On the biochemical background of cimetidine's anti-ulcerogenic effect in the rat.

It was published earlier that during gastric ulceration an elevation in the antral and fundic mucosal cAMP/cGMP ratios can be encountered in rats. This phenomenon was interpreted as a probable sign of the reparative, antiulcerogenic processes. This assumption received further evidence when the experimental animals were treated with prostacyclin. According to the present investigations the H2-receptor blocker cimetidine, in such small dose which does not interfere with the gastric acid secretion, evoked the same cAMP/cGMP ratio elevating effect. On the basis of the performed investigations the questions raised, namely: Does the cytoprotective (anti-ulcerogenic) effect of different drugs have a common molecular basis in the (rat) gastric mucosa? If so, what is the possible role of the mucosal cyclic nucleotides in this process? Further studies are being needed in this field to clarify all details.     

The effect of different prostaglandins on gastric mucosal intracellular second messenger system in the rat.

After an oral administration of 100 micrograms/kg dose, the investigated prostaglandins: PGF2 alpha, PGE2 and a synthetic PGE2 derivative: FCE-20700, exerted a significant effect on cAMP and cGMP content of both parts (antral and fundic) of gastric mucosa, resulting in an elevated cAMP/cGMP ratio, while 6-keto-PGF1 alpha, the stable break-down product of prostacyclin, was inactive. Since the above-mentioned phenomenon seems to be proportionate to the cytoprotective (anti-ulcerogenic) property of the investigated prostaglandins, this cAMP/cGMP ratio "shift" is interpreted as a probable (molecular) sign of the reparative, (anti-ulcerogenic) processes.     

On a possible new intracellular signal-system in rat gastric mucosa.

It is known that cAMP and cGMP, as an "intracellular second messenger system" play a significant role as a signal system, in the mechanism of action of anti-ulcerogenic (cytoprotective) drugs. According to our present, preliminary investigations it seems that during different experimental circumstances the gastric mucosal 3'-5'-cyclic-cytidine-mono-phosphate (cCMP) 3'-5'-cyclic-uridine-monophosphate and (cUMP) levels were changed--similarly to CAMP and cGMP--and these changes might be a possible indicator of a further, most probably secondary, signal- system role.     

The effect of different prostaglandins on cysteamine-induced duodenal ulcer in the rat

Most of the experiments have verified the cytoprotective effect of prostaglandins on the gastric mucosa and only few observations are known to deal with "cytoprotection" exerted on the duodenal or intestinal mucosa. Duodenal ulcers were produced by cysteamine (CEA). The treatment with different prostaglandins (PGI2, PGF2 alpha, PGE2) was carried out every six hours during the 48-hour experimental period. The obtained results indicate that neither prostacyclin nor prostaglandin-F2 alpha has any significant effect on CEA-induced duodenal ulceration, while prostaglandin-E2 exerts a slight anti-ulcerogenic effect. It is possible that cytoprotection is not the result of the same process in the duodenal and gastric mucosa: alternatively, three prostaglandins are not involved in the pathomechanism of CEA-induced duodenal ulceration.     

Effect of atropine, PGF2 alpha and cimetidine on the beta-carotene induced cytoprotection in ethanol-treated rats

The aim of the study was to evaluate the influence of atropine, PGF2 alpha and cimetidine on the gastric cytoprotective effect of beta-carotene. Mucosal damage was produced by intragastric (i.g.) addition of 96% ethanol in CFY-strain rats of both sexes weighing 180-220 g. Gastric cytoprotection caused by i. g. pretreatment with 1.0 mg/kg beta-carotene 30 minutes before ethanol administration, was observed after 1 hour. Atropine (0.5 mg/kg), cimetidine (50 mg/kg) and PGF2 alpha (200 micrograms/kg) were given intraperitoneally (i.p.) 30 minutes before ethanol administration with and without beta-carotene and the changes in the number and severity of the gastric ulcers were detected. PGF2 alpha did not influence the gastric cytoprotective effect of beta-carotene meanwhile it was inhibited by atropine and markedly by cimetidine. Deleterious effect of cimetidine on the beta-carotene-induced cytoprotection may be explained perhaps by the adverse effect of the two compounds on ATP-cAMP transformation hereby counteracting one another, but more data are needed to the better understanding of drug interactions relating to mucosal cytoprotection.     

