Histological study of mechanisms of adaptive cytoprotection on ethanol-induced mucosal damage in rat stomachs

Adaptive cytoprotection in the gastric mucosa could be induced by exposure to low concentrations of noxious agents. However, experimental results reported so far were based on macroscopic studies. We aimed to investigate the phenomenon of gastric adaptive cytoprotection of mild irritants and its correlation with intramucosal mucus at the histological level. It was found that histological damage induced by ethanol had a negative correlation with the length of the mucus-secreting layer in the gastric mucosa. Mild irritants such as 20% ethanol and 5% NaCl preserved the 100% ethanol-induced intramucosal mucus depletion, but only the former agent demonstrated a cytoprotective effect against the histological damage, indicating that preservation of intramucosal mucus may not necessarily play a permissive role in adaptive cytoprotection. The capsaicin-sensitive sensory afferent neurons, sensory chemoreceptors, muscarinic receptors, alpha2-adrenoceptors and peripheral dopamine D2-receptors were found to be the components of the autonomic nervous system involved in the cytoprotective processes of 20% ethanol. Endogenous mediators including nitric oxide, prostaglandins, and possibly nonprotein sulfhydryl compounds also seemed to participate in such protection. Nevertheless, 0.3 M HCl did not show any effect either on mucosal damage or depletion of intramucosal mucus induced by absolute ethanol. These findings suggest that only 20% ethanol shows histological cytoprotection, which would involve various components of the autonomic nervous system and endogenous mediators. Furthermore, this investigation also implies a new perspective: that in order to study a true adaptive cytoprotection, histological examination of the gastric mucosa should be performed.     

Phosphatidylcholines as mediators of adaptive cytoprotection of the rat duodenum

BACKGROUND/AIMS: Surfactant phospholipids impede diffusion of acid through the gastric mucus, but their relevance in the defense of the duodenum against luminal acid is not known. METHODS: Duodenal resistance to acid was tested in anesthetized rats by instillation of HCl using a tube implanted in the proximal duodenum. The effects of a detergent (Brij 35; Sigma, St. Louis, MO) and a lipid mixture flushed through the luminal surface on duodenal resistance to acid were studied. The lipid content in the mucus and the effects of acid, prostaglandins, and indomethacin on the lipid layer were also analyzed. RESULTS: Instillation of 100 mumol HCl or 5 micrograms/kg 16,16-dimethyl prostaglandin E2 increased resistance to acid, preventing duodenal lesions induced by 500 mumol HCl. However, 100 mumol HCl or 16,16-dimethyl prostaglandin E2 did not prevent lesions induced by 500 mumol HCl in rats undergoing perfusions with 5% Brij 35. Indomethacin suppressed acid-induced protection. A mixture of tripalmitin and dipalmitoyl-phosphatidylcholine protected against 500 mumol HCl, and the effect was also observed in rats receiving indomethacin. Finally, 100 mumol HCl increased the phosphatidylcholine content in the duodenal mucus but not in rats receiving 5% Brij 35 or indomethacin. CONCLUSIONS: Surface-active phospholipids are critical for adaptive cytoprotection to acid in the rat duodenum.     

The antiulcer effect of corticosteroids and the blood flow in the gastric mucosa

Owing to the short wavelengths of X-radiation X-ray microscopes allow higher resolution than optical microscopes. In contrast to electron microscopes, X-radiation can be used to study relatively thick aqueous specimens in their natural environment. X-ray microscopes require intense X-radiation, which is best provided by electron storage rings, as well as efficient X-ray optics. X-ray microscopes with zone plate optics are installed at the storage ring BESSY in Berlin for studies in the fields of biology, medicine, biophysics, colloid chemistry, and soil sciences.     

