Resistance to apoptosis is a mechanism of adaptation of rat stomach to aspirin

Barbara M. Alderman, Gregory A. Cook, Mary Familari, Neville D. Yeomans, and Andrew S. Giraud

University of Melbourne, Department of Medicine, Western Hospital, Footscray, Victoria 3011, Australia


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Adaptation of the gastric mucosa to nonsteroidal anti-inflammatory drug-induced injury is a well-documented phenomenon, but the mechanisms are not known. We investigated whether changes in stress protein expression and apoptosis play roles in adaptation of rat stomach to aspirin. RT-PCR and Western blotting techniques were used to analyze mRNA and protein expression of HSP72 and HSP90 and cleavage of caspase 3 protein. Apoptosis was detected by the TUNEL method and quantified. HSP72 mRNA and protein expression was unchanged in adapted mucosa, whereas HSP90 mRNA and protein levels decreased. Caspase 3 protein was activated, and the number of apoptotic cells increased in mucosa after one aspirin dose. However, in adapted mucosa after aspirin, activated caspase 3 and the number of apoptotic cells had returned to basal levels. Induction of the stress response was found not to be a mechanism of mucosal adaptation against multiple doses of aspirin. Our results lead us to propose instead that resistance to aspirin-induced apoptosis plays a role in the protective phenomenon of adaptation.

stress proteins; caspase 3; nonsteroidal anti-inflammatory drugs; gastric mucosa


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

NONSTEROIDAL ANTI-INFLAMMATORY drugs (NSAIDs) are used extensively to treat a wide variety of medical conditions. Aspirin is also prescribed as a prophylactic treatment against coronary artery disease and stroke. Although NSAIDs have many beneficial effects, each dose is known to cause measurable damage to the gastric mucosa (11). A phenomenon called adaptation has been recognized for more than 25 years (21, 45) and is characterized by a marked reduction in gastric mucosal injury after repeated administration of a damaging agent over several days (45). The mechanisms of adaptation are not known, and many of the publications reporting changes in NSAID-adapted mucosa are contradictory. The most consistent findings to date are that adapted stomachs have increased expression of the growth factor transforming growth factor (TGF)-alpha (9, 36), increased infiltration of granulocytes (24, 44, 47), and increased cellular proliferation (10, 24, 26, 49, 51).

The stress (or heat shock) proteins are a large family of proteins that are upregulated in cells after they have been damaged by a variety of agents, including heat, toxic metals, and alcohol. The stress proteins are also expressed constitutively and are known to play a fundamental role in a number of biological processes, including the proper folding and translocation of many cellular proteins. One of the stress proteins, HSP90, is generating much interest as its importance in the control of signal transduction pathways (by assisting shape changes in some kinases and steroid hormone receptors) is unraveled (1, 8, 32, 34, 38).

Prior induction of heat shock proteins has been widely demonstrated to protect cells against damage either by the same agent that induced the stress response or by other damaging agents (22). In a similar way, gastric mucosa that has adapted to NSAIDs is not only protected from damage caused by the same NSAID but is also protected against damage caused by other gastric mucosal toxins (4, 41). For these reasons, induction of the stress response has been suggested to be one of the ways by which the gastric mucosa might protect itself against ongoing damage (14, 20, 31).

We postulated that induction of stress proteins might mediate mucosal adaptation to NSAIDs. This article examines whether any changes occur in the expression of two major members of the stress protein family, HSP72 and HSP90, during gastric adaptation to aspirin in rats.

