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MUCOSAL BIOLOGY

Ethanol induced NF-B activation protects against cell injury in cultured rat gastric mucosal epithelium

Department of Surgery, Helsinki University Central Hospital, Helsinki, Finland

Submitted 20 June 2006 ; accepted in final form 6 March 2007

	ABSTRACT

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Ethanol is a well-established irritant inducing inflammation in gastric mucosa, but the effects at the cellular level remain unclear. This study investigates NF-B activation in gastric mucosal cells by ethanol and assesses the effects of heat shock pretreatment in this ulcerogenic situation. Rat gastric mucosal epithelia were exposed to ethanol for different time periods. Heat shock was induced by incubating the cells at 42°C for 1 h prior to the experiments. For evaluation of NF-B activation, the nuclear fraction of the cell lysates was analyzed with an EMSA or an ELISA-based assay. Caspase-3 (a promoter of apoptosis) activity was measured with a time-resolved fluorescence based assay, cell viability with a tetrazolium assay, and cell membrane integrity with a LDH assay. Ethanol (1–5%) induced NF-B activation, reaching a maximum after 3 h, and also led to moderately increased COX-2 expression. Heat shock pretreatment and the intracellular calcium chelator BAPTA were able to inhibit ethanol-induced NF-B activation. Heat shock pretreatment decreased ethanol-induced caspase-3 activation, decreased cell membrane damage, and retained cellular viability. Inhibition of NF-B activation by NEMO-binding peptide, by decreasing RelA expression, or by inhibiting COX-2 activity by CAY-14040 promoted the effects of ethanol, such as increased caspase-3 activity and decreased cell viability. In conclusion, ethanol induces NF-B activation via a calcium-dependent pathway and induces COX-2 expression. Inhibition of the NF-B activation or COX-2 activity potentiates apoptosis and cell damage induced by ethanol, suggesting a protective role for NF-B activation and COX-2 expression.

heat shock pretreatment; NF-B inhibition; gastric mucosa; caspase-3; apoptosis

Heat shock proteins are stress-induced proteins that are involved in protection against various cellular injuries. They are molecular chaperones providing protection against apoptosis and repairing damaged proteins (15). The major heat shock proteins are in the HSP27, HSP60, HSP70, and HSP90 families. The HSP27, HSP70, and HSP90 families usually inhibit apoptosis directly or indirectly by inhibiting effector caspase-3 activity (24). HSP synthesis is regulated by heat shock factor-1 (HSF-1), and in their normal state HSP70, HSP90, and other chaperones bind to transcription factor HSF-1, keeping it cytoplasmic. Under activation, the molecular chaperones prefer to bind stress-induced misfolded proteins and release HSF-1 for nuclear translocation. HSF-1 forms a trimer and binds to the heat shock consensus sequence, thereby activating a heat shock response together with nuclear machinery.

This study investigates NF-B activation by ethanol in gastric mucosal cells and assesses the effects of heat shock pretreatment in this ulcerogenic situation, because both of these factors are involved in the adaptive response to various pathological insults but their mutual interplay is not well established in gastric mucosa. We found out that ethanol induces NF-B activation and that inhibition of this activation enhances ethanol-induced apoptotic and necrotic cell death. On the other hand, heat shock pretreatment opposed the actions induced by ethanol, leading to decreased apoptotic and necrotic activity.

	MATERIALS AND METHODS

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Rat gastric mucosal cell line cells (RGM-1) were grown in cell culture medium to confluent monolayers at 37°C in a humidified atmosphere containing 5% CO₂ in air. The medium contained an equal mixture of Ham's F-12 and DMEM (DMEM-F-12) supplemented with inactivated 20% fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.125 µg/ml amphotericin. The cells were exposed for different time intervals to different ethanol concentrations at 37°C. Heat shock was induced by incubating the cells in the cell culture medium at 42°C for 1 h and thereafter at 37°C for 1 h prior to exposure to ulcerogenic agents. For evaluation of NF-B activation and IB degradation, nuclear and cytoplasmic protein extracts were prepared as described by Schreiber et al. (23), and protein concentrations were determined by colorimetric Bradford assays (3).

