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Truncal vagotomy is known to aggravate the damaging effects of alcohol-induced gastric injury and prevent the occurrence of adaptive cytoprotection against such injury by a mild irritant. This study was undertaken to determine whether aberrations in glutathione (GSH) metabolism were responsible for these vagotomy-induced effects. Fasted rats (6-8/group) were subjected to truncal vagotomy and pyloroplasty or sham vagotomy and pyloroplasty. One week later they were given 2 ml of oral saline or the mild irritant, 25% ethanol (EtOH). Thirty minutes following such treatment, animals were either sacrificed or orally received 2 ml of 100% EtOH and then were sacrificed 5 min later. At sacrifice, in each experimental group, stomachs were removed and either evaluated macroscopically for the degree of injury involving the glandular gastric epithelium or samples of the mucosa were prepared for measurement of total GSH levels or GSH peroxidase (GPX) and GSH reductase (GRT) activity. In nonvagotomized animals, saline treatment prior to 100% EtOH exposure resulted in injury to the glandular epithelium involving approximately 18%. Treatment with 25% EtOH prior to 100% EtOH exposure virtually abolished this injury. In vagotomized animals, 100% EtOH elicited almost three times the amount of injury observed in the nonvagotomized state and the protective effect of 25% EtOH pretreatment was prevented. Effects of the various treatment modalities on GPX and GRT activity were not significantly different from control values. When mucosal GSH results were plotted against the presence or absence of gastric injury among the various groups studied, no significant correlation was apparent.(ABSTRACT TRUNCATED AT 250 WORDS)

A significant fraction of orally consumed ethanol is metabolized by the alcohol dehydrogenase (ADH) enzyme present in the gastric mucosa. Human studies have shown that this “first pass metabolism” of ethanol correlates quite closely with gastric ADH activity which has been demonstrated to be greater in men than women. The present study was undertaken to determine if gender influences the magnitude of ethanol-induced injury in rat gastric mucosa and whether any differences can be linked to altered levels of ADH activity. Since prostaglandins (PGs) have been shown to markedly attenuate the severity of gastric injury induced by ethanol in the rat stomach, a further goal of this study was to determine whether the efficiency of PG's protective action was in any way influenced by gender. Accordingly, both male and female rats were pretreated subcutaneously with 16,16-dimethyl PGE2 (10 μg/kg) or saline 30 minutes prior to administering an oral dose of 50% ethanol in saline or saline alone. They were then sacrificed 5 minutes later. In a portion of animals (n = 6 per group), samples of mucosa from the glandular stomach were obtained and kinetic activity of ADH determined. In another portion of animals (n = 6 per group), gastric tissue samples from the glandular mucosa were examined by light microscopy and the magnitude of mucosal injury quantified. Alcohol-treated females showed significantly (p < 0.05) less superficial and more deep mucosal injury than male counterparts. Further, ADH kinetic activity in female rats was significantly less than that observed in male counterparts of similar weight (83% of males; p < 0.04). Despite these differences in ADH activity, and the magnitude of mucosal injury, the efficiency of protection by PG was equivalent in both sexes and involved primarily the deep epithelium. The results of this study indicate that ethanol damage to the gastric mucosa in female rats is more severe than that in corresponding males, and may be linked to the lower ADH kinetic activity present in female gastric mucosa. Despite these differences in ethanol oxidation and gastric injury, PG's protective action was equally efficatious in both sexes indicating that gender differences in ADH activity were of no importance in mediating this protection.

In an earlier study1 we observed that alcohol-induced gastric damage in the rat was exacerbated by truncal vagotomy. We further noted that the mild irritant, 30% ethanol, which significantly reduced the magnitude of injury when mucosa was subsequently exposed to 100% alcohol, was no longer capable of eliciting this protective effect under conditions of vagal denervation. These observations suggested that vagal integrity played an important role in mucosal defense and was necessary for adaptive cytoprotection to occur, at least under conditions of injury induced by alcohol. While the mechanism(s) responsible for these findings has remained elusive, the possibility that capsaicin-sensitive fibers may play a role in mediating these vagotomy effects must be considered. This contention is based on the fact that as many as 80% of fibers comprising the gastric vagus are afferent in nature, being composed of unmyelinated and thinly myelinated afferent neurons2, and capsaicin, the pungent ingredient of red peppers, is able to impede the conducting activity of such primary afferent neurons when administered in high doses3,4,5. Further, functional ablation of capsaicin-sensitive fibers has been shown to enhance gastric injury and ulcer formation in a number of experimental models including those in which gastric damage was induced by pylorus ligation6, acid distension of the stomach6, and in response to various noxious agents such as indomethacin7 and ethanol7.