A correlative study on the mechanism of adaptive cytoprotection against ethanol-induced gastric lesion formation in rats

The protective effect of mild irritants against the subsequent gastric injury induced by necrotizing agents has been termed 'adaptive cytoprotection'. In this study, the possible pathway and mechanisms of adaptive cytoprotection induced by 20% ethanol were investigated. An ex-vivo gastric chamber preparation was used. The gastric mucosa was exposed to 20% ethanol before subsequent administration of 100% ethanol 15 min later. Subdiaphragmatic vagotomy or drug pretreatment was carried out in order to elucidate the mechanisms of adaptive cytoprotection by 20% ethanol. The results showed that 20% ethanol pre-exposure significantly protected the gastric mucosa against damage caused by 100% ethanol. This protective action was completely abolished by atropine or lidocaine pretreatment, whereas vagotomy and hexamethonium failed to have a significant influence. The cytoprotective effect, however, was independent of the gastric secretory volume, titratable acid content, luminal soluble mucus level and gastric mucosal blood flow. Exposure of only half the gastric mucosa to the mild irritant resulted in the protection of both sides of the mucosa. All these findings indicate that the adaptive cytoprotection of 20% ethanol involves the participation of chemoreceptors and muscarinic receptors in the gastric mucosa. An internal enteric reflex arc, with transmission of signals within the gastric mucosa, may also contribute to the cytoprotective process of the mild irritant.     

Gastric intramucosal acidosis in mechanically ventilated patients: role of mucosal blood flow

To examine the roles of Arg-vasopressin (AVP)- and vasoactive intestinal peptide (VIP)-containing neurons in the suprachiasmatic nucleus (SCN) in production of circadian rhythmicity of locomotor activity, variations in the contents of AVP and VIP in punched-out SCN tissue and locomotor activity were measured under a light-dark cycle as well as under conditions of constant light for up to 3 weeks. Under the light-dark cycle, contents of AVP and VIP, and locomotor activity showed marked circadian rhythmicity. Under constant light, AVP content showed circadian rhythmicity until 3 weeks, while VIP rhythm disappeared from the first week with decreases in its content. Locomotor activity showed a free-running circadian rhythm for more than 3 weeks under constant light conditions in most cases. These results suggest that AVP but not VIP in the SCN may be involved in the generation of locomotor activity rhythm under conditions of constant light.     

Mucosal irritation, adaptive cytoprotection, and adaptation to topical ammonia in the rat stomach

BACKGROUND: The urease-ammonia (NH4OH) system has been proposed to play a major role in the pathogenesis of the Helicobacter pylori-associated gastritis, but the mechanism of the mucosal damage has not been fully explained. This study was designed to examine possible adaptive cytoprotection and the adaptation of rat gastric mucosa to the irritant action of NH4OH and urease. METHODS AND RESULTS: Single application of NH4OH alone in various concentrations (15-500 mM) caused concentration-dependent mucosal damage starting with 30 mM and reaching a maximum at 250 mM NH4OH, similar to that obtained with 100% ethanol; it was accompanied by a decrease in gastric blood flow (GBF) to approximately 30% of the normal value. When the mucosa was first exposed to the low, non-damaging concentration (15 mM) of NH4OH and then insulted with 100% ethanol, the extent of ethanol damage was greatly attenuated as compared with that caused by ethanol alone. This adaptive cytoprotection was accompanied by the rise in GBF and reversed, in part, by the pretreatment with indomethacin, an inhibitor of prostaglandin (PG)-cyclooxygenase; with L-NAME, a blocker of NO-synthase; or with capsaicin deactivating the sensory nerves. Damaging concentrations of NH4OH (125 mM) caused widespread mucosal damage after the first application, but with repeated insults with 125 mM NH4OH a gradual reduction in the mucosal lesions, accompanied by enhanced mucosal cell proliferation and over-expression of epidermal growth factor (EGF) (using immunocytochemistry) and mRNA of EGF (using trans-reverse polymerase chain reaction), were observed. CONCLUSIONS: NH4OH alone damages gastric mucosa only at the concentration exceeding that found in H. pylori-infected stomachs, whereas at lower concentrations it acts as 'mild' irritant to induce adaptive cytoprotection. This adaptive cytoprotection appears to be mediated, in part, by endogenous PG, sensory nerves, and an arginine-NO-dependent pathway, and repeated applications of NH4OH induce gastric adaptation, probably mediated by enhanced expression of EGF and its receptors and by an increased cell proliferation.     

