Brigitta M. Peskar

Effects of inhibition of prostaglandin endoperoxide synthase-2 in chronic gastro-intestinal ulcer models in rats

1 In the stomach, prostaglandins protect the gastric mucosa against injuries. One rate‐limiting step in prostaglandin synthesis is mediated by prostaglandin endoperoxide synthase (PGHS), the target enzyme of non‐steroidal anti‐inflammatory drugs (NSAIDs). Two isoforms of PGHS exist: a constitutive (PGHS‐1) and an inducible (PGHS‐2) enzyme. PGHS‐1 is the major source of gastric prostaglandins under physiological conditions. Inhibition of prostaglandin synthesis by traditional NSAIDs such as indomethacin and diclofenac which non‐selectively inhibit both PGHS‐1 and PGHS‐2, causes gastric and intestinal ulceration and delays gastric ulcer healing in chronic models. It has been shown that selective PGHS‐2 inhibitors such as L‐745,337 (5‐methanesulphonamide‐6‐(2,4‐difluorothio‐phenyl)‐1‐indanone) are not ulcerogenic and do not inhibit gastro‐intestinal prostaglandin synthesis. However, minimal information is available on the long‐term effects of PGHS‐2 inhibitors on the healing of previously established gastric injuries. We assessed the cellular localization and expression of PGHS‐1 and PGHS‐2 during gastric ulcer healing and assessed the effects of L‐745,337 on previously established cryoulcers in the rat gastric stomach. 2 PGHS‐1 and PGHS‐2 were located and quantified by immunohistochemistry during experimental gastric ulcer healing. PGHS‐2 immunoreactivity was only negligible in the normal gastric wall, but after gastric ulcerations, it was strongly detected in monocytes, macrophages, fibroblasts and endothelial cells below and between the regenerative glands. PGHS‐1 immunoreactivity detected in normal gastric mucosa, disappeared after gastric ulceration in the mucosa adjacent to the ulcer crater. However, it reappeared in the regenerative glands from day 5 onwards. Thus, PGHS‐1 and PGHS‐2 were located at different sites and their maximal expression followed a different time‐sequence. 3 We assessed the effects of L‐745,337, indomethacin and diclofenac on gastric ulcer healing and histological healing parameters in rats. L‐745,337, indomethacin and diclofenac dose‐dependently decreased the healing of gastric ulcers. L‐745,337, indomethacin and diclofenac decreased epithelial cell proliferation in the ulcer margin and microvessel density in the ulcer bed on day 8 and increased the thickness of the granulation tissue below the ulcer crater and the gap between both edges of the muscularis mucosae on day 15. Indomethacin and diclofenac, but not L‐745,337, decreased synthesis of 6‐keto‐PGF1α and PGE2 in tissue fragments from the stomach and terminal ileum and decreased platelet thromboxane B2 synthesis in clotting whole blood. 4 Dose‐response curves for the inhibition of chronic gastric ulcer healing by L‐745,337 (administered twice daily intragastrically) showed an ID50 value of 1.7 mg (4.3 μmol) kg−1. Dose‐response curves for the inhibition of PGE2 synthesis in inflammatory exudates in the acute carrageenin sponge rat model, showed ID50 values of 1.1  mg (3.1  μmol) kg−1 and 1.3 (3.3  μmol) mg kg−1 for indomethacin and L‐745,337, respectively. Thus, inhibition of chronic gastric ulcer healing by L‐745,337 occurs within a potentially therapeutic dose‐range. 5 In summary, PGHS‐2 is markedly accumulated after gastric ulceration in monocytes, macrophages, fibroblasts and endothelial cells in regions of maximal repair activity. Selective inhibition of PGHS‐2 by L‐745,337 delayed gastric ulcer healing though interference with epithelial cell proliferation, angiogenesis and maturation of granulation tissue in a potentially therapeutic dose range. PGHS‐2‐derived prostaglandins seem to have an important role in gastric ulcer healing.

