It is known that calcium ion has an important role in the cellular function. For this reason, calcium channel blockers may have a protective action against gastric ...
Digestive Diseases and Sciences, Vol. 47, No. 4 (April 2002), pp. 850 – 855 (© 2002)
Antioxidant Effect of T-Type Calcium Channel Blockers in Gastric Injury ˘ LU,* AHMET KIZILTUNC DILEK BILICI,* Z. NUR BANOG ¸ ,† BAHATTIN AVCI,† ˘ LU,‡ and SEFA BILICI§ AKIF C ¸ IFTC ¸ IOG
It is known that calcium ion has an important role in the cellular function. For this reason, calcium channel blockers may have a protective action against gastric injury which is induced by various stimuli. In this study, the influence of mibefradil on ethanol-induced gastric injury was investigated in rats. Mibefradil was given at a dose 50 mg/kg intraperitoneally 30 min before administration of 1 ml absolute ethanol given by gavage. We compared this effect of mibefradil with that of omeprazol. Ethanol-induced mucosal damage was evaluated using three different approaches: analysis of biochemical parameters and pathologic and macroscopic investigation. It was found that pretreatment with mibefradil significantly reduced ethanol-induced macroscopic, pathologic, and biochemical changes in the gastric mucosa. In conclusion, it is speculated that this findings may prove important in the development of new and improved therapies for the treatment and prevention of gastric ulcers in humans. KEY WORDS: free radicals; mibefradil; rat stomach; ethanol; glutathione; glutathione reductase.
Peptic ulceration, including acute erosions, is a multifactorial disease involving, well-known factors such as trauma, stress, sepsis, hemorrhagic shock, burns, pulmonary and liver diseases, and drugs such as reserpine, epinephrine, and steroids (1). Ethanol rapidly penetrates the gastroduodenal mucosa causing membrane damage, exfoliation of cells, and erosion. The subsequent increase in mucosal permeability together with the release of vasoactive products from mast cells, macrophages, and other blood cells may lead to vascular injury, necrosis, and ulcer formation (2). Oxygen radicals have recently been implicated as potential mediators of gastric mucosal injury caused by ischemia–reperfusion (3, 4), cold–restraint (5), ethanol (6, 7), and nonsteroidal antiinflamatory drugs (6, 8). Ethanol-induced gastric mucosal damage is
associated with a significant reduction in the nonprotein sulfhydryl concentration in the rat and human (4, 6, 9, 10, 11 12, 13). Calcium channel blockers are widely used in the treatment of hypertension, chronic stable angina pectoris, and cardiac arrhytmias. Despite their desirable cardiac effect, the effects of the use of calcium channel blockers in patients with gastric ulcers is unknown. Mibefradil is the prototype of a new class of calcium antagonists that selectively block T-type voltage-gated plasma membrane calcium channels in vascular smooth muscle. The drug is structurally and pharmacologically different from traditional calcium antagonists (14). In the present study, we examined the protective effect of mibefradil against ethanolinduced gastric damage.
Manuscript received March 14, 2001; accepted August 2, 2001. From the *Department of Pharmacology, †Department of Biochemistry, and ‡Department of Pathology, Medical School, Atatu ¨rk University, Erzurum, Turkey; and §Numune Hospital TR25240 Erzurum, Turkey. ¨ niversiAddress for reprint requests: Dr. Dilek Bilici, Atatu ¨rk U tesi Tıp Faku ¨ltesi, Farmakoloji anabilim dalı, 25240, Erzurum, Turkey
MATERIALS AND METHODS
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Animals. For these studies 32 adult Sprague-Dawley male rats (200 –250 g) were used. They were housed in Plexiglas cages with three animals per cage. The animal rooms were windowless with automatic temparature (22 ⫾ 1°C) and lighting controls (lights on at 07:00 hr and off at Digestive Diseases and Sciences, Vol. 47, No. 4 (April 2002)
0163-2116/02/0400-0850/0 © 2002 Plenum Publishing Corporation
