Agents Actions, 35 (1992)
0065-4299/92/020130-05 $1.50+ 0.20/0 9 1992 Birkh~iuser Verlag, Basel
Calcium channel blockers prevent stress-induced ulcers in rats * Berrak C. Yegen 1, Inci Alican 1, A. Stiha Yal~in 2, Sule Oktay 3 Departments of Biochemistry a, Pharmacology 3 and Physiology 1, Marmara University, School of Medicine, 81326, Haydarpasa, Istanbul, Turkey
Abstract
Gastric mucosal damage induced by cold and restraint stress caused increase in gastric lipid peroxidation (LP) and decrease in gastric glutathione levels. Two calcium-channel blockers, verapamil and nicardipine, prevented stress-induced increase in gastric LP, as well as ulcer formation. Both calcium-channel blockers protected against stress-induced ulcers, and inhibition of LP may be among their mechanisms of action.
Introduction
The discovery of calcium channel blockers has started a considerable research effort in basic and applied health sciences to reexamine calcium-dependent processes [1]. Recently attention has been directed toward the gastrointestinal effects of calcium channel blockers [2, 3]. Hypercalcemia was found to be associated with increased gastric acid secretion [3, 4]. On the other hand, pentagastrin- and cholinergically stimulated acid output were decreased in the absence of calcium [5]. Although nitrendipine and verapamil were reported to decrease basal gastric acid secretion, effects of calcium channel blockers on gastric ulcer formation due to different stimuli are variable, and depend on both the ulcerogenic stimulus and the type of calcium channel blocker [2, 3, 6]. Cold-restraint treatment causes gastric stress ulcers in rats [7]. We have recently reported that * In memory of Dr. Ilker Ayka~ whom we have started with. 3 Author for correspondence.
changes in gastric lipid peroxidation and gastric glutathione levels may be included among the etiopathogenic factors leading to stress-induced gastric ulcers [8]. Some calcium-channel blockers were reported to have strong in vitro antioxidant effects [9-11]. Recently, nifedipine and diltiazem treatment was shown to prevent brain toxicity of lithium caused by lipid peroxidation in mice [12]. In the present study, we have attempted to investigate the protective effects of the two calcium-channel blockers, verapamil and nicardipine on cold-restraint-induced gastric ulcer formation, lipid peroxidation and glutathione levels.
Materials and methods
1. Animals Albino rats of both sexes (200-250 g) were fed a standard diet and water ad libitum. The control group (n = 6) was deprived of food for 48 h prior to sacrifice. Animals in the stress group (n = 13) were
Agents Actions, 35 (1992)
131
barbituric acid reactive substance formation as described previously [14]. Lipid peroxide levels were expressed in terms of malondialdehyde (MDA) equivalents using an extinction coefficient of 1.56 x 105 M - 1 c m - 1.
immobilized in plastic restraining devices at 2 4 ~ for 3 h following a 48 h starvation period. 2. Cold-restraint stress-induced gastric lesions Following stress procedures, all rats were decapitated and the stomachs were rapidly removed, opened along the greater curvature, washed and examined macroscopically to measure the length of ulcers (ulcer index) and % ulcer occurrence was recorded.
4. Pretreatment with calcium channel blockers Rats were pretreated with either verapamil or nicardipine (Knoll and Sandoz, respectively) 1 h before cold-restraint administration (0.01, 0.1, 1 and 10 mg/kg; i.p.; n = 1 0 - 1 5 for each dose).
3. Determination of glutathione and lipid peroxide levels
5. Statistical analysis Data were expressed as m e a n + S.E.M. and analyzed by Student's unpaired t-test.
Tissue samples were homogenized in ice-cold trichloracetic acid (1 g tissue plus 10 ml 10% TCA) in an Ultra Turrax tissue homogenizer. Glutathione measurements were performed using a modification of the Ellman procedure [13]. Briefly, after centrifugation at 3000 rev./min for 10 min, 0.5 ml of supernatant was added to 2 m l of 0 . 3 M Na2HPO4.2H20 solution. A 0.2-ml solution of dithiobisnitrobenzoate (0.4 mg/ml 1% sodium citrate) was added and the absorbance at 412 nm was measured immediately after mixing. Glutathione levels were calculated using an extinction coefficient of 13 600 M - 1 c m - 1. The degree of lipid peroxide formation was assayed by monitoring thio-
Results 1. Gastric ulcers
Cold-restraint administration caused multiple gastric ulcers in 92% of animals. The ulcer index was calculated as 13.5_+ 3.3 mm. In the control group no ulcers were observed. Both verapamil and nicardipine significantly prevented gastric ulcerogenesis induced by cold-restraint, in a dose-dependent manner (Fig. 1). Gastric ulcers developed on-
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0065-4299/92/020130-05 $1.50+ 0.20/0 9 1992 Birkh~iuser Verlag, Basel
Calcium channel blockers prevent stress-induced ulcers in rats * Berrak C. Yegen 1, Inci Alican 1, A. Stiha Yal~in 2, Sule Oktay 3 Departments of Biochemistry a, Pharmacology 3 and Physiology 1, Marmara University, School of Medicine, 81326, Haydarpasa, Istanbul, Turkey
Abstract
Gastric mucosal damage induced by cold and restraint stress caused increase in gastric lipid peroxidation (LP) and decrease in gastric glutathione levels. Two calcium-channel blockers, verapamil and nicardipine, prevented stress-induced increase in gastric LP, as well as ulcer formation. Both calcium-channel blockers protected against stress-induced ulcers, and inhibition of LP may be among their mechanisms of action.