Ethanol stimulates formation of leukotriene C4 in rat gastric mucosa

Ethanol-induced gastric mucosal damage is characterized by microcirculatory changes such as stasis and plasma leakage. Sluggish blood flow and stasis have also been observed after administration of exogenous leukotriene (LT) C4. The effect of ethanol on the release of LTC4 from rat gastric mucosa was therefore investigated. It was found that intragastric instillation of ethanol increases gastric mucosal release of LTC4 in a dose- and time-dependent manner parallel to the production of gastric lesions. The lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA) and the anti-ulcer drug carbenoxolone (CX) inhibited mucosal release of LTC4 and simultaneously protected against gastric damage caused by ethanol. It is concluded that increased formation of LTC4 and/or other 5-lipoxygenase-derived products of arachidonate metabolism may be involved in ethanol-induced gastric damage. Furthermore, inhibition of the 5-lipoxygenase pathway may be an important mechanism of action of gastric protective drugs.

Afferent nerve-mediated protection against deep mucosal damage in the rat stomach☆

Intragastric capsaicin protects against ethanol-induced gross mucosal lesion formation by stimulation of afferent nerve endings in the rat stomach. The aims of the present study were to examine histologically the protective effect of capsaicin and to test whether this effect is related to changes in mucosal eicosanoid formation and mucosal blood flow. Intragastric capsaicin (160 microM) significantly reduced gross mucosal lesion formation induced by 25% ethanol. Light microscopy revealed that the depth of erosions was attenuated likewise. However, capsaicin did not prevent ethanol from causing superficial damage to the mucosa as observed by light and scanning electron microscopy. The protective action of capsaicin against ethanol remained unchanged by a dose of indomethacin that reduced the ex vivo formation of prostaglandin E2 and 6-oxo-prostaglandin F1 alpha in the gastric mucosa by about 90%. Capsaicin alone did not affect the ex vivo formation of these prostaglandins and of leukotriene C4. Intragastric capsaicin (160 microM) enhanced gastric mucosal blood flow by 89% as measured by the hydrogen gas clearance technique. This effect was also observed when capsaicin was administered together with 25% ethanol. These data indicate that afferent nerve stimulation by intragastric capsaicin protects against deep mucosal damage in response to ethanol, an effect that seems related to an increase in mucosal blood flow but not to eicosanoid formation.

Aspirin-like drugs may block pain independently of prostaglandin synthesis inhibition

Using flurbiprofen, a chiral anti-inflammatory and analgesic 2-arylpropionic acid derivative, the enantiomers of which are not converted to each other (less than 5%) in rats or man, we obtained evidence that prostaglandin synthesis inhibition is primarily mediating the anti-inflammatory activity but prostaglandin synthesis independent mechanisms contribute to the analgesic effects. Thus, the S-form inhibited prostaglandin synthesis, inflammation and nociception in rats. The R-form had much less effect on prostaglandin synthesis and did not affect inflammation. It did, however, block nociception in rats almost as potently as the S-form. S-flurbiprofen, in contrast to the R-form, was clearly ulcerogenic in the gastrointestinal mucosa. These results indicate additional molecular mechanisms of analgesia and suggest the use of R-arylpropionic acids as analgesics.

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

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. 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. SC‐560 (20 mg kg−1) inhibited gastric 6‐keto‐prostaglandin (PG) F1α by 86±5% and platelet thromboxane (TX) B2 formation by 89±4% comparable to indomethacin (20 mg kg−1). Rofecoxib (20 mg kg−1) did not inhibit gastric and platelet eicosanoids. 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. 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. Gastric injurious effects of rofecoxib and DFU correlated with inhibition of inflammatory PGE2. 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.

Release of calcitonin gene-related peptide induced by capsaicin in the vascularly perfused rat stomach

It has been suggested that capsaicin-induced gastric mucosal protection results from the local release of vasodilator peptides such as calcitonin gene-related peptide (CGRP) from afferent nerve endings within the stomach, since CGRP is able to reduce gastric lesion formation. This concept is supported by the present finding that capsaicin (10(-5) M), administered to the vascularly perfused stomach of the rat, produces a more than 30-fold rise of the CGRP content of the venous effluent. High-pressure liquid chromatography revealed only one peak of immunoreactivity coeluting with synthetic CGRP.

Role of calcitonin gene-related peptide and nitric oxide in the gastroprotective effect of capsaicin in the rat.