Direct cellular effects of some mediators,hormones and growth factor-like agents on denervated (isolated) rat gastric mucosal cells

The brain-gut axis has an important role in the mechanism of gastric cytoprotection in vivo. The aim of this study was to evaluate the in vitro effect of protective agents without any central and peripheral innervation. A mixed population of rat gastric mucosal cells was isolated by the method of Nagy et al (Gastroenterology (1994) 77, 433–443). Cells were incubated for 60 min with cytoprotective drugs such as prostacyclin, histamine, pentagastrin and PL-10 substances (synthesized parts of BPC). At the end of this incubation cells were treated by 15% ethanol for 5 min. Cell viability was tested by trypan blue exclusion test and succinic dehydrogenase activity. The following results were obtained: 1) prostacyclin, histamine and pentagastrin had no direct cytoprotective effect on isolated cells; and 2) PL-10 substances significantly protected the cells against ethanol-induced cellular damage. This led to the following conclusions: 1) in the phenomenon of gastric cytoprotection only the growth factor-like agents have a direct cellular effect; and 2) the intact peripheral innervation is basically necessary for the development of mediators and hormone-induced gastric cytoprotection.     

The key-role of vagal nerve and adrenals in the cytoprotection and general gastric mucosal integrity.

BACKGROUND: Our laboratory group observed earlier that the gastric mucosal cytoprotective effect of prostacyclin (PGI(2)) disappeared after surgical vagotomy in rats. Similarly to this, the beta-carotene induced gastric cytoprotection disappeared in adrenalectomized rats too. AIMS: In these studies we aimed to investigate the possible role of vagal nerve and adrenals in the development of gastric mucosal lesions induced by exogenously administered chemicals (ethanol, HCl, NaOH, NaCl and indomethacin), and on the effects of cytoprotective and antisecretory drugs (atropine, cimetidine), and scavengers (vitamin A and beta-carotene). METHODS: The observations were carried out in fasted CFY strain rats. The gastric mucosal lesions were produced by intragastric (i.g.) administration of narcotising agents (96% ethanol; 0.6 M HCl; 0.2 M NaOH; 25% NaCl) or subcutaneously (s.c.) administered indomethacin (20 mg/kg) in intact, surgically bilaterally vagatomized, and adrenalectomized rats without or with glucocorticoid supplementation (Oradexon, 0.6 mg/kg given i.m. for 1 week). The gastric mucosal protective effect of antisecretory doses of atropine (0.1-0.5-1.0 mg/kg i.g.) and cimetidine (10-25-50 mg/kg i.g.), and vitamin A and beta-carotene (0.01-0.1-1.0-10 mg/kg i.g.) was studied. The number and severity of mucosal gastric lesions was numerically or semiquantitatively measured. In other series of observations the gastric acid secretion and mucosal damage were studied in 24 h pylorus-ligated rats without and with acute bilateral surgical vagotomy. RESULTS: It was found that: (1) the chemical-induced gastric mucosal damage was enhanced in vagotomized and adrenalectomized rats, meanwhile the endogenous secretion of gastric acid, and the development of mucosal damage can be prevented by surgical vagotomy; (2) the gastric cyto- and general protection produced by the drugs and scavengers disappeared in vagotomized and adrenalectomized rats; (3) the gastric mucosal protective effects of drugs and of scavengers returned after sufficient glucocorticoid supplementation of the rats. CONCLUSION: It has been concluded that the intact vagal nerve and adrenals have a key role in the gastric mucosal integrity, and in drugs- and scavengers-induced gastric cyto- and general mucosal protection.     