MIBEFRADIL AND GASTRIC PROTECTION 21:00 hr; 14 h of light–10 h of dark). The rat received standart laboratory chow and water ad libitum. Animal Treatment. Twelve hours before the experiment, the rats were fasted but allowed water ad libitum on the day of the experiment. Animals were divided into four groups of eight animals each. Gastric hemorrhagic lesions were induced by administration of 1 ml of 96% ethanol to rat. The control group received 1 ml distilled water by gavage with a metal orogastric tube. Group 2 received 1 ml of absolute ethanol by gavage. Group 3 was given mibefradil (50 mg/kg, orally) followed 30 min later by ethanol. Group 4 received omeprazol (20 mg/kg, orally) followed 30 min later by ethanol. One hour after the administration of ethanol, animals were killed by decapitation. The stomach was removed, opened along the greater curvature, and washed in physiological saline. Measurement of Macroscopic Damage. For the measurement of gross gastric lesions, the freshly excised stomach was laid flat and the mucosal lesions were traced on clear acetate paper. Gross mucosal lesions were recognized as hemorrhage or linear breaks (erosions) with damage to the mucosal surface. The area of gross lesions was calculated in a blind manner by planimetry. The area of mucosal lesion was expressed as a percentage of the control. Tissue Preparation and Assays. For pathological examination, all samples were fixed in formaldehyde buffer and dehydrated in graded concentrations of ethanol (70, 80, 90, and 100%). The tissues were embedded in paraffin, and pathological sections were cut along the longitudinal axis. From each sample, 5-m-thick sections were obtained and stained hematoxylin– eosin to evalute gastric morphology. PMNs were counted in 10 microscopic fields (⫻100). To analyze for the amount of total glutathione (tGSH) and glutathione reductase (GSSG-Rd) activity, the stomach was frozen on solid CO2 and stored ⫺80°C until the day of the assay. tGSH was measured by the method of Griffith (15). GSSG-Rd activity was measured as described by Okpodu and White (16). Chemicals. 5,5⬘-Dithio-bis-2-nitrobenzoic acid (DTNB), reduced glutathione (GSH), GSSG-Rd, NADPH, and tetrasodium salt were obtained from Sigma. Mibefradil (provided by S. Hoffmann-La Roche, Ltd., Basel, Switzerland) was dissolved in distilled water. Statistical Analyses. Data were analyzed by one-way analysis of variance (ANOVA) and Duncan’s multiple comparison test. All results are presented as the mean ⫾ SD of
Fig 1. Effect of mibefradil and omeprazole on hemorhogic mucosal lesions induced by ethanol in the rat. Values are mean ⫾ SD; lesion areas were analyzed by one way ANOVA followed by Duncan’s test (N ⫽ 8 per group). *P ⬍ 0.01 vs control
the mean. The level of significance was accepted at P ⬍ 0.01. Institutional Review. This study was conducted so as to prevent pain and to minimize suffering in all animals that were employed. All experimental protocols were previously approved by the Ethic Committe of the University of Atatu ¨rk Medical School at Erzurum prior to initiating studies.
RESULTS The administration of 96% ethanol produced severe hemorrhagic necrotic lesions in the gastric mucosa. When ethanol alone administrated, the ulcer area was 155.93 (P ⬍ 0.01). However, no gastric lesions appeared in rats treated with mibefradil plus ethanol. We observed only hyperemia in the gastric mucosa (Figure 1, Table 1). Pathologically, the damaged areas included desquamation of the surface epithelium, deep necrosis, edema, and PMN infiltration in the submucosa. One hour after ethanol administration, there was a significant improvement in the gastric lesion because the damage was greatly reduced by mibefradil (Table 1). Mibefradil decreased PMN infiltration, ulcer area,
TABLE 1. MIBEFRADIL PROTECTION ON ETHANOL-INDUCED MACROSCOPIC AND PATHOLOGICAL GASTRIC DAMAGE*
Control EtOH MBF ⫹ EtOH OMEP ⫹ EtOH
Ulcer area
Lesions (N/stomach)
mm2
%
PMN
0.00 ⫾ 0.00 7.55 ⫾ 0.44† 0.00 ⫾ 0.00 4.11 ⫾ 2.61†
0.00 ⫾ 0.00 155.93 ⫾ 5.16† 0.00 ⫾ 0.00 44.94 ⫾ 6.53†
0 100 0 29
113.00 ⫾ 2.21 714.33 ⫾ 2.18† 403.00 ⫾ 2.00† 340.21 ⫾ 0.59†
*The damaged area is expressed as percentage versus control. PMN were counted in 10 microscopic fields. Data were analyzed by one way analysis of variance followed by Duncan’s test. Results are expressed as mean values ⫾ SD. N ⫽ 8 rats per study group. EtOH, etanol: MBF, mibefradil; OMEP, omeprazole. †P ⬍ 0.01 vs. control. Digestive Diseases and Sciences, Vol. 47, No. 4 (April 2002)
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BILICI ET AL TABLE 2. EFFECTS OF MIBEFRADIL AND OMEPRAZOLE ON TOTAL GLUTATHIONE (TGSH) CONCENTRATION AND GLUTATHIONE REDUCTASE (GSSG-RD) ACTIVITY*
Control EtOH EtOH ⫹ MBF EtOH ⫹ OMEP
tGSH (mol/g tissue)
GSSG-Rd (arbitrary units)
3.004 ⫾ 0.01 1.95 ⫾ 0.04† 2.94 ⫾ 0.06 2.06 ⫾ 0.007†
8.34 ⫾ 0.05 5.30 ⫾ 0.08† 8.02 ⫾ 0.06 6.02 ⫾ 0.19†
*Data were analysed by one-way analysis of variance followed by Duncan’s test. Result are expressed as mean values ⫾ SD. N ⫽ 8 rats per study group. †P ⬍ 0.01 vs. control. Abbreviations as in Table 1.