Histological effect of (R)-alpha-methylhistamine on ethanol damage in rat gastric mucosa: influence on mucus production

(R)-alpha-Methylhistamine, a selective agonist of histamine H3 receptors, prevents macroscopically visible gastric lesions by absolute ethanol in the rat. A further insight into its activity was the aim of our study. Rats were given saline or (R)-alpha-methylhistamine (100 mg/kg) intragastrically. After 30 min, absolute ethanol was given and gastric mucosa was sampled 60 min later. Histologic damage and intracellular and adherent mucus were quantified. Luminal surface and mucous cells were examined by scanning and transmission electron microscopy. (R)-alpha-Methylhistamine reduced the extent of lesions by ethanol from 96 to 18%. Surface mucous cells and mucous neck cells were increased in volume and number, packaging of intracellular mucus was modified, and the secretory processes were promoted by (R)-alpha-methylhistamine itself, although these modifications were mostly evident in stomachs subsequently exposed to ethanol. Adherent mucus layer thickness was increased by (R)-alpha-methylhistamine only after ethanol exposure. It is concluded that (R)-alpha-methylhistamine predisposes mucous cells to react to ethanol.     

Effect of marzulene on the restitution of rat gastric mucosa after NaOH-induced injury

BACKGROUND/AIMS: The purpose of this study was to assess the effect of marzulene (L-glutamine plus azulene) on the repair of NaOH-induced gastric mucosal injury in rats. METHODOLOGY: Gastric mucosal injury was induced with intragastric instillation of 3.0 ml of 5% NaOH for 1 minute. From 2 days after the operation, the rats were orally given chow pellets containing 0%, 0.25%, or 0.5% of marzulene for 25 weeks. RESULTS: Oral administration of marzulene at both dosages significantly increased the mucosal heights of the fundic and antral mucosa at week 25. Marzulene also increased the labeling indices of the fundic and antral epithelial cells, but not the mucosal blood flow. CONCLUSIONS: These findings indicate that marzulene stimulates repair mechanisms of rat gastric mucosa after NaOH injury. This effect of marzulene may be associated with a stimulation of gastric epithelial cell proliferation.     

Selective effects of the endogenous cannabinoid arachidonylethanolamide (anandamide) on regional cerebral blood flow in the rat

Chronic focal epilepsy is associated with synaptic plasticity and growth of new connections. Brain-derived neurotrophic factor (BDNF) is associated with each of these processes in normal brain and shows acute up-regulation in models of generalized epilepsy. Here, using an experimental model of focal epilepsy, we show persistent up-regulation of BDNF mRNA, independent of that of other growth factors, in association with the development and persistence of chronic seizures. In situ hybridization histochemistry revealed that rats perfused within 2-3 days after seizure onset had widespread increases in BDNF mRNA levels in the neocortex. Rats perfused at later times, however, showed focal up-regulation of BDNF mRNA at the injection site and down-regulation in a surrounding cortical zone. Nerve growth factor and neurotrophin-3 mRNAs were not significantly altered. These reciprocal changes in BDNF gene expression in the epileptic focus and the cortical surround may contribute to plastic changes in epileptic neuronal circuits that accompany the transition from acute to chronic epilepsy. BDNF down-regulation in the surround is likely to be associated with the inhibitory surround that hampers seizure spread, but facilitates the persistence of a chronic epileptic focus.     

The adaptation-protective effect of corticosteroids on the rat gastric mucosa and its mechanism

The influence of stress-induced corticosteroid production on gastric ulceration, blood flow velocity in gastric microvessels and blood pressure was studied in rats. The role of plasma corticosteroids was investigated by means of blockade of the pituitary-adrenocortical system (PACS) and following corticosterone replacement therapy (400 mu/100 g b.w.). The blockade which was induced by Fi. hydrocortisone administration (7 days before stress, 30 mg/100 g b. w.) resulted in an insufficient corticosteroid production. To evaluate the influence of corticosteroids on blood flow velocity in gastric microvessels of muscular, submucosal and mucosal coats it was used intravital microfilming by means of a dark-field contact epiobjective. Stress (water immersion + restraint) induces an ulceration, a decrease in the systemic arterial blood pressure (3 h after stress onset) and a decrease in blood flow velocity in the gastric microvessels (3 h after stress onset). In rats with insufficient corticosteroid production stress-induced ulceration, a decrease in blood pressure and gastric blood velocity were more greater than in rats with intact PACS. Replacement corticosterone therapy corrected all parameters. The results revealed that antiulcerogenic effect of stress-induced glucocorticoid production is realised owing to normalisation of gastric blood supply which is provided by an increase in systemic blood pressure.     