EMSA. A DIG Gel Shift kit (Roche, Mannheim, Germany) was used for EMSA assays. The oligonucleotide probe (59-AGTTGAGGGGACTTTCCCAGGC-39; Promega, Madison, WI) containing the B binding motif was end-labeled with DIG-ddUTP. This labeling was accomplished by adding 4 µl 5x labeling buffer, 5 mM CoCl₂, 0.05 mM DIG-ddUTP, and 2.5 U/µl terminal transferase to 3.85 pmol double-stranded oligonucleotide (in 10 µl double-distilled water) and incubating the mixture at 37°C for 30 min. These agents are included in the DIG Gel Shift kit, except the oligonucleotide. After incubation, the reaction was stopped by adding 2 µl 0.2 M EDTA (pH 8.0), and thereafter 3 µl double-distilled water was added. The final concentration of the labeled oligonucleotide in the binding reaction was 0.4 ng/µl. For the binding reaction, 9 µg of the sample protein in 20 µl binding buffer was incubated for 20 min at room temperature with 0.5 µg poly(dI-dC), 0.1 µg poly L-lysine, and 0.8 µg labeled oligonucleotide. DNA-protein complexes were resolved in a 7% nondenaturing polyacrylamide gel in a TBE buffer (22.5 mM Tris, 22.5 mM boric acid, and 0.5 mM EDTA, pH 8.3) at 100 V for 1.5–2 h at 4°C. The samples were transferred to positively charged nylon membranes (Roche) by electroblotting for 30 min with a 0.4 A constant current. After being blotted, the oligonucleotides were cross-linked for 3 min under ultraviolet light. Thereafter the membrane was washed with a solution containing 100 mM maleic acid, 150 mM NaCl, and 0.3% Tween 20 at pH 7.5 for a few minutes and the membrane was moved to the blocking solution (DIG Gel Shift kit) for 40 min. Thereafter the membrane was incubated in the blocking solution with anti-digoxigenin-AP Fab fragments (1:1,000; Roche) for 30 min, then it was washed in washing solution for 30 min. The membrane was moved to detection buffer containing 100 mM Tris·HCl and 100 mM NaCl at pH 9.5. The detection buffer was supplemented with 100 µg/ml disodium 3-{4-metoxyspiro[1,2'-(5'-chloro)tricyclo(3.3.1.1 [EC] ^3.7)decan]-4-yl}phenylphosphate (CSPD), and this CSPD working solution was applied to the membrane. The membrane was incubated for 5 min at room temperature and further at 37°C for 10 min, after which it was exposed to an X-ray film for 40 min. NF-B bands from films were quantitated by using an image-analysis program (ImageJ; National Institutes of Health, Bethesda, MD). NF-B DNA-binding activity of a sample was calculated in proportion to NF-B DNA binding of controls and TNF--stimulated tissues [100 x (D_sample – D_control)/(D_TNF- – D_control)%, where D = density].

Western blot analysis. Equal amounts of cytoplasmic protein extracts (5–10 µg) were diluted in Laemmli sample buffer with 5% mercaptoethanol. After incubation for 5 min at 95°C, the samples were resolved in 10% polyacrylamide gels in Tris-glycine-SDS buffer. The gels were transferred to nitrocellulose membranes and blocked in 5% nonfat dry milk in PBS, pH 7.5, containing 0.1% vol/vol Tween 20 (PBST-milk). Blots were then incubated overnight with polyclonal rabbit anti-IB (catalog no. sc-371), rabbit-anti-p65 (catalog no. sc-372), goat-anti-HSP27 (catalog no. sc-1048), goat-anti-HSP70 (catalog no. sc-1060), or goat anti-heme oxygenase-1 (catalog no. sc-1797) antibodies from Santa Cruz Biotechnology (Santa Cruz, CA) or rabbit anti-COX-2 (catalog no. 160126) or rabbit anti-iNOS (catalog no. 160862) from Cayman Chemical (Ann Arbor, MI) at 1:1,000 vol/vol dilution in PBST-milk at 4°C. The membranes were washed in PBST and were incubated for 1 h with horseradish peroxidase-conjugated anti-rabbit IgG (catalog no. sc-2004) or anti-goat IgG (catalog no. sc-2033) (Santa Cruz Biotechnology) at 1:5,000 vol/vol dilution in PBST-milk. After the washing, protein bands in the membranes were visualized by enhanced chemiluminescence (Pierce, Rockford, IL).

Detection of specific transcription factor activities with ELISA. A BD Mercury TransFactor assay kit (p65; BD Bioscience Clontech, Palo Alto, CA) was used to detect different transcription factors from nuclear extracts. In short, wells coated with the consensus binding sequences for the NF-B transcription factor were blocked for 15 min with the TransFactor blocking buffer. The blocking buffer was removed, and 30 µg of nuclear extract were added to the wells in TransFactor buffer and was incubated for 60 min. Positive-control cell extracts, mutant DNA-coated wells, and competitive oligo controls were used as positive and negative controls. Thereafter the wells were washed four times, incubated with the primary antibody (rabbit anti-p65, 60 min), washed four times, and incubated with the secondary antibody (anti-rabbit-horseradish peroxidase, 30 min). After the final washing, tetramethyl benzidine substrate was added to the wells for 10 min, the reaction was stopped with stopping buffer, and the absorbance was read at 655 nm with a Victor² plate reader (Perkin-Elmer, Turku, Finland).

