Pharmacological Research, Vol. 23, No. 1, 1991
71
CALCIUM AND ETHANOL-INDUCED GASTRIC MUCOSAL DAMAGE IN RATS S. H. WONG, C. H. CHO and C. W. OGLE
Department of Pharmacology, Facultyof Medicine, Universityof Hong Kong, Hong Kong Received in final form 4 May 1990
SUMMARY
The effects of graded doses of ethanol on stomach mucosal damage and calcium levels were studied in rats. The influence of verapamil and/or calcium chloride on these changes was also investigated. Orally administered ethanol (20, 50 or 80% v/v) markedly decreased gastric glandular tissue calcium and it concentration dependently produced mucosal lesions. Pretreatment with verapamil (2.5 or 5 mg/kg, i.p.) dose dependently lessened glandular wall calcium levels and worsened ethanol-induced mucosal damage. Calcium chloride (50 mg/kg, i.p.) significantly prevented ethanol-induced gastric calcium depletion; it also dose dependently antagonized the damaging effect of ethanol as well as the lesionintensifying action of verapamil. The findings that verapamil potentiated, whereas calcium chloride prevented, ethanol-induced glandular mucosal damage and tissue calcium changes indeed suggest that altered gastric cell calcium levels could be closely related to the mucosal lesions produced by ethanol in rats. KEY WORDS:ethanol, gastric lesions, calcium, verapamil, calcium chloride.
INTRODUCTION Calcium regulates many different types of cellular activity [1]. It is necessary for the synaptic release of acetycholine, following an action potential in the nerve, which leads to muscle contraction [2]. The involvement of calcium in stimulus-secretion coupling has also been demonstrated in a variety of tissues [3]. The release of histamine from mast cells and of catecholamines from adrenergic neurones has been shown to require the presence of calcium [4, 5]. Cholinergic stimulation of acid secretion likewise seems to depend on calcium [6]. Hypercalcaemia induces gastric hypersecretion in man, whereas hypocalcaemia leads to lowered secretion [7]. Recently, calcium channel blockers have been found either to prevent [8] or worsen [9, 10] gastric damage by ethanol. Thus, it is conceivable that calcium participates in the pathogenesis of ethanol-induced mucosal lesions. However, the Correspondence: Dr C. H. Cho, Department of Pharmacology, Faculty of Medicine, University of Hong Kong, 5 Sassoon Road, Hong Kong. 1043-6618/91/010071-09/S03.00/0
© 1991 The ltalian Pharmacological Society
72
Pharmacological Research, Vol. 23, No. 1, 1991
effects of ethanol and of calcium channel blockers on calcium levels in the gastric tissue have not yet been investigated. The present study, therefore, examines the hypothesis that disturbances in gastric tissue calcium may be involved in stomach mucosal damage produced by oral administration of ethanol in rats.
