Investigation of gastroprotective compounds at subcellular level in isolated gastric mucosal cells

Lajos Nagy, Romeo E. Morales, Martin Beinborn, Peter Vattay, and Sandor Szabo

Chemical Pathology Research Division, Departments of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We tested the hypothesis that recognized gastroprotective agents exert direct protection against ethanol-induced injury in isolated rat gastric mucosal cells in vitro. If protection exists, we also wanted to identify subcellular targets in the reversible and/or irreversible stages of cell injury. Ethanol-induced cell injury was quantified by measuring plasma membrane leakage (trypan blue exclusion and lactate dehydrogenase release), mitochondrial integrity (succinic dehydrogenase), and nuclear damage (ethidium bromide-DNA fluorescence). Initial cell viability and responsiveness were estimated by the effects of carbachol, carbachol + atropine, or 16,16-dimethyl-PGE2 on chief cell pepsinogen secretion. Enriched parietal cells were stimulated by histamine, carbachol, or histamine + IBMX. Preincubation of cells with PG, sucrose octasulfate, or the sulfhydryl compounds N-acetylcysteine, taurine, or cysteamine increased cell resistance <= 21% against ethanol. Similar protection was found with low histamine concentrations, but a higher concentration aggravated ethanol toxicity. Other naturally occurring or synthetic gastroprotective agents offered partial protection or aggravated ethanol-induced cell injury. Only a few in vivo gastroprotective agents demonstrated in vitro direct cytoprotection, which involved mainly the reversible stage of cell injury (e.g., plasma membrane changes) and, less often, irreversible (e.g., mitochondrial and nuclear) damage. Our findings also indicate that a major part of the beneficial effect of gastroprotective agents is expressed at the tissue level.

ethanol-induced cell injury; plasma membrane; mitochondria; nuclear damage; direct cytoprotection


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE TERM "CYTOPROTECTION" was introduced into gastrointestinal pathophysiology and pharmacology by A. Robert to describe the prevention of acute hemorrhagic gastric erosions produced by concentrated ethanol, hydrochloric acid, sodium hydroxide, sodium chloride, or boiling water by non-antisecretory doses of PGs in rats (28, 29). Although the basic biochemical mechanisms of gastric mucosal injury and protection remain unclear and controversial, the list of natural and synthetic "cytoprotective" compounds has been increasing. In vivo, chemically induced hemorrhagic mucosal lesions (HML) were also decreased by sulfhydryl (SH) compounds (10, 30, 39, 42), protease inhibitors (23, 39, 40), somatostatin (43), growth factors (14, 47), histamine (26), dopamine and related drugs (17, 27), gangliosides (35, 37), carotenoids (22), sucralfate and its derivatives (12, 36), antacids (24, 44), and spasmolytics (11, 32).

The original concept of cytoprotection has been criticized because of the incomplete protection by exogenous PG against chemically induced gastric HML. That is, histological and electron microscopic investigations revealed that only the deep hemorrhagic erosions could be reduced by PG or other cytoprotective compounds, whereas the surface cell damage was not decreased (10, 12, 16, 30, 44). Thus the terms "histoprotection" and "organoprotection" were suggested to reflect protection at the tissue and organ levels (41, 43). Tarnawski and colleagues (45) found a limited but statistically significant direct protection by PG of human isolated gastric glands against indomethacin- or ethanol-induced damage. Ivey and co-workers (31, 46) demonstrated that the chemically induced damage was diminished by PG and SH derivatives in cultured and transformed epithelial surface cells. Because the gastric mucosa contains numerous cell types, data are lacking concerning the possible direct protection of parietal, chief, and neuroendocrine cells by cytoprotective agents. Thus we tested the hypothesis that old and new cytoprotective compounds might exert direct protection on a mixed population of gastric mucosal cells (GMC). We also wanted to identify subcellular targets in the interaction of gastroprotective agents and ethanol. For this purpose, a method was adapted in our laboratory for the isolation of a mixed population of rat GMC with long viability and preserved membrane receptor sensitivity using minimal amounts of pronase and calcium-binding EGTA (13, 25). The new method not only includes the measurement of plasma membrane damage by the usual dye exclusion tests, e.g., by trypan blue (TB) exclusion, leakage of lactate dehydrogenase (LDH), or total protein from cytosol, but we can also assess mitochondrial integrity by measuring the activity of mitochondrial succinic dehydrogenase (SDH) and nuclear damage by fluorescence induced by ethidium bromide (EB)-DNA binding (25).

The main purpose of this study was to evaluate the possible direct cellular effects of established and newly synthesized gastroprotective compounds in isolated mixed rat GMC at the levels of the plasma membrane, mitochondria, or nuclei alone or against a moderate uniform cell injury after a short incubation with ethanol.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Reagents and Chemicals