and number of lesions much more than did omeprazole (Figure 2). Biochemical Parameters. The corresponding gastric mucosal tGSH levels are shown in Table 2. In the stomach, ethanol administration markedly decreased the mucosal tGSH level (P ⬍ 0.01) with this effect being totally prevented by mibefradil treatment. Omeprazole did not reduce the loss in tGSH as well as mibefradil (P ⬍ 0.01) (Figure 3). As shown in Table 2, GSSG-Rd activity was significantly decreased by ethanol administration in the stomach (P ⬍ 0.01) with mibefradil reversing this effect (Figure 4). However, omeprazole did not increase GSSG-Rd activity as well as mibefradil (P ⬍0.01). The protection conferred by omeprazole against ethanol-induced biochemical changes was less effective compared with that of mibefradil (Table 2). There was no a meaningful distinction between control group and the group receiving mibefradil.
Fig 3. Effect of mibefradil and omeprazole on gastric total glutathione level decreased by ethanol. Values are mean ⫾ SD. Data were analyzed by one-way ANOVA followed by Duncan’s test (N ⫽ 8 per group). *P ⬍ 0.01 vs control.
The aim of the present study is to compare the therapeutic and antioxidant effects of mibefradil and
omeprazole on ethanol-induced gastric lesions. It has been suggested that one of the mechanisms responsible for ethanol-induced gastroduodenal damage is the generation of the free radicals (3, 6, 7, 11, 17). Results obtained in studies using various inorganic compounds confirm the possible central role of superoxide (䡠O2⫺) and hydroxyl (䡠OH) radicals in the development of ethanol-induced gastric mucosal damage (18). In vivo measurement of oxygen radical release is technically difficult. Most evidence for a role of oxygen radicals in the pathogenesis of acute gastric mucosal lesions, however, is indirect and largely based on the protective effect provided by agent that the proposed biochemical pathway of oxygen radical generation and by removing oxygen radicals with various scavenging agent. For example, superoxide dismutase and catalase, two endogenous antioxidants that protect cells against superoxide anion and hydroperoxide, respectively, decrease mucosal damage caused by ethanol administration (3, 6, 7). Much less information is available on the pharmacology of calcium channel blockers in systems such as
Fig 2. Effect of mibefradil and omeprazole on gastric PMNs increased by ethanol in the rat stomach. Values are mean ⫾ SD. Data were analyzed by one-way ANOVA followed by Duncan’s test (N ⫽ 8 per group). *P ⬍ 0.01 vs control.
Fig 4. Mibefradil protection against ethanol-induced changes in glutathione reductase activity. Values are mean ⫾ SD. Data were analyzed by one-way ANOVA followed by Duncan’s test (N ⫽ 8 per group). *P ⬍ 0.01 vs control.
DISCUSSION
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the gastrointestinal tract. Voltage and use-dependent block of L-type calcium channels have been extensively investigated for a variety of organic calcium entry blockers. In contrast, little is known about Ttype calcium channels in the stomach in both physiological and pathophysiological states. Despite their desirable cardiac effect, the effects of these calcium channel blockers in patients with gastric ulcer are unknown. Mibefradil is a novel calcium antagonist from a new chemical structural class. It is the first calcium antagonist that at therapeutic dosages selectively blocks the T-type (transient, low-voltage-activated) calcium channel, whereas the L-type (long-lasting, highvoltage-activated) calcium channels are blocked by approximately 30% (14). Calcium antagonists are reported to have protective effects against gastric lesions induced by various stimuli (19 –23). In contrast, little is known about T-type calcium channels in the stomach in both physiological and pathophysiological states. Recent research has been directed toward the gastrointestinal effects of calcium channel modulators. Calcium channel blockers inhibit the entry of calcium into cells and/or its mobilization from intracellular stores, with resultant inhibition of calcium effects. As to the mechanisms of their protective effects against chemically induced gastric lesions, several direct or indirect possible mechanism exist. There is evidence that they can decrease gastric contractility (24 –26), motility, increase gastric blood flow, inhibit mast cell degranulation (23), suppress the lipoxygenase pathway (19), and exert certain antioxidant effect (27–29). Calcium influx seems to play an essential role in the stimulation-secretion coupling in mammalian oxyntic cells (30), an effect that can be inhibited by calcium channel blockers. Clinically, the relationship between calcium, gastric acid secretion, and gastric ulcer formation is best represented in patients with primary hyperparathyroidism. The elevated serum calcium levels found in such patients are thought to account for the high incidence of peptic ulcer disease due to hypersecretion of gastric acid (19). It was indicated that calcium channel blockers can inhibit the contractility of gastric muscle as well as basal and stimulated gastric acid secretion (26). In contrast to this, mibefradil does not have inhibitory effects on the gastrointestinel smooth muscle at the therapeutic dosage (14). On the other hand, calcium channel blockers inhibit the lipoxygenase pathway. Thus it is possible that calcium channel blockers may protect against chemically induced gastric lesions by Digestive Diseases and Sciences, Vol. 47, No. 4 (April 2002)
