacin
demonstrated significantly increased clearance to BSA compared with
control cells. However, increased permeability of the epithelial cell
monolayer was not observed in cells treated with 100 µM ibuprofen
(1.06 ± 0.04 nl · h
1 · cm
2)
or 500 µM ASA (1.08 ± 0.03 nl · h
1 · cm
2).
Interestingly, the changes in BSA clearance in AGS cells exposed to 50 and 100 µM indomethacin were not evident when extracellular Ca2+ was removed. These data are
depicted in Fig. 7 and suggest that 100 µM indomethacin treatment, although not directly associated with
cellular injury, significantly increases cellular permeability, an
effect that appears to be dependent on extracellular
Ca2+.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
It is generally accepted that GI complications induced by NSAIDs are a direct result of the systemic effects of PG synthesis inhibition. The current study suggests that while pretreatment of human gastric cells with indomethacin is itself not damaging, it is associated with elevated [Ca2+]i and increased cellular permeability. Furthermore, indomethacin pretreatment significantly predisposes human gastric cells to injury induced by two dissimilar agents (DC and A-23187), both of which are associated with intracellular Ca2+ accumulation.
Our initial assumption was that the effects of indomethacin were related to its inhibitory effect on basal PG production. However, subsequent data support the premise that indomethacin initiates additional mechanisms of action that appear to be responsible for the disruption of gastric cellular integrity. In support of this contention we noted that the addition of exogenous PGs did not reverse the predisposition to cell injury induced by indomethacin pretreatment. Second, the observed effects elicited by indomethacin were directly dependent on the concentration employed and not on its ability to inhibit PG synthesis. Finally, similar effects were not duplicated by two other common cyclooxygenase inhibitors (ibuprofen and ASA) when employed at equipotent concentrations.
Several earlier studies have suggested that NSAID gastric damage correlates with inhibition of PG synthesis (25, 30, 44). Although it was initially felt that NSAIDs (especially ASA) may act as topical irritants, subsequent work demonstrated that NSAID injury is similar with oral, parenteral, or rectal administration, thus further implicating a more important effect of systemic PG inhibition (16, 30).
In contrast to the aforementioned hypothesis, recent data support the existence of other mechanisms of NSAID action, with regard to both clinical efficacy and GI injury. To date, few studies have related the degree of PG synthesis inhibition with anti-inflammatory effect, and it has been shown that significantly higher doses of NSAIDs are required to suppress inflammation than to inhibit PG synthesis (1). Interestingly, in patients with arthritis, salicylate, a weak cyclooxygenase inhibitor, appears to be as effective as ASA in both controlling pain and limiting inflammation (29). Similarly, PGs alone have been shown to inhibit inflammation in other animal models of arthritis (50).
Whittle (47) investigated the temporal relationship between PG inhibition and small bowel lesion formation in rats. His data suggested that indomethacin may cause intestinal damage through a PG-independent process. Human trials have failed to demonstrate a correlation between tissue PG concentration and the gross or microscopic appearance of gastric lesions. Redfern et al. (31) administered oral doses of indomethacin (sufficient to reduce mucosal PGs) to humans and observed no gastric lesions. However, upon increasing the dosage, they documented no further decrease in PG synthesis but did detect significant gastric ulceration. Recent observations in cyclooxygenase 1 knockout mice further support our contention that indomethacin elicits gastric injury through mechanisms independent of PG synthesis inhibition. These mice did not spontaneously develop gastric ulceration but did demonstrate significant GI injury when exposed to indomethacin (24).
Previous investigators have reported that the concentration of indomethacin utilized in the current study (100 µM) is noninjurious under in vitro conditions (8, 11). Our findings are clinically relevant because data from both human and rat experimentation suggest that serum indomethacin concentrations are frequently greater than 20 µM and may even approach 150 µM (2, 7). Furthermore, in vitro concentrations may also be significantly less than that attainable under in vivo conditions because NSAIDs, in acidic environments such as the stomach, tend to penetrate lipid membranes and accumulate within cells (27).
