Cysteamine induced-duodenal ulcers are associated with a selective depletion in gastric and duodenal calcitonin gene-related peptide-like immunoreactivity in rats.

We have measured the endogenous levels of gastric and duodenal calcitonin gene-related peptide (CGRP)-, neurokinin A (NKA)-, galanin-vasoactive intestinal polypeptide (VIP)- and neuropeptide Y (NPY)-like immunoreactivity (li) in relation to cysteamine-induced gastric lesions and duodenal ulcers in rats. CGRP-li but not NKA-, galanin-, VIP- or NPY-li was decreased in gastric and duodenal samples following a single ulcerogenic dose of cysteamine (900 mg/kg p.o.). Temporal relationships of this phenomenon showed that CGRP-li was selectively decreased (stomach 45%, duodenum 68% as compared to controls, respectively after 24 h) concomitantly to the formation of acute gastric lesions and duodenal ulcers. Animals bearing healed ulcers 12 days after cysteamine, had gastroduodenal CGRP-li similar to control values. Pretreatment with the selective sensory neurotoxin capsaicin decreased gastroduodenal CGRP-li but not NKA-, galanin-, VIP- or NPY-li, showing that CGRP might be considered a marker of the afferent innervation of the gastroduodenal tract. The residual gastroduodenal CGRP-li levels in capsaicin-pretreated animals were not decreased by cysteamine administration, indicating that the effect of cysteamine is restricted to a peptide pool of primary afferent origin. Duodenal CGRP-li is selectively decreased by the duodenal ulcerogen cysteamine during the acute phase of ulcers formation and might be among the local mediators which afford protection against the ulcerogenic stimuli.     

Selective decrease of duodenal phosphoprotein phosphatase activity is a general property of duodenal ulcerogens

A phosphoprotein phosphatase (PPPase) is inhibited in rat duodenal mucosal cells very early after a single s. c. injection of the duodenal ulcerogens cysteamine, propionitrile and mepirizole. The effect seems to be independent of their effects on gastric acid secretion and on duodenal suppression of alkaline secretion. The enzyme inhibition is dose- and time-dependent under in vivo conditions. The inhibition of the PPPase activity is enzyme-selective at the level of mucosal cells in the duodenum. Under in vitro conditions, none of the duodenal ulcerogens inhibited PPPase activity. The results indicate that the effect of the ulcerogens on PPPase activity is probably exerted indirectly. When given simultaneously in vivo, propionitrile attenuated the inhibitory effects of cysteamine on the PPPase activity. However, both propionitrile and cysteamine potentiated the effect of mepirizole on PPPase depletion. These data indicate that cysteamine and propionitrile may act through the same mechanism when depleting PPPase activity. The mechanism of the decrease of duodenal protein phosphatase activity by mepirizole is probably different from the other duodenal ulcerogens.     

Effect of 3,4-toluenediamine on output from in situ rat Brunner's glands pouches.

Duodenal and gastric glandular mucosal damage have been observed 24 hr following single subcutaneous doses of 3,4-TDA in fed, unrestrained rats. 3,4-TDA significantly reduced secretion from in situ Brunner's glands pouches in pentobarbital anesthetized rats. The reduction in volume output with a definitely duodenal ulcerogenic dose of this compound was more than twice that observed with a minimally ulcerogenic dose, suggesting a correlation between the duodenal ulcerogenic and duodenal anti-secretory activities of this compound. The animal model described in this communication should facilitate experimentation to establish the inhibitory effect of compounds on the output of protective fluids from the proximal duodenum.     

Regulation of somatostatin-14 and gastrin I binding sites in rat gastrointestinal mucosa by ulcerogenic dose of cysteamine

A single duodenal ulcerogenic dose of cysteamine administered into rats induced time-dependent depletion of immunoreactive somatostatin in the gastric corporeal, antral, and duodenal mucosa with a parallel increase (up-regulation) of somatostatin binding sites. The concentration of somatostatin binding sites returned to the control level in the corporeal mucosa when measured at 24 hrs; however, in the duodenal mucosa there was only a partial return to the control level. Somatostatin binding sites in the antral mucosa did not return to control level even after 24 hrs. Except for the duodenum mucosal immunoreactive gastrin level was unaffected by cysteamine administration, but corporeal mucosal gastrin I binding sites were diminished (down-regulation) after 24 hrs.     

