Br. J. Pharmacol.
Br.
J.
Pharmacol.
(I992), 105, 912 918 (1992),
105,
'."
912-918
©
Macmillan Press Ltd, 1992
Macmillan
Press
Ltd,
1992
Dexamethasone-induced gastric mucosal damage in the rat: possible role of platelet-activating factor l*tJanos G. Filep,
tFerenc
Herman, *tEva Foldes-Filep, TFerenc Schneider & **Pierre Braquet
*Department of Pharmacology, Faculty of Medicine, University of Sherbrooke, Sherbrooke, P.Q., Canada J1H 5N4, tDepartment of Pathophysiology, Semmelweis University Medical School, Budapest, 1445 Hungary and **Institut Henri Beaufour, Le Plessis Robinson, 92350 France 1 The aim of the present experiments was to study the possible role of platelet-activating factor (PAF) in mediating gastric mucosal damage induced by dexamethasone in the rat by measuring gastric tissue levels of PAF during dexamethasone-treatment and by investigating the effects of specific PAF receptor antagonists on dexamethasone-induced gastric lesions. PAF-like bioactivity extracted from the rat glandular stomach was determined by a platelet aggregation assay. 2 Dexamethasone treatment (0.4- 4 mg kg-', daily for 1- 6 days) produced time- and dose-dependent damage to the glandular mucosa of the stomach as characterized by extensive, uniform hyperaemia with multiple, focal petechiae and erosions. 3 These changes were accompanied by a time-, and dose-dependent increase in PAF content of the glandular stomach. Control rat stomach contained small amounts of PAF (0.14 ± 0.04 ng per g wet weight), which increased over 40 fold in response to dexamethasone treatment (4 mg kg-1, daily for 6 consecutive days). The presence of PAF-like material in the stomach extract was ascertained by thin-layer chromatography, high performance liquid chromatography and by alkaline hydrolysis. 4 Pretreatment of the animals with one or other of the structurally unrelated PAF receptor antagonists, BN 52021 (10mg kg-', i.p.) or BN 50727 (1 mg kg-', i.p.) significantly reduced dexamethasone-induced gastric damage. In these animals neither petechiae nor erosions were observed. 5 These observations suggest that PAF is a likely endogenous mediator of glucocorticoid-induced gastric mucosal damage in the rat. Keywords: PAF; dexamethasone; glucocorticoid-induced gastric ulcer; PAF antagonists; BN 52021 (ginkgolide B); BN 50727
Introduction Although there is a long-standing debate over whether corticosteroid therapy leads to peptic ulcer disease in man (recently reviewed by Piper et al., 1991), it is well established that administration of glucocorticoids to laboratory animals can result in an acute gastric erosion formation (Robert & Nezamis, 1958; Nobuhara et al., 1985; Wallace, 1987). The mechanism of this damaging action of glucocorticoids is, however, far from being understood. Glucocorticoids have been shown to inhibit synthesis of gastric mucus (Menguy & Masters, 1963), to induce G cell and parietal cell hyperplasia (Delaney et al., 1979) and to affect epithelial cell turnover (Eastwood, 1984). Dexamethasone and prednisolone have been shown to be ineffective in blocking prostaglandin (Whittle, 1978; Wallace, 1987), but not leukotriene C4 (Wallace, 1987) synthesis in the rat stomach, suggesting that their ulcerogenic action may not be related to decreased prostaglandin formation. Platelet-activating factor (PAF) is a phospholipid mediator of allergy and inflammation with a wide variety of biological activities including platelet aggregation, activation of neutrophil granulocytes, contraction of smooth muscle, increasing vascular permeability and inducing hypotension (for a recent review see Braquet et al., 1987). Administration of picomol doses of PAF into the gastric artery induced substantial gastric mucosal hyperaemia, haemorrhage and erosions (Rosam et al., 1986; Wallace & Whittle, 1986; Whittle et al., 1986; Espluques & Whittle, 1988), changes similar to those observed following glucocorticoid administration (Rob-
I Author for correspondence at: Department of Pharmacology, Faculty of Medicine, University of Sherbrooke, Sherbrooke, P.Q., Canada J1H 5N4.
ert & Nezamis, 1958; Nobuhara et al., 1985; Wallace, 1987).
These findings raised the possibility that release of endogenous PAF may underlie or contribute to certain forms of gastric ulceration. The aim of the present experiments was to study the possible role of PAF in mediating gastric damage to dexamethasone by measuring the PAF content in the rat stomach during dexamethasone treatment and by investigating the effects of two structurally unrelated, specific PAF receptor antagonists, BN 52021 (ginkgolide B) (Braquet et al., 1985; Foldes-Filep et al., 1987) and the benzodiazepine derivative, BN 50727 (Braquet & Esanu, 1991) on dexamethasone-induced gastric lesions.
Methods Male, Wistar rats weighing 160-205 g were housed in groups of 4 and were fed standard laboratory chow and tap water ad libitum.