To date, several studies have reported the contribution of apoptosis to NSAID-induced damage to the gastric mucosa. After a single damaging dose of an NSAID, including aspirin, there is an increase in the number of cells undergoing apoptosis in the stomach (42, 50). We wanted to investigate whether changes in apoptosis occur in the mucosa after adaptation to aspirin. We measured two parameters of apoptosis after single and multiple aspirin doses: 1) TUNEL staining to detect cells with nuclei characteristic of programmed cell death and 2) caspase 3 cleavage from the precursor form to the active cell death effector.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Adaptation of rat stomach to aspirin. Male Long Evans rats weighing 220 ± 45 g were maintained on a controlled feeding regimen to enable the animals to be dosed daily in a fasted state. Rats were dosed orally by gavage at 24-h intervals with either 1% methylcellulose vehicle (pH 5.0) or aspirin suspended in 1% methylcellulose vehicle (pH 3.1). The dosing schedule was as follows: 6 daily doses of 1% methylcellulose vehicle (control); 5 daily doses of vehicle and 1 dose of 120 mg/kg aspirin (single-dose); or 6 daily doses of 120 mg/kg aspirin (adapted). Four hours after the last dose, rats were killed under Fluothane anesthesia and the stomachs were immediately removed, opened along the lesser curvature, and pinned out flat. They were gently rinsed with saline and photographed. The fundic (i.e., corpus) portion of each stomach was divided into sections for histology and extraction of RNA and protein.

Assessment of macroscopic damage. Macroscopic gastric mucosal injury was measured using a digital planimeter to trace areas of damage on each photograph (with an observer blinded to treatment groups). Damage was expressed as a percentage of the total fundic area and was defined as hemorrhagic regions of the mucosa that did not clear after rinsing.

Histology. Rat gastric body sections were immersion fixed in phosphate-buffered formalin. The tissues were then dehydrated and embedded in wax. Four-micrometer sections were cut, dewaxed, and stained with hematoxylin and eosin.

Stress protein expression in rat gastric body explant culture. Rat stomachs were taken immediately after death, and small sections (~0.1 cm2) of gastric body were placed immediately into 37°C standard DMEM containing 10% FCS. The stomach samples were incubated at either 37°C (control) or 42°C (heat shock) for 1.5 h. RNA was extracted, and RT-PCR was performed and quantified for HSP72, HSP90, and the housekeeper gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH; parameters described below).

RT-PCR. Total RNA was extracted from rat gastric body using the acid-phenol method (5). Three micrograms of total RNA was reverse transcribed into single-stranded cDNA using 200 units Moloney murine leukemia virus (Promega) primed with 0.3 µg oligo(dT) in 40 mM Tris · HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol, and 1 mM dNTPs in a 40-µl volume at 37°C. Rat sense and antisense primer sequences were designed on the basis of GenBank entries and are as follows: HSP72, sense 5'-TCGAGGAGGTGGATTAGAG, antisense 5'-GGGATGCAAGGAAAAAAC; HSP90, sense 5'-ACATCATCCCCAACCCTC, antisense 5'- TCCACCAGCAGAAGACTCC; GAPDH, sense 5'-TGTCAGCAATGCATCCTGCA, antisense 5'-CTGTTGAAGTCACAGGAGAC. The optimum number of cycles for PCR of each primer pair was determined by adding fourfold serial dilutions of the cDNA to the PCR reactions until a linear response was obtained. The optimum number of cycles for each primer pair were found to be HSP72, 29 cycles, HSP90, 24 cycles, and GAPDH, 26 cycles. Parameters for each cycle were as follows: denaturation at 95°C for 40 s, annealing at primer-specific temperature (HSP72, 57°C; HSP90, 61°C; GAPDH, 45°C) for 40 s, and extension at 72°C for 1 min. Reaction conditions were 10 mM Tris · HCl, 50 mM KCl, 2 mM MgCl2, 200 µM dNTPs, 25 pmol each of sense and antisense primer, and 1.25 units Taq polymerase (Promega) in a 50-µl volume. Control reactions to ensure that RNA samples and the RT-PCR reagents did not contain contaminating DNA were performed for each sample and primer pair. PCR products were resolved on 2% agarose gels containing ethidium bromide and were visualized under ultraviolet light. Photographs of each gel were scanned, and the intensities of the bands were measured using the ImageMASTER software system (Pharmacia Biotech).