MATERIALS AND METHODS
General Male Sprague-Dawley rats, weighing 250-280g, were housed in an airconditioned room in which the temperature was maintained at 22 +__I°C and humidity at 65-70%. They were fed a balanced pellet diet (Ralston Purina Co.) and given ordinary tap water to drink. Solid food was withheld 2 4 h before experiments, but the rats were allowed free access to tap water which was removed 1 h before they were used. Verapamil (Knoll; 2.5 or 5 mg/kg), calcium chloride (BDH; 25 or 50 mg/kg) or 0.9% v/v NaC1 solution (saline; 1 ml/kg) was injected i.p. 30 min before oral administration of 50% v/v ethanol (BDH) (10 ml/kg) via a plastic gastric tube. All rats were killed by a blow on the head 1 h after ethanol treatment. The severity of mucosal damage was evaluated, using a single-blind method, by measuring the area of the lesions with a transparent grid (divided into 1 nllll 2 areas) placed on the glandular mucosal surface [11]. In the case of petechiae, five such lesions were taken as the equivalent of 1 mm 2. The sum of the lesion areas in each group of rats was divided by the number of animals and expressed as the mean lesion index. The effects of ethanol (20, 50 or 80% v/v; 10 ml/kg, p.o.), verapamil (2.5 or 5 mg/kg, i.p.), calcium chloride (25 or 50 mg/kg, i.p.), saline (1 ml/kg, i.p.) or distilled water (10 ml/kg, p.o.) on the glandular mucosal calcium content were also assessed. All rats were killed by a blow on the head 15, 30 or 60 rain after drug treatment; their stomachs were then immediately removed, opened along the greater curvature and rinsed thoroughly with deionized water. The glandular segments were excised and weighed before determination of their calcium content. Measurement of gastric tissue calcium levels The gastric tissue calcium levels were determined by the method developed by Koo et al. [12] but with higher oven temperature and lanthanum chloride concentration. The weighed glandular parts of the stomachs were placed individually into crucibles and ashed in an oven (Naber, model N11) at 700°C for 24 h. Two empty crucibles were also exposed to the same temperature for a similar period of time. After 24 h of ashing, all the crucibles were taken out and allowed to cool before 2 ml of 2 M nitric acid in 0.8% w/v lanthanum chloride were added to each (including the two empty crucibles) to obtain a clear solution of the ashed tissue. The amount of calcium present in these solutions was then determined by using an atomic absorption spectrophotometer (Perkin-Elmer, model 103) set at a wavelength of 495 nm. The acetylene flow rate had been presented and the burner ignited for 10 rain before readings were taken, in order to achieve temperature equilibrium; zero readings were set during aspiration of the lanthanum chloride
Pharmacological Research, Vol. 23, No. 1, 1991
73
solution which acted as the blank. Calcium (BDH) solutions of various concentrations (10, 20, 30, 40 and 50/.tmol/ml) were also made up with lanthanum chloride solution and used to prepare the standard curve. Readings were taken with each sample alternating with a deionized water wash; zero readings were checked after every five sample determinations. The gastric tissue calcium content was then calculated from a standard curve which was freshly prepared whenever determinations were carried out. After subtracting the averaged residual calcium found in the empty crucibles, the values were expressed as ktmol of calcium per unit mass of tissue.
Statistical analysis of the data The data were expressed as the means +SEM. Differences were analysed for statistical significance by Student's unpaired two-tailed t-test.
RESULTS Ethanol given p.o. dose dependently decreased the stomach glandular wall calcium content and produced mucosal damage at 15, 30 and 60 rain after administration (Table I). No haemorrhagic lesions or changes in calcium content were observed in the rumenal segment of the stomach (data not shown). Veraparnil itself dose dependently lessened the glandular wall calcium content (Table II); this reduction was seen 15 min after treatment and was maintained throughout the 60-min observation period. In contrast, calcium chloride did not alter the glandular calcium level when compared to the saline-treated control (Table II). No haemorrhagic lesions were observed after verapamil or calcium chloride treatment. In Table III, as already seen, the higher pretreatment dose (5 mg/kg) of verapamil alone significantly decreased the calcium level in the glandular segment. Verapamil pretreatment (30 min beforehand) markedly and dose dependently enhanced the severity of the gastric damage produced by ethanol; statistical significance was reached with both pretreatment doses of verapamil. This effect of the calcium channel blocker was accompanied by reduced glandular calcium levels. In contrast, calcium chloride pretreatment (30 min beforehand), which itself did not affect the basal calcium values in the glandular segment, was able to reverse the calciumdepleting action of ethanol (Table 11I). Pretreatment with calcium chloride markedly lessened ethanol-induced gastric mucosal damage and prevented the ethanol-lesion-intensifying action of verapamil (Table IV). Calcium chloride also lessened the calcium-lowering action of verapamil in ethanol-treated animals. DISCUSSION This study shows that ethanol lowers the glandular wall calcium content, thus, pointing to the possibility that calcium depletion in the gastric glandular segment during ethanol exposure could be an important factor involved in lesion formation.
