Inhibition of angiogenesis by nonsteroidal anti-inflammatory drugs: insight into mechanisms and implications for cancer growth and ulcer healing.

Angiogenesis, the formation of new capillary blood vessels, is essential not only for the growth and metastasis of solid tumors, but also for wound and ulcer healing, because without the restoration of blood flow, oxygen and nutrients cannot be delivered to the healing site. Nonsteroidal anti-inflammatory drugs (NSAIDs) such as aspirin, indomethacin and ibuprofen are the most widely used drugs for pain, arthritis, cardiovascular diseases and, more recently, the prevention of colon cancer and Alzheimer disease. However, NSAIDs produce gastroduodenal ulcers in about 25% of users (often with bleeding and/or perforations) and delay ulcer healing, presumably by blocking prostaglandin synthesis from cyclooxygenase (COX)-1 and COX-2 (ref. 10). The hypothesis that the gastrointestinal side effects of NSAIDs result from inhibition of COX-1, but not COX-2 (ref. 11), prompted the development of NSAIDs that selectively inhibit only COX-2 (such as celecoxib and rofecoxib). Our study demonstrates that both selective and nonselective NSAIDs inhibit angiogenesis through direct effects on endothelial cells. We also show that this action involves inhibition of mitogen-activated protein (MAP) kinase (ERK2) activity, interference with ERK nuclear translocation, is independent of protein kinase C and has prostaglandin-dependent and prostaglandin-independent components. Finally, we show that both COX-1 and COX-2 are important for the regulation of angiogenesis. These findings challenge the premise that selective COX-2 inhibitors will not affect the gastrointestinal tract and ulcer/wound healing.

On the synthesis of prostaglandins by human gastric mucosa and its modification by drugs.

1. Specific radioimmunoassays for the prostaglandins E2, A2 and F2alpha were used to study the synthesis of prostaglandins by gastroscopically obtained small biopsy specimens of human gastric corpus mucosa. 2. Both prostaglandin E2 and prostaglandin F2alpha were found to be synthesized from arachidonic acid by themicrosomal fraction of human gastric mucosa. The synthesis of prostaglandin E2 exceeded that of prostagladin F2alpha by a factor of about 10. 3. Synthesis of prostaglandin A2 or prostaglandin B2 was not observed under the same incubation conditions. 4. Indometacin effectively inhibited synthesis of both prostaglandin E2 (ID50 4.2 microng/ml) and prostaglandin F2alpha (ID50 1.8 microng/ml) by human gastric mucosa, while paracetamol even in a concentration of 310 microng/ml did not influence prostaglandin synthesis. The anti-ulcer agent carbenoxolone, which has been shown to inhibit prostaglandin inactivation, at the same concentration only slightly inhibited (about 20%) prostaglandin synthesis. 5. The results support the hypothesis that the gastro-intestinal effects or side effects of several drugs are mediated by an influence on the enzymes of prostaglandin synthesis or inactivation.

On the metabolism of prostaglandins by human gastric fundus mucosa.

1. Specific radioimmunoassays for the prostaglandins E2, F2alpha and A2 and the metabolites 13,14-dihydro-15-keto-prostaglandin E2, 15-keto-prostaglandin F2alpha and 13,14-dihydro-15-keto-prostaglandin F2alpha were used to study the metabolism of prostaglandins by gastroscopically obtained small biopsy specimens of human gastric fundus mucosa. 2. Three prostaglandin-metabolizing enzymes were found in the 100 000 X g supernatant of human gastric fundus mucosa, 15-hydroxy-prostaglandin-dehydrogenase, delta13-reductase and delta9-reductase. The specific activity was highest for 15-hydroxy-prostaglandin-dehydrogenase and lowest for delta9-reductase. 3. Formation of prostaglandin A2 (or B2) was not observed under the same conditions. 4. None of the three enzyme activities detected in the 100 000 X g supernatant was found in the 10 000 X g and 100 000 X g pellets of human gastric fundus mucosa. 5. The results indicate that high speed supernatant derived from human gastric mucosa can rapidly metabolize prostaglandin E2 and prostaglandin F2alpha to the 15-keto and 13,14-dihydro-15-keto-derivatives. Furthermore, prostaglandin E2 can be converted to prostaglandin F2alpha, the biological activity of which, on gastric functions, differs from that of prostaglandin E2.

