Effects of specific inhibition of cyclo-oxygenase-1 and cyclo-oxygenase-2 in the rat stomach with normal mucosa and after acid challenge.

1. Effects of the cyclo-oxygenase (COX)-1 inhibitor SC-560 and the COX-2 inhibitors rofecoxib and DFU were investigated in the normal stomach and after acid challenge. 2. In healthy rats, neither SC-560 nor rofecoxib (20 mg kg(-1) each) given alone damaged the mucosa. Co-treatment with SC-560 and rofecoxib, however, induced severe lesions comparable to indomethacin (20 mg kg(-1)) whereas co-administration of SC-560 and DFU (20 mg kg(-1) each) had no comparable ulcerogenic effect 5 h after dosing. 3. SC-560 (20 mg kg(-1)) inhibited gastric 6-keto-prostaglandin (PG) F(1alpha) by 86+/-5% and platelet thromboxane (TX) B(2) formation by 89+/-4% comparable to indomethacin (20 mg kg(-1)). Rofecoxib (20 mg kg(-1)) did not inhibit gastric and platelet eicosanoids. 4. Intragastric HCl elevated mucosal mRNA levels of COX-2 but not COX-1. Dexamethasone (2 mg kg(-1)) prevented the up-regulation of COX-2. 5. After acid challenge, SC-560 (5 and 20 mg kg(-1)) induced dose-dependent injury. Rofecoxib (20 mg kg(-1)), DFU (5 mg kg(-1)) and dexamethasone (2 mg kg(-1)) given alone were not ulcerogenic but aggravated SC-560-induced damage. DFU augmented SC-560 damage 1 but not 5 h after administration whereas rofecoxib increased injury after both treatment periods suggesting different time courses. 6. Gastric injurious effects of rofecoxib and DFU correlated with inhibition of inflammatory PGE(2). 7. The findings show that in the normal stomach lesions only develop when both COX-1 and COX-2 are inhibited. In contrast, during acid challenge inhibition of COX-1 renders the mucosa more vulnerable suggesting an important role of COX-1 in mucosal defence in the presence of a potentially noxious agent. In this function COX-1 is supported by COX-2. In the face of pending injury, however, COX-2 cannot maintain mucosal integrity when the activity of COX-1 is suppressed.

Selective cyclo-oxygenase-2 inhibitors aggravate ischaemia-reperfusion injury in the rat stomach.

1. Effects of indomethacin, the selective cyclo-oxygenase (COX)-2 inhibitors NS-398 and DFU, and dexamethasone on gastric damage induced by 30 min ischaemia followed by 60 min reperfusion (I-R) were investigated in rats. Modulation of gastric levels of COX-1 and COX-2 mRNA by I-R was evaluated using Northern blot and reverse transcription-polymerase chain reaction. 2. I-R-induced gastric damage was dose-dependently aggravated by administration of indomethacin (1 - 10 mg kg(-1)), NS-398 (0.4 - 4 mg kg(-1)) or DFU (0.02 - 2 mg kg(-1)) as assessed macroscopically and histologically. 3. Likewise, administration of dexamethasone (1 mg kg(-1)) significantly increased I-R damage. 4. Low doses of 16, 16-dimethyl-prostaglandin(PG)E(2), that did not protect against ethanol-induced mucosal damage, reversed the effects of the selective COX-2 inhibitors, indomethacin and dexamethasone. 5. I-R had no effect on gastric COX-1 mRNA levels but increased COX-2 mRNA levels in a time-dependent manner. Dexamethasone inhibited the I-R-induced expression of COX-2 mRNA. 6. I-R was not associated with a measurable increase in gastric mucosal formation of 6-keto-PGF(1alpha) and PGE(2). PG formation was substantially inhibited by indomethacin (10 mg kg(-1)) but was not significantly reduced by NS-398 (4 mg kg(-1)), DFU (2 mg kg(-1)) or dexamethasone (1 mg kg(-1)). 7. The findings indicate that selective COX-2 inhibitors and dexamethasone markedly enhance gastric damage induced by I-R. Thus, whereas COX-2 has no essential role in the maintenance of gastric mucosal integrity under basal conditions, COX-2 is rapidly induced in a pro-ulcerogenic setting and contributes to mucosal defence by minimizing injury. This suggests that in certain situations selective COX-2 inhibitors may have gastrotoxic effects.

Increased production of vascular endothelial growth factor by intestinal mucosa of patients with inflammatory bowel disease.