Failure of prostacyclin, beta-carotene, atropine and cimetidine to produce gastric cyto- and general mucosal protection in surgically vagotomized rats

Different chemicals (such as ethanol, HCl, drugs) produce gastric mucosal injury. A special type of gastric mucosal defense, which differed from the inhibition of gastric acid secretion, was discovered in response to small doses of prostaglandins. This phenomenon was termed "gastric cytoprotection". Later, the existence of gastric cytoprotection was proved using different compounds, such as vitamin A and other carotenoids, prostacyclin, small doses of anticholinergic and H2-blocking agents. These compounds produce cyto-protection by different mechanisms. In this study we tested the role of vagus nerve on the development of these different types of gastric cytoprotection. These compounds prevent ethanol-induced gastric mucosal injury in rats with intact vagus nerve, but their cyto- and mucosal protective effects disappear in surgically vagotomized rats. These results indicate that the intact vagus nerve is basically necessary for the overproduction of HCl and pepsin secretion, and for the development of gastric cytoprotection, produced by different compounds (e.g. prostacyclin, beta-carotene, small doses of atropine and cimetidine) acting without the presence of inhibition of gastric acid secretion.     

Interrelationships between the development of the gastric cytoprotective effects of prostacyclin, atropine, cimetidine and the gastric mucosal superoxide dismutase activity in rats

Gastric mucosal damage was produced by the intragastric administration of 96% ethanol or 0.6 M HCl. The cytoprotective doses of prostacyclin (PGI2) (5 micrograms/kg), atropine (0.025 mg/kg) or cimetidine (2.5 mg/kg) were given intraperitoneally 30 min before the administration of the necrotizing agents. The animals were killed 1 hr later. The number and severity of gastric mucosal lesions (ulcer) were recorded. At the time of the sacrifice of the animals, superoxide dismutase (SOD) was prepared from the gastric fundic mucosa and its activity was measured. It was found that PGI2 (5 micrograms/kg), atropine (0.025 mg/kg) and cimetidine (2.5 mg/kg) significantly decreased the number and severity of gastric mucosal lesions (ulcers) produced by the intragastric administration of 96% ethanol a 0.6 M HCl, PGI2, atropine, cimetidine, given in cytoprotective doses, significantly mounted the ethanol-induced increase of gastric mucosal SOD activity; PGI2, atropine, cimetidine, given them in cytoprotective doses significantly shunted the HCl-induced decrease of gastric mucosal SOD activity. It has been concluded that; chemically different cytoprotective agents (PGI2, atropine, cimetidine) give rise to similar tendencies in the changes of gastric mucosal SOD activity; both the significant decrease (in the ethanol-model) and the significant increase (in the HCl-model) of this enzyme seem to be involved in the development of gastric mucosal protection by PGI2, atropine and cimetidine.     

Evidence for the development of tolerance to PGF2 alpha on the ethanol-induced gastric mucosal damage

PGF2 alpha, 100 micrograms/kg intraperitoneally, applied 30 min before 1.0 ml intragastric ethanol (96%) exerts cytoprotective effect on the gastric mucosal membrane. After a week long pretreatment of the animals with 0.25; 0.5 and 1.0 mg/day PGF2 alpha resulted in a diminishing cytoprotective effect. The gastric tissue cAMP level raised simultaneously and after the PGF2 alpha pretreatment with the taming cytoprotection the cAMP level diminished parallel in a dose dependent manner. It is assumed that after PGF2 alpha pretreatment the density of the cellular PGF2 alpha receptors decreases, according to the observed phenomenon.     

一氧化氮和cGMP参与神经降压素的肝细胞保护作用

本工作在原代培养的小鼠肝细胞上观察了一氧化氮,ｃＧＭＰ和ｃＡＭＰ的变化与神经降压素肝细胞保护作用之间的关系。结果如下：向培养液中加入醋氨酚１２ｈ后,ＧＯＴ和ＧＰＴ漏出明显增加;在加醋酚之前给予神经降压素则使液氨酶漏出明显减轻。给予ＮＯ合成酶阻断剂Ｌ－ＮＡＭＥ可完全阻断神经降压素的保护作用。     

Role of endogenous prostacyclin in gastric ulcerogenic and healing responses--a study using IP-receptor knockout mice.