74
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71
CALCIUM AND ETHANOL-INDUCED GASTRIC MUCOSAL DAMAGE IN RATS S. H. WONG, C. H. CHO and C. W. OGLE
Department of Pharmacology, Facultyof Medicine, Universityof Hong Kong, Hong Kong Received in final form 4 May 1990
SUMMARY
The effects of graded doses of ethanol on stomach mucosal damage and calcium levels were studied in rats. The influence of verapamil and/or calcium chloride on these changes was also investigated. Orally administered ethanol (20, 50 or 80% v/v) markedly decreased gastric glandular tissue calcium and it concentration dependently produced mucosal lesions. Pretreatment with verapamil (2.5 or 5 mg/kg, i.p.) dose dependently lessened glandular wall calcium levels and worsened ethanol-induced mucosal damage. Calcium chloride (50 mg/kg, i.p.) significantly prevented ethanol-induced gastric calcium depletion; it also dose dependently antagonized the damaging effect of ethanol as well as the lesionintensifying action of verapamil. The findings that verapamil potentiated, whereas calcium chloride prevented, ethanol-induced glandular mucosal damage and tissue calcium changes indeed suggest that altered gastric cell calcium levels could be closely related to the mucosal lesions produced by ethanol in rats. KEY WORDS:ethanol, gastric lesions, calcium, verapamil, calcium chloride.
INTRODUCTION Calcium regulates many different types of cellular activity [1]. It is necessary for the synaptic release of acetycholine, following an action potential in the nerve, which leads to muscle contraction [2]. The involvement of calcium in stimulus-secretion coupling has also been demonstrated in a variety of tissues [3]. The release of histamine from mast cells and of catecholamines from adrenergic neurones has been shown to require the presence of calcium [4, 5]. Cholinergic stimulation of acid secretion likewise seems to depend on calcium [6]. Hypercalcaemia induces gastric hypersecretion in man, whereas hypocalcaemia leads to lowered secretion [7]. Recently, calcium channel blockers have been found either to prevent [8] or worsen [9, 10] gastric damage by ethanol. Thus, it is conceivable that calcium participates in the pathogenesis of ethanol-induced mucosal lesions. However, the Correspondence: Dr C. H. Cho, Department of Pharmacology, Faculty of Medicine, University of Hong Kong, 5 Sassoon Road, Hong Kong. 1043-6618/91/010071-09/S03.00/0
© 1991 The ltalian Pharmacological Society
72
Pharmacological Research, Vol. 23, No. 1, 1991
effects of ethanol and of calcium channel blockers on calcium levels in the gastric tissue have not yet been investigated. The present study, therefore, examines the hypothesis that disturbances in gastric tissue calcium may be involved in stomach mucosal damage produced by oral administration of ethanol in rats.