The following reagents were purchased from Sigma Chemical (St. Louis, MO): N-acetyl-L-cysteine (NAC), L-alanine, D-arginine, L-arginine, atropine sulfate, brilliant cresyl blue (BCB), BSA, bovine hemoglobin, carbamycholine chloride (carbachol), DMSO, EB, EGTA, Folin and Ciocalteau's phenol reagent, fumaric acid, D-glucose, L-glutamine, glycine sodium salt, histamine dihydrochloride, HEPES, 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-diphenyltetrazolium chloride (INT), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), IBMX, MTT formazan, NAD+, Percoll, L(+)lactic acid, phenazine metasulfate (PMS), protease (type XXV, pronase E) from Streptomyces griseus, pyruvic acid sodium salt, Trizma base, TB, and urea. Cysteamine (2-aminoethanethiol) hydrochloride was purchased from Aldrich Chemical (Milwaukee, WI). 16,16-Dimethyl-PGE2 (dmPGE2) was a gift from Upjohn (Kalamazoo, MI), ganglioside GM1 from Angio-Medical (New York, NY), potassium sucrose octasulfate (SOS) from Marion Laboratories (Kansas City, MO), and KT1-32 from Kotobuki Seiyaku (Nagano, Japan), whereas pinaverium and KC-3-10667 were obtained from Kali-Chemie Pharma (Hannover, Germany). Taurine was purchased from National Biochemicals (Cleveland, OH). Protein measurement utilized the Bradford reagent (Pierce, Rockford, IL). Salts and other reagent grade chemicals were purchased from Fisher Scientific (Pittsburgh, PA).

Preparation of Mixed GMC

GMC from one or two nonfasted Sprague-Dawley rats (180-210 g; Taconic Farms, Germantown, NY) were obtained by sequential incubation with a low concentration of pronase E in calcium-free medium (EGTA) as we previously described (13, 25). Cells were counted in an improved Neubauer counting chamber (Hemocytometer; Fisher Scientific), dispersed, and kept in warm (37°C) HEPES-buffered salt solution (0.159 M, pH 7.4) produced fresh in our laboratory with the following ingredients (in mM): 98.0 NaCl, 5.8 KCl, 2.54 NaH2PO4, 5.1 Na pyruvate, 6.9 Na fumarate, 2.0 L-glutamine, 24.5 HEPES-Na, 1.0 Tris base, 11.1 D-glucose, and 1.0 CaCl2 with 2.0 mg/ml (wt/vol) BSA. The volume of GMC suspension was adjusted to a density of 1.5 × 107 cells/ml for incubations, TB, and biochemical assays. The numbers of viable and nonviable cells indicating initial cell viability (CV) were also counted by TB exclusion test (% of unstained cells) (25).

Secretory Studies

The responsiveness of rat mixed GMC and the membrane receptor sensitivity of both chief and parietal cells were also measured in separate experiments using secretory agents.

Pepsinogen secretion. Freshly isolated and dispersed (1 ml) rat GMC (5 × 106 cells/ml, CV = 94 ± 8%; chief cells = 42 ± 6%) were incubated with carbachol (10-1-10-4 M) or dmPGE2 (10-4-10-8M) alone as well as in combination with carbachol (10-1M) + atropine (10-4M) for 15 min at 37°C. Pepsinogen was determined by a modified Berstad method at pH 2.0 (HCl-KCl buffer, 0.1 M) using 3.0 M urea-denaturated bovine hemoglobin (1.0%) as substrate and incubated for 10 min at 37°C (5). The enzymic products were quantified with freshly diluted Folin and Ciocalteu's phenol reagent spectrophotometrically (A = 578 nm) with a Gilford 2400-2 spectrophotometer, calculated from a pepsin (Merck, Rahway, NJ) dose-response curve and expressed as micrograms of pepsin per minute per 5 × 106 GMC.

Enrichment of parietal cells and measurement of [14C]aminopyrine accumulation. Freshly isolated and dispersed rat GMC (1-2 × 108) were separated in a Beckman 5.0 elutriation system (600 rpm, 10°C) in Hanks' balanced salt solution supplemented with 0.1% (wt/vol) BSA. Parietal cells (60-65% of total GMC) were collected by continuous Percoll density gradient centrifugation at a density of 1.06 mg/ml (15). The implication of HCl production in enriched rat parietal cells is based on accumulation of 14C-labeled aminopyrine ([14C]AP) in acidic tubulovesicles of parietal cells on stimulation (3, 33, 34). Parietal cells (106/ml) were suspended in Hanks' balanced salt solution (containing 106 cells/ml 0.1% albumin) and incubated at 37°C in an orbital shaker (100 oscillations/min). Incubations were carried out in a total volume of 1.0 ml with 0.05 Ci of [14C]AP in the absence or presence of receptor stimuli such as carbachol (10-4 M), histamine (10-4 M), IBMX (10-4 M), or their combination. The washed cells were dissolved in 1.0 M NaOH, and neutralized aliquots were used for liquid scintillation counting.

Treatment of Rat Isolated GMC

All compounds for incubation were freshly dissolved and diluted in HEPES-buffered salt solution containing albumin (0.2% wt/vol). The pH was adjusted to 7.4, and the agents were used immediately. Initially, 30% ethanol was freshly prepared and subsequently diluted in the reaction tube containing GMC and test substances. The final concentration was 15% ethanol (25).