Endogenous prostaglandins (PGs) play an important role in the cytoprotective and healing responses in the stomach, by altering various functions, i.e., an increase of the mucosal blood flow, yet the role of prostacyclin (PGI(2)) and its receptor (IP-receptor) in these responses remains unclarified. In the present study, we used IP-receptor knockout mice [IP (-/-)] and examined the importance of IP-receptors in gastric ulcerogenic, cytoprotective and healing responses in these animals. The studies included the ulcerogenic response to cold-restraint stress, the cytoprotective response to a mild irritant (20 mM taurocholate: TC) and capsaicin, and the healing response of chronic gastric ulcers induced by thermo-cauterization. We first checked the absence of IP-receptors by examining the effect of cicaprost (a PGI(2) agonist, topical mucosal application) on gastric mucosal blood flow and found that this agent increased the mucosal blood flow in wild-type [WT (+/+)] mice but not in IP (+/-) mice. Cold-restraint stress (4 h) induced gastric lesions in both groups of mice, but the severity of damage was significantly greater in IP (-/-) mice. Prior p.o. administration of both TC and capsaicin exhibited a marked cytoprotection against HCl/ethanol-induced gastric damage in WT (+/+) mice, both responses being significantly mitigated in the presence of indomethacin. The adaptive cytoprotection induced by TC was similarly observed in IP (-/-) mice, while the capsaicin protection was totally attenuated in the animals lacking IP receptors. On the other hand, the healing of gastric ulcers was significantly delayed by daily administration of indomethacin in WT (+/+) mice. However, this process was not altered in IP (-/-) mice. These results suggest that endogenous PGI(2) is involved in the gastric ulcerogenic response to stress, but not in the healing of pre-existing gastric ulcers. In addition, PGI(2) and its receptors may play a crucial role in capsaicin-induced gastric protection but not in the adaptive cytoprotection-induced by mild irritants.     

Mechanisms of action of retinoids in gastrointestinal mucosal protection in animals, human healthy subjects and patients.

Retinoids prevent chemically induced gastric mucosal damage without inhibiting gastric acid secretion ("nutritional gastric cytoprotection"). The gastroprotective effects of retinoids do not depend on 1) vitamin A activity; 2) number of unsaturated double bonds; 3) the presence of a characteristic chemical structure of their terminal components; however, they depend on 1) intact vagal nerve and 2) adrenals in experimental animals. The gastric cytoprotective effect of retinoids produces a dose-dependent inhibition of ATP-transformation into ADP. It also increases the transformation of ATP into cAMP. Other features of these gastric cytoprotective effects of retinoids include: 1) The retinoid-induced gastric mucosal protection differs from that of PGs; 2) The cAMP is an intracellular signal in the development of gastric mucosal damage produced by chemicals (e.g., ethanol, HCl, indomethacin) and in the protection of gastric mucosa induced by retinoids (but not by PGs); 3) The gastric mucosal protection induced by retinoids and gastric mucosal permeability can be separated in time. The existence of gastric mucosal protection can be demonstrated in healthy persons (against indomethacin treatment), in patients with gastric ulcer (GU) and duodenal ulcer (DU) without any inhibition of gastric acid secretion. The serum levels of vitamin A and zeaxanthin were significantly decreased in patients with chronic gastrointestinal (GI) inflammatory diseases (e.g., terminal ileitis, ulcerative colitis), colorectal polyposis, and different (e.g., esophageal, gastric, pancreatic, hepatocellular and colorectal) malignant diseases. The serum levels of vitamin A provitamins were unchanged and their GI mucosal protective effects do not depend on vitamin A activity. Conclusions: 1) Abundant experimental and human observations clearly proved the defensive role of retinoids in the GI tract; 2) There is a correlation between the a) scavenger properties of retinoids vs. intact vagal nerve; b) scavenging properties vs. intact adrenals. 3) The GI mucosal protective effect of retinoids is correlated with biochemical changes in the GI mucosa.     