Our data indicate that indomethacin, while it remains noninjurious in the concentrations we employed, still predisposes cells to injury subsequently induced by bile salts. Because the gastric mucosa is continuously exposed to noxious irritants such as acid, refluxed bile salts, and ethanol, this observation has clinical relevance. Other investigators have reported similar findings. Indomethacin has been implicated in aggravating bile-induced mucosal lesions in rats and dogs (45, 46). Yamada et al. (49) investigated indomethacin-induced enteritis in IEC-18 (rat ileal epithelial) cells. They noted that neither indomethacin (exceeding concentrations of 200 µM) nor rat bile exposure individually induced mucosal cell injury. However, when administered in combination they observed significant cytotoxicity. Whittle et al. (48) explored gastric mucosal injury in rats and reported that indomethacin pretreatment alone did not cause significant mucosal injury but did elicit enhanced damage by NG-monomethyl-L-arginine (a nitric oxide synthase inhibitor) or capsaicin treatment.
Predisposition to mucosal injury induced by indomethacin appears to involve a profound disturbance in Ca2+ homeostasis. Our work also reinforces that of Tepperman and Soper (36), who proposed that injury induced by indomethacin in rabbit gastric mucosal cells appeared to involve extracellular Ca2+. We have recently investigated the role of extracellular Ca2+ and the relationship between intracellular Ca2+ accumulation and injury induced by both DC and A-23187 in AGS cells. Our data indicate that sustained Ca2+ accumulation resulting from the influx of extracellular Ca2+ precedes cellular injury induced by both agents. However, transient changes in intracellular Ca2+ content as observed in a Ca2+-free environment do not appear to significantly affect cellular injury (23). Proposed mechanisms whereby sustained elevations in intracellular Ca2+ cause cellular toxicity include disruption of the cytoskeleton, phospholipid hydrolysis, and protease and endonuclease activation (28).
Several mechanisms (independent of PG synthesis inhibition) have been proposed whereby NSAIDs potentially decrease inflammation and elicit GI mucosal injury. NSAIDs are lipophilic and insert into lipid bilayers, thereby uncoupling protein-protein interactions and potentially disrupting signal transduction mechanisms (1, 3). Other reported cellular effects of NSAIDs include disturbances in oxidative phosphorylation, active transport, transmembrane ion fluxes, and cell-to-cell adhesion (3, 13, 14). These alternate mechanisms may also partially account for the observed ability of indomethacin to disrupt Ca2+ homeostasis.
The current study supports the premise that indomethacin exhibits properties independent of other cyclooxygenase inhibitors (ibuprofen and ASA) that predispose human gastric cells to injury. Data from human studies suggest that indomethacin has greater ulcerogenic potential than ibuprofen and other NSAIDs (12, 17). Clinically, indomethacin is still extensively used in obstetrics to delay uterine contractions and in the neonatal unit to facilitate patent ductus arteriosus closure. However, the major significance of the current study is that indomethacin is still commonly used to indirectly deduce the physiological role of PGs in a given process being studied.
Our findings suggest that indomethacin may have additional properties that significantly affect mucosal integrity. Thus data resulting from studies (either in vitro or in vivo) employing only indomethacin as a PG synthesis inhibitor should be interpreted with caution. As an example, the majority of the early work investigating adaptive cytoprotection demonstrated that this protective response was blocked with indomethacin pretreatment (4). It was thus proposed that adaptive cytoprotection was in large part mediated by endogenous PGs. Although many subsequent studies, including those in our laboratory, confirmed Robert's (4) original hypothesis that indomethacin blocked adaptive cytoprotection, the role of endogenous PGs remained controversial. We and others were unable to demonstrate that mild irritant pretreatment correlated with enhanced endogenous PG synthesis (6, 21, 22, 35). The current work may provide a partial explanation for these observations in that indomethacin, by disturbing intracellular Ca2+ homeostasis and predisposing human gastric cells to injury through mechanisms independent of PG synthesis, may negate the protective effects conferred by the mild irritant.
![]() |
ACKNOWLEDGEMENTS |
---|
The current study was supported by the National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-25838.
![]() |
FOOTNOTES |
---|
Portions of this work were presented at the 1997 Annual Meeting of the American College of Surgeons, Chicago, IL, and at the 1998 Annual Meeting of the American Gastroenterological Association, New Orleans, LA, and have been published in abstract form (Surg. Forum 48: 166-168, 1997; Gastroenterology 114: A184, 1998).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
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 27 January 1998; accepted in final form 21 May 1998.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Abramson, S. B.,
and
G. Weissmann.
The mechanisms of action of nonsteroidal antiinflammatory drugs.