XTT assay. The tetrazolium salt sodium 3'-[1-(phenylaminocarbonyl)-3,2.tetrazolium]-bis (4-methoxy-6-nitro) benzene sulfonic acid hydrate (XTT) is cleaved by mitochondrial dehydrogenases to formazan salts. Formazan salts can be detected with spectrophotometric absorbance measurements. Thus the mitochondrial activity can be measured with this technique, and it has been used as a cell proliferation and cytotoxicity assay (21). RGM-1 cells were grown on 96-well plates to confluent monolayers as above at 37°C in a humidified atmosphere containing 5% CO₂ in air. Test compounds of 0–10% vol/vol ethanol in the cell culture medium were added to the cells. The total volume in each well was 150 µl, including 50 µl XTT labeling mixture from the Cell Proliferation kit II (Roche), giving a final XTT concentration of 0.3 mg/ml. The plate was incubated for 2 h with test compounds and thereafter for 2 h with XTT reagents and test compounds in an incubator (37°C, 5% CO₂), and the absorbance was measured with a Victor² plate reader at 490 nm. The reference absorbance at 650 nm was subtracted from the results. The percentage of mitochondrial activity was calculated from the absorbance measurements as 100 x (cells treated with test agent – untreated cells)/(untreated cells).

LDH assay. The release of LDH from damaged cells was measured with CytoTox-ONE (Promega) homogenous membrane integrity assay. Release of LDH from damaged cells is measured by supplying lactate, NAD+, and resazurin as substrates in the presence of diaphorase. Generation of the fluorescent resorufin is proportional to the amount of LDH released. RGM-1 cells were grown on 96-well plates to confluent monolayers at 37°C in humidified atmosphere containing 5% CO₂ in air. Test compounds of 0–10% vol/vol ethanol were added to the cells in the cell culture medium for 4 h. Maximum LDH release was determined by adding 2 µl of the CytoTox-ONE lysis buffer to 11 wells for 10 min. The assay was performed in black 384-well plates by adding 25 µl of the sample supernatant and 25 µl of CytoTox-ONE reagent, after which the plate was shaken for 10 s. After 10 min of incubation, 12.5 µl CytoTox-ONE stop solution was added and the plate was again shaken for 10 s. The fluorescent signal was measured with a Victor² plate reader with 546 nm excitation and 615 nm emission. The percentage of membrane disintegration was calculated from the fluorescence intensities as 100 x (cells treated with test agent – untreated cells)/(lysed cells – untreated cells).

Caspase-3 assay. Caspase-3 activity was measured with a LANCE caspase-3 kit (Wallac, Turku, Finland). The caspase-3 substrate in this assay is a hexapeptide (CDEVDK) with a fluorescent europium chelate coupled to one end and a quencher of europium fluorescence (QSY 7) coupled to the other end via lysine. Active caspase-3 will cleave this substrate and the quencher, and europium chelate will be separated. For the activity assay, 5 µl of samples were added to 15 µl of diluted caspase-3 substrate solution (dilution in reaction buffer from the LANCE caspase-3 kit with 10 mM DTT). The final substrate concentration in the assay was 200 nM. After 2 min of slow shaking, the samples were incubated for 1 h at 37°C and were measured with time-resolved Victor² fluorometer using 340 nm excitation and 615 nm emission wavelengths. Black 384-well plates were used for this assay. The fluorescence values of blank samples were subtracted from the results obtained for the samples. Caspase inhibitor N-acetyl-Asp-Glu-Val-Asp-CHO (aldehyde) (Pharmingen, San Diego, CA) was used (100 nM) as a negative control.

Short interfering RNA experiments. Short interfering RNA (siRNA) experiments were performed to reduce p65 expression in RGM-1 cells. RGM-1 cells cultured on multiwell plates were transfected with a siGENOME SMARTpool rat RelA siRNA pool (100 nM, catalog no. M-080033-00-0010; Dharmacon, Lafayette, CO) with DharmaFECT 4 reagent. In short, 2 µM siRNA was diluted with an equal volume of DMEM-F-12, and in a separate tube Dharma FECT 4 was diluted by 1/25 with DMEM-F-12. After 5 min of incubation, these dilutions were mixed and incubated for 20 min at room temperature. The epithelia were incubated with the transfection solution for 3 days before any experiments were performed. Transfections with Dharma FECT 4 alone or with nonfunctional siRNA (100 nM; si CONTROL Non-Targeting siRNA 1, catalog no. D-001210-01-05; Dharmacon) were used as negative controls. Positive controls were obtained by transfecting with Lamin A/C siRNA (100 nM; catalog no. D-001050-01-05; Dharmacon) and following the Lamin A/C expression in these cells.