Leukotriene synthesis by human gastrointestinal tissues.

The prostaglandin and leukotriene synthesizing capacity of human gastrointestinal tissues obtained at surgery was investigated using radioimmunoassay for prostaglandin E2, leukotriene B4 and sulfidopeptide leukotrienes. The leukotriene immunoassay data were validated by high-pressure liquid chromatography (HPLC). During incubation at 37 degrees C, fragments of human gastric, jejuno-ileal and colonic mucosa released considerably larger amounts of prostaglandin E2 than of leukotriene B4 and sulfidopeptide leukotrienes. Gastrointestinal smooth muscle tissues released even larger amounts of prostaglandin E2, but smaller amounts of leukotrienes than the corresponding mucosal tissues. Adenocarcinoma tissue released larger amounts of leukotriene B4, sulfidopeptide leukotrienes and prostaglandin E2 than normal colonic mucosa. Ionophore A23187 (5 micrograms/ml) did not stimulate release of prostaglandin E2 from any of the tissues investigated, but enhanced release of leukotriene B4 and sulfidopeptide leukotrienes. HPLC analysis demonstrated that immunoreactive leukotriene B4 co-chromatographed almost exclusively with standard leukotriene B4, while immunoreactive sulfidopeptide leukotrienes consisted of a mixture of leukotrienes C4, D4 and E4. Leukotriene synthesis by human gastrointestinal tissues was inhibited by the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA) and the dual enzyme inhibitor BW755C (3-amino-1-(trifluoromethylphenyl)-2-pyrazoline hydrochloride). Synthesis of prostaglandin E2 was inhibited by the cyclooxygenase inhibitor indomethacin as well as by BW755C. Incubation of gastrointestinal tissues in the presence of glutathione decreased the amounts of leukotrienes D4 and E4, while release of leukotriene C4 was simultaneously increased. On the other hand, incubation of tritiated leukotriene C4 with incubation media from human gastric or colonic mucosa resulted in conversion of the substrate to [3H]leukotriene D4 and [3H]leukotriene E4. The results indicate the capacity of human gastrointestinal tissues to synthesize the 5-lipoxygenase-derived products of arachidonate metabolism, leukotriene B4 and sulfidopeptide leukotrienes, in addition to larger amounts of prostaglandin E2. Furthermore, considerable activities of the sulfidopeptide leukotriene-metabolizing enzymes gamma-glutamyl transpeptidase and dipeptidase were detected in human gastrointestinal tissues. These enzymes might play an important role in biological inactivation and/or change of biological profile of sulfidopeptide leukotrienes generated in the human gastrointestinal tract.

Radioimmunoassay for leukotriene E4. Use for determination of total sulfidopeptide-leukotriene release from rat gastric mucosa.

A conjugate of leukotriene (LT) E4 and bovine serum albumin (BSA) was prepared by covalently linking the free amino group of the hapten to the protein using dimethyl pimelindiimidate (DMP) as coupling reagent. Anti-LTE4 antibodies were raised in rabbits immunized with the conjugate. Binding of [3H]LTE4 to the antibodies is inhibited by 50% with 0.63 ng LTE4, while the relative cross-reaction of LTC4 and LTD4 is 46.3% and 12.6%, respectively. Using the radioimmunoassay release of sulfidopeptide-LT (SP-LT) from rat gastric mucosa incubated in vitro was determined after quantitative enzymatic conversion of SP-LT to LTE4. It could be demonstrated that this method is suitable for determination of SP-LT in biological material.

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.

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.

Effects of non-steroidal anti-inflammatory drugs on rat gastric mucosal leukotriene C4 and prostanoid release: relation to ethanol-induced injury.