Roles of endogenous prostaglandins and nitric oxide in gastroduodenal ulcerogenic responses induced in rats by hypothermic stress.

We examined the roles of endogenous prostaglandins (PGs) and nitric oxide (NO) in the gastroduodenal ulcerogenic responses to hypothermic stress (28 approximately 30 degrees C) in anesthetized rats. Lowering body temperature provoked damage in the gastroduodenal mucosa, with an increase of gastric acid secretion and motility. These responses were completely abolished by bilateral vagotomy or atropine, while 16,16-dimethyl PGE2 decreased the mucosal ulcerogenic response with no effect on acid secretion. The non-selective COX inhibitors, indomethacin or aspirin, worsened these lesions with enhancement of gastric motility and no effect on acid secretion, while the selective COX-2 inhibitor NS-398 did not affect any of these responses. On the other hand, the non-selective NOS inhibitor L-NAME but not aminoguanidine (a relatively selective inhibitor of iNOS), significantly potentiated the acid secretory and mucosal ulcerogenic responses in the stomach but reduced the duodenal damage in response to hypothermia, the effects being antagonized by co-administration of L-arginine. Hypothermia itself decreased duodenal HCO3- secretion under both basal and mucosal acidification-stimulated conditions. Both indomethacin and aspirin further decreased the HCO3- response to the mucosal acidification, while L-NAME significantly increased the HCO3- secretion even under hypothermic conditions, similar to 16,16-dimethyl PGE2. These results suggest that 1) hypothermic stress caused an increase of acid secretion and motility as well as a decrease of duodenal HCO3-secretion, resulting in damage in both the stomach and duodenum, 2) the COX-1 but not COX-2 inhibition worsened these lesions by enhancing gastric motility and further decreasing duodenal HCO3- response, 3) the cNOS but not iNOS inhibition worsened gastric lesions by increasing acid secretion but decreased duodenal damage by increasing HCO3- secretion. Thus, it is assumed that the gastroduodenal ulcerogenic and functional responses to hypothermic stress are modified by cNOS/NO as well as COX-1/PGs.     

Enhanced ghrelin secretion in rats with cysteamine-induced duodenal ulcers

Ghrelin, produced and secreted by the A-like cells of the stomach, stimulates growth hormone secretion, gastric motility, and food intake. Cysteamine inhibits the release of somatostatin and induces the formation of duodenal ulcers in rats. The present study was conducted to investigate the dynamics of ghrelin secretion in rats treated with cysteamine. Male Wistar rats (7 wk old) were administered three doses of cysteamine (400 mg/kg) orally; at 50 h after the first dose, duodenal ulcers were induced, and the plasma level of somatostatin and gastric density of somatostatin-immunoreactive cells were significantly reduced. The plasma total and active ghrelin levels were significantly higher in the cysteamine-treated rats than in the control rats, whereas the gastric ghrelin levels, number of gastric ghrelin-immunoreactive cells, and preproghrelin mRNA expression levels were significantly lower. Even at the time points of 2 and 10 h after the first dose of cysteamine, at which time no significant ulcer formation or antral neutrophil accumulation was yet noted, a significant increase in the plasma ghrelin level and decrease in the gastric ghrelin level were observed. Furthermore, although lansoprazole treatment attenuated the duodenal ulceration induced by cysteamine, the increase in the plasma level of ghrelin could still be demonstrated. Because an inverse correlation was found between the plasma ghrelin and somatostatin levels, the inhibition of somatostatin secretion may be associated with the increased ghrelin secretion. In conclusion, an increase in the plasma ghrelin level precedes the formation of duodenal ulcers in rats treated with cysteamine.     