BACKGROUND/AIMS: Vascular endothelial growth factor (VEGF) is a heparin-binding glycoprotein with potent angiogenic, mitogenic and vascular permeability-enhancing activities specific for endothelial cells. Recent studies have shown significantly increased VEGF serum levels in patients with active Crohn's disease and ulcerative colitis. The origin of the circulating VEGF is not yet completely described. The present investigation examines the VEGF production of colonic mucosa in consideration of mucosal disease activity in patients with inflammatory bowel disease. METHODOLOGY: Fifteen patients with inflammatory bowel disease were studied, 9 patients with Crohn's disease and 6 patients with ulcerative colitis. Biopsies were taken from endoscopically inflamed and non-inflamed colonic mucosa. Therefore, an analysis of the spontaneous VEGF production of cultured biopsies without stimulus and of the histological grade of inflammation scored on a scale of 0-3 (normal mucosa--severe chronic colitis) were performed. Eight patients with irritable bowel syndrome served as controls. VEGF levels in the supernatant of cultured mucosal biopsies were measured using an enzyme linked immunosorbent assay. RESULTS: VEGF production is expressed as pg/mg wet weight of the biopsies. Inflamed mucosa of patients with active ulcerative colitis (16.27 +/- 10.39, p = 0.003, n = 6) and active Crohn's disease (9.88 +/- 5.98, p < 0.012, n = 9) showed a significantly higher spontaneous production of VEGF by colonic mucosa than normal mucosa of controls (3.16 +/- 1.63, n = 8). In addition, there was an increased unstimulated VEGF production by cultured inflamed mucosa of patients with Crohn's disease compared with non-inflamed mucosa (3.88 +/- 3.66, p < 0.015, n = 9). In both Crohn's disease and ulcerative colitis, there was no significant difference between VEGF production by non-inflamed mucosa and normal mucosa of controls. CONCLUSIONS: The present study identifies the intestinal mucosa as one of the origins of the elevated VEGF serum levels in patients with active inflammatory bowel disease and verifies the findings of recent studies about the importance of VEGF in Crohn's disease and ulcerative colitis.

Peptidergic and cholinergic neurons and mediators in peptone-induced gastroprotection: role of cyclooxygenase-2.

This study investigates the neural pathways, mediators, and cyclooxygenase isoenzymes involved in the gastroprotection conferred by peptone in rats. Intragastric perfusion with 8% peptone protected against gross and histological damage induced by subsequent perfusion with 50% ethanol. The gastroprotective effect of peptone was near maximally inhibited by gastrin immunoneutralization, inactivation of capsaicin-sensitive afferent neurons, calcitonin gene-related peptide (CGRP) immunoneutralization, blockade of gastrin receptors, CGRP, bombesin/gastrin-releasing peptide (GRP), or somatostatin receptors, and by the nitric oxide (NO) synthase inhibitor NG-nitro-L-arginine methyl ester and was partially (46%) counteracted by atropine. Indomethacin and the selective cyclooxygenase-2 inhibitors NS-398 and L-745,337 dose dependently (50% inhibitory dose, 4.2, 0.8, and 1.5 mg/kg, respectively) attenuated the peptone-induced protection. Dexamethasone was ineffective. These results indicate that protective effects of peptone involve endogenous gastrin and possibly somatostatin and are mediated by capsaicin-sensitive afferent, cholinergic, and bombesin/GRP neurons. CGRP, NO, and prostaglandins participate as essential mediators. The study provides evidence that prostaglandins derived from a constitutive cyclooxygenase-2 contribute to mucosal defense in the presence of ulcerogens and thus participate in homeostatic functions of the stomach.

Selective cyclo-oxygenase-2 inhibitors and their influence on the protective effect of a mild irritant in the rat stomach.