Western blotting. Stomach sections were weighed and homogenized in 10 volumes of ice-cold 1% NP-40 in 50 mM Tris (pH 8.8) buffer. The homogenates were centrifuged, and the supernatants were transferred into sterile tubes. Fifteen-microgram protein aliquots of each sample, as determined by the Coomassie blue method, were resolved on 15% SDS-polyacrylamide gels and transferred onto nitrocellulose membranes. Membranes were blocked with BSA before being incubated with primary antibody overnight at 4°C [antibodies used were kind gifts from Dr. Susan Lindquist of University of Chicago (HSP90), Dr. David Walsh of University of Sydney (monoclonal rat HSP72), and Dr. Don Nicholson of Merck-Frosst (caspase 3)]. Membranes were rinsed and then incubated for 1 h with peroxidase-conjugated secondary antibody. Immunoreactive proteins were visualized using the enhanced chemiluminescence system (Amersham). The films were scanned, and the intensities of the bands were measured using the ImageMASTER software system (Pharmacia Biotech).

In situ detection and quantification of apoptotic cells. Apoptotic cells were detected using a modified terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay (Promega DeadEnd colorimetric apoptosis detection system). Horseradish peroxidase-labeled streptavidin was bound to incorporated biotinylated nucleotides, and positively labeled cells were detected using hydrogen peroxide and diaminobenzidine. Nuclei that stained dark brown and showed morphological signs indicative of apoptosis (i.e., condensed chromatin and/or fragmented nuclei) were considered positive. Apoptotic cells were counted (observer blinded to treatment groups) in two separate gastric mucosal compartments: the surface epithelial compartment and the glandular compartment, the junction of which was defined by the highest parietal cell. Ten areas per compartment were counted for each sample and expressed as the number of apoptotic cells per 105 µm2. Only areas of intact surface epithelium were included in the assessment of apoptosis; areas containing lesions were omitted due to the increased density of staining and difficulty of accurately determining whether nuclei were exhibiting characteristics indicative of apoptosis. Cells that were detached from the epithelium were not counted.

Statistical analysis of data. All data are expressed as means ± SE. Comparisons between groups were performed using the one-way ANOVA followed by the Student-Newman-Keuls test or (where parametric tests were inappropriate) the Kruskal-Wallis one-way ANOVA on ranks followed by the Dunnett's method of all pairwise multiple comparison.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Macroscopic damage. Adaptation of the gastric mucosa to aspirin is characterized by a decrease in the amount of damage to the mucosa after successive doses of this NSAID. Figure 1 compares the level of macroscopic damage in the gastric mucosa of control (6 daily doses of 1% methylcellulose vehicle), single-dose (5 daily doses of vehicle and 1 dose of 120 mg/kg aspirin), and adapted (6 daily doses of 120 mg/kg aspirin) rats 4 h after the last dose. Both control (0.04 ± 0.03) and adapted rats (0.35 ± 0.17) sustained significantly less damage than rats exposed to a single dose of aspirin (1.30 ± 0.33). Overall, there was a 73% reduction in damage in the adapted group (P < 0.05). Two of nine rats in this group showed moderate adaptation, whereas the remaining seven were markedly protected (mean reduction 93%).


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Fig. 1.   Comparison of macroscopic damage in rat stomach given either 6 daily doses of 1% methylcellulose vehicle (control), 5 daily doses of vehicle and 1 dose of 120 mg/kg aspirin (single dose), or 6 daily doses of 120 mg/kg aspirin (adapted). Stomachs were excised 4 h after last dose. Damage was assessed by digital planimetry (2 observers, 1 blinded). Results are expressed as means ± SE (n = 9). * P < 0.05 compared with control; # P < 0.05 compared with single dose (Kruskal-Wallis one-way ANOVA on ranks followed by Dunnett's method of all pairwise multiple comparisons).