1. The effects of oral and subcutaneous administration of the non-steroidal anti-inflammatory drugs sodium salicylate, aspirin and indomethacin on ex vivo gastric mucosal release of leukotriene C4 (LTC4) prostaglandin E2 (PGE2), 6-oxo-PGF1 alpha and thromboxane B2 (TXB2) were investigated in rats under basal conditions as well as after challenge with ethanol. 2. Basal release of PGE2, 6-oxo-PGF1 alpha and TXB2 was inhibited by oral administration of aspirin (0.6-400 mgkg-1) and indomethacin (4 or 20 mgkg-1), but not by sodium salicylate (up to 400 mgkg-1), in a dose-dependent manner. Oral administration of aspirin in the dose range 3.2-400 mgkg-1 and of indomethacin (20 mgkg-1) additionally inhibited release of LTC4, while sodium salicylate (up to 400 mgkg-1) had no effect. Indomethacin (20 mgkg-1) and aspirin (400 mgkg-1) administered subcutaneously inhibited generation of cyclo-oxygenase products of arachidonate metabolism, but did not significantly affect LTC4 synthesis. 3. Oral instillation of ethanol caused gastric mucosal damage and simultaneously induced a selective increase in the ex vivo release of LTC4 from rat gastric mucosa, while release of cyclo-oxygenase products of arachidonate metabolism was not significantly affected. Oral pretreatment of rats with sodium salicylate protected the gastric mucosa and simultaneously inhibited the ethanol-stimulated gastric mucosal LTC4 release in a dose-dependent manner. Sodium salicylate had no effects on the release of PGE2 and TXB2, while that of 6-oxo-PGF1 alpha was slightly increased. 4. Pretreatment with indomethacin (4 or 20mg kg- p.o.) or aspirin in doses up to 25mg kg-1 p.o. prior to oral instillation of ethanol did not inhibit gastric mucosal damage and had no effect on the stimulatory action of ethanol on LTC4 release. Higher doses of aspirin (100mgkg-1 or 400mgkg-1 p.o.) reduced the mucosal damaging effect of ethanol and simultaneously inhibited LTC4 release. 5. The results suggest that aspirin and indomethacin in concentrations higher than those necessary to inhibit the cyclo-oxygenase pathway of arachidonate metabolism additionally inhibit gastric mucosal LTC4 synthesis under basal conditions, while sodium salicylate has no such effect. On the other hand, sodium salicylate, but not indomethacin or low doses of aspirin (up to 25mg kg 1), by an unknown mechanism inhibits stimulation of LTC4 biosynthesis by ethanol and simultaneously protects the gastric mucosa against ethanol-induced damage. Similar effects of high oral doses (> 100mgkg- 1) of aspirin might be due to significant formation of salicylate. These results suggest that there is a causal relationship between enhanced LTC4 biosynthesis and the development of ethanol-induced gastric injury.

Effect of carbenoxolone on the biological activity of nitric oxide: relation to gastroprotection.

1. The interactions between carbenoxolone and nitric oxide (NO) were examined by investigating their effects on human platelet aggregation, on rat aortic strips precontracted by phenylephrine and on protection of rat gastric mucosa against ethanol-induced injury. 2. Carbenoxolone (100-300 microM) caused a significant and concentration-dependent potentiation of rat peritoneal neutrophil (RPN)- 3-morpholino-syndnonimine (SIN-1)- or iloprost-induced inhibition of platelet aggregation. Higher concentrations (500 microM) of carbenoxolone alone markedly inhibited platelet aggregation. Pretreatment with carbenoxolone (100-300 microM) antagonized the reversal of the RPN- or SIN-1-induced antiaggregatory effect by oxyhaemoglobin (10 microM). 3. Rat aortic strips with intact endothelium precontracted by phenylephrine (0.1-0.3 microM) were relaxed by carbenoxolone (100-300 microM) in a concentration-dependent manner. Relaxations were abolished by mechanical removal of the endothelium or by incubation with methylene blue (10 microM) or NG-nitro-L-arginine (L-NNA, 100 microM). Sodium nitroprusside (10 nM)-induced relaxations of endothelium-denuded rat aortic strips were potentiated by carbenoxolone (100 microM). . The carbenoxolone (200 mg kg-1, p.o.)-induced gastroprotection against ethanol was antagonized by L-NNA (5-40 mg kg-1) in a dose-dependent manner. Pretreatment of rats with indomethacin (10 mg kg-1, s.c.) increased the effect of L-NNA. 5. The results suggest that the activity of carbenoxolone in the experimental systems tested is due to phosphodiesterase inhibition, although radical scavenging properties of the drug could contribute to some of the effects observed. In the rat gastric mucosa both increased prostaglandin levels and effects on the NO system could contribute to the protective action of carbenoxolone.