1. The effects of the non-selective cyclo-oxygenase (COX) inhibitor indomethacin and the selective COX-2 inhibitors, N-[2-(cyclohexyloxy)-4-nitrophenyl] methanesulphonamide (NS-398), 5-methanesulphonamido-6-(2,4-difluorothio-phenyl)-1-indan one (L-745,337) and 5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulphonyl) phenyl-2(5H)-furanone (DFU), on the protection induced by the mild irritant 20% ethanol were investigated in the rat stomach. 2. Instillation of 20% ethanol (1 ml, p.o.) effectively protected against gastric mucosal injury induced by subsequent instillation of 70% or 96% ethanol (1 ml, p.o.). 3. Oral administration of indomethacin (1.25-20 mg kg[-1]) dose-dependently counteracted the protective effect of 20% ethanol (ID50: 3.5 mg kg[-1]). 4. Likewise, NS-398 (0.1-1 mg kg[-1]), L-745,337 (0.2-2 mg kg[-1]) and DFU (0.02-0.2 mg kg[-1]) inhibited the protective effect of 20% ethanol in a dose-dependent manner with ID50 values of 0.3 mg kg(-1), 0.4 mg kg(-1) and 0.06 mg kg(-1), respectively. 5. Inhibition of mild irritant-induced protection was also found when NS-398 (1 mg kg[-1]) was administered s.c. or when 96% ethanol was used to damage the mucosa. 6. Pretreatment with 16,16-dimethyl-prostaglandin (PG)E2 at 4 ng kg(-1), a dose that did not protect against ethanol (70%)-induced mucosal damage when given alone, completely reversed the effect of the selective COX-2 inhibitors on the mild irritant-induced protection. 7. Pretreatment with dexamethasone (3 mg kg(-1), 24 and 2 h before instillation of 20% ethanol) did not affect the protective activity of the mild irritant, indicating that enzyme induction is not involved. 8. Indomethacin (20 mg kg(-1), p.o.) did not prevent the protection conferred by sodium salicylate (100 mg kg[-1]), dimercaprol (30 microg kg[-1]), iodoacetamide (50 mg kg[-1]) and lithium (20 mg kg[-1]). Likewise, the protective effect of these agents was not counteracted by NS-398 (1 mg kg(-1), p.o.). 9. Whereas indomethacin (20 mg kg(-1), p.o.) near-maximally inhibited gastric mucosal formation of PGE2, 6-keto-PGF1alpha and thromboxane (TX) B2 as well as platelet TXB2 release, the selective COX-2 inhibitors were ineffective. 10. The findings show that selective COX-2 inhibitors, although lacking in ulcerogenic activity, prevent the protection conferred by a mild irritant. Prostaglandis generated by a constitutive COX-2 could thus contribute to physiological functions involved in gastric homeostasis, although at present a non-COX-2-related mechanism underlying the effect of the selective COX-2 inhibitors tested on mild irritant-induced protection cannot be completely excluded.

Na+/K+ pump inhibition and positive inotropic effect of digitoxigenin and some C-22-substituted derivatives in sheep cardiac preparations.

Affinity labeling might be used to localize the binding site(s) of the lactone ring of cardioactive steroids on the Na+/K+-ATPase. The aim of the experiments described below was to identify C-22-substituted derivatives of digitoxigenin suitable for this purpose. The positive inotropic effect of digitoxigenin, 22-benzoyloxy-digitoxigenin, 22-acetoxy-digitoxigenin, 22-allyl-digitoxigenin, and 22-hydroxy-digitoxigenin was studied in sheep cardiac Purkinje fibres. In addition, the inhibition of the Na+/K+ pump by these drugs was investigated by means of simultaneous measurements of membrane current and intracellular Na+ concentration in voltage-clamped Purkinje fibres and by means of whole-cell recording in isolated sheep Purkinje cells. The experiments were performed at 5.4 mM K+ and 30 to 33 degrees C. All compounds exerted a reversible positive inotropic effect. The concentrations required for the half maximal effect (EC50 value) amounted to approximately 5 x 10(-7) M digitoxigenin, 22-acetoxy-digitoxigenin or 22-hydroxy-digitoxigenin. The EC50 values for 22-benzoyloxy-digitoxigenin and 22-allyl-digitoxigenin were estimated to be 1.3 x 10(-6) M and 1.1 x 10(-5) M, respectively. From measurements on voltage-clamped Purkinje fibres the concentrations required for half maximal Na+/K+ pump inhibition (K'D value) were calculated to be approximately 10(-6) M for digitoxigenin, 22-acetoxy-digitoxigenin or 22-hydroxy-digitoxigenin. The K'D value for 22-benzoyloxy-digitoxigenin was 10 times larger. The K'D value for 22-allyl-digitoxigenin was even larger and amounted to approximately 4 x 10(-5) M. The K'D values of the drugs derived from whole-cell recording on single Purkinje cells tended to be smaller by a factor 2 to 8. Measurements of drug binding and unbinding revealed that the apparent association rate constant of 22-benzoyloxy-digitoxigenin (approximately 9 x 10(2) s(-1) M[-1]) was smaller than the association rate constant of digitoxigenin (approximately 2 x 10(4) s(-1) M[-1]), whereas the apparent dissociation rate constants of both compounds were similar (approximately 4 x 10(-3) s[-1]). Compared to digitoxigenin 22-allyl-digitoxigenin displayed a lower association rate constant (approximately 3 x 10(3) s(-1) M[-1]) and a larger dissociation rate constant (approximately 8 x 10(-2) M[-1]). The structure-activity relationships of the drugs are discussed. We conclude that esters derived from 22-hydroxy-digitoxigenin might be suitable to localize the binding site(s) of the lactone moiety on the Na+/K+ pump by affinity labeling.