GMC (1.5 × 107 cells/tube) were tested for direct cytoprotection by 60-min incubation (1.0 ml of cell suspension plus 1.0 ml of drug solution) before addition of 2.0 ml of 30% ethanol for 5 min. The tested compounds were also used in the parallel tubes without ethanol treatment. The following gastroprotective compounds were tested for direct cytoprotection: glycine (10-3-10-6 M), D-arginine (10-3-10-6 M), L-arginine (10-3-10-6 M), L-alanine (10-3-10-6 M), dmPGE2 (10-4-10-8 M), cysteamine (10-2-10-4 M), NAC (10-3-10-5 M), taurine (10-3-10-5 M), histamine (2 × 10-3-2 × 10-7 M), GM1 (10-3-10-6 M), sucrose octasulfate (10-2-10-4 M), nitecapone (10-5-10-7 M), pinaverium (10-3-10-6 M), and KC-10667 (10-3-10-6 M).

After incubations the cells and the supernatant containing ethanol were separated by careful centrifugation (500 g, 8 min). The cells were resuspended in another 2.0 ml of buffer and incubated for 10 min in a shaking water bath. The ethanol-free supernatant was used for measurement of LDH leakage. The cells were redispersed in 1.5 ml of buffer (107 cells/ml) and immediately distributed for simultaneous biochemical assays.

TB Dye Exclusion Test

The TB dye exclusion test was carried out as we described previously (25). Briefly, 106 cells (0.1 ml of cell suspension) were mixed with 0.1 ml of 0.4% TB, and 5 min later the number of stained (dead) and unstained (viable) cells were counted in a hemocytometer and the yield of viable cells and CV (% of unstained cells) were calculated.

LDH Assay

LDH assay was carried out in samples of both supernatants alone and with 106 sonicated cells/0.1 ml (Sonifer cell disruptor 200; Branson, Danbury, CT). The colorimetric assay is based on reduction of 1.5 mM NAD+ to NADH catalyzed by LDH in the presence of lactate (50 mM) as substrate in a 1.0-ml final volume at 37°C in a 10-min incubation, as described previously (4). A color product was rapidly formed by reductions of PMS (1.6 mM) and INT (0.8 mM) in the same tube and measured spectrophotometrically at 500 nm. The specific LDH activity was measured and calculated as milliunits per minute per microgram of protein.

Succinic Dehydrogenase Assay

Mitochondrial integrity was tested in 2 × 106 previously treated and redispersed GMC at 37°C after incubation (90 min) with MTT (2.4 mM) in buffer (pH 7.4) in 1.0 ml of total reaction volume. The color formazan product was quantified spectrophotometrically at 500 nm after its dissolution in 2.0 ml of DMSO (21). Specific activity of SDH was calculated as nanomoles of formazan per minute per microgram of protein.

EB-DNA Fluorescence Assay

Nuclear damage of GMC was assessed by nuclear fluorescence of cells due to the EB-DNA binding as described previously (9). GMC suspension (1 ml containing 107 cells) was mixed with 2.0 ml of 25 µM EB solution, and fluorescence intensity was measured in a Perkin-Elmer fluorescence spectrophotometer (Hitachi Instruments) at 325-585 nm (excitation-emission). The results were expressed as arbitrary fluorescence units/107 cells.

Protein Concentration

Protein concentration in the supernatants and sonicated cells was determined by Bradford's method (Bio-Rad Laboratories, Richmond, CA; Ref. 6). Absorption of samples at 595 nm was measured against blanks and known standards of BSA.

Data Presentation and Statistical Evaluation

Data from three to eight experiments were pooled and expressed as means ± SE. Comparisons were performed by Student's t-tests (paired or unpaired) and nonparametric Mann-Whitney U-tests. Values were considered statistically significant at P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Yield, Composition, and Viability Of Isolated Rat GMC

A large number of mixed GMC (1.2-1.6 × 108) can be isolated from a single rat glandular stomach. The ratio of parietal, chief, and mucous epithelial cells using supravital staining with 0.05% BCB was 23.0 ± 2.1, 42.1 ± 4.9, and 34.1 ± 3.7%, respectively. The average initial CV of 28 cell isolations was 90.5 ± 3.1% measured by TB dye exclusion. High CV (85-95%) was maintained for 7 h after cell isolation using our cell harvesting method and physiological solution at 37°C.

Secretory Studies

Pharmacological investigations with chief cells in mixed rat GMC revealed that the muscarinic receptor agonist carbachol significantly stimulated pepsinogen secretion from 3.15 ± 0.56 to 7.50 ± 0.75 µg · min-1 · 5 × 106 GMC-1 (P < 0.01). Atropine given in combination with carbachol almost totally inhibited carbachol-induced increase in pepsinogen secretion (P < 0.001) (Table 1). In addition, dmPGE2 also increased pepsinogen secretion from isolated chief cells from 8.75 ± 0.35 to 10.10 ± 0.30 µg · min-1 · 5 × 106 GMC-1 (P < 0.01; Fig. 1).

                              
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Table 1.   Effects of carbachol and carbachol + atropine on pepsinogen secretion of isolated rat gastric mucosal cells



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Fig. 1.   Pepsinogen secretion from isolated chief cells in a mixed suspension of rat gastric mucosal cells (GMC) containing peptic cells. Dose response for 16,16-dimethyl-PGE2 (dmPGE2) in a 15-min incubation at 37°C is shown. Peptic activity of GMC medium was assayed at pH 2.0 using hemoglobin as substrate. Results shown are means ± SE of 6 experiments (6 different cell preparations). *P < 0.05; **P < 0.01.

Uptake of [14C]AP in enriched (65%) rat parietal cells was stimulated by either histamine or carbachol (P < 0.05). The histamine-induced increase in HCl production in parietal cells was considerably potentiated by the phosphodiesterase inhibitor IBMX (Fig. 2).