Role of endogenous prostacyclin in gastric ulcerogenic and healing responses—a study using IP-receptor knockout mice

Endogenous prostaglandins (PGs) play an important role in the cytoprotective and healing responses in the stomach, by altering various functions, i.e., an increase of the mucosal blood flow, yet the role of prostacyclin (PGI₂) and its receptor (IP-receptor) in these responses remains unclarified. In the present study, we used IP-receptor knockout mice [IP (−/−)] and examined the importance of IP-receptors in gastric ulcerogenic, cytoprotective and healing responses in these animals. The studies included the ulcerogenic response to cold-restraint stress, the cytoprotective response to a mild irritant (20 mM taurocholate: TC) and capsaicin, and the healing response of chronic gastric ulcers induced by thermo-cauterization. We first checked the absence of IP-receptors by examining the effect of cicaprost (a PGI₂ agonist, topical mucosal application) on gastric mucosal blood flow and found that this agent increased the mucosal blood flow in wild-type [WT (+/+)] mice but not in IP (+/−) mice. Cold-restraint stress (4 h) induced gastric lesions in both groups of mice, but the severity of damage was significantly greater in IP (−/−) mice. Prior p.o. administration of both TC and capsaicin exhibited a marked cytoprotection against HCl/ethanol-induced gastric damage in WT (+/+) mice, both responses being significantly mitigated in the presence of indomethacin. The adaptive cytoprotection induced by TC was similarly observed in IP (−/−) mice, while the capsaicin protection was totally attenuated in the animals lacking IP receptors. On the other hand, the healing of gastric ulcers was significantly delayed by daily administration of indomethacin in WT (+/+) mice. However, this process was not altered in IP (−/−) mice. These results suggest that endogenous PGI₂ is involved in the gastric ulcerogenic response to stress, but not in the healing of pre-existing gastric ulcers. In addition, PGI₂ and its receptors may play a crucial role in capsaicin-induced gastric protection but not in the adaptive cytoprotection-induced by mild irritants.     

Correlation between the cytoprotective effect of beta-carotene and its gastric mucosal level in indomethacin (IND) treated rats with or without acute surgical vagotomy.

As to earlier observations that beta-carotene prevents the development of gastric mucosal injury produced by different noxious agent, however, its cytoprotective effect can be abolished by acute surgical vagotomy. The aim of this study was to evaluate the possible correlation between the gastric mucosal cytoprotective effect of beta-carotene and its gastric mucosal level in rats treated with IND. The gastric mucosal damage was produced by the administration of IND (20 mg/kg s.c.). The instillation of beta-carotene and acute surgical vagotomy (ASV) or SHAM operation were carried out 30 min before IND treatment. The rats were sacrificed 4 h after IND application, and the number and severity of gastric mucosal erosions were noted. The blood rats was collected quantitatively, the liver and the gastric mucosa were removed, and the beta-carotene and vitamin A level of the gastric mucosa, serum and liver were measured with HPLC. It was found that: 1. Beta-carotene induced gastric cytoprotection in SHAM-operated rats treated with IND but its effect disappeared after ASV. 2. Although the beta-carotene level of the gastric mucosa increased its concentration was not elevated in the serum of intact and vagotomized animals either. 3. Vitamin A Formation was not detected in the liver of animals with or without ASV. It was concluded that the lack of intake of beta-carotene into the gastric mucosa can not play etiologic role in the failure of gastric cytoprotection of rats with acute bilateral surgical vagotomy.     

Effects of capsaicin on the development of gastric mucosal damage by different necrotizing agents and of gastric cytoprotection by PGI2 atropine and cimetidine on rats

Capsaicin desensitization was used as a tool to reveal the role of neurogenic inflammation in the gastric mucosal lesions induced by intragastric application of four different noxious chemical agents (96% ethanol, 0.6 M HCl, 0.2 M NaOH, 25% NaCl). In capsaicin desensitized rats the number of lesions did not differ from that of the controls one hour after the application. There was, however, a significant reduction in the severity of the mucosal damage. These findings provide the first evidence for the participation of neurogenic inflammation in the gastric mucosal damage induced by aggressive chemicals. Gastrocytoprotection induced by prostacyclin (PGI2, 5 micrograms/kg), atropine (25 micrograms/kg) or cimetidine (2.5 mg/kg) was not inhibited in capsaicin desensitized rats. The number of lesions was not altered, while the severity of damage was more effectively reduced in the desensitized group. These findings indicate that the cytoprotective effect of these drugs is not mediated through capsaicin-sensitive sensory-efferent local tissue reactions.     