Stimulation of HCO3- secretion by the prostaglandin E2 analog enprostil: studies in human stomach and rat duodenum

This study investigated the action of enprostil, a synthetic analog of PGE2, on gastric HCO3- secretion in humans and on duodenal HCO3- secretion in the anesthetized rat. A previously validated 2-component model was used to calculate gastric HCO3- and H+ secretion in 10 human subjects. Compared to placebo, a single 70 micrograms oral dose of enprostil increased basal gastric HCO3- secretion from 1810 +/- 340 to 3190 +/- 890 mumol/hr (P less than 0.05). In addition, enprostil reduced basal gastric H+ secretion from 5240 +/- 1140 to 1680 +/- 530 mumol/hr (P less than 0.02). Enprostil also increased HCO3- secretion and reduced H+ secretion during intravenous pentagastrin infusion. In the rat, duodenal HCO3- secretion was measured by direct titration in situ using perfused segments of duodenum just distal to the Brunner gland area and devoid of pancreatic and biliary secretions. Addition of enprostil (10 micrograms/ml) to the duodenal bathing solution increased duodenal HCO3- secretion from 6.3 +/- 1.3 to 15.1 +/- 2.0 mumol/cm X hr (P less than 0.01, n = 6). The stimulatory action of enprostil on duodenal HCO3- secretion at 10 micrograms/ml was comparable in magnitude and duration to that of 10 micrograms/ml natural PGE2. In summary, the PGE2 analog enprostil stimulated gastroduodenal HCO3- secretion, effects which may be beneficial in protection of the gastroduodenal mucosa against luminal acid.     

Role of gastrin in duodenal mechanisms of inhibition of gastric secretion in the dog and man

Gastrin serum levels after acidification of the second portion of the duodenum were studied, in dogs and humans, while simultaneously measuring secretin levels and gastric acid secretion. After duodenal acidification in dogs, a 50% inhibition of gastric acid secretion with parallel 100% increases in the serum secretin levels was noted whereas gastrin serum levels did not change (after duodenal acidification). In humans, a 25% inhibition of gastric acid secretion with parallel 50% (not significative) increases in the secretin serum levels was noted. In the entire group gastrin levels did not change, but in 35.2% of the subjects a little increment without statistical significance was noted. It is concluded that the inhibition mechanism of gastric acid secretion after duodenal acidification is more important in dog than in man, and that, probably, gastrin does not play an important role in this mechanism.     

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.     

Influence of duodenal acidification on the sensorimotor function of the proximal stomach in humans

Decreased acid clearance and increased exposure to acid of the duodenum have been reported in a subset of functional dyspepsia patients. However, the mechanism by which increased duodenal acid exposure may affect symptoms is unclear. The aim of the present study was to investigate the effects of duodenal acidification on proximal gastric tone and mechanosensitivity in humans. An infusion tube with a pH electrode attached was positioned in the second part of the duodenum, and a barostat bag was located in the gastric fundus. In 12 healthy subjects, fundic tone and sensitivity to distensions were assessed before and during duodenal infusion of 0.1 N hydrochloric acid or saline in a randomized, double-blind design. In 10 healthy subjects, meal-induced accommodation was measured during duodenal infusion of acid or saline. Acid infusion in the duodenum significantly increased fundic compliance and decreased fasting fundic tone. This was accompanied by a significant decrease in the pressures and the corresponding wall tensions at the thresholds for discomfort. During infusion of acid, significantly higher perception and symptom scores were obtained for the same distending pressures. The meal-induced fundic relaxation was significantly smaller during acid infusion compared with saline infusion. In conclusion, duodenal acidification induces proximal gastric relaxation, increases sensitivity to gastric distension, and inhibits gastric accommodation to a meal. Through these mechanisms, increased duodenal acid exposure may be involved in the pathogenesis of dyspeptic symptoms.     

Interrelation between the analgesic and ulcerogenic action of cysteamine

Interrelationship was studied between the influence of cysteamine on pain threshold and ulcerogenic effect on the duodenum. Cysteamine (350 mg/kg) induced analgesia in mice which was prevented by naloxone (1.5 mg/kg). In rats, cysteamine produced duodenal ulcers with concomitant analgesia. The intensity of ulceration was higher in animals with lower basal pain threshold. The correlation between central and peripheral effects of endogenous opioids in the development of experimental duodenal ulcers is discussed.     