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Fig. 2.   Implication of HCl production in parietal cells in a mixed suspension of rat GMC containing enriched (65%) parietal cells of total GMC using 14C-labeled aminopyrine ([14C]AP) accumulation. Incubations of 106 parietal cells with 0.05 Ci [14C]AP were performed in absence or presence of carbachol, histamine, or their combinations for 45 min at 37°C. Results represent means ± SE of 4 experiments. *P < 0.05; ***P < 0.001.

Ethanol-Induced Cellular Injury: An Assay for Cytoprotection

The average values obtained by all methods used to measure indicators of damage before and after 5-min incubation with ethanol at 37°C are shown in Table 2. Ethanol (15% vol/vol) significantly decreased CV by TB dye exclusion from 90.3 ± 3.1 to 20.9 ± 1.4 (P < 0.001) and increased LDH leakage from cytosol into media via damaged cell membrane (P < 0.001). It induced severe mitochondrial and nuclear lesions as detected by marked decrease in SDH activity from 5.7 ± 0.3 to 1.5 ± 0.1 nmol formazan · min-1 · mg protein-1 and elevation of EB-DNA fluorescence intensity from 4.2 ± 0.5 to 17.3 ± 1.1 fluorescence units/107 GMC.

                              
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Table 2.   Ethanol-induced injury in isolated gastric mucosal cells after 5-min incubation

Effects of Thiol Compounds on Ethanol-Induced Cell Injury

NAC, taurine, or cysteamine alone did not induce any injury in mixed GMC (data not presented). The effects of 60-min incubation of isolated rat GMC with NAC against ethanol-induced cellular injury are shown in Fig. 3. Cell membrane damage quantified by TB dye exclusion and LDH leakage, mitochondrial SDH activity, and EB-DNA fluorescence revealed a mild (12-21%) protection against ethanol-induced damage (P < 0.05).


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Fig. 3.   Effects of increasing concentrations of N-acetyl-L-cysteine (NAC) on ethanol-induced lesions in rat GMC. Freshly isolated mixed GMC (1.5 × 107) were preincubated with NAC for 60 min at 37°C and incubated with 15% ethanol for 5 min at 37°C. Values of cell viability (trypan blue exclusion), lactate dehydrogenase (LDH) leakage from cytosol into media (released and remaining LDH), mitochondrial lesion [succinic dehydrogenase (SDH) activity], and nuclear injury [ethidium bromide (EB)-DNA fluorescence] are presented for control (untreated) cells (open bars) and for preincubated + ethanol-treated cells (hatched bars). Results represent means ± SE of 6 separate experiments. *P < 0.05, statistically significant difference compared with placebo preincubated + ethanol-treated group.

As shown in Fig. 4, a similar (10-20%) increase in cell resistance was detected after 60-min preincubation with taurine (2-aminoethanesulfonic acid) against ethanol injury, and the difference was statistically significant with LDH leakage and EB-DNA fluorescence (P < 0.05). The protection of mitochondria and nuclei reached the level of significance (P < 0.05).


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Fig. 4.   Effects of increasing concentrations of taurine on ethanol-induced lesions in rat GMC. Freshly isolated mixed GMC (1.5 × 107) were preincubated with taurine for 60 min at 37°C and incubated with 15% ethanol for 5 min at 37°C. Values of cell viability (trypan blue exclusion), LDH leakage from cytosol into media (released and remaining LDH), mitochondrial lesion (SDH activity), and nuclear injury (EB-DNA fluorescence) are presented for control (untreated) cells (open bars) and for preincubated + ethanol-treated cells (hatched bars). Results represent means ± SE of 7 separate experiments. *P < 0.05, statistically significant difference compared with placebo preincubated + ethanol-treated group.

In contrast, cysteamine moderately aggravated (P < 0.05) the ethanol-induced cell membrane and mitochondrial damage measured by TB exclusion and SDH, respectively. However, some concentration-dependent nuclear protection (P < 0.01) was measured by EB-DNA fluorescence. No major changes were found in LDH leakage from cysteamine-treated GMC after incubation with 15% ethanol (Fig. 5).


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Fig. 5.   Effects of increasing concentrations of cysteamine on ethanol-induced lesions in rat GMC. Freshly isolated mixed GMC (1.5 × 107) were preincubated with cysteamine for 60 min at 37°C and incubated with 15% ethanol for 5 min at 37°C. Values of cell viability (trypan blue exclusion), LDH leakage from cytosol into media (released and remaining LDH), mitochondrial lesion (SDH activity), and nuclear injury (EB-DNA fluorescence) are presented for control (untreated) cells (open bars) and for preincubated + ethanol-treated cells (hatched bars). Results represent means ± SE of 6 separate experiments. *P < 0.05, statistically significant difference compared with placebo preincubated + ethanol-treated group.

Effects of dmPGE2 and Ethanol

Concentration-dependent protection (15-21%) against ethanol-induced injury was found after 60-min incubation with dmPGE2 as measured by TB exclusion (P < 0.05), LDH release (P < 0.05), and changes in activity of SDH from 1.95 ± 0.11 to 3.31 ± 0.21 (P < 0.05). Only a slight increase (P > 0.05) in nuclear resistance was observed after incubation with dmPGE2 in ethanol-treated GMC (Fig. 6).