Arthritis Rheum.
32:
1-9,
1989[Medline].
2.
Alvan, G.,
M. Orme,
L. Bertilsson,
R. Ekstrand,
and
L. Palmer.
Pharmokinetics of indomethacin.
Clin. Pharmacol. Ther.
18:
364-373,
1975[Medline].
3.
Brooks, P. M.,
and
R. O. Day.
Nonsteroidal antiinflammatory drugs: differences and similarities.
N. Engl. J. Med.
324:
1716-1725,
1991[Medline].
4.
Chaudhury, T. K.,
and
A. Robert.
Prevention by mild irritants of gastric necrosis produced in rats by sodium taurocholate.
Dig. Dis. Sci.
25:
830-836,
1980[Medline].
5.
Cima, R. R.,
I. Cheng,
M. E. Klingensmith,
N. Chattopadhyay,
O. Kifor,
S. C. Hebert,
E. M. Brown,
and
D. I. Soybel.
Identification and functional assay of an extracellular calcium-sensing receptor in Necturus gastric mucosa.
Am. J. Physiol.
273 (Gastrointest. Liver Physiol. 36):
G1051-G1060,
1997
6.
Dempsey, D. T.,
D. W. Mercer,
B. Deb,
A. Sauter,
and
W. P. Ritchie.
Adaptive cytoprotection of gastric surface epithelial cells against injury by physiologic concentrations of bile acid.
Surgery
108:
348-355,
1990[Medline].
7.
Duggan, D. E.,
A. F. Hogans,
K. C. Kwan,
and
F. G. McMahon.
The metabolism of indomethacin in man.
J. Pharmacol. Exp. Ther.
181:
563-575,
1972[Medline].
8.
Durbin, T.,
A. Tarnawski,
T. G. Douglass,
S. Wu,
and
J. Osman.
Acute injury of cultured gastric cells (KATO III) by indomethacin is partly dependent on calcium but independent of prostaglandin synthesis inhibition (Abstract).
Gastroenterology
100:
A57,
1991.
9.
Farber, J. L.
The role of calcium in cell death.
Life Sci.
29:
1289-1295,
1981[Medline].
10.
Fries, J. F. NSAID gastropathy: the second most
deadly rheumatic disease? Epidemiology and risk appraisal.
J. Rheumatol. 28, Suppl.: 6-10, 1991.
11.
Fujiwara, Y.,
A. Tarnawski,
K. Fujiwara,
I. Thilai,
T. Arakawa,
and
K. Kobayashi.
Indomethacin inhibits proliferation of KATO III cells in basal condition and in response to EGF (Abstract).
Gastroenterology
102:
A621,
1993.
12.
Garcia-Rodriguez, L. A.,
and
H. Jick.
Risk of upper gastrointestinal bleeding and perforation with individual non-steroidal anti-inflammatory drugs.
Lancet
343:
769-772,
1994[Medline].
13.
Glarborg Jorgensen, T.,
U. S. Weis-Fogh,
H. H. Nielsen,
and
H. P. Olesen.
Salicylate- and aspirin-induced uncoupling of oxidative-phosphorylation in mitochondria isolated from mucosal membrane of stomach.
Scand. J. Clin. Lab. Invest.
36:
649-654,
1976[Medline].
14.
Glarborg Jorgensen, T.,
U. S. Weis-Fogh,
and
H. P. Olesen.
The influence of acetylsalicylic acid (aspirin) on gastric mucosal content of energy rich phosphate bond.
Scand. J. Clin. Lab. Invest.
36:
771-777,
1976[Medline].
15.
Graham, D. Y.
Nonsteroidal anti-inflammatory drugs, Helicobacter pylori, and ulcers. Where do we stand?
Am. J. Gastroenterol.
91:
2080-2086,
1996[Medline].
16.
Graham, D. Y.
Prevention of gastroduodenal injury induced by chronic nonsteroidal anti-inflammatory drug therapy.
Gastroenterology
96:
675-681,
1989[Medline].
17.
Henry, D.,
A. Dobson,
and
C. Turner.
Variability in the risk of major gastrointestinal complications from nonaspirin nonsteroidal anti-inflammatory drugs.
Gastroenterology
105:
1078-1088,
1993[Medline].
18.
Johnson, J. E.
Methods for studying cell death and viability in primary neuronal cultures.