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-inda none) 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-PGF1alpha 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 micromol) 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 micromol) kg(-1) and 1.3 (3.3 micromol) 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.

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.

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.

Mediation by CCKB receptors of the CCK-evoked hyperaemia in rat gastric mucosa.

1. Cholecystokinin octapeptide (CCK-8) and gastrin-17 augment gastric mucosal blood flow in the rat. The present study examined whether the gastric vasodilator effect of these peptides is mediated by CCKA or CCKB receptors. 2. Intravenous injection of CAM-1481 (1 mg kg-1), a dipeptoid antagonist of CCKA receptors, or CAM-1028, a dipeptoid CCKB receptor antagonist (1 mg kg-1), had no effect on basal gastric mucosal blood flow as determined by the clearance of hydrogen in urethane-anaesthetized rats. 3. Intravenous infusion of CCK-8 or gastrin-17 (8-200 pmol min-1) increased gastric mucosal blood flow in a dose-dependent fashion. The CCKB receptor antagonist, CAM-1028, significantly attenuated the hyperaemic response to CCK-8 and gastrin-17 whereas the CCKA receptor antagonist, CAM-1481, did not antagonize CCK-8 but caused a slight attenuation of the vasodilator response to gastrin-17. 4. The selectivity of the two antagonists was proved by the findings that CAM-1028, but not CAM-1481, inhibited gastric acid secretion evoked by CCK-8 or gastrin-17 (CCKB receptor assay) while CAM-1481, but not CAM-1028, inhibited the CCK-8-induced contraction of guinea-pig isolated gall bladder strips (CCKA receptor assay). 5. These data show that the actions of CCK-8 and gastrin-17 to increase mucosal blood flow in the rat stomach are primarily mediated by CCKB receptors.

Role of peptidergic sensory neurons in gastric mucosal blood flow and protection.

The present findings have revealed a new aspect of how mechanisms of gastric mucosal resistance to injury are called into effect and are coordinated by the nervous system. Capsaicin-sensitive sensory neurons in the stomach play a physiological role in monitoring acid influx into the superficial mucosa. Once activated, they strengthen gastric mucosal defense against deep injury, with a key process in this respect being an increase in blood flow through the gastric mucosa. This concept opens up completely new perspectives in the physiology and pathophysiology of the gastric mucosa if we consider that the long-term integrity of the gastric mucosa may be under the subtle control of acid-sensitive sensory neurons and that, vice versa, improper functioning of these neutral control mechanisms may predispose to gastric ulcer disease. The present observations also indicate that some of the peptides contained in gastric sensory nerve endings might fulfill a transmitter or mediator role in controlling gastric mucosal blood flow and integrity. Whereas substance P and neurokinin A are unlikely to play a role in the regulation of gastric mucosal blood flow, there is severalfold evidence that CGRP is very important in this respect. This peptide, which in the rat gastric mucosa originates exclusively from spinal sensory neurons, is released upon stimulation of sensory nerve endings and is extremely potent in facilitating gastric mucosal blood flow and in protecting the mucosa from injurious factors. Selective ablation of spinal sensory neurons containing CGRP weakens the resistance of the gastric mucosa against acid injury, which is most likely due to inhibition of protective vasodilator reflexes. We now aim at providing direct pharmacological evidence that antagonism of endogenously released CGRP results in similar pathophysiological consequences as ablation of capsaicin-sensitive sensory neurons.