diverting arachidonic acid metabolism toward the synthesis of prostoglandins, which are known to be cytoprotective against chemically induced gastric lesions and decreasing leukotriene formation by inhibiting the lipoxygenase pathway (19). However, controversy exists regarding their effects on ethanolinduced injury. Some authors report gastroprotection (19, 22, 23) while others describe worsening of the ulcers by different calcium antogonists (28, 31–34). In this study mibefradil significantly reduced ethanolinduced gastric lesions. The efficiency of endogenous antioxidant defense mechanism may be of critical importance in protecting against the development of acute gastric mucosal injury. The tissue level of nonprotein sulfhydryls (mainly reduced glutathione) is high in organs (liver, stomach, lung) potentially exposed to free-radical reactions (5). Cytoprotective sulfhydryls are efficient free-radical scavengers (4, 6). In vitro experiments have shown that GSH is essential for the protection of endothelial cells and gastric chief cells against oxidative stress (4, 9). Ethanol-induced gastric mucosal damage is associated with a significant reduction in the nonprotein sulfhydryl concentration in the rat and humans (6, 10, 11, 13). There is evidence that glutathione depletion in hepatocytes results in an increase in cytosolic free calcium concentration. Therefore, glutathione depletion in the gastric mucosa may cause gastric mucosal lesions indirectly by triggering an increase in the intracellular calcium concentration in oxyntic cells. Thus, the gastroprotective effect, obtained with mibefradil may be due to the decrease in the intracelluler calcium concentration in oxyntic cells. In our study, ethanol significantly decreased gastric glutathione concentration. This reduction may be due to the oxidation of GSH because of ethanol-induced generation of toxic oxygen metabolites (6) or binding of glutathione to acetaldehyde generated through the oxidation of ethanol by the gastric alcohol dehydrogenase activity (10). Alternatively, a block in the synthesis of the tripeptide induced by ethanolgenerated free radicals may occur. Ethanol treatment has been shown to decrease the level of gastric cysteine by 50% (10). Thus, it is likely that oxygen free radicals, generated by ethanol administration, converted gastric cysteine to cystine thereby inhibiting the synthesis of GSH. Moreover, the impairment of GSSG-Rd activity, which would decrease the rate of conversion of GSSG to GSH, could contribute to GSH depletion. The possibility that the decrease in glutathione brought about by ethanol is the result of
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leakage from damaged mucosa cannot be excluded. Little is known about the antioxidant function of mibefradil. In this study, mibefradil significantly corrected by ethanol-induced GSH depletion. Hirashi et al demonstrated that GSSG-Rd is much more potent than endogenous catalase in protecting cultured gastric mucosal cell from oxidative stress (35). The impairment of GSSG-Rd activity induced by ethanol administration is probably due to the generation of free radicals at a rate that overwhelms the naturally occurring defense mechanism of the cell. In this study, too, GSSG-Rd activity was decreased in the stomach by ethanol administration. Mibefradil completely or partially overcame ethanol-induced changes in GSSG-Rd activity in the stomach. Thus, mibefradil’s antioxidative effects can be direct or by stimulation of other antioxidants. The antioxidant and cytoprotective effect or mibefradil was described by Mason et al (29). Mibefradil not only protected against macroscopic gastric damage and altered GSH methabolism but also reduced PMN infiltration into the stomach. This is important because the number of PMNs is proportional to the extent of the gastric damage following ethanol administration. PMNs have been proposed as alternative sources of superoxide radicals (36 –38). The respiratory burst of activated PMNs causes the univalent enzymatic reduction of molecular oxygen to the superoxide anion radical, which, in turn can give rise to further generation of more reactive oxygen species (39). The results of our study demonstrate that T-type calcium channel blockers would have antioxidant effect and have therapeutic importance in peptic ulcer. This evidence suggests it may be safe in patients with peptic ulcer, although continuing studies are necessary before definitive conclusion can be made.
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