Methods Cell Biol.
46:
254-261,
1995.
19.
Kao, J. P. Y.
Practical aspects of measuring [Ca2+] with fluorescent indicators.
Methods Cell Biol.
40:
155-181,
1994[Medline].
20.
Kao, J. P. Y.,
A. T. Harootunian,
and
R. Y. Tsein.
Photochemically generated cytosolic calcium pulses and their detection by fluo 3.
J. Biol. Chem.
264:
8179-8184,
1989
21.
Kauffman, G. L., Jr.
Putative mediators of adaptive cytoprotection.
Prostaglandins
41:
201-205,
1991[Medline].
22.
Kokoska, E. R.,
G. S. Smith,
A. B. Wolff,
Y. Deshpande,
C. L. Rieckenberg,
A. Banan,
and
T. A. Miller.
The role of calcium in adaptive cytoprotection and cell injury induced by deoxycholate in human gastric cells.
Am. J. Physiol.
275 (Gastrointest. Liver Physiol. 38):
G322-G338,
1998
23.
Kokoska, E. R.,
G. S. Smith,
C. L. Rieckenberg,
Y. Deshpande,
A. Banan,
and
T. A. Miller.
Adaptive cytoprotection against deoxycholate-induced injury in human gastric cells in vitro: is there a role for endogenous prostaglandins?
Dig. Dis. Sci.
43:
806-815,
1998[Medline].
24.
Langenbach, R.,
S. G. Morham,
H. F. Tiano,
C. D. Loftin,
B. I. Ghanayem,
P. C. Chulada,
J. F. Mahler,
C. A. Lee,
E. H. Goulding,
K. D. Kluckman,
H. S. Kim,
and
O. Smithies.
Prostaglandin synthase 1 gene disruption in mice reduces arachidonic acid-induced inflammation and indomethacin-induced gastric ulceration.
Cell
83:
483-492,
1995[Medline].
25.
Lanza, F. L. A review of gastric ulcer and
gastroduodenal injury in normal volunteers receiving aspirin and other
nonsteroidal anti-inflammatory drugs. Scand.
J. Gastroenterol. 24 Suppl.: 24-31, 1989.
26.
Miller, T. A.
Protective effects of prostaglandins against gastric mucosal damage: current knowledge and proposed mechanisms.
Am. J. Physiol.
245 (Gastrointest. Liver Physiol. 8):
G601-G623,
1983
27.
Minta, J. O.,
and
M. D. Williams.
Some nonsteroidal anti-inflammatory drugs inhibit the generation of superoxide anions by activated polymorphs by blocking ligand-receptor interactions.
J. Rheumatol.
12:
751-757,
1985[Medline].
28.
Orrenius, S.,
D. J. McConkey,
G. Bellomo,
and
P. Nicotera.
Role of Ca2+ in toxic cell killing.
Trends Pharmacol. Sci.
10:
281-285,
1989[Medline].
29.
Preston, S. J.,
M. H. Arnold,
E. M. Beller,
P. M. Brooks,
and
W. W. Buchanan.
Comparative analgesic and anti-inflammatory properties of sodium salicylate and acetylsalicylic acid (aspirin) in rheumatoid arthritis.
Br. J. Clin. Pharmacol.
27:
607-611,
1989[Medline].
30.
Rainsford, K. D.,
and
C. Willis.
Relationship of gastric mucosal damage induced in pigs by anti-inflammatory drugs to their effects on prostaglandin production.
Dig. Dis. Sci.
27:
624-635,
1982[Medline].
31.
Redfern, J. S.,
E. Lee,
and
M. Feldman.
Effect of indomethacin on gastric mucosal prostaglandins in humans. Correlation with mucosal damage.
Gastroenterology
92:
969-977,
1987[Medline].
32.
Schepp, W.,
B. Steffen,
V. Schusdziarra,
and
M. Classen.
Calcium, calmodulin, and cyclic adenosine monophosphate modulate prostaglandin E2 release from isolated human gastric mucosal cells.
J. Clin. Endocrinol. Metab.
63:
886-891,
1986[Abstract].
33.
Sei, Y.,
and
P. K. Arora.
Quantitative analysis of calcium (Ca2+) mobilization after stimulation with mitogens or anti-CD3 antibodies.
J. Immunol. Methods
137:
237-244,
1991[Medline].