The effect of parenteral fish oil on leukocyte membrane fatty acid composition and leukotriene-synthesizing capacity in patients with postoperative trauma.

The incorporation of omega-3 and omega-6 fatty acids (FAs) into leukocyte membranes and the leukotriene (LT)B4-, LTB5 -, LTC4-, and LTCs-synthesizing capacity in stimulated leukocytes were measured following parenteral omega-3 FA nutrition in 20 postoperative patients. Total parenteral nutrition (TPN) over 5 days postoperatively was isonitrogenous (0.24 g N x kg-1 x d1) and isoenergetic (92 kJ/22 kcal x kg-1 x d-1), containing 0.15 g fish oil and 0.85 g soybean oil per kg-1 x d-1 (FO) or 1.0 g soybean oil x kg-1 x d-1 (SO). Following 5 days' FO administration, the content of eicosapentaenoic acid (EPA) was increased 2.5-fold, LTB5 1.5-fold, and LTC5 sevenfold. With SO nutrition, EPA and LTB5 generation remained unaltered, whereas LTC5 doubled. The production of LTB4 and LTC4 was not affected in any of the groups. We conclude that a 5-day parenteral fish oil supplementation has an immunomodulatory effect on lipid-mediator generation in human leukocytes in postoperative trauma.

Role of cyclooxygenase isoforms in gastric mucosal defence.

A complex system of interacting mediators exists in the gastric mucosa to strengthen its resistance against injury. In this system prostaglandins play an important role. Prostaglandin biosynthesis is catalysed by the enzyme cyclooxygenase (COX), which exists in two isoforms, COX-1 and COX-2. Initially the concept was developed that COX-1 functions as housekeeping enzyme, whereas COX-2 yields prostaglandins involved in pathophysiological reactions such as inflammation. In the gastrointestinal tract, the maintenance of mucosal integrity was attributed exclusively to COX-1 without a contribution of COX-2 and ulcerogenic effects of non-steroidal anti-inflammatory drugs (NSAIDs) were believed to be the consequence of inhibition of COX-1. Recent findings, however, indicate that both COX-1 and COX-2 either alone or in concert contribute to gastric mucosal defence. Thus, in normal rat gastric mucosa specific inhibition of COX-1 does not elicit mucosal lesions despite near-maximal suppression of gastric prostaglandin formation. When a selective COX-2 inhibitor which is not ulcerogenic when given alone is added to the COX-1 inhibitor, severe gastric damage develops. In contrast to normal gastric mucosa which requires simultaneous inhibition of COX-1 and COX-2 for breakdown of mucosal resistance, in the acid-challenged rat stomach inhibition of COX-1 alone results in dose-dependent injury which is further increased by additional inhibition of COX-2 enzyme activity or prevention of acid-induced up-regulation of COX-2 expression by dexamethasone. COX-2 inhibitors do not damage the normal or acid-challenged gastric mucosa when given alone. However, when nitric oxide formation is suppressed or afferent nerves are defunctionalized, specific inhibition of COX-2 induces severe gastric damage. Ischemia-reperfusion of the gastric artery is associated with up-regulation of COX-2 but not COX-1 mRNA. COX-2 inhibitors or dexamethasone augment ischemia-reperfusion-induced gastric damage up to four-fold, an effect abolished by concurrent administration of 16,16-dimethyl-PGE(2). Selective inhibition of COX-1 is less effective. Furthermore, COX-2 inhibitors antagonize the protective effect of a mild irritant or intragastric peptone perfusion in the rat stomach, whereas the protection induced by chronic administration of endotoxin is mediated by COX-1. Finally, an important function of COX-2 is the acceleration of ulcer healing. COX-2 is up-regulated in chronic gastric ulcers and inhibitors of COX-2 impair the healing of ulcers to the same extent as non-selective NSAIDs. Taken together, these observations show that both COX isoenzymes are essential factors in mucosal defence with specific contributions in various physiological and pathophysiological situations.