Drug treatment Between 09 h 00 min and 10 h 00 min each day the animals were given an intraperitoneal injection of the PAF receptor antagonists, BN 52021 (10 mg kg-') or BN 50727 (1 mg kg-1), or their vehicle, and 30 min later they received a subcutaneous injection of dexamethasone sodium phosphate or vehicle. The effectiveness of BN 52021 and BN 50727 treatments was tested in preliminary experiments in anaesthetized (Inactin, 100 mg kg', i.p.) male rats by injecting 25 and 50 ng kg-' PAF into the right femoral vein 30 min before and 30 min after administration of either BN 52021, 10 mg kg-', i.p., or BN 50727, 1 mg kg-', i.p. Mean arterial
PAF, DEXAMETHASONE AND GASTRIC MUCOSAL DAMAGE
blood pressure was monitored through a catheter inserted into the right femoral artery by an electromanometer (EM 61, Medicor, Budapest, Hungary) using a Statham P23 dB pressure transducer (Hato Ray, Puerto Rico). The following sets of experiments were performed: Series I The animals received 0.4, 1 or 4mgkg-' of dexamethasone or its vehicle daily for 6 consecutive days.
Series 2 The animals were given 4 mg kg- ' of dexamethasone each day for 1, 2, 3, 4, 5 or 6 consecutive days. Series 3 The animals were divided into four groups: (a) control (receiving vehicle only), (b) BN 52021 (i.e. BN 52021, 10 mg kg-', i.p. plus vehicle s.c.), (c) dexamethasone (vehicle i.p. plus dexamethasone, 4mg kg-' s.c. ) and (d) BN 52021, 10 mg kg-' plus dexamethasone, 4 mg kg-l. The treatments were continued for 6 consecutive days.
Series 4 In these series of experiments a protocol similar to that of Series 3 was followed except that another PAF receptor antagonist, BN 50727 (1 mg kg-', i.p.) was used instead of BN 52021. Depending on the experimental protocol used, on days 1, 2, 3, 4, 5 or 6 the animals were anaesthetized with sodium pentobarbitone (40 mg kg-', i.p.) S h after the second injection. The animals were deprived of food, but not water, for 18-20 h before being anaesthetized. The stomach was immediately removed, opened along the greater curvature and examined. The extent of mucosal damage was scored by two observers in a randomized, blind manner using a 0 to 3 scoring system (Rosam et al., 1986; Wallace, 1987). A score of 0 was assigned if the mucosa appeared normal. A stomach
exhibiting hyperaemia or haemorrhage was assigned a score of 1 (limited to diffuse superficial hyperaemia), 2 (severe, extensive hyperaemia or focal haemorrhage) or 3 (multiple sites of bleeding or erosion). The correlation between the scores assigned by the two observers was greater than 0.860 (n=20, P
Br.
J.
Pharmacol.
(I992), 105, 912 918 (1992),
105,
'."
912-918
©
Macmillan Press Ltd, 1992
Macmillan
Press
Ltd,
1992
Dexamethasone-induced gastric mucosal damage in the rat: possible role of platelet-activating factor l*tJanos G. Filep,
tFerenc
Herman, *tEva Foldes-Filep, TFerenc Schneider & **Pierre Braquet
*Department of Pharmacology, Faculty of Medicine, University of Sherbrooke, Sherbrooke, P.Q., Canada J1H 5N4, tDepartment of Pathophysiology, Semmelweis University Medical School, Budapest, 1445 Hungary and **Institut Henri Beaufour, Le Plessis Robinson, 92350 France 1 The aim of the present experiments was to study the possible role of platelet-activating factor (PAF) in mediating gastric mucosal damage induced by dexamethasone in the rat by measuring gastric tissue levels of PAF during dexamethasone-treatment and by investigating the effects of specific PAF receptor antagonists on dexamethasone-induced gastric lesions. PAF-like bioactivity extracted from the rat glandular stomach was determined by a platelet aggregation assay. 2 Dexamethasone treatment (0.4- 4 mg kg-', daily for 1- 6 days) produced time- and dose-dependent damage to the glandular mucosa of the stomach as characterized by extensive, uniform hyperaemia with multiple, focal petechiae and erosions. 3 These changes were accompanied by a time-, and dose-dependent increase in PAF content of the glandular stomach. Control rat stomach contained small amounts of PAF (0.14 ± 0.04 ng per g wet weight), which increased over 40 fold in response to dexamethasone treatment (4 mg kg-1, daily for 6 consecutive days). The presence of PAF-like material in the stomach extract was ascertained by thin-layer chromatography, high performance liquid chromatography and by alkaline hydrolysis. 4 Pretreatment of the animals with one or other of the structurally unrelated PAF receptor antagonists, BN 52021 (10mg kg-', i.p.) or BN 50727 (1 mg kg-', i.p.) significantly reduced dexamethasone-induced gastric damage. In these animals neither petechiae nor erosions were observed. 5 These observations suggest that PAF is a likely endogenous mediator of glucocorticoid-induced gastric mucosal damage in the rat. Keywords: PAF; dexamethasone; glucocorticoid-induced gastric ulcer; PAF antagonists; BN 52021 (ginkgolide B); BN 50727