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Fig. 6.   Effects of increasing concentrations of dmPGE2 on ethanol-induced lesions in rat GMC. Freshly isolated mixed GMC (1.5 × 107) were preincubated with dmPGE2 for 60 min at 37°C and incubated with 15% ethanol for 5 min at 37°C. Values of cell viability (trypan blue exclusion), LDH leakage from cytosol into media (released and remaining LDH), mitochondrial lesion (SDH activity), and nuclear injury (EB-DNA fluorescence) are presented for control (untreated) cells (open bars) and for preincubated + ethanol-treated cells (hatched bars). Results represent means ± SE of 8 separate experiments. *P < 0.05, statistically significant difference compared with placebo preincubated + ethanol-treated group.

Effects of SOS and Ethanol

Incubation of GMC for 60 min with SOS dose-dependently decreased the ethanol-induced LDH release and increased CV measured by TB dye exclusion; the differences reached statistical significance (P < 0.05) at 10-3 M of SOS (Fig. 7). The effect of SOS on mitochondrial and nuclear membranes detected by changes in the activity of SDH and EB-DNA binding was not statistically significant.


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Fig. 7.   Effects of increasing concentrations of sucrose octasulfate (SOS) on ethanol-induced lesions in rat GMC. Freshly isolated mixed GMC (1.5 × 107) were preincubated with SOS for 60 min at 37°C and incubated with 15% ethanol for 5 min at 37°C. Values of cell viability (trypan blue exclusion), LDH leakage from cytosol into media (released and remaining LDH), mitochondrial lesion (SDH activity), and nuclear injury (EB-DNA fluorescence) are presented for control (untreated) cells (open bars) and for preincubated + ethanol-treated cells (hatched bars). Results represent means ± SE of 6 separate experiments. *P < 0.05, statistically significant difference compared with placebo preincubated + ethanol-treated group.

Effects of Various Gastroprotective Compounds on Ethanol Toxicity

The effects of different agents that in vivo exert gastroprotection in isolated GMC with or without incubation with 15% ethanol are summarized in Table 3. Only the statistically significant changes in TB dye exclusion and biochemical assays are listed, indicating cell injury alone or aggravation of ethanol-induced cellular damage. Most of these compounds (i.e., GM1, nitecapone, pinaverium, KC-10667, and glycine) aggravated the ethanol-induced injury in GMC in a concentration-dependent fashion and, administered alone (i.e., without ethanol exposure), also exerted some direct cell toxicity in GMC. Ethanol-induced cell injury was aggravated by L-arginine, D-arginine, and L-alanine, but these compounds alone did not induce damage. Histamine had a biphasic effect in the rat GMC: low concentration protected but high concentration aggravated the ethanol-induced cell injury.

                              
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Table 3.   Effects of various gastroprotective compounds alone and in presence of 15% ethanol on isolated rat gastric mucosal cells


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Our study demonstrates that preincubation of isolated rat gastric mucosal cells with dmPGE2, SOS, or the SH compounds NAC, taurine, or cysteamine alone did not cause any measurable cell injury, and these agents slightly increased resistance (up to 21%) against ethanol-induced cellular damage. Similar protection was found with a low concentration of histamine, but at a higher concentration it aggravated ethanol toxicity. Partial or general aggravation of ethanol injury was detected on GMC membranes after preincubation with wide concentration ranges of amino acids such as L-arginine, D-arginine, glycine, L-alanine, and D-alanine or in vivo gastroprotective compounds such as nitecapone, ganglioside GM1, the spasmolytic pinaverium, and KC-10667. Some of these agents alone (i.e., without ethanol) exerted some direct cellular toxicity. Our results also revealed that the isolated chief cells (pepsinogen secretion) or enriched parietal cells (HCl production) have an excellent membrane receptor-mediated responsiveness to pharmacological stimuli such as cholinergic and histaminergic stimulators or PG. These results are in accordance with other studies in cultured and isolated parietal cells (3, 33, 34) or peptic cells (2, 8, 18, 19).

Acute gastric mucosal injury is a complex process because of the heterogenous structure and multiple functions of the stomach wall. In the pathways of direct and indirect chemical injury, vascular damage, inflammatory processes, free radicals, and proteases are involved (41, 45). The list of gastroprotective agents has been growing since the introduction of the concept of gastric cytoprotection, without a proportionate increase in our understanding of the mechanisms of gastric mucosal injury and protection. We nevertheless know that most of the mucosal protection is relative and indirect. Namely, despite the initial destruction of superficial epithelial cells in rats given ethanol intragastrically after pretreatment with gastroprotective agents, the histological integrity of the gastric mucosa is restored by rapid epithelial restitution and the organ structure and function are maintained.