Pharmacological agents. For the inhibition of intracellular Ca²⁺ signaling in calcium-signaling experiments, the cell monolayers were loaded with BAPTA-AM (10 µM, Sigma, St. Louis, MO) for 30 min at 37°C prior to the experiments. To inhibit NF-B activation in some experiments, the monolayers were preincubated for 4 h at 37°C prior to the experiments either with NEMO-binding domain peptide (50 µM, H-DRQIKIWFQNRRMKWKK-TALDWSWLQTE-OH) or with its negative control peptide (50 µM; H-DRQIKIWFQNRRMKWKK-TALDASALQTE-OH), which is mutated (Trp to Ala) at two different locations (Calbiochem, La Jolla, CA). NEMO-binding peptide blocks the association of NEMO/IKK- with the IKK complex and thereby prevents the dissociation of IB from the NF-B complex, leading thus to inhibition of NF-B activation (14). For the inhibition of COX-2, enzyme CAY-14040 (0.1–1 µM; Cayman Chemical) was used. Lipopolysaccharides (0.05–0.5 µg/ml, Sigma) and IL-1 were used as positive controls for COX-2 and inducible nitric oxide synthase (iNOS) expression.

Statistics. The results are expressed as means ± 95% confidence intervals. Student's unpaired t-test and ANOVA were used for statistical analysis of the raw data. P values <0.05 were considered statistically significant. In multiple comparisons, the Bonferroni correction was used by lowering the critical significance probability to P < 0.05/n, where n is the number of multiple comparisons. The EMSA and Western blot gels shown are representative of at least three such gels prepared from independent experiments.

	RESULTS

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The effects of ethanol on NF-B activation and the effect of heat shock pretreatment on ethanol-induced NF-B activation. NF-B activation was measured in ethanol-exposed rat gastric epithelial cells with or without heat shock pretreatment with either an EMSA- or an ELISA-based technique. Exposure of cultured RGM-1 to 5% ethanol for 4 h induced NF-B activation (82.3 ± 5.6% of TNF--induced NF-B activation; Fig. 1). Lower ethanol concentration (4%) caused lesser activation (Fig. 1B). Intracellular calcium chelator BAPTA prevented ethanol-induced NF-B activation (from 82.3 ± 5.6% to 34.3 ± 7.0% with 5% ethanol; P = 0.03; Fig. 1C). Likewise, heat shock pretreatment also prevented ethanol-induced NF-B activation (from 82.3 ± 5.6% to 33.2 ± 3.1% with 5% ethanol; P = 0.02; Fig. 1C). The ethanol-induced NF-B activation was concentration dependent (Fig. 2A), and the maximal activation was detected at 3 h of incubation (Fig. 2B). Heat shock pretreatment induced synthesis of heat shock proteins HSP27 and HSP70 (Fig. 3). On the other hand, 3 h of ethanol exposure did not significantly induce heat shock protein synthesis (Fig. 3A). Together, these results indicate that ethanol induces the activation of the NF-B pathway in a calcium-dependent manner and that heat shock pretreatment prevents this NF-B activation.

View larger version (24K): [in this window] [in a new window]   Fig. 1. Effects of ethanol (ETOH) on NF-B activation in rat gastric mucosal (RGM-1) cells. A: EMSAs were performed to show NF-B DNA-binding activity. Cytoplasmic IB and nuclear p65 protein levels are shown below the NF-B DNA-binding activity assay. Controls were incubated in normal serum-free DMEM-Ham's F-12 (DMEM-F-12) medium at 37°C in 5% CO2. Ethanol samples were incubated for 4 or 6 h with 4 or 5% vol/vol ethanol in serum-free DMEM-F-12 medium at 37°C in 5% CO2. TNF- samples were incubated for 30 min in 10 ng/ml TNF- in serum-free DMEM-F-12 medium at 37°C in 5% CO2. Heat shock-pretreated samples (HS) were incubated in normal serum-free DMEM-F-12 medium at 42°C in 5% CO2 for 1 h, after which they were incubated for 1 h at 37°C in 5% CO2 prior to further experimentation. The BAPTA samples were incubated with 20 µM BAPTA-AM in serum-free DMEM-F-12 medium at 37°C in 5% CO2 for 30 min prior to further experimentation. B and C: NF-B DNA-binding activity was quantified densitometrically from 5 EMSA assays as shown in A for 4% (B) and 5% (C) ethanol. TNF--induced NF-B DNA-binding activity was set to 100%, and that of the controls was set to 0%. *P < 0.05 compared with ethanol with Bonferroni correction.