MATERIALS AND METHODS
General Male Sprague-Dawley rats, weighing 250-280g, were housed in an airconditioned room in which the temperature was maintained at 22 +__I°C and humidity at 65-70%. They were fed a balanced pellet diet (Ralston Purina Co.) and given ordinary tap water to drink. Solid food was withheld 2 4 h before experiments, but the rats were allowed free access to tap water which was removed 1 h before they were used. Verapamil (Knoll; 2.5 or 5 mg/kg), calcium chloride (BDH; 25 or 50 mg/kg) or 0.9% v/v NaC1 solution (saline; 1 ml/kg) was injected i.p. 30 min before oral administration of 50% v/v ethanol (BDH) (10 ml/kg) via a plastic gastric tube. All rats were killed by a blow on the head 1 h after ethanol treatment. The severity of mucosal damage was evaluated, using a single-blind method, by measuring the area of the lesions with a transparent grid (divided into 1 nllll 2 areas) placed on the glandular mucosal surface [11]. In the case of petechiae, five such lesions were taken as the equivalent of 1 mm 2. The sum of the lesion areas in each group of rats was divided by the number of animals and expressed as the mean lesion index. The effects of ethanol (20, 50 or 80% v/v; 10 ml/kg, p.o.), verapamil (2.5 or 5 mg/kg, i.p.), calcium chloride (25 or 50 mg/kg, i.p.), saline (1 ml/kg, i.p.) or distilled water (10 ml/kg, p.o.) on the glandular mucosal calcium content were also assessed. All rats were killed by a blow on the head 15, 30 or 60 rain after drug treatment; their stomachs were then immediately removed, opened along the greater curvature and rinsed thoroughly with deionized water. The glandular segments were excised and weighed before determination of their calcium content. Measurement of gastric tissue calcium levels The gastric tissue calcium levels were determined by the method developed by Koo et al. [12] but with higher oven temperature and lanthanum chloride concentration. The weighed glandular parts of the stomachs were placed individually into crucibles and ashed in an oven (Naber, model N11) at 700°C for 24 h. Two empty crucibles were also exposed to the same temperature for a similar period of time. After 24 h of ashing, all the crucibles were taken out and allowed to cool before 2 ml of 2 M nitric acid in 0.8% w/v lanthanum chloride were added to each (including the two empty crucibles) to obtain a clear solution of the ashed tissue. The amount of calcium present in these solutions was then determined by using an atomic absorption spectrophotometer (Perkin-Elmer, model 103) set at a wavelength of 495 nm. The acetylene flow rate had been presented and the burner ignited for 10 rain before readings were taken, in order to achieve temperature equilibrium; zero readings were set during aspiration of the lanthanum chloride
Pharmacological Research, Vol. 23, No. 1, 1991
73
solution which acted as the blank. Calcium (BDH) solutions of various concentrations (10, 20, 30, 40 and 50/.tmol/ml) were also made up with lanthanum chloride solution and used to prepare the standard curve. Readings were taken with each sample alternating with a deionized water wash; zero readings were checked after every five sample determinations. The gastric tissue calcium content was then calculated from a standard curve which was freshly prepared whenever determinations were carried out. After subtracting the averaged residual calcium found in the empty crucibles, the values were expressed as ktmol of calcium per unit mass of tissue.
Statistical analysis of the data The data were expressed as the means +SEM. Differences were analysed for statistical significance by Student's unpaired two-tailed t-test.
RESULTS Ethanol given p.o. dose dependently decreased the stomach glandular wall calcium content and produced mucosal damage at 15, 30 and 60 rain after administration (Table I). No haemorrhagic lesions or changes in calcium content were observed in the rumenal segment of the stomach (data not shown). Veraparnil itself dose dependently lessened the glandular wall calcium content (Table II); this reduction was seen 15 min after treatment and was maintained throughout the 60-min observation period. In contrast, calcium chloride did not alter the glandular calcium level when compared to the saline-treated control (Table II). No haemorrhagic lesions were observed after verapamil or calcium chloride treatment. In Table III, as already seen, the higher pretreatment dose (5 mg/kg) of verapamil alone significantly decreased the calcium level in the glandular segment. Verapamil pretreatment (30 min beforehand) markedly and dose dependently enhanced the severity of the gastric damage produced by ethanol; statistical significance was reached with both pretreatment doses of verapamil. This effect of the calcium channel blocker was accompanied by reduced glandular calcium levels. In contrast, calcium chloride pretreatment (30 min beforehand), which itself did not affect the basal calcium values in the glandular segment, was able to reverse the calciumdepleting action of ethanol (Table 11I). Pretreatment with calcium chloride markedly lessened ethanol-induced gastric mucosal damage and prevented the ethanol-lesion-intensifying action of verapamil (Table IV). Calcium chloride also lessened the calcium-lowering action of verapamil in ethanol-treated animals. DISCUSSION This study shows that ethanol lowers the glandular wall calcium content, thus, pointing to the possibility that calcium depletion in the gastric glandular segment during ethanol exposure could be an important factor involved in lesion formation.
74
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