Adaptation of the stomach to aspirin is not only characterized by a reduction in macroscopic damage caused by successive doses but also by decreased severity and depth of lesions in the mucosa when assessed histologically. Figure 2 shows representative sections of control (A), single-dose (B), and adapted (C) gastric mucosa 4 h after the last dose of vehicle or aspirin. After a single dose of aspirin (Fig. 2B), acute erosions are present in some areas of the mucosa and are characterized by almost complete ablation of the surface epithelium and loss of gastric pit structure. The damage caused by aspirin in the adapted stomach (Fig. 2C) is far more superficial, with some loss of surface epithelial cells into the lumen but with the gastric pits remaining essentially intact.


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Fig. 2.   Histology of representative sections of rat gastric mucosa 4 h after last dose of vehicle or aspirin in control (A), single-dose (B), and adapted (C) rats. Sections are stained with hematoxylin and eosin. Magnification = ×200.

RT-PCR of stress proteins in normal and heated stomach explant cultures. Stomach explant cultures incubated at either 37°C or 42°C were used to determine the heat inducibility of HSP72 and HSP90 in rat gastric mucosa. After RNA extraction and RT-PCR, the products were resolved on an agarose gel alongside a 100-bp ladder to confirm that the sizes of the products were as predicted: HSP72, 307 bp; HSP90, 269 bp; and GAPDH, 425 bp (data not shown). Quantification of the HSP72 and HSP90 bands (standardized to GAPDH) is shown in Fig. 3, and as expected HSP72 gene transcription increased in the gastric mucosa after incubation at 42°C. No significant change in HSP90 gene transcription was observed between the control and heat-shocked samples.


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Fig. 3.   Quantification of RT-PCR products of stress proteins HSP72 and HSP90 in rat gastric body explant culture exposed to either 37°C (control) or 42°C (heat shock) for 1.5 h. Results are expressed as means ± SE (n = 4) standardized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). * P < 0.05 compared with control group.

Analysis of HSP72 mRNA and protein expression. Levels of HSP72 mRNA and protein in control, single-dose, and adapted gastric mucosa 4 h after the last dose of vehicle or aspirin were assessed by semiquantitative RT-PCR and Western blotting, respectively (Fig. 4). The level of HSP72 mRNA in adapted stomach was unchanged compared with the control and single-dose groups (Fig. 4A). HSP72 protein expression (Fig. 4B) in adapted stomach was also unchanged compared with the control and single-dose groups.


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Fig. 4.   Comparison of HSP72 mRNA and protein levels in control, single-dose, and adapted rat stomachs 4 h after last dose of vehicle or aspirin (see Fig. 1). A: RT-PCR of stomach total RNA comparing HSP72 message levels between groups as indicated. Optical densitometric (OD) values are expressed as means ± SE (n = 9). Three examples of PCR products from each group are shown above corresponding graph. B: Western blot analysis comparing HSP72 protein levels between groups as indicated. Values are expressed as means ± SE (n = 9). Two examples of immunoreactive proteins from each group are shown above corresponding graph.

Analysis of HSP90 mRNA and protein expression. HSP90 mRNA and protein levels in the gastric mucosa 4 h after the last dose of either vehicle (control) or aspirin (single dose, adapted) were assessed using semiquantitative RT-PCR and Western blotting, respectively (Fig. 5). After a single dose of aspirin, no change in HSP90 mRNA expression was detected compared with control. Adapted stomachs showed reduced HSP90 mRNA expression (0.64 ± 0.03) compared with both the single-dose stomachs (0.79 ±0.06) and controls (0.89 ± 0.05) (Fig. 5A). In accordance with this, HSP90 protein was markedly reduced in adapted stomachs after aspirin (426 ± 55) compared with both single-dose stomachs (921 ± 73) and controls (1067 ± 74) (Fig. 5B).


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Fig. 5.   Comparison of HSP90 mRNA and protein levels in control, single-dose, and adapted stomachs 4 h after last dose of vehicle or aspirin (n = 9 per group). A: RT-PCR of stomach total RNA comparing HSP90 message levels between groups as indicated. # P < 0.05 compared with control and single-dose groups. Three examples of PCR products from each group are shown above corresponding graph. B: Western blot analysis comparing HSP90 protein levels between groups as indicated. # P < 0.05 compared with control and single-dose groups. Two examples of immunoreactive proteins from each group are shown above corresponding graphs.