View larger version (14K): [in this window] [in a new window]   Fig. 2. Ethanol-induced activation of NF-B was concentration- and time-dependent. RGM-1 cells were grown to confluence and were exposed to different concentrations of ethanol (A, n = 5) or to the same concentration for different exposure times (B, n = 6). The nuclear fractions were analyzed by ELISA-based transcription factor assays. TNF- is a potent activator of NF-B and was used as a positive control. Ethanol-induced activation of NF-B was concentration-dependent (A; ANOVA, P < 0.05) and reached maximum at 3 h of incubation (B). *P < 0.05 compared with control with Bonferroni correction.

View larger version (16K): [in this window] [in a new window]   Fig. 3. The effect of heat shock pretreatment on heat shock proteins and the effect of RelA short interfering RNA (siRNA) transfections on RelA protein expression. A: RGM-1 cells were grown to confluence, pretreated with heat shock, and exposed to ethanol. The cytoplasmic fractions were analyzed by Western blot, demonstrating an increase in heat shock proteins after 1 h pretreatment with heat shock at 42°C. The ethanol exposure alone (3 h) did not have an effect on heat shock protein levels. B: effect of RelA siRNA transfection on the expression of RelA (NF-B p65) proteins. MOCK is transfected only with the transfection media, NF is transfected with nonfunctional siRNA. The expression of p65 was decreased with increasing dose of RelA siRNA.

The effects of ethanol, heat shock pretreatment, and inhibition of NF-B activation on apoptosis.

View larger version (21K): [in this window] [in a new window]   Fig. 4. Heat shock pretreatment inhibits ethanol-induced caspase-3 activity. RGM-1 cells were grown to confluence and either pretreated with 1 h heat shock at 42°C or left untreated. The epithelia were exposed to different doses of ethanol for 3 h, after which the cells were lysed and caspase-3 activity was measured with a time-resolved fluorescence-based assay. A: ethanol dose-dependently increased caspase-3 activity (P < 0.05, ANOVA). Inhibition of NF-B activation by NEMO-binding peptide further increased the caspase-3 activity (P < 0.05, ANOVA; n = 25). The negative control NEMO-binding peptide (neg. NEMO) did not have this effect. Reducing RelA expression with the siRNA technique gave similar results to inhibition of NF-B activation (NF, nonfunctional siRNA). Inset: caspase-3 activity was followed temporally during 5% ethanol exposure (n = 9). B: heat shock pretreatment decreased ethanol-induced caspase-3 activity (n = 14). *P < 0.05 compared with Bonferroni correction to respective ethanol concentration in control series or in respective negative control series (NEMO-binding peptide or nonfunctional siRNA series).

View larger version (21K): [in this window] [in a new window]   Fig. 5. Effects of NF-B inhibitor NEMO-binding peptide and heat shock pretreatment on cell viability in ethanol-exposed epithelium. Cell viability was measured with tetrazolium salt-based assay from RGM-1 cells grown to confluence. A: epithelia were pretreated with NF-B inhibitor NEMO-binding peptide, with its negative control binding peptide, transfected with RelA siRNA, transfected with nonfunctional siRNA, or left untreated. Thereafter the cells were exposed to different concentrations of ethanol, and after 4 h of incubation the cell viability was assessed (n = 11). B: epithelia were either pretreated with 1 h heat shock at 42°C or left untreated, after which the epithelia were exposed to different concentrations of ethanol for 4 h (n = 15). *P < 0.05 compared with Bonferroni correction against respective ethanol control or in respective negative control series (NEMO-binding peptide or nonfunctional siRNA series).

View larger version (8K): [in this window] [in a new window]   Fig. 6. Assessment of cell membrane integrity. RGM-1 cells were grown to confluence, and cell membrane integrity was measured with a LDH-based assay. The epithelia were either pretreated with heat shock for 1 h at 42°C or left untreated, after which the epithelia were exposed to different concentrations of ethanol for 4 h. Ethanol induced membrane damage in both control and heat-shocked epithelia. *P < 0.05 compared with respective ethanol control (n = 16).