N-acetyl-cysteine (NAC), when given orally, has been shown to prevent gastric damage induced by ethanol, but when administered intraperitoneally, it appears to potentiate such damage. In an effort to resolve these seemingly discordant findings, fasted rats (six per group) received 1 ml of saline or 20% NAC orally or intraperitoneally (ip). Two hours or 15 min later, they received 1 ml of 100% ethanol orally. At sacrifice 5 min later, rats receiving oral pretreatment with 20% NAC at both 15 and 120 min prior to ethanol exposure demonstrated a significant reduction in the magnitude of gastric injury when compared with saline controls. In contrast, actual promotion of ethanol damage was noted when NAC was given intraperitoneally, but was more pronounced when NAC was administered 15 min prior to exposing the mucosa to 100% ethanol. In all animals receiving intraperitoneal NAC, large amounts of peritoneal fluid (4-6 ml/rat) were recovered at the time of sacrifice, most of which occurred within 15 min of NAC administration; these more pronounced peritoneal effects at 15 min after NAC correlated with the more severe injury from ethanol at this time period compared to 120 min after intraperitoneal NAC. Saline controls had no peritoneal fluid. Mucosal glutathione (GSH) levels generally paralleled these results in that a significant decrease in tissue GSH occurred at 15 min following intraperitoneal NAC when compared with controls; at 120 min after intraperitoneal NAC, GSH levels were similar to control values. Additional experiments demonstrated that within 15 min following NAC administration, systemic blood pressure dropped by approximately 20% and basically remained unchanged over the next 2 hr; intraperitoneal saline had no sustained adverse effects on blood pressure. It was concluded that the inability of NAC to prevent ethanol injury when given intraperitoneally in contrast to orally is related to the drop in blood pressure secondary to NAC's peritoneal irritant effects, which presumably altered gastric mucosal blood flow, thus obivating its ability to prevent ethanol damage under these conditions. Furthermore, the decreased levels in mucosal GSH following the hypotension induced by intraperitoneal NAC suggest that perturbations in GSH metabolism may also have contributed to the decreased resistance to ethanol injury.

Using macroscopic criteria for injury, prostaglandins have been alleged to possess potent antiulcer properties despite meager histologic evidence for this cytoprotective action. This time-sequence study used light, scanning, and transmission electron microscopy to evaluate the effects of 16,16-dimethyl prostaglandin E 2 on gastric mucosal integrity after exposure to 100% ethanol. Macroscopically, virtually complete protection against injury to the glandular mucosa of the in vivo rat stomach was noted in animals receiving 10 μg/kg body wt of prostaglandin subcutaneously before oral ethanol administration when killed at 5, 20, and 60 min after ethanol exposure compared with oral ethanol after saline injection. On light microscopy the length of injured epithelium in prostaglandin/ethanol- and saline/ ethanol-treated tissues was not significantly different at all time periods studied. Although the depth of injury extended into gastric glands in both groups killed at 5 min, the deep pit surface mucus cells in prostaglandin/ethanol mucosa were less damaged and necrotic lesions were virtually absent when compared with saline/ethanol mucosa. At 20 and 60 min, cellular injury could still be identified in prostaglandin/ethanol-treated mucosa but the depth of injury became even less pronounced over time in contrast to mucosa exposed to ethanol without prostaglandin. Scanning electron microscopy and transmission electron microscopy confirmed these differences. Despite the macroscopic findings, these results indicate that prostaglandin does not prevent superficial surface mucus cell necrosis in ethanol-exposed mucosa even though it does spare cells in the pit base. The reduction in damaged cells over time in prostaglandin/ethanol-treated mucosa, in contrast to saline/ethanol-treated mucosa, supports the hypothesis that the reepithelialization of the lamina propria is initiated by spared deep-lying pit cells.