Aspirin-like drugs, ethanol-induced rat gastric injury and mucosal eicosanoid release.

The effect of oral administration of various non-steroidal antiinflammatory drugs on ethanol-induced rat gastric injury and mucosal release of leukotriene C4, 6-keto-prostaglandin F1 alpha and 15-hydroxy-5,8,11,13-eicosatetraenoic acid was investigated. It was found that besides sodium salicylate and high doses of aspirin, other salicylate-type drugs, such as diflunisal, 4-aminosalicylic acid, 2,4-dihydroxybenzoic acid and methyl salicylate, and several non-acidic compounds, such as proquazone, benzydamine and paracetamol, were gastroprotective. All these drugs inhibited ex vivo leukotriene C4 formation by ethanol-stimulated gastric mucosa. However, naproxen, lonazolac, ibuprofen, gentisic acid and 5-aminosalicylic acid also inhibited leukotriene C4 formation, but were not protective. Gastroprotection was independent of 6-keto-prostaglandin F1 alpha formation. Both protective and non-protective drugs inhibited the ethanol-stimulated, but not the basal, release of 15-hydroxy-5,8,11,13-eicosatetraenoic acid. The results indicate that the differential effects of various non-steroidal antiinflammatory drugs on gastroprotection against ethanol are not correlated with specific effects on mucosal cyclooxygenase, 5-lipoxygenase or 15-lipoxygenase activity.

Nitric oxide as mediator of the gastroprotection by cholecystokinin-8 and pentagastrin.

Cholecystokinin-8 and pentagastrin protect against ethanol-induced gastric mucosal lesions in rats. The protective effect is antagonized by the nitric oxide (NO) synthase inhibitor, NG-nitro-L-arginine. The inhibitory action of NG-nitro-L-arginine is reversed by L-arginine, but not D-arginine. The findings suggest that NO is involved in the gastroprotection induced by both peptides.

Interaction of 5-aminosalicylic acid with nitric oxide on rat aortic strips and human platelets.

We have examined the interactions of 5-aminosalicylic acid with nitric oxide (NO). Phenylephrine-precontracted rat aortic strips with intact endothelium were further contracted by 5-aminosalicylic acid (50-200 microM) in a concentration-dependent manner. Removal of endothelium, inhibition of guanylate cyclase by methylene blue, inhibition of NO biosynthesis by NG-nitro-L-arginine as well as in inactivation of NO by oxyhemoglobin abolished the effect of 5-aminosalicylic acid. The antiaggregatory effects of 3-morpholinosydnonimine and rat peritoneal neutrophils, which are due to release of NO, were diminished in a concentration-dependent manner by 5-aminosalicylic acid (50-250 microM). In both experimental models the effects of 5-aminosalicylic acid were significantly reduced by superoxide dismutase in a concentration which alone exhibited no effect. Since NO might act as a cytotoxic and vasodilating mediator, our results suggest that inactivation of NO by 5-aminosalicylic acid could contribute to the therapeutic activity of the drug in inflammatory bowel disease.

A monoclonal antibody to calcitonin gene-related peptide abolishes capsaicin-induced gastroprotection.

Calcitonin gene-related peptide (CGRP) released from vasodilator nerves is implicated in the gastroprotective action of capsaicin. This experimental paradigm was used to prove the effectiveness of a monoclonal anti-CGRP antibody. The experiments were performed in anaesthetized rats in which intragastric capsaicin (0.5 mg/kg) reduced gastric injury due to ethanol (50%) by 72%. The protective effect of capsaicin was abolished by close arterial administration of the anti-CGRP antibody #4901 (5 mg) to the stomach. A monoclonal antibody to keyhole limpet haemocyanin was without effect. These data establish anti-CGRP antibody #4901 as a tool to neutralize endogenously released CGRP and show that CGRP is indispensable for the gastroprotective action of capsaicin.