Analysis of intact and cleaved caspase 3 levels. Concentrations of both the intact (inactive precursor) and cleaved (active enzyme) products of the apoptosis-associated caspase 3 protein were measured by Western blotting (Fig. 6). After aspirin, the amount of the 32-kDa precursor form of caspase 3 in adapted stomach (373 ± 64) was reduced compared with both single-dose stomachs (885 ± 112) and controls (981 ± 80) (Fig. 6A). The changes observed in the levels of the 17-kDa active caspase 3 were quite different from that of the 32-kDa precursor form. The amount of active caspase 3 in normal mucosa given a single dose of aspirin (660 ± 80) increased compared with mucosa given vehicle only (423 ± 79). However, in adapted stomachs the amount of active caspase 3 after aspirin (368 ± 60) remained approximately the same as that in control stomachs that received vehicle alone (Fig. 6B).


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Fig. 6.   Western blot analysis of caspase 3 protein levels in gastric mucosa of control, single-dose, and adapted rats (n = 9 per group). Stomachs were excised 4 h after last dose of vehicle or aspirin. A: comparison of 32-kDa caspase 3 precursor protein levels between groups as indicated. # P < 0.05 compared with control and single-dose groups. B: comparison of 17-kDa caspase 3 active protein levels between groups as indicated. * P < 0.05 compared with control; # P < 0.05 compared with single dose. Examples of immunoreactive proteins from each group are shown above corresponding graphs.

Quantification of apoptosis. Apoptotic cells in the control, single-dose, and adapted stomachs were detected using a modified TUNEL assay and quantified as outlined in MATERIALS AND METHODS. In all three sample groups, the majority of apoptotic cells were located in the surface epithelial compartment of the gastric mucosa. Apoptotic cells found in the glandular compartment of the mucosa were located mainly in the area immediately below the first parietal cell or at the base of the glands. Sections showing cells with morphological signs indicative of apoptosis are shown in Fig. 7. Quantification revealed that at 4 h after a single dose of aspirin the number of apoptotic cells (per 105 µm2) increased in both the surface epithelial (111 ± 11) (Fig. 8A) and glandular compartments (4.3 ± 0.5) (Fig. 8B) compared with controls (76 ± 14 and 2.8 ± 0.2, respectively). In adapted mucosa the number of apoptotic cells detected in both the surface epithelial (66 ± 5) and glandular compartments (3.5 ± 0.3) was very similar to that of controls and substantially lower than after a single aspirin dose, particularly in the surface epithelial compartment.


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Fig. 7.   Sections of rat gastric mucosa stained using TUNEL method. A: surface epithelial compartment; B: glandular compartment. Cells showing morphological signs indicative of apoptosis are indicated by arrows. Magnification = ×400.



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Fig. 8.   Quantification of apoptosis in control, single-dose, and adapted gastric mucosa 4 h after last dose of vehicle or aspirin (n = 9 per group). A: comparison of apoptosis in surface epithelial compartment of gastric mucosa between groups as indicated. B: comparison of apoptosis in glandular compartment of gastric mucosa between groups as indicated. * P < 0.05 compared with control; # P < 0.05 compared with single dose.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The aim of the studies described here was to ascertain whether changes in expression of the stress proteins HSP72 and HSP90 occur in rat stomach after single or multiple oral doses of the NSAID aspirin and therefore contribute to the cytoprotective phenomenon of adaptation. The stress (or heat shock) proteins are both constitutively and inducibly expressed in all tissues. Their inducibility is dependent on many factors, including cell type, tissue environment, type of stressor, and the level of basal constitutive expression. The expression and inducibility of the HSP72 stress protein also varies considerably between in vitro preparations of cells and whole tissue in vivo. In this study we have demonstrated that inducible HSP72 is expressed in rat stomach under basal conditions and that its expression can be modestly increased (almost twofold) by heat shock. This result conforms with other in vivo experiments in which induction of HSP72 after various forms of stress, such as whole body hypo/hyperthermia and water restraint, results in only a modest two- to fourfold increase in HSP72 expression in a variety of tissue types, including gut (7, 14, 28, 46). This is in contrast with most in vitro cell culture studies (and our unpublished data), which demonstrate almost no basal expression and large increases in expression of HSP72 after stress. We found that constitutive expression of HSP90 in the stomach is also high but it is not inducible after a short exposure to heat. The detection of high tissue concentrations of both HSP72 mRNA and protein in undamaged gastric mucosa suggests that the rat stomach has an augmented and sustained inducible HSP70 response compared with other tissues that lack an ongoing harsh extracellular environment, such as that of the gastric lumen. This conclusion is supported by previous studies in our laboratory, which have shown that a major site of HSP72 expression in the stomach is the gastric epithelium (50).