View larger version (12K): [in this window] [in a new window]   Fig. 7. Expression of COX-2 in epithelia exposed to ethanol. Changes in expression of COX-2 were measured with Western blotting from confluent RGM-1 epithelia exposed to 5% ethanol. A: sample Western blot showing moderately increased COX-2 level in ethanol-exposed epithelia. B: densitometric results corrected with GADPH expression (n = 5). *P < 0.05 compared with respective ethanol control.

View larger version (19K): [in this window] [in a new window]   Fig. 8. Effect of inhibiting COX-2 activity during simultaneous exposure to ethanol on caspase-3 activity and cell viability. Confluent RGM-1 monolayers were exposed for 4 h simultaneously to different concentrations of ethanol and CAY-14040. Caspase-3 activity and cell viability were measured. CAY-14040 increased ethanol-induced caspase-3 activity and further decreased ethanol-affected cell viability. *P < 0.05 compared with respective ethanol control with Bonferroni correction.

	DISCUSSION

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The present findings indicate that ethanol concentration-dependently induces NF-B activation. This activation is calcium-dependent, since intracellular calcium-chelating agent BAPTA prevented ethanol-induced NF-B activation. Heat shock pretreatment also prevented NF-B activation, which may represent an alternative mechanism of defense of gastric mucosa against ethanol insult. We therefore further studied the apoptosis, cell viability, and cell membrane integrity in gastric mucosal cells exposed to ethanol. These studies have been performed with immortalized rat gastric mucosal epithelia, and hence the results should be confirmed with in vivo experiments.

Apoptosis can be divided into three different phases: initiation, signal transduction, and execution (24). Ethanol exposure dose-dependently increased caspase-3 activity, which is activated in the execution phase of apoptosis and leads eventually to apoptosis. The inhibition of NF-B activation directly by NEMO-binding peptide or by reducing RelA expression with the siRNA technique increased ethanol-induced caspase-3 activity even more and decreased cell viability, suggesting antiapoptotic action rather than induction of programmed cell death by NF-B activation. Ethanol-induced NF-B activation led to moderately increased COX-2 expression during ethanol exposure. On the other hand, the inhibition of COX-2 enzyme activity during ethanol exposure led to increased caspase-3 activity and decreased cell viability, suggesting a protective role of COX-2 during the ulcerogenic situation.

In this study, high concentrations of ethanol clearly caused a necrotic cell death rather than apoptotic cell death, because the cell membranes were profoundly damaged and cell viability was already severely affected during the first 4 h. Interestingly, pretreatment with heat shock was able to oppose necrotic cell death. Because ethanol is soluble to cell membranes and the plasma membranes are very permeable to it, and because ethanol probably affects plasma membrane fluidity, it would be tempting to suggest that the disruption of the plasma membranes results from a direct effect of ethanol on the plasma membrane structure. We have previously shown that the plasma membrane first becomes permeable to small marker molecules and thereafter to larger molecules, such as LDH, during the first minutes of ethanol exposure, probably demonstrating a direct fluidizing influence of ethanol on plasma membranes (17). The present findings indicate that during exposure to low concentrations of ethanol (1–5%), cell membrane damage also increases dose-dependently in a heat shock-pretreated epithelium. Obviously, the increasing ethanol concentration affects the plasma membrane directly, but in the wake of the increasing ethanol concentration other necrotic processes in the cells also emerge, which can be counteracted with heat shock pretreatment.

We have recently shown that ethanol exposure moderately increases intracellular free calcium concentration in primary cultured rabbit gastric surface epithelial cells (18) and in immortal RGM-1 cells (17). In these studies, inhibition of calcium signaling either totally with BAPTA or partially with specific inhibitors [3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester to inhibit intracellular calcium release and lanthanum to block plasma membrane calcium channels] also inhibited the target actions under investigation in these studies, i.e., opening of basolateral potassium ion channels with resultant cell volume shrinkage and closure of gap-junctional channels. It is noteworthy that the effects described above are relatively fast, occurring within a few minutes after ethanol exposure (16–18), whereas NF-B activation following ethanol exposure requires a few hours, as shown by the present study, but it also seems to be dependent on calcium signaling. Overall, calcium signaling seems to be crucial in many aspects of gastric defense against ethanol insult.