BACKGROUND Capsaicin-sensitive neurons contain various peptides including calcitonin gene-related peptide. This study examines (1) whether calcitonin gene-related peptide is involved in capsaicin-induced gastroprotection and (2) whether nitric oxide and prostaglandin are required for calcitonin gene-related peptide to prevent mucosal injury. METHODS Gastroprotection by capsaicin or calcitonin gene-related peptide against ethanol-induced gross and histological damage was studied after pretreatment with the calcitonin gene-related peptide receptor antagonist, human calcitonin gene-related peptide8-37, anti-calcitonin gene-related peptide antibodies, and NG-nitro-L-arginine. RESULTS Protection by capsaicin was dose-dependently (50% inhibitory dose, 305 pmol.kg-1.min-1) antagonized by human calcitonin gene-related peptide8-37 and significantly attenuated by anti-calcitonin gene-related peptide antibodies. NG-nitro-L-arginine dose-dependently inhibited the protective effect of calcitonin gene-related peptide (50% inhibitory dose, 0.9 mg/kg), 3 mg/kg completely blocking protection. L-Arginine reversed the effects of NG-nitro-L-arginine. Protection by calcitonin gene-related peptide was neither associated with increased prostaglandin formation nor inhibited by indomethacin. CONCLUSIONS The results suggest that calcitonin gene-related peptide is an essential mediator of the protection elicited by stimulation of capsaicin-sensitive neurons and that the protective effect of calcitonin gene-related peptide is lost after blockade of the nitric oxide system but not the prostaglandin system.

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‐indanone (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 μg 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‐PGF1α 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.

Stimulation of prostaglandin biosynthesis mediates gastroprotective effect of rebamipide in rats

The concept that gastroprotection by agents such as mild irritants, antacids, or sucralfate is prostaglandin (PG)-mediated has been challenged recently. These agents do not reproducibly stimulate prostaglandin formation, and indomethacin does not effectively attenuate their protective potency. Rebamipide is a novel antiulcer compound. This study was designed to clarify whether eicosanoids contribute to the gastroprotective activity of the drug. In the rat stomach, rebamipide (100 and 500 mg/kg, intraperitoneally) slightly increased release of PGE2, 6-keto-PGF1 α thromboxane B2, and the metabolite 15-keto-13,14-dihydro-PGE2 from mucosal fragments incubated ex vivo and significantly enhanced secretion of these products into the lumen, resulting in gastric juice eicosanoid levels exceeding those in controls several-fold. Mucosal formation of leukotriene (LT) C4 was not affected by rebamipide. Rebamipide caused substantial protection against gastric damage produced by ethanol, which was antagonized by pretreatment with indomethacin (0.1–5 mg/kg, subcutaneously). The dose-response relationship of indomethacin for inhibition of prostaglandin formation and rebamipide-induced protection correlated well and 5 mg/kg indomethacin completely prevented the protective effect of rebamipide. The results indicate that: (1) in contrast to most other protective agents, protection by rebamipide involves the endogenous prostaglandin system; (2) the increase in prostaglandin formation results from stimulation of biosynthesis, and not inhibition of degradation; (3) gastroprotection by rebamipide occurs despite increased thromboxane formation and is not associated with reduced generation of LTC4; and (4) determinations of gastric juice eicosanoids seem to be particularly useful to evaluate effects of agents increasing formation of cyclooxygenase products in the stomach.

Release of leukotriene C4 from human polymorphonuclear leucocytes as determined by radioimmunoassay

Rabbits were immunized with a conjugate of leukotriene (LT) C4 and bovine serum albumin prepared by coupling the single free amino group of the hapten to the protein using gluteraldehyde. Binding of [3H]LTC4 to the antibodies obtained is inhibited by 50% with 1.5 ng LTC4. The relative cross‐reaction of LTD4 is 16% and of LTC4‐methyl ester 3.6%. The validity of the radioimmunoassay was demonstrated by comparison with bioassay using the isolated guinea pig ileum. Using the radioimmunoassay it could be shown that endogenous LTC4 is released in a dose‐dependent manner by human polymorphonuclear leucocytes stimulated with the divalent cation ionophore A23187.