Although basal HSP72 expression is high in the stomach, and can be modestly elevated by heat shock, no increase in gastric HSP72 mRNA or protein was detected in adapted animals or those given a single damaging dose of aspirin, compared with controls. Although pretreatment with an NSAID has been shown to augment the stress response after heat shock (2, 12), it has been demonstrated in systems as diverse as yeast and fruit flies (17, 23, 48) that salicylate itself does not increase HSP72 gene transcription or protein levels. Our observations suggest that the same may be true for the stomach.

In contrast to HSP72 expression, single or multiple (adapting) doses of aspirin resulted in a progressive reduction in expression of HSP90 mRNA and protein, so that steady-state synthesis was reduced by ~60% in the adapted group. Little is known of the factors that regulate HSP90 gene expression, and to our knowledge this is the first report of inhibition of HSP90 in an in vivo system. HeLa cells transfected with the heat shock transcription factor (HSF4) were found to synthesize less HSP90 while maintaining HSP72 expression, suggesting that HSF4 may contribute to the transcriptional control of this stress protein (30). HSP90 inhibition in a monoblastoid cell line has implicated it in cell cycle control and differentiation (15), and these same cells were shown to be resistant to TNF-alpha -induced apoptosis (16). Aspirin-induced damage in the gastric mucosa has been shown to be TNF-alpha -dependent, with both types of TNF-alpha receptors, R1 and R2, independently involved in modulating caspase activity (13). Since HSP90 is known to play an important role in receptor signaling, with reduced HSP90 levels inhibiting the activation of steroid receptors (32), it is possible that the decreased HSP90 level in adapted stomach acts to inhibit activation of the TNF-alpha receptors, thereby reducing subsequent caspase cleavage and apoptosis.

Apoptosis, or programmed cell death, plays an important role in maintaining normal tissue homeostasis in most organs. In the stomach, apoptotic processes serve to maintain cell number by ensuring that cell division is balanced by cell death (19). The pathogenesis of gastric mucosal injury induced by damaging agents such as NSAIDs, ethanol, and Helicobacter pylori, has been correlated with increased apoptosis (18, 27, 29, 33, 40, 42, 52), and induction of apoptosis has been proposed as the mechanism by which NSAIDs prevent colorectal tumorigenesis (3, 40). Conversely, attenuation of gastric mucosal injury has been shown to be accompanied by decreased apoptosis (18, 33, 42, 52). In addition, it is known that induction of TGF-alpha correlates with reduced programmed cell death (35, 37, 39), and we and others have shown that the epidermal growth factor receptor ligand TGF-alpha is induced in the adapted gastric mucosa (9, 36). Together, these data suggest that acute damage produces increased apoptosis, and adaptation might lead to inhibition of apoptotic processes.