Large doses of ethanol induce heat shock protein synthesis in gastric mucosa (12, 22, 28), and pretreatment with heat shock promotes a protective action against ethanol-induced damage (26). Furthermore, heat shock or ethanol pretreatment inhibits spontaneous apoptotic DNA fragmentation in the cultured guinea pig gastric mucosal cells (27), and pretreatment with low doses of ethanol also prevents ethanol-induced apoptosis (26). Helicobacter pylori infection may contribute to the protection against ethanol-induced damage, since injection of Helicobacter-derived lipopolysaccharide into rats induced iNOS, COX-2, and HSP70 synthesis in gastric mucosa, thereby increasing gastric mucosal resistance against ethanol insult and water immersion stress (4). Heat shock proteins have a multifunctional role in the control of apoptosis. These proteins are mainly known to exert antiapoptotic actions, but in some instances they might even promote apoptosis. For example, HSP90 is necessary for apoptotic signal propagation from the plasma membrane to cytoplasm in TNF--induced apoptosis in a monoblastoid cell line (U937) (24). In the present study, a short-term (3 h), low-dose (5%) exposure to ethanol did not induce synthesis of heat shock proteins. Presumably, induction of heat shock protein synthesis by ethanol requires a longer exposure time and/or higher ethanol concentration, after which it might contribute to adaptive cytoprotection against repeated ethanol-induced damage. The adaptive cytoprotection is a complex phenomenon but has been demonstrated previously, e.g., with repeated ethanol exposure in gastric mucosa (6, 8, 11).

In conclusion, our findings indicate that ethanol exposure activates the NF-B pathway and a moderate increase in COX-2 expression, plausibly with the aim of generating defensive mechanisms, which lead to decreased apoptotic signaling. This protection mechanism was overcome by induction of heat shock proteins with heat shock pretreatment, which opposes ethanol-induced caspase-3 activation as well as cell membrane damage, thereby presumably protecting gastric mucosa against ethanol injury.

	GRANTS

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This work was supported by the EVO Research Foundation of Helsinki University Central Hospital, the Jenny and Antti Wihuri Foundation (Helsinki, Finland), and the Biomedicum Research Foundation (Helsinki, Finland).

	ACKNOWLEDGMENTS

 
We thank Paula Kokko and Sanna Vainionpää for excellent technical assistance.

	FOOTNOTES

 

Address for reprint requests and other correspondence: H. Mustonen, Dept. of Surgery, Helsinki Univ. Central Hospital, Box 700, 00029 HUS, Helsinki, Finland (e-mail: harri.mustonen{at}helsinki.fi)

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. Section 1734 solely to indicate this fact.