The role of nitric oxide and cGMP in platelet adhesion to vascular endothelium

The inhibition of platelet adhesion by nitric oxide (NO) and prostacyclin and their mechanism of action was studied. Platelet adhesion to collagen fibrils and endothelial cell matrix was inhibited completely by NO but only partially by prostacyclin. Adhesion of platelets to endothelial cell monolayers was inhibited by bradykinin. This effect of bradykinin was unaffected by aspirin, and was accounted for by the amounts of NO released by the endothelial cells. Inhibition of platelet adhesion by NO and prostacyclin was potentiated by selective inhibitors of cGMP phosphodiesterase, but not of cAMP phosphodiesterase, indicating that elevation of cGMP regulates platelet adhesion.     

Cytoprotection and intracellular calcium.

It seems that prostacyclin has an increasing effect on gastric mucosal (antral and fundic) calmodulin level in rats. Using either the calcium channel blocker verapamil or anti-calmodulin drugs (diazepam, trifluoperazine,) the cytoprotective effect of prostacyclin can be inhibited. Therefore, it is probable that calcium ions and calcium-activated calmodulin play a role in the effect of prostacyclin.     

Interleukin-1 is cytoprotective, antisecretory, stimulates PGE2 synthesis by the stomach, and retards gastric emptying

Human recombinant interleukin 1 beta (IL-1) administered intraperitoneally to rats produced the following gastric effects: 1. It was cytoprotective, preventing gastric mucosal necrosis produced by oral administration of one ml of absolute ethanol to fasted animals. The ED50 was 1200 units/kg (110 ng per animal). IL-1 was 125 times more potent than prostaglandin E2 (on a weight basis), and 6,000 times more potent (on a molar basis). 2. The cytoprotective effect of IL-1 was blocked by indomethacin (inhibitor of prostaglandin synthesis) and by IRAP (a specific interleukin-1 receptor antagonist protein). IRAP did not inhibit cytoprotection induced by PGE2. 3. IL-1 prevented the formation of gastric erosions induced by aspirin. 4. IL-1 inhibited gastric secretion (volume, acid concentration and output), in the pylorus-ligated rat, with an ED50 of 300 units/kg (3.2 ng per animal). 5. Indomethacin and IRAP blocked the antisecretory effect of IL-1. 6. IL-1 retarded gastric emptying, an effect blocked by IRAP, but not by indomethacin. 7. IL-1 increased synthesis of prostaglandin E2 by the gastric mucosa by 111%. IL-1 is the most potent of known agents that are gastric cytoprotective, antiulcer, antisecretory, and delay gastric emptying. It appears to act mostly by stimulating the synthesis of prostaglandins by the stomach. These studies suggest that the stomach possesses IL-1 receptors. These are probably located on parietal cells (that produce acid), on prostaglandin-producing cells, on smooth muscle cells (responsible for gastric emptying), and on as yet unidentified cells involved in gastric cytoprotection. Both IL-1 and IRAP, being natural substances, may play a physiological role in the maintenance of gastric mucosal integrity, and in the regulation of acid secretion and gastric motility.     

Effect of piracetam, a nootropic agent, on experimental gastric ulcers in rat

Piracetam, used clinically for cognitive disorders, was found to have significant anti-ulcerogenic activity against immobilization stress- and aspirin-induced gastric ulcers in rats. The anti-ulcer effect of piracetam was exerted by augmentation of mucosal resistance. This was indicated by the significant attenuation of the decrease in total carbohydrate: protein ratio induced by aspirin. It also reversed the marked increase in gastric juice protein and DNA induced by aspirin, indicating that piracetam attenuated the augmented mucosal cell exfoliation induced by the ulcerogen. The drug also increased gastric mucosal serotonin concentrations. Piracetam, thus appears to have a profile of activity associated with cytoprotective agents.