The effect of 16,16 dimethyl prostaglandin E2 (PG) upon tight junctions (TJs) of adjacent surface mucous cells (SMCs) as a possible mechanism by which PGs mediate their protective effects was studied using transmission electron microscopy (TEM) and freeze fracture (FF) techniques. Fasted rats were subcutaneously injected with 10 micrograms/kg of PG or an equal volume of saline, followed 30 min later by 1 ml of oral 100% ethanol or saline. Ten or sixty minutes later, animals were sacrificed and stomach blocks were prepared for TEM or FF using standard techniques. Electron micrographs (X60,000) were obtained and the distance between SMC inner membrane leaflets was measured with a micrometer and expressed as TJ width. Stomach blocks for FF were stored at 4 degrees C, cryoprotected, freeze fractured, and photographed by TEM (X30,000). At 0.5-micron intervals, measurements of TJ strand number and depth were made. No statistical differences were found in TJ width or strand number of SMCs among the various experimental groups when compared with controls at each sacrifice time. At the 60 but not 10 min sacrifice time, TJ depth was greatly increased in cells treated with 10 micrograms/kg PG prior to ethanol exposure (P less than 0.025) in contrast to control mucosae. We conclude that 16,16-dimethyl PGE2 induces no changes in the structural composition of TJs as a possible explanation for its protective properties. The increase in TJ depth observed in ethanol exposed mucosa following PG pretreatment at the 60 min sacrifice time is most likely related to the architectural restructuring that occurs during reconstitution of damaged surface epithelium.

Using [14C]aminopyrine clearance as a measure of mucosal blood flow, 16,16-dimethyl PGE2 was administered orally as a single bolus and its effects on resting and histamine-stimulated gastricmucosal blood flow assessed in conscious dogs with vagally denervated gastric pouches. No effect on resting mucosal flow was observed with any of the doses (ie, 2, 10, and 50 microgram/kg) of prostaglandin given. In histamine-stimulated experiments, intravenous infusion of histamine dihydrochloride (1 mg/hr) elicited a significant (P0.0005) increase from basal in gastric acid production, volume output, and gastric mucosal blood flow which remained unchanged for the duration of the study. Prostaglandin (50 microgram/kg) significantly decreased (P0.0005) these secretory responses and markedly inhibited (P0.005) mucosal blood flow without altering the ratio (R) of flow to the volume rate of secretion. The observation that R was unchanged suggests that this inhibitory action of 16,16-dimethyl PGE2 on gastric secretion and blood flow was mediated through direct effects on gastric parietal cells rather than any primary alteration in the gastric microcirculation. We conclude that 16,16-dimethyl PGE2 given orally has no effect on the resting gastric mucosal circulation and that any reduction in mucosal flow during histamine stimulation is secondary to its inhibitory effects on gastric acid secretion.

We examined the role of the vagus nerve in mediating the protective effects of subcutaneous 16,16-dimethyl prostaglandin E 2 (16,16-dmPGE 2 ) or of the mild irritant 30% ethanol (topically) against gastric mucosal injury induced by concentrated solutions of ethanol. Anesthetized rats underwent either truncal vagotomy or sham truncal vagotomy and were studied acutely or 7 days later. Under acute conditions, in rats with intact vagi, oral saline followed by 100% ethanol produced severe gastric hemorrhagic and necrotic lesions throughout the glandular gastric mucosa. Oral 30% ethanol or pretreatment with 16,16-dmPGE 2 (5, 10, or 25 μg/kg) before giving oral saline significantly reduced the magnitude of injury when mucosa was subsequently exposed to 100% ethanol. In animals with truncal vagotomy, the protective effect of 16,16-dmPGE 2 or 30% ethanol was not observed. Similar results were noted when animals were studied 7 days after sham or truncal vagotomy. In other studies, the ability of 16,16-dmPGE 2 to prevent gastric injury induced by 50% ethanol or 80 mM aspirin in acid solution (160 mM HCl) with and without prior vagotomy was compared. Although 16,16-dmPGE 2 (5 or 25 μg/kg) pretreatment significantly reduced the degree of gastric damage induced by both agents in the nonvagotomized state, and by aspirin under vagotomized conditions, only partial protection by 16,16-dmPGE 2 against ethanol injury was observed in the vagotomized state. These results suggest that the mechanisms whereby prostaglandins mediate their protective effects against aspirin and ethanol may be different and that the vagus nerve influences the ability of 16,16-dmPGE 2 and of the mild irritant 30% ethanol to prevent alcohol-induced gastric injury in the rat stomach.