	REFERENCES

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1. Altura BM, Gebrewold A, Zhang A, Altura BT. Ethanol induces rapid lipid peroxidation and activation of nuclear factor-kappa B in cerebral vascular smooth muscle: relation to alcohol-induced brain injury in rats. Neurosci Lett 325: 95–98, 2002.[CrossRef][ISI][Medline]
2. Barkett M, Gilmore TD. Control of apoptosis by Rel/NF-kappaB transcription factors. Oncogene 18: 6910–6924, 1999.[CrossRef][ISI][Medline]
3. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254, 1976.[CrossRef][ISI][Medline]
4. Brzozowski T, Konturek PC, Moran AP, Kwiecien S, Pajdo R, Konturek SJ, Drozdowicz D, Ptak A, Pawlik W, Hahn EG. Enhanced resistance of gastric mucosa to damaging agents in the rat stomach adapted to Helicobacter pylori lipopolysaccharide. Digestion 67: 195–208, 2003.[ISI][Medline]
5. Davis RL, Syapin PJ. Ethanol increases nuclear factor-kappa B activity in human astroglial cells. Neurosci Lett 371: 128–132, 2004.[CrossRef][ISI][Medline]
6. Dinda PK, Wasan S, Beck IT, Kossev P. Adaptive cytoprotection against ethanol-induced small intestinal mucosal injury. Can J Physiol Pharmacol 74: 598–602, 1996.[CrossRef][ISI][Medline]
7. Halsted CH, Robles EA, Mezey E. Distribution of ethanol in the human gastrointestinal tract. Am J Clin Nutr 26: 831–834, 1973.[Abstract]
8. Jacobson ED. Direct and adaptive cytoprotection. Dig Dis Sci 31: 28S–31S, 1986.[CrossRef][Medline]
9. Jobin C, Sartor RB. The I kappa B/NF-kappa B system: a key determinant of mucosal inflammation and protection. Am J Physiol Cell Physiol 278: C451–C462, 2000.[Abstract/Free Full Text]
10. Jonsson AS, Palmblad JE. Effects of ethanol on NF-kappaB activation, production of myeloid growth factors, and adhesive events in human endothelial cells. J Infect Dis 184: 761–769, 2001.[CrossRef][ISI][Medline]
11. Ko JK, Cho CH. Endogenous mediators in adaptive cytoprotection against ethanol-induced gastric gland damage in rabbits. Life Sci 56: L1–L5, 1995.[CrossRef]
12. Konturek PC, Brzozowski T, Kania J, Konturek SJ, Hahn EG. Nitric oxide-releasing aspirin protects gastric mucosa against ethanol damage in rats with functional ablation of sensory nerves. Inflamm Res 52: 359–365, 2003.[CrossRef][ISI][Medline]
13. Mandrekar P, Catalano D, Szabo G. Inhibition of lipopolysaccharide-mediated NFkappaB activation by ethanol in human monocytes. Int Immunol 11: 1781–1790, 1999.[Abstract/Free Full Text]
14. May MJ, D'Acquisto F, Madge LA, Glockner J, Pober JS, Ghosh S. Selective inhibition of NF-kappaB activation by a peptide that blocks the interaction of NEMO with the IkappaB kinase complex. Science 289: 1550–1554, 2000.[Abstract/Free Full Text]
15. Mosser DD, Caron AW, Bourget L, Meriin AB, Sherman MY, Morimoto RI, Massie B. The chaperone function of hsp70 is required for protection against stress-induced apoptosis. Mol Cell Biol 20: 7146–7159, 2000.[Abstract/Free Full Text]
16. Mustonen H, Kivilaakso E. Effect of luminal ethanol on epithelial resistances and cell volume in isolated Necturus gastric mucosa. Dig Dis Sci 48: 2037–2044, 2003.[CrossRef][ISI][Medline]
17. Mustonen H, Kiviluoto T, Paimela H, Puolakkainen P, Kivilaakso E. Calcium signaling is involved in ethanol induced volume decrease and gap junction closure in cultured rat gastric mucosal cells. Dig Dis Sci 50: 103–110, 2005.[CrossRef][ISI][Medline]
18. Mustonen H, Kiviluoto T, Puolakkainen P, Kivilaakso E. Ethanol induces volume changes and gap junction closure via intracellular Ca²⁺ signalling pathway in cultured rabbit gastric epithelial cells. Scand J Gastroenterol 39: 104–110, 2004.[CrossRef][ISI][Medline]
19. Mustonen H, Kiviluoto T, Puolakkainen P, Kivilaakso E. Heat shock inhibits ethanol induced NF-kappaB activation in cultured rat gastric mucosal cells. Gastroenterology 128: A599, 2005.
20. Perkins ND. The Rel/NF-kappa B family: friend and foe. Trends Biochem Sci 25: 434–440, 2000.[CrossRef][ISI][Medline]
21. Roehm NW, Rodgers GH, Hatfield SM, Glasebrook AL. An improved colorimetric assay for cell proliferation and viability utilizing the tetrazolium salt XTT. J Immunol Methods 142: 257–265, 1991.[CrossRef][ISI][Medline]
22. Saika M, Ueyama T, Senba E. Expression of immediate early genes, HSP70, and COX-2 mRNAs in rat stomach following ethanol ingestion. Dig Dis Sci 45: 2455–2462, 2000.[CrossRef][ISI][Medline]
23. Schreiber E, Matthias P, Muller MM, Schaffner W. Rapid detection of octamer binding proteins with ‘mini-extracts’, prepared from a small number of cells. Nucleic Acids Res 17: 6419, 1989.[Free Full Text]
24. Sreedhar AS, Csermely P. Heat shock proteins in the regulation of apoptosis: new strategies in tumor therapy: a comprehensive review. Pharmacol Ther 101: 227–257, 2004.[CrossRef][ISI][Medline]
25. Szabo G, Mandrekar P. Ethanol-mediated regulation of transcription factors in immunocompetent cells. Front Biosci 7: a80–a89, 2002.[CrossRef][ISI][Medline]
26. Takano T, Tsutsumi S, Tomisato W, Hoshino T, Tsuchiya T, Mizushima T. Geranylgeranylacetone protects guinea pig gastric mucosal cells from gastric stressor-induced apoptosis. Dig Dis Sci 47: 1546–1553, 2002.[CrossRef][ISI][Medline]
27. Tsutsumi S, Rokutan K, Tsuchiya T, Mizushima T. Geranylgeranylacetone suppresses spontaneous apoptotic DNA fragmentation in cultured guinea pig gastric pit cells. Biol Pharm Bull 22: 886–887, 1999.[ISI][Medline]
28. Ueyama T, Saika M, Senba E. Distinct gene expression in the stomach following stress and alcohol exposure. Kaibogaku Zasshi 76: 435–441, 2001.[Medline]
29. Wilkes JM, Ballard HJ, Dryden DT, Hirst BH. Proton permeability and lipid dynamics of gastric and duodenal apical membrane vesicles. Am J Physiol Gastrointest Liver Physiol 256: G553–G562, 1989.[Abstract/Free Full Text]

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