To proceed, programmed cell death requires the activation of the intracellular protease family of caspases (6). These proteolytic enzymes participate in a cascade in which upstream members cleave and activate those immediately downstream, which in turn cleave substrate proteins, leading to the biochemical and morphological changes characteristic of apoptosis. Caspase 3 (also known as CPP32 or apopain) is one of the more downstream caspases of the apoptotic cascade and is activated by the cleavage of an inert 32-kDa precursor to yield a 17-kDa active enzyme. Increased expression of the active enzyme correlates with an elevated incidence of apoptotic cell death. In the stomach, immunohistochemical analysis of caspase 3 expression in vivo has shown that caspase 3 is highly expressed by foveolar cells lining the neck region of the gastric pits, but only a low level of caspase 3 expression was found in the parietal and chief cells (25). Recent studies have reported that aspirin-induced apoptosis in the gastric mucosa is mediated by TNF-alpha -dependent activation of caspases, including caspase 3 (13, 43). Our results revealed that the level of active 17-kDa caspase 3 enzyme is increased almost twofold 4 h after a single damaging dose of aspirin, whereas the level of the 32-kDa zymogen is modestly decreased. However, in adapted stomachs after aspirin, there was no increase in the level of active caspase 3, whereas expression of the inactive zymogen was found to have depleted by ~70%. This observation suggests that synthesis of the caspase 3 precursor is chronically inhibited in aspirin adaptation, perhaps contributing to increased resistance to apoptosis.

To further test whether adaptation may induce resistance to apoptosis, the numbers of apoptotic nuclei in gastric mucosal sections from control, single-dose, and aspirin-adapted rats were quantified by TUNEL staining and cell morphology. Quantification of apoptotic cells was carried out on randomly chosen regions of multiple sections; however, areas of frank erosion were excluded due to the difficulty in distinguishing between cells undergoing programmed cell death or necrosis and because apoptotic cells were likely to have been lost from the eroded surface. It is possible therefore that the magnitude of the increase in apoptotic areal density after a single aspirin dose was actually underestimated in this study. However, in adapted stomachs in which erosions were few, any such underestimation would have been much less. By TUNEL staining, the majority of apoptotic nuclei were located in the surface/pit compartment, consistent with the reported morphological analysis of caspase 3 expression (25) and with the fact that surface and pit epithelial cells have a faster turnover rate than any other cell in the gastric epithelium. In both the surface epithelial and glandular compartments, substantially more apoptotic cells were observed 4 h after a single aspirin dose compared with controls. This result is consistent with reports that gastric epithelial cell apoptosis after NSAIDs is readily detectable within several hours of dosing using a number of detection methods, including DNA laddering (13, 43).

In adapted mucosa after aspirin, the number of apoptotic cells was equivalent to the number in control mucosa that received vehicle alone (correlated with the return of active caspase 3 to the control level). The reduction in apoptosis after aspirin in adapted stomach was more marked in the surface epithelial compartment.

The onset and offset of adaptation in rats has been shown to take ~3-5 days (41, 45), which correlates closely with mammalian surface epithelial cell turnover time. It has been proposed that adaptation may be attributable to the generation of new surface epithelial cells that appear after damage and may have developed the capacity to resist further damage (21, 41). The results of the quantification of apoptotic cells in adapted mucosa presented here suggests that this hypothesis is credible since the most significant decrease in apoptosis after aspirin in adapted stomach was demonstrated in the surface epithelial compartment.

To date, investigation into the possible mechanisms of gastric adaptation to injury has focused on changes in growth factor expression and the subsequent enhancement of wound healing. However, it is becoming increasingly clear that gastric mucosal defense and repair mechanisms are complex, and while enhanced wound healing undoubtedly contributes to adaptation, changes in the expression and associations of intracellular proteins like the stress proteins and caspases are also likely to be involved. Combining the studies reported here with what we and others have previously reported on adaptation, we conclude that a major contributor to mucosal adaptation to chronic aspirin application may be the development of resistance to programmed cell death.


    ACKNOWLEDGEMENTS

This work was supported by a grant from the National Health and Medical Research Council of Australia.


    FOOTNOTES

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 and other correspondence: A. S. Giraud, Dept. of Medicine, Western Hospital, Footscray, Victoria, 3011 Australia (E-mail: a.giraud{at}medicine.unimelb.edu.au).

Received 2 September 1999; accepted in final form 5 January 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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