Modulatory role of 5-HT3 receptors in gastric function and ethanol-induced mucosal damage in rat stomachs

The involvement of 5-hydroxytryptamine (5-HT) in gastric function and mucosal damage has been defined. 5-HT also potentiates lesion formation in animals. The current study investigated further whether these actions are mediated through 5-HT3 receptors in rats. Ondansetron, a 5-HT3 receptor antagonist, was given subcutaneously, 2 or 4 mg/kg, 30 min before the gastric parameters were measured. The higher dose of ondansetron, 4 mg/kg, significantly increased gastric mucosal blood flow (GMBF) and also basal acid and Na+ secretion. However, it did not affect pepsin output. 5-HT time dependently reduced GMBF and pepsin secretion, but not that of acid and Na+. These actions were not altered by ondansetron pretreatment. The drug, however, dose dependently reduced ethanol-induced gastric mucosal lesions in the 5-HT-treated animals. These findings indicate that 5-HT3 receptors regulate not only basal GMBF, but also acid and Na+ secretion in stomachs. However, the depressive action of 5-HT on GMBF and pepsin secretion is most likely not mediated through 5-HT3 receptors. Ondansetron also modulates the toxicities of ethanol in the stomach and this action is likely to be mediated through the preservation of GMBF.     

Effect of pibutidine hydrochloride on nitric oxide production in rat gastric mucosa

The aim of the study was to determine blood pressures in young heterozygous transgenic rats harbouring the Ren-2 mouse gene, TGR(mREN2)27, and to examine the association between blood pressure and plasma renin-angiotensin parameters during the conscious state. Two litters of heterozygous TGR(mREN2)27 rats and their normotensive Sprague-Dawley reference animals were bred in our laboratory. Cardiovascular phenotypes and blood, for the evaluation of plasma renin-angiotensin-system parameters, were obtained via arterial catheters in conscious rats 4 weeks after birth. Blood pressure values showed a high degree of interindividual variability and significant litter differences in the group of transgenic animals ranging from normotensive tohypertensive values. Plasma prorenin levels were markedly increased in TGR(mREN2)27, whereas both active renin and angiotensin II plasma concentrations were reduced independently from hypertension. Plasma prorenin levels in litter 2 of transgenic animals which exhibited significantly elevated blood pressures were significantly higher as compared to those of litter 1 which showed blood pressures that were not significantly different from SD. These data demonstrate that the development of hypertension in heterozygous TGR(mREN2)27 takes place in the fourth week of postnatal life and exhibits large interindividual variability. Moreover, development of early hypertension in young heterozygous TGR(mREN2)27 is associated with higher plasma prorenin levels.     

Effect of fasting on mucosal blood flow in antrum and corpus of the stomach

Gastric mucosal blood flow in the rat was determined by the indicator fractionation technique 86RbCl. Antral and corpus flow rates were determined after increasing periods of fasting, and the effect of vagotomy was assessed. The physiological stimulus of food in the stomach markedly increased antral as well as corpus blood flow, and after vagotomy lower flow rates were recorded in all parts of the stomach. With progressive starvation, the antral:corpus flow ratio decreased. The findings suggest that the antrum plays an active role in response to a physiological stimulus and may, as has been suggested by others, contribute to the control of parietal cell blood flow and secretion.     