One of the new possibilities for assessing the phenomenon of direct gastric cytoprotection is to investigate in vitro the possible protection of isolated GMC by naturally occurring or exogenous compounds against chemically induced and measurable cell damage. We have recently developed and optimized morphological and biochemical methods for harvesting mixed populations of rat GMC to investigate reversible and irreversible cell damage and protection at the level of cell membranes in organelles (13, 25). Selective and parallel assessment of chemical injury and protection of plasma membrane, mitochondria, and nuclei can be investigated with sensitive measurements such as TB exclusion, LDH leakage, SDH activity, or EB-DNA binding. These targets were selected because of their critical roles in reversible and irreversible cell injury. That is, the extent of plasma membrane and mitochondrial damage is currently accepted as the rate-limiting step between reversible and irreversible cell damage, whereas nuclear damage is an indicator of cell death by either necrosis or apoptosis (7, 38). This short incubation (5 min) of rat GMC with a low concentration of ethanol induced reversible and/or irreversible cell membrane, mitochondrial, or nuclear injury that can be measured by morphological and biochemical assays (25). The short exposure is meant to imitate the in vivo conditions when fluids like ethanol are rapidly emptied from the stomach and mucosal lesions develop within minutes (10, 16, 20, 40). Our previous experiments revealed only minimal or no protection of isolated GMC against ethanol injury after preincubation with PG for 30 min, whereas the present results indicate a moderate increase in cellular resistance of GMC after 1-h incubation with dmPGE2.

The SH compounds constitute one of the groups of endogenous mediators of acute gastroprotection (10, 20, 30, 39, 42, 44). Only a moderate but statistically significant direct gastroprotection was observed in rat GMC against ethanol-induced cytotoxicity by preincubation with NAC (in all organelles examined) or taurine (in mitochondria and nuclei). Taurine is a metabolite of L-cysteine. In vivo, it protects several organs against chemical injury, and we recently found gastroprotection against ethanol in rats (unpublished data). Cysteamine, another SH-containing agent, has also been shown to exert in vivo gastroprotection (42). Romano et al. (31) demonstrated that cysteamine has a direct protective effect in vitro against damage induced by taurocholate or indomethacin in gastric epithelial monolayers derived from a human cell line. In our study with freshly isolated rat GMC, cysteamine did not induce significant enhancement of cell resistance.

SOS, a derivative of sucralfate, had a moderate protective effect against ethanol injury in isolated rat GMC. This direct cellular protection might be one of the mechanisms of acute gastroprotection by sucralfate (36).

GM1, a ganglioside and sialic acid-containing glycosphingolipid, exerts acute gastric mucosal protection against ethanol (37, 48). Gangliosides play a role in the regulation of transmembrane signaling, cellular differentiation and proliferation, membrane fluidity, and ion transport (35). In the present study, exogenous GM1 was not able to protect against ethanol-induced damage in isolated GMC.

Amino acids such as L- or D-arginine, glycine, and D- or L-alanine are either modulators of neural transmissions or sources of vasoactive nitric oxide. They are also gastroprotective against ethanol injury in rats (unpublished data), and they reduce tubular cell damage in the kidney (1). In our study they did not have any direct cytoprotective effect in rat GMC against ethanol.

Our findings show the following. 1) A large number of mixed GMC with high and long viability and preserved membrane receptor sensitivity can be isolated from rat stomach. 2) Selective examination of ethanol injury and protection of plasma, mitochondrial, and/or nuclear membranes may be demonstrated biochemically and morphologically. 3) Only a slight or moderate concentration-dependent protection was detected by thiol compounds, dmPGE2, or SOS. Of the few chemicals that exerted direct cytoprotection in vitro (e.g., NAC, taurine, dmPGE2, SOS), all decreased plasma membrane damage as revealed by TB exclusion and/or LDH release, whereas only taurine and NAC diminished nuclear damage, i.e., cell death. This implies that it is much easier to offer protection against the reversible than against the irreversible stage of cell injury. 4) In contrast, a large number of naturally occurring or synthetic in vivo gastroprotective compounds have no direct cytoprotective effects against ethanol cytotoxicity in a mixed population of GMC. 5) Consequently, minimal or no correlation could be demonstrated between in vitro cytoprotection and in vivo gastroprotection by most compounds investigated. These findings indicate that a major part of the beneficial effect of gastroprotective agents seems to be mediated via complex mechanisms at the tissue and organ levels.


    ACKNOWLEDGEMENTS

We thank Joanne M. Jenkins, Ken Green, and Gabor Nagy for technical assistance and acknowledge the contributions of Eileen Holman and Rozalia Nagy in the preparation of the manuscript.


    FOOTNOTES

Address for reprint requests and other correspondence: S. Szabo, Pathology & Laboratory Medicine Service, 5901 East 7th St., VA Med. Ctr., Long Beach, CA 90822 (E-mail: sandor.szabo{at}med.va.gov).

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.

Received 29 July 1999; accepted in final form 14 June 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Baines, AD, Shaikh N, and Ho P. Mechanism of perfused kidney cytoprotection by alanine and glycine. Am J Physiol Renal Fluid Electrolyte Physiol 259: F80-F86, 1990[Abstract/Free Full Text].

2.   Beinborn, M, Beil W, Bersimbae RI, Hennies S, and Sewing KF. Histamine-receptor mediated pepsinogen release in porcine chief cells (Abstract). Gastroenterology 96: A36, 1989.

3.   Berglindh, T, Helander HF, and Obrink KJ. Effects of secretagogues on oxygen consumption, aminopyrine accumulation and morphology in isolated gastric glands. Acta Physiol Scand 97: 401-414, 1976[ISI][Medline].

4.   Bergmeyer, HU, and Bernt E. Colorimetric assay with L(+)lactate, NAD, phenazine methosulphate and INT. In: Methods in Enzymatic Analysis, edited by Bergmeyer HU.. New York: Academic, 1974, vol. 1, p. 579-582.