Using an in vivo canine chambered stomach preparation, the clearance of [ 14 C]aminopyrine across mucosa when intravenously infused and the backdiffusion of this substance from gastric lumen to mucosa when topically applied to gastric epithelium were evaluated in aspirin-damaged gastric epithelium. In mucosa damaged by either 20 mM or 40 mM aspirin, the recovery of [ 14 C]aminopyrine, when topically mixed with acid (pH = 1.1) perfusate solution, was not significantly different from nondamaged control mucosa. In addition, the degree of "trapping" of this substance from back-diffusion was not different in damaged mucosa from that observed in nondamaged epithelium. In contrast, when [ 14 C]aminopyrine was intravenously infused, its clearance was significantly impaired in aspirindamaged mucosa when compared with control studies, as evidenced by the increased "trapping" of this substance in injured epithelium. These findings indicate that movement of aminopyrine from plasma to gastric lumen is impaired in damaged epithelium, making the aminopyrine clearance technique an unreliable method to accurately measure absolute gastric blood flow in this experimental setting.

Using a canine chambered stomach preparation, the effect of topical 16,16-dimethyl prostaglandin E 2 (dmPGE 2 ) in neutral solution (150 m N NaCl) on gastric mucosal bicarbonate (HCO 3 − ) secretion was assessed. Compared to control studies in which neutral solution alone bathed the epithelium, dmPGE 2 (0.5, 1.0, 2.0, and 4.0 μg/ml), when applied to gastric mucosa, significantly increased the output of gastric HCO 3 − in a stepwise and dose-related fashion. Accompanying these effects in HCO 3 − output were similar increases in sodium, potassium, chloride, and gastric perfusate volume. In other studies, the effects of intravenous atropine and close intraarterial tetrodotoxin on this PG-induced HCO 3 − secretion were evaluated. Both agents completely prevented the stimulation of HCO 3 − output induced by dmPGE 2 (2 μg/ml). It is concluded that dmPGE 2 , when topically applied to canine gastric epithelium, is a potent stimulant of bicarbonate output that is dose-dependent. The ability of atropine and tetrodotoxin to prevent this secretion suggests that a cholinergic mechanism may be involved and that dmPGE 2 mediates its effects on HCO 3 − output through acetylcholine release which in turn stimulates cholinergic nerve endings.

Fasted rats were given either 16,16-dimethyl-PGE2 (dmPGE2) (1 microgram/kg) or normal saline subcutaneously followed by the oral administration of 1 ml of 100% ethanol or saline 30 min later. At 1, 3, 6, and 24 hr later, animals were sacrificed, their stomachs examined for necrotic ulcerations, and the incorporation of [3H]thymidine into DNA as well as tissue levels of DNA, RNA, and protein content of glandular mucosa determined. Compared with control animals, severe ulcerations of 70-80% of the glandular mucosa were observed in rats given 100% ethanol at all time periods. Accompanying these ulcerations were marked depressions in tissue levels of DNA and RNA at 1, 3, 6, and 24 hr after exposure to ethanol, and protein at 1, 3, and 6 hr following ethanol. In rats pretreated with dmPGE2 before ethanol administration, these alterations in tissue levels of DNA, RNA, and protein were prevented as were ulcerations of the glandular stomach at each time period. Synthesis of mucosal DNA was not significantly different from control rats in any of the groups studied. It is concluded that (1) gastric mucosal damage by alcohol is associated with a decrease in tissue levels of DNA, RNA, and protein; (2) dmPGE2 maintains normal tissue levels of DNA, RNA, and protein by preventing the shedding of mucosal cells by alcohol; and (3) the ability of dmPGE2 to prevent gastric damage by alcohol is not mediated through stimulation of DNA synthesis.

Using an in vitro canine gastric mucosal preparation, this study evaluated the effects of 1 m M aspirin in a buffered Ringer solution (pH = 7.4), with and without concomitant prostaglandin (PG) treatment, on net sodium transport (mucosa to serosa) across gastric epithelium. Administration of aspirin to the mucosal bathing solution for 2 hr significantly decreased the potential difference (PD), short circuit current (I sc ), and net sodium transport (net J-Na + ) when compared with untreated control mucosa. In mucosa treated with 16,16-dimethyl PGE 2 (8 × 10 −6 M ) in the serosal bathing solution 40 min after aspirin exposure and for 80 min thereafter, the initial inhibitory effects on PD, I sc , and net J-Na + induced by aspirin were completely reversed within 40 min of PG treatment, having returned to control values. Histologically, mucosa exposed to aspirin alone showed evidence of diffuse cellular injury involving 50–60% of the surface epithelium. In contrast, mucosa treated with prostaglandin in conjunction with aspirin exposure demonstrated damage involving only 20–30% of the epithelium. These findings suggest that stimulation of sodium transport by PG may play a role in mediating the cytoprotective effects of PGs against aspirin-induced gastric mucosal injury.