Introduction
The discovery of calcium channel blockers has started a considerable research effort in basic and applied health sciences to reexamine calcium-dependent processes [1]. Recently attention has been directed toward the gastrointestinal effects of calcium channel blockers [2, 3]. Hypercalcemia was found to be associated with increased gastric acid secretion [3, 4]. On the other hand, pentagastrin- and cholinergically stimulated acid output were decreased in the absence of calcium [5]. Although nitrendipine and verapamil were reported to decrease basal gastric acid secretion, effects of calcium channel blockers on gastric ulcer formation due to different stimuli are variable, and depend on both the ulcerogenic stimulus and the type of calcium channel blocker [2, 3, 6]. Cold-restraint treatment causes gastric stress ulcers in rats [7]. We have recently reported that * In memory of Dr. Ilker Ayka~ whom we have started with. 3 Author for correspondence.
changes in gastric lipid peroxidation and gastric glutathione levels may be included among the etiopathogenic factors leading to stress-induced gastric ulcers [8]. Some calcium-channel blockers were reported to have strong in vitro antioxidant effects [9-11]. Recently, nifedipine and diltiazem treatment was shown to prevent brain toxicity of lithium caused by lipid peroxidation in mice [12]. In the present study, we have attempted to investigate the protective effects of the two calcium-channel blockers, verapamil and nicardipine on cold-restraint-induced gastric ulcer formation, lipid peroxidation and glutathione levels.
Materials and methods
1. Animals Albino rats of both sexes (200-250 g) were fed a standard diet and water ad libitum. The control group (n = 6) was deprived of food for 48 h prior to sacrifice. Animals in the stress group (n = 13) were
Agents Actions, 35 (1992)
131
barbituric acid reactive substance formation as described previously [14]. Lipid peroxide levels were expressed in terms of malondialdehyde (MDA) equivalents using an extinction coefficient of 1.56 x 105 M - 1 c m - 1.
immobilized in plastic restraining devices at 2 4 ~ for 3 h following a 48 h starvation period. 2. Cold-restraint stress-induced gastric lesions Following stress procedures, all rats were decapitated and the stomachs were rapidly removed, opened along the greater curvature, washed and examined macroscopically to measure the length of ulcers (ulcer index) and % ulcer occurrence was recorded.
4. Pretreatment with calcium channel blockers Rats were pretreated with either verapamil or nicardipine (Knoll and Sandoz, respectively) 1 h before cold-restraint administration (0.01, 0.1, 1 and 10 mg/kg; i.p.; n = 1 0 - 1 5 for each dose).
3. Determination of glutathione and lipid peroxide levels
5. Statistical analysis Data were expressed as m e a n + S.E.M. and analyzed by Student's unpaired t-test.
Tissue samples were homogenized in ice-cold trichloracetic acid (1 g tissue plus 10 ml 10% TCA) in an Ultra Turrax tissue homogenizer. Glutathione measurements were performed using a modification of the Ellman procedure [13]. Briefly, after centrifugation at 3000 rev./min for 10 min, 0.5 ml of supernatant was added to 2 m l of 0 . 3 M Na2HPO4.2H20 solution. A 0.2-ml solution of dithiobisnitrobenzoate (0.4 mg/ml 1% sodium citrate) was added and the absorbance at 412 nm was measured immediately after mixing. Glutathione levels were calculated using an extinction coefficient of 13 600 M - 1 c m - 1. The degree of lipid peroxide formation was assayed by monitoring thio-
Results 1. Gastric ulcers
Cold-restraint administration caused multiple gastric ulcers in 92% of animals. The ulcer index was calculated as 13.5_+ 3.3 mm. In the control group no ulcers were observed. Both verapamil and nicardipine significantly prevented gastric ulcerogenesis induced by cold-restraint, in a dose-dependent manner (Fig. 1). Gastric ulcers developed on-
15
100
E E 10
n,, I.d 0 ...I
x b.J r'.,,
50 4c
Z
a: w
i
5
0 ._1
I
CR
0.01 0.1
*
1
10
0.01 0.1
1