5.   Berstad, A. A modified hemoglobin substrate method for the estimation of pepsin in gastric juice. Scand J Gastroenterol 5: 343-348, 1970[ISI][Medline].

6.   Bradford, MM. A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254, 1976[ISI][Medline].

7.   Cotran, RS, Kumar S, and Robbinson SL. Robin's Pathologic Basis of Disease (4th ed.). Philadelphia: Saunders, 1989.

8.   Defize, J, and Hunt RH. Effect of hydrochloric acid and prostaglandins on pepsinogen synthesis and secretion in canine gastric chief cell monolayer cultures. Gut 30: 774-781, 1989[Abstract].

9.   Dey, CS, and Majumder GC. A simple quantitative method of estimation of cell-intactness based on ethidium bromide fluorescence. Biochem Int 17: 367-374, 1987[ISI].

10.   Dupuy, D, Raza A, and Szabo S. The role of endogenous nonprotein and protein sulfhydryls in gastric mucosal injury and protection. In: Ulcer Disease. New Aspects of Pathogenesis and Pharmacology, edited by Szabo S, and Pfeiffer CJ.. Boca Raton, FL: CRC, 1989, p. 421-434.

11.   Hagel, J, Renner H, and Hirsch M. Gastric cytoprotection by antacids and papaverine in rats. Hepatogastroenterology 29: 271-274, 1982[ISI][Medline].

12.   Hollander, D, Tarnawski A, Gergely H, and Zipser RD. Sucralfate protection of the gastric mucosa against ethanol-induced injury: a prostaglandin-mediated process? Scand J Gastroenterol 19: S97-S102, 1984.

13.   Johnson, BR, Morales RE, Nagy L, Glavin GB, and Szabo S. Rat gastric mucosal cells: optimal conditions for cell harvesting and measures of viability (Abstract). Dig Dis Sci 35: 1013, 1990.

14.   Konturek, S, Brzozowski T, Dembinski A, Warzecha Z, and Yamazaki J. Gastric protective and ulcer healing action of epidermal growth factor. In: Advances in Drug Therapy of Gastrointestinal Ulceration, edited by Garner A, and Whittle BJR. New York: Wiley, 1989, p. 261-273.

15.   Kromer, W, Baron E, Beinborn M, Boer R, and Eltze M. Characterization of the muscarinic receptor type on paracrine cells activated by MLN-A-343 in the mouse isolated stomach. Naunyn Schmiedebergs Arch Pharmacol 341: 165-170, 1990[ISI][Medline].

16.   Lacy, ER, and Ito S. Microscopic analysis of ethanol damage to rat gastric mucosa after treatment with a prostaglandin. Gastroenterology 83: 619-625, 1982[ISI][Medline].

17.   MacNaughton, WK, and Wallace JL. A role for dopamine as an endogenous protective factor in the rat stomach. Gastroenterology 96: 972-980, 1989[ISI][Medline].

18.   Matsumoto, H, Dickinson KEJ, Hirakawa T, Komiyama K, and Hirschowitz BI. Synergism between cellular messenger and agonist combinations in pepsinogen secretion. Am J Physiol Gastrointest Liver Physiol 253: G557-G565, 1987[Abstract/Free Full Text].

19.   Matsumoto, H, Dickinson KEJ, Anderson W, and Hirschowitz BI. Pepsinogen secretion from perfused frog peptic glands: rapid transients detected with a modified pepsin assay. Life Sci 42: 1234-1244, 1988.

20.   Miller, TA. Protective effects of prostaglandins against gastric mucosal damage: current knowledge and proposed mechanisms. Am J Physiol Gastrointest Liver Physiol 245: G601-G623, 1983[Abstract/Free Full Text].

21.   Mosmann, T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65: 5-63, 1983.

22.   Mózsik, G, Pihan G, Szabo S, Jávor T, Czeglédi B, Tigyi A, Tárnok F, and Zsoldos T. Free radicals, nonsulfhydryl antioxidants, drugs and vitamins in acute gastric mucosal injury and protection. In: New Pharmacology of Ulcer Disease: Experimental and New Therapeutic Approaches, edited by Szabo S, and Mozsik G.. New York: Elsevier, 1987, p. 197-207.

23.   Nagy, L, Johnson BR, Saha B, LeQuesne P, Neumeyer JL, Plebani M, and Szabo S. Correlation between gastroprotection and inhibition of cysteine proteases by new maleimide derivatives (Abstract). Dig Dis Sci 35: 1037, 1990.

24.   Nagy, L, Mózsik G, Vincze A, Sut G, Hunyady B, Rinfel J, Past T, and Jávor T. Effects of a novel Hungarian antacid containing Al and Mg (Tisacid) on mucosal prostaglandin generation and oxygen free radicals in normal rats. Drugs Exp Clin Res 16: 197-203, 1990[ISI][Medline].

25.   Nagy, L, Szabo S, Morales RE, Plebani M, and Jenkins JM. Identification of subcellular targets and sensitive test of ethanol-induced damage in isolated rat gastric mucosal cells. Gastroenterology 107: 907-914, 1994[ISI][Medline].