Potent CNS action of calcitonin to inhibit cysteamine-induced duodenal ulcers in rat

Intracisternal injection of calcitonin (0.01-5 micrograms) dose dependently prevented the development of duodenal ulcers induced by cysteamine in female rats. By contrast, intravenous infusion of the peptide at a dose 50 times higher than an effective intracisternal dose, had no effect. Intracisternal injection of calcitonin increased by three fold the generation of 6-keto-PGF1 alpha, the stable hydrolysis product of PGI2, in the duodenal mucosa. These studies demonstrated that calcitonin acts within the brain to potently suppress duodenal ulcers induced by cysteamine. The mechanisms of the antiulcer effect may involve changes in prostaglandin generation along with alterations of gastrointestinal secretion and motility associated the central injection of calcitonin. Growing evidence suggests that salmon calcitonin may act as a neuromodulator or neurotransmitter in the central nervous system. Specific binding sites have been demonstrated for calcitonin in the hypothalamus, brain stem and dorsal horn of the spinal cord using homogenate and membrane preparations or in vitro autoradiography methods. The peptide injected into the cerebrospinal fluid (CSF) produces a wide spectrum of biological effects including analgesia, hyperthermia, changes in pituitary hormone release, decrease in food and water intake, locomotor activity, and blood pressure. Numerous studies also demonstrated that calcitonin acts within the brain to markedly influence gastrointestinal secretory and motor function in rats and dogs and gastric ulceration in rats. In particular, intracisternal injection of salmon calcitonin was found very potent to selectively inhibit gastric ulcers elicited by stress, aspirin and central thyrotropin-releasing factor but not by necrotizing agents. In the present study, we further investigated the antiulcer effect of salmon calcitonin using the well established cysteamine experimental model to induce duodenal ulcers in rats. Part of this work has been reported in abstract form.     

Effect of stobadine on indomethacin- and ethanol-induced stomach lesions and gastric secretion.

Stobadine was found to inhibit the ulcerogenic activity of indomethacin in relation to the dose but was ineffective against the direct necrotizing action of ethanol. It also inhibited gastric acid secretion when administered intraduodenally. Although stobadine is considered to be a scavenger of free radicals, our results indicate that, under the given experimental conditions, it is rather the inhibition of gastric acid secretion that is responsible for its antiulcerogenic effect. The preliminary results do not allow the exclusion of other mechanisms for explaining its antiulcerogenic effect.     

Cysteamine-colon and cysteamine-duodenum lesions in rats. Attenuation by gastric pentadecapeptide BPC 157, cimetidine, ranitidine, atropine, omeprazole, sulphasalazine and methylprednisolone.