34.
Smalley, W. E.,
W. A. Ray,
J. R. Daugherty,
and
M. R. Griffin.
Nonsteroidal anti-inflammatory drugs and the incidence of hospitalizations for peptic ulcer disease in elderly persons.
Am. J. Epidemiol.
141:
539-545,
1995[Abstract].
35.
Smith, G. S.,
S. I. Myers,
L. L. Bartula,
and
T. A. Miller.
Adaptive cytoprotection against alcohol in the rat stomach is not due to increased prostanoid synthesis.
Prostaglandins
41:
207-223,
1991[Medline].
36.
Tepperman, B. L.,
and
B. D. Soper.
Effect of extracellular Ca2+ on indomethacin-induced injury to rabbit dispersed gastric mucosal cells.
Can. J. Physiol. Pharmacol.
72:
63-69,
1994[Medline].
37.
Tepperman, B. L.,
S. Y. Tan,
and
B. J. R. Whittle.
Effects of calcium-modifying agents on integrity of rabbit isolated gastric mucosal cells.
Am. J. Physiol.
261 (Gastrointest. Liver Physiol. 24):
G119-G127,
1991
38.
Thibault, N.,
and
F. Ballet.
Effect of bile acids on intracellular calcium in isolated rat hepatocyte couplets.
Biochem. Pharmacol.
45:
289-293,
1993[Medline].
39.
Tripp, M. A.,
and
B. L. Tepperman.
Role of calcium in nitric oxide-mediated injury to rat gastric mucosal cells.
Gastroenterology
111:
65-72,
1996[Medline].
40.
Unno, N.,
M. J. Menconi,
M. Smith,
and
M. P. Fink.
Nitric oxide mediates interferon-gamma-induced hyperpermeability in cultured human intestinal epithelial monolayers.
Crit. Care Med.
23:
1170-1176,
1995[Medline].
41.
Vane, J. R.
Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs.
Nature
231:
232-235,
1971.
42.
Wallace, J. L.,
K.-E. Arfors,
and
G. W. McKnight.
A monoclonal antibody against the CD18 leukocyte adhesion molecule prevents indomethacin-induced gastric damage in the rabbit.
Gastroenterology
100:
878-888,
1991[Medline].
43.
Wallace, J. L.,
C. M. Keenan,
and
D. N. Granger.
Gastric ulceration induced by nonsteroidal anti-inflammatory drugs is a neutrophil-dependent process.
Am. J. Physiol.
259 (Gastrointest. Liver Physiol. 22):
G462-G467,
1990
44.
Wallace, J. L.,
D. M. McCafferty,
L. Carter,
W. McNight,
and
D. Argentieri.
Tissue selective inhibition of prostaglandin synthesis in rat by tepoxalin: anti-inflammatory without gastropathy.
Gastroenterology
105:
1630-1636,
1993[Medline].
45.
Walus, K. M.,
J. D. Fandacaro,
and
E. D. Jacobson.
Hemodynamic and metabolic changes during stimulation of ileal motility.
Dig. Dis. Sci.
26:
1069-1077,
1980.
46.
Whittle, B. J. R.
Temporal relationship between cyclooxygenase inhibition, as measured by prostacyclin biosynthesis, and the gastrointestinal damage induced by indomethacin in the rat.
Gastroenterology
80:
94-98,
1981[Medline].
47.
Whittle, B. J. R.
Mechanisms underlying gastric mucosal damage induced by indomethacin and bile salts, and the actions of prostaglandins.
Br. J. Pharmacol.
60:
455-460,
1977[Abstract].
48.
Whittle, B. J. R.,
J. Lopez-Belmonte,
and
S. Moncada.
Regulation of gastric mucosal integrity by endogenous nitric oxide: interactions with prostanoids and sensory neuropeptides in the rat.
Br. J. Pharmacol.
99:
607-611,
1990[Abstract].
49.
Yamada, T.,
E. Deitch,
R. D. Specian,
M. A. Perry,
R. B. Sartor,
and
M. B. Grisham.
Mechanisms of acute and chronic intestinal inflammation induced by indomethacin.
Inflammation
17:
641-662,
1993[Medline].
50.
Zurier, R. B.,
and
F. Quagliata.
Effect of prostaglandin E1 on adjuvant arthritis.
Nature
234:
304-305,
1971[Medline].