26.   Palitzsch, KD, Kusterer K, Zendler S, and Usedal KH. Histamine protects rat gastric mucosa against ethanol-induced injury by maintaining blood flow while cimetidine does not (Abstract). Gastroenterology 98: A103, 1990.

27.   Piotrowski, J, Ismail A, Rajiyah G, Yamaki K, Slomiany A, and Slomiany BL. Nitecapone protection against gastric mucosal injury is not prostaglandin mediated (Abstract). Gastroenterology 100: A142, 1991.

28.   Robert, A. Cytoprotection by prostaglandins. Gastroenterology 77: 761-767, 1979[ISI][Medline].

29.   Robert, A, Nezamis JE, Lancaster C, and Hanchar AJ. Cytoprotection by prostaglandins in rats. Prevention of gastric necrosis produced by alcohol, HCl, NaOH, hypertonic NaCl, and thermal injury. Gastroenterology 77: 433-443, 1979[ISI][Medline].

30.   Rogers, C, Brown A, and Szabo S. Gastric mucosal protection by new aryl sulfhydryl drugs. Dig Dis Sci 33: 324-329, 1988[ISI][Medline].

31.   Romano, M, Razandi M, Raza A, Szabo S, and Ivey J. Cysteamine protects gastric epithelial cell monolayers against drug induced damage: evidence for direct cellular protection by sulphydryl compounds. Gut 33: 30-38, 1992[Abstract].

32.   Ruppin, H, Hagel J, Kachel G, Domschke S, and Domschke W. Gastric cytoprotection in rat and man by various drugs: contrasting and competitive results. In: New Pharmacology of Ulcer Disease: Experimental and New Therapeutic Approaches, edited by Szabo S, and Mozsik G.. New York: Elsevier, 1987, p. 57-67.

33.   Sewing, KF, Harms P, Schultz G, and Hanneman H. Effect of substituted benzimidazoles on acid secretion in isolated and enriched guinea pig parietal cells. Gut 24: 557-560, 1980[Abstract].

34.   Sewing, KF, Beil W, and Beinborn H. Measurement of 14C-aminopyrine accumulation in isolated parietal cells (Abstract). Gastroenterology 96: 1625, 1989[ISI][Medline].

35.   Slomiany, BL, Piotrowski J, Ismail A, Klibaner M, Murty VLN, and Slomiany A. GM1 ganglioside protection against ethanol-induced gastric mucosal injury. Alcohol Clin Exp Res 15: 196-204, 1991[ISI][Medline].

36.   Szabo, S. The mode of action of sucralfate: the 1 × 1 × 1 mechanism of action. Scand J Gastroenterol 26: 7-12, 1991[ISI].

37.   Szabo, S, DiPietro S, Morales RE, Lippe TI, and Rogers C. Gangliosides are novel cytoprotective and antiulcer agents (Abstract). Gastroenterology 96: 498, 1989.

38.   Szabo, S, and Kovacs K. Causes and mechanisms of cell and tissue injury in endocrine glands. In: Functional Endocrine Pathology, edited by Kovacs K, and Asa L.. Boston: Blackwell Scientific, 1991, p. 914-933.

39.   Szabo, S, Nagy L, and Johnson BR. Protein and nonprotein sulfhydryls as targets of gastroprotection and antiulcer agents (Abstract). Dig Dis Sci 35: 1041, 1990.

40.   Szabo, S, Nagy L, and Plebani M. Glutathione, protein sulfhydryls and cysteine proteases in gastric mucosal injury and protection. Clin Chim Acta 206: 95-105, 1992[ISI][Medline].

41.   Szabo, S, and Szelenyi I. Cytoprotection in gastrointestinal pharmacology. Trends Pharmacol Sci 8: 149-154, 1987[ISI].

42.   Szabo, S, Trier JS, and Frankel PW. Sulfhydryl compounds may mediate gastric cytoprotection. Science 214: 200-202, 1981[ISI][Medline].

43.   Szabo, S, and Usadel KH. Cytoprotection-organoprotection by somatostatin in gastric and hepatic lesions. Experientia 38: 254-255, 1982[ISI][Medline].

44.   Szelenyi, I, and Brune K. Possible role of sulfhydryls in mucosal protection induced by aluminum hydroxide. Dig Dis Sci 31: 1207-1210, 1986[ISI][Medline].

45.   Tarnawski, A, Brzozowski T, and Sarfeh IJ. Prostaglandin protection of human isolated glands against indomethacin and ethanol injury. Evidence for direct cellular action of prostaglandin. J Clin Invest 81: 1081-1089, 1988[ISI][Medline].

46.   Terano, A, Ota S, Mach T, Hiraishi H, Stachura J, Tarnawski A, and Ivey KJ. Prostaglandin protects against taurocholate-induced damage to rat gastric mucosal cell culture. Gastroenterology 92: 669-677, 1987[ISI][Medline].

47.   Vattay, P, and Szabo S. Growth factors as novel pharmacologic agents (Abstract). Acta Biomedica Hungarica Americana 2: 6, 1990.

48.   Zeller, CB, and Marchase RB. Gangliosides as modulators of cell function. Am J Physiol Cell Physiol 262: C1341-C1355, 1992[Abstract/Free Full Text].


Am J Physiol Gastrointest Liver Physiol 279(6):G1201-G1208
0193-1857/00 $5.00 Copyright © 2000 the American Physiological Society




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