Recently, we showed cysteamine-duodenal lesions without gastric acid, since they were induced also in gastrectomized rats, as in naive rats, and they were inhibited by the novel stomach pentadecapeptide BPC 157 as well as standard antiulcer drugs (i.e. cimetidine, ranitidine, omeprazole, bromocriptine, atropine). Therefore, as an advantage of considering cysteamine as a directly acting cytotoxic agent and mentioned agents as direct cytoprotective agents, the present focus was on the ulcerogenic effect of cysteamine and protective effect of gastroduodenal antiulcer agents outside upper gastrointestinal tract (i.e. in colon). Intrarectal administration of the cysteamine (200 or 400 mg/kg b.w) produced severe colon lesions (i.e. transmural inflammation with serosal involvement) in rats (30 min-72 h-experimental period), apparently distinctive from smaller lesions after non-specific irritant enema [diluted HCl solution, pH 3.8 (adjusted to pH of cysteamine solution (pH 3.8)]. All of the tested antiulcer agents were applied simultaneously with cysteamine enema (8 cm from the anus, in a volume of the 1.0 ml/rat) intraperitoneally (i.p.), intragastrically (i.g.) or intrarectally (i.r.). Pentadecapeptide BPC 157 (10 microg or 10 ng/kg b.w.), given in either regimen, previously shown to have, besides others, a particular beneficial activity just in the intestinal mucosa, inhibited these cysteamine colon lesions (assessed after 30 min, 60 min, 180 min, 24 h, 48 h, 72 h following cysteamine in a dose of either 200 or 400 mg/kg i.r.). Cysteamine-colon lesions were also attenuated by standard antiulcer agents (mg/kg b.w.), given i.p., i.g., or i.r., such as ranitidine (10), cimetidine (50), omeprazole (10), atropine (10), together with methylprednisolone (1), and sulphasalazine (50, i.r.), assessed 30 min following application of 200 mg of cysteamine. Finally, standard cysteamine duodenal lesions (assessed 24 h after a subcutaneous application of 400 mg/kg of cysteamine) were also attenuated by these agents application (given in the same doses, i.p., 1 h before cysteamine), with only exception to sulphasalazine. Thus, the extended cysteamine specific ulcerogenic effect, cysteamine colon/duodenum lesion-link and an extenuation of agents protection from upper to lower part of gastrointestinal tract (i.e. stomach pentadecapeptide BPC 157, standard antiulcer agents, cimetidine, ranitidine, atropine, omeprazole) and vice versa (remedies for inflammatory bowel disease) evidenced in the present study may be potentially important for both further experimental and clinical research.     

Growth hormone and somatostatin interaction in the ulcerogenic effect of cysteamine in female rats

It is known that cysteamine's ulcerogenic effect depends, among others, on a depletion of somatostatin (SRIH). Since growth hormone (GH) affects the release of hypothalamic SRIH, we have studied the influence of GH and the GH-SRIH interaction on the severity of gastric mucosa lesions induced by cysteamine. Female rats of the Sprague-Dawley strain were pretreated with GH (1 mg/kg) and subjected to cysteamine-induced gastric lesions. We found that these animals showed an increased mortality and severity of gastric lesions. Pretreatment with SRIH (25 or 50 μg/kg) was followed by a decrease in mortality and of incidence and severity of gastric mucosa lesions as compared to those found in control animals pretreated with saline. The dose of 5μg/kg was ineffective in this respect. The combined administration of GH and SRIH revealed that cysteamine ulcerogenic action remained unchanged. It is possible that high levels of plasma GH, as induced by exogenous GH administration, may decrease the release of gastro-intestinal SRIH and this in turn may potentiate the ulcerogenic activity of cysteamine.     

The influence of cysteamine and propionitrile on duodenal phosphoprotein phosphatase in rats

Cysteamine and propionitrile cause severe duodenal ulcers with perforation within 24-48 h after a single injection in rats. These animal models were used to gain insight into the early, preulcerogenic biochemical changes in the duodenal mucosa. The results indicate that a single sc injection of cysteamine and propionitrile induced dose- and time-dependent decreases in the activity of phosphoprotein phosphatase (PPPase) in homogenate and particulate fractions of rat duodenal mucosa. The decrease in enzyme activity was detectable 4 h after the injection of the ulcerogens, it was maximal at 12 h, and hardly detectable at 24 h. No effect on the enzyme activity was found under in vitro conditions. PPPase activity in the liver was not influenced by either cysteamine or propionitrile. Furthermore, the toxic but nonulcerogenic derivative of cysteamine ethanolamine had no effect on PPPase in the duodenum. Thus, the effect of the duodenal ulcerogens on PPPase activity was indirect and organ specific, related only to the target organ (i.e., duodenal mucosa). The effect of the drugs was also selective at the level of mucosal cells: both duodenal ulcerogens depleted protein and alkaline phosphatase but not lysosomal acid phosphatase. The decrease of PPPase activity could be a general property of the duodenal ulcerogens since it is independent of their effect on endogenous somatostatin.     

Comparison of the ulcerogenicity of indomethacin and the gastric secretion in verapamil-treated rats

The authors studied the effect of different doses of verapamil on the ulcerogenic activity of indomethacin (20 mg.kg-1) in rats. This was compared with the effect of verapamil on total gastric juice secretion, the amount of acid and the pH. It was found that, as distinct from total secretion and the amount of HCl, which verapamil reduced in correlation to the dose, ulcerogenicity after indomethacin was inhibited the most by a dose of 10 mg.kg-1 verapamil. Larger doses (20 and 30 mg.kg-1) did not increase the anti-ulcerogenic effect any further. This implies that verapamil-induced inhibition of the ulcerogenicity of indomethacin is not related directly to inhibition of total and acid gastric juice secretion.     

A novel small molecule CFTR inhibitor attenuates HCO3- secretion and duodenal ulcer formation in rats

The cystic fibrosis (CF) transmembrane conductance regulator (CFTR) plays a crucial role in mediating duodenal bicarbonate (HCO(3)(-)) secretion (DBS). Although impaired DBS is observed in CF mutant mice and in CF patients, which would predict increased ulcer susceptibility, duodenal injury is rarely observed in CF patients and is reduced in CF mutant mice. To explain this apparent paradox, we hypothesized that CFTR dysfunction increases cellular [HCO(3)(-)] and buffering power. To further test this hypothesis, we examined the effect of a novel, potent, and highly selective CFTR inhibitor, CFTR(inh)-172, on DBS and duodenal ulceration in rats. DBS was measured in situ using a standard loop perfusion model with a pH stat under isoflurane anesthesia. Duodenal ulcers were induced in rats by cysteamine with or without CFTR(inh)-172 pretreatment 1 h before cysteamine. Superfusion of CFTR(inh)-172 (0.1-10 microM) over the duodenal mucosa had no effect on basal DBS but at 10 microM inhibited acid-induced DBS, suggesting that its effect was limited to CFTR activation. Acid-induced DBS was abolished at 1 and 3 h and was reduced 24 h after treatment with CFTR(inh)-172, although basal DBS was increased at 24 h. CFTR(inh)-172 treatment had no effect on gastric acid or HCO(3)(-) secretion. Duodenal ulcers were observed 24 h after cysteamine treatment but were reduced in CFTR(inh)-172-pretreated rats. CFTR(inh)-172 acutely produces CFTR dysfunction in rodents for up to 24 h. CFTR inhibition reduces acid-induced DBS but also prevents duodenal ulcer formation, supporting our hypothesis that intracellular HCO(3)(-) may be an important protective mechanism for duodenal epithelial cells.     

Effects of endothelin-1 on duodenal bicarbonate secretion and mucosal integrity in rats

Effects of endothelin-1 on gastric acid secretion, duodenal HCO3- secretion, and duodenal mucosal integrity were investigated in anesthetized rats, in comparison with those of TY-10957, a stable analogue of prostacyclin. A rat stomach mounted on an ex-vivo chamber or a proximal duodenal loop was perfused with saline, and gastric acid or duodenal HCO3- secretion was measured using a pH-stat method and by adding 100 mM NaOH or 10 mM HCl, respectively. Duodenal lesions were induced by mepirizole (200 mg/kg) given subcutaneously. Intravenous administration of endothelin-1 (0.6 and 1 nmol/kg) caused an increase of duodenal HCO3- secretion with concomitant elevation of blood pressure; this effect was antagonized by co-administrahon of BQ-123 (ET(A) antagonist; 3 mg/kg, i.v.) and significantly mitigated by vagotomy. Likewise, endothelin-1 caused a significant decrease in histamine-stimulated acid secretion, and this effect was also significantly antagonized by BQ-123. Although TY-10957 (10 and 30 mg/kg, i.v.) produced a temporal decrease of blood pressure, this agent caused not only an increase of duodenal HCO3- secretion, independent of vagal nerves, but also a decrease of acid secretion as well. In addition, both endothelin-1 and TY-10957 significantly prevented mepirizole-induced duodenal lesions at the doses that caused an increase of duodenal HCO3- secretion and a decrease of gastric acid secretion. These results suggest that endothelin-1 affects the duodenal mucosal integrity by modifying both gastric acid and duodenal HCO3- secretions, the effects being mediated by ET(A) receptors.