Introduction Although there is a long-standing debate over whether corticosteroid therapy leads to peptic ulcer disease in man (recently reviewed by Piper et al., 1991), it is well established that administration of glucocorticoids to laboratory animals can result in an acute gastric erosion formation (Robert & Nezamis, 1958; Nobuhara et al., 1985; Wallace, 1987). The mechanism of this damaging action of glucocorticoids is, however, far from being understood. Glucocorticoids have been shown to inhibit synthesis of gastric mucus (Menguy & Masters, 1963), to induce G cell and parietal cell hyperplasia (Delaney et al., 1979) and to affect epithelial cell turnover (Eastwood, 1984). Dexamethasone and prednisolone have been shown to be ineffective in blocking prostaglandin (Whittle, 1978; Wallace, 1987), but not leukotriene C4 (Wallace, 1987) synthesis in the rat stomach, suggesting that their ulcerogenic action may not be related to decreased prostaglandin formation. Platelet-activating factor (PAF) is a phospholipid mediator of allergy and inflammation with a wide variety of biological activities including platelet aggregation, activation of neutrophil granulocytes, contraction of smooth muscle, increasing vascular permeability and inducing hypotension (for a recent review see Braquet et al., 1987). Administration of picomol doses of PAF into the gastric artery induced substantial gastric mucosal hyperaemia, haemorrhage and erosions (Rosam et al., 1986; Wallace & Whittle, 1986; Whittle et al., 1986; Espluques & Whittle, 1988), changes similar to those observed following glucocorticoid administration (Rob-
I Author for correspondence at: Department of Pharmacology, Faculty of Medicine, University of Sherbrooke, Sherbrooke, P.Q., Canada J1H 5N4.
ert & Nezamis, 1958; Nobuhara et al., 1985; Wallace, 1987).
These findings raised the possibility that release of endogenous PAF may underlie or contribute to certain forms of gastric ulceration. The aim of the present experiments was to study the possible role of PAF in mediating gastric damage to dexamethasone by measuring the PAF content in the rat stomach during dexamethasone treatment and by investigating the effects of two structurally unrelated, specific PAF receptor antagonists, BN 52021 (ginkgolide B) (Braquet et al., 1985; Foldes-Filep et al., 1987) and the benzodiazepine derivative, BN 50727 (Braquet & Esanu, 1991) on dexamethasone-induced gastric lesions.
Methods Male, Wistar rats weighing 160-205 g were housed in groups of 4 and were fed standard laboratory chow and tap water ad libitum.
Drug treatment Between 09 h 00 min and 10 h 00 min each day the animals were given an intraperitoneal injection of the PAF receptor antagonists, BN 52021 (10 mg kg-') or BN 50727 (1 mg kg-1), or their vehicle, and 30 min later they received a subcutaneous injection of dexamethasone sodium phosphate or vehicle. The effectiveness of BN 52021 and BN 50727 treatments was tested in preliminary experiments in anaesthetized (Inactin, 100 mg kg', i.p.) male rats by injecting 25 and 50 ng kg-' PAF into the right femoral vein 30 min before and 30 min after administration of either BN 52021, 10 mg kg-', i.p., or BN 50727, 1 mg kg-', i.p. Mean arterial
PAF, DEXAMETHASONE AND GASTRIC MUCOSAL DAMAGE
blood pressure was monitored through a catheter inserted into the right femoral artery by an electromanometer (EM 61, Medicor, Budapest, Hungary) using a Statham P23 dB pressure transducer (Hato Ray, Puerto Rico). The following sets of experiments were performed: Series I The animals received 0.4, 1 or 4mgkg-' of dexamethasone or its vehicle daily for 6 consecutive days.
Series 2 The animals were given 4 mg kg- ' of dexamethasone each day for 1, 2, 3, 4, 5 or 6 consecutive days. Series 3 The animals were divided into four groups: (a) control (receiving vehicle only), (b) BN 52021 (i.e. BN 52021, 10 mg kg-', i.p. plus vehicle s.c.), (c) dexamethasone (vehicle i.p. plus dexamethasone, 4mg kg-' s.c. ) and (d) BN 52021, 10 mg kg-' plus dexamethasone, 4 mg kg-l. The treatments were continued for 6 consecutive days.
Series 4 In these series of experiments a protocol similar to that of Series 3 was followed except that another PAF receptor antagonist, BN 50727 (1 mg kg-', i.p.) was used instead of BN 52021. Depending on the experimental protocol used, on days 1, 2, 3, 4, 5 or 6 the animals were anaesthetized with sodium pentobarbitone (40 mg kg-', i.p.) S h after the second injection. The animals were deprived of food, but not water, for 18-20 h before being anaesthetized. The stomach was immediately removed, opened along the greater curvature and examined. The extent of mucosal damage was scored by two observers in a randomized, blind manner using a 0 to 3 scoring system (Rosam et al., 1986; Wallace, 1987). A score of 0 was assigned if the mucosa appeared normal. A stomach
exhibiting hyperaemia or haemorrhage was assigned a score of 1 (limited to diffuse superficial hyperaemia), 2 (severe, extensive hyperaemia or focal haemorrhage) or 3 (multiple sites of bleeding or erosion). The correlation between the scores assigned by the two observers was greater than 0.860 (n=20, P