This study was undertaken to determine the possible role that prostaglandins (PGs) may play in enhancing epithelial repair in ethanol-damaged gastric mucosa. Fasted rats were injected subcutaneously with 10 micrograms/kg of 16,16-dimethyl PGE2 or saline and 30 min later received an oral bolus of 1 ml of absolute ethanol or saline. At 5 min and 2, 8, and 24 hr after ethanol exposure tissues were prepared from identical regions of the glandular mucosa for microscopic evaluation. Normal, injured, and repairing tissues were differentiated and quantitated. The length of injured surface epithelium was the same in ethanol-treated tissues with and without PG pretreatment when evaluated 5 min after ethanol exposure, but the deeper epithelium was protected from injury in animals receiving PG pretreatment. Although the repair process itself was identical in the two experimental groups, in PG-treated tissues repair was initiated earlier, was more widespread, and was much more rapid than in tissues exposed to ethanol without such treatment. At the end of 24 hr of observation, only 5.5% of the surface epithelium was considered normal histologically in mucosa exposed to ethanol alone without PG pretreatment. This is in marked contrast to PG-treated tissues in which 82.7% of the gastric surface was normal at 24 hr. The mechanism responsible for our findings is unknown but may be related to PG's ability to spare the cellular pool in the gland isthmus from damage, enhancement of cellular migration from this pool to resurface the damaged epithelium, or a combination of both of these processes.

Using a canine chambered stomach preparation, the effects of three 30-min exposures of the gastric mucosa to 20% ethanol in 100 mN HCl on gastric mucosal barrier disruption and ulcer formation were assessed. The interval between exposures was 30 min. Following an initial exposure to 20% ethanol, the net fluxes of H+, Na+, and K+ ions and perfusate volume induced by a second and third exposure of the gastric epithelium to this damaging agent were significantly reduced. Only minimal ulceration was observed following the first exposure which did not worsen with subsequent exposure to ethanol. If indomethacin was given intravenously either before or immediately after the first ethanol exposure, recovery of barrier function was significantly lessened after this challenge, and the resistance to barrier disruption was significantly decreased during the two subsequent exposures to ethanol when compared to experiments in which mucosa was exposed to 20% ethanol without concomitant administration of indomethacin. In addition, marked mucosal ulceration was observed during the second and third ethanol exposures if indomethacin was given. These studies suggest that the first alcohol challenge may have elicited the synthesis and release of tissue prostaglandins and thereby enhanced resistance of the gastric mucosa to subsequent challenge by this damaging agent. When prostaglandin synthesis was blocked by indomethacin, the increased resistance to gastric injury did not occur.

This study compared the ability of topical 16,16-dimethyl prostaglandin E 2 in a dose range of 0.3–3.0 μg/ml to prevent aspirin-induced injury in the canine stomach under both in vivo and in vitro conditions. For in vitro studies, isolated strips of oxyntic mucosa were exposed to 10 or 20 mM aspirin (ASA) at pH 1–4, with and without treatment with 16,16-dimethyl prostaglandin E 2 . For in vivo experiments, a portion of the oxyntic stomach was mounted between the rings of a Lucite chamber, with splenic vessels intact, such that the mucosa was divided into halves. Both sides were exposed to 20 mM ASA at pH 1 or 2, and one side also received concomitant treatment with 16,16-dimethyl prostaglandin E 2 . After ASA exposure, tissue samples were prepared for quantitative microscopic analysis of the degree of injury. Under both experimental conditions, the magnitude of gastric injury by ASA was pH-related, being most pronounced at pH 1; this damage was worse under in vitro conditions, and both concentrations of ASA were equally damaging in this setting. 16,16-Dimethyl prostaglandin E 2 failed to prevent ASA injury in vitro at any pH and ASA concentration tested, but markedly reduced the magnitude of injury in vivo. The most effective protective dose of 