Gastric mucosal blood flow response to stress in streptozotocin diabetic rats: regulatory role of nitric oxide

To investigate cytoprotection against mucosal injuries of the stomach in patients with diabetes, we investigated gastric mucosal blood flow (GMBF), its response to a burn stress, and the involvement of nitric oxide (NO) in streptozotocin (STZ) diabetic rats. GMBF was measured by laser-Doppler velocimetry (LDV) and by the hydrogen gas clearance technique (HGC). The steady-state GMBF of STZ rats decreased according to the duration of diabetes, and insulin treatment blocked this decrease. Burn stress caused a rapid decrease in the GMBF. Reduction of the GMBF and gastric mucosal leakage of Evans blue (EB) after the burn stress were greater in the STZ rats than in the controls, but insulin treatment completely blocked this increase in EB leakage in the STZ rats. There was a significant negative correlation between the percent GMBF 3 h after the burn stress and EB leakage at the same time point. In the controls and the insulin-treated STZ rats, N-nitro-L-arginine (L-NNA), an NO synthase inhibitor, enhanced the decrease in postburn GMBF and EB leakage, but was without effect in the STZ rats. These results suggest that NO may be involved in the regulation of GMBF, and that persistent hyperglycemia may impair this regulation. These findings suggest that patients with diabetes have reduced cytoprotection against a variety of gastric mucosal injuries.     

Chronic nicotine intake causes vascular dysregulation in the rat gastric mucosa

Chronic cigarette smoking has adverse effects on peptic ulcer disease because the healing of ulcers is delayed and the incidence of relapses is enhanced. Short term intake of nicotine induces vascular damage in the rat gastric mucosa, but the pathophysiological mechanisms of nicotine's action in the stomach are largely unknown. In this study rats were treated with nicotine, added to their drinking water, for 50 days. They were then anaesthetised and their stomachs perfused with acidified acetylsalicylic acid (ASA). Chronic nicotine treatment failed to change the effects of acidified ASA to induce gastric mucosal acid back diffusion, haemorrhagic damage and bleeding. Basal blood flow in the gastric mucosa was also unchanged by chronic nicotine intake, whereas the mucosal hyperaemia evoked by ASA induced acid back diffusion was averted. The concentrations of sulfidoleukotrienes were significantly augmented in the gastric wall of nicotine treated rats. These data show that chronic nicotine intake causes dysregulation of the gastric microcirculation, an effect that is associated with biochemical changes in the stomach. This study thus substantiates the adverse effects of smoking on gastric mucosal pathophysiology. These data suggest that inappropriate regulation of gastric mucosal blood flow inhibits recovery from gastric mucosal injury in smokers.     

Effects of excitatory amino acids on cerebral oxygen consumption and blood flow in rat

The oncogene Tpr-Met is a constitutively active form of the hepatocyte growth factor/scatter factor (HGF/SF) receptor Met. It comprises the intracellular moiety of Met linked to the dimerization domain of the nuclear envelope protein Tpr, thus functioning as a constitutively activated Met. HGF/SF is responsible for various biological processes including angiogenesis and wound healing, in which secreted serine protease urokinase-type plasminogen activator (uPA) is implicated. The action of HGF/SF on cells is mediated by the autophosphorylation of Met on two carboxyterminal tyrosine residues, Y1349VHVNATVY1356VNV. The two tyrosine residues provide docking sites for various effector molecules, suggesting that multiple signaling pathways are activated to exert biological effects of HGF/SF [Ponzetto et al., Cell (1994) 77: 261]. We found that Tpr-Met efficiently activates the uPA gene via a SOS/Ras/extracellular signal regulated kinase (ERK)-dependent signaling pathway. Mutation of Y1356, which abrogates GRB2 binding, reduced the induction to half of the control level, while mutation of Y1349 showed little effect on uPA induction, suggesting an important but partly replaceable role for GRB2 in Met-dependent uPA gene induction. Mutation of both Y1349VHV and Y1356VNV into optimal PI 3-kinase sites resulted in a residual induction of about one quarter of the control level, suggesting a potential role for PI 3-kinase. Dose-response analysis of the Tpr-Met showed a biphasic curve. These results suggest that the interplay among different signaling molecules on the receptor is important for full induction of the pathway leading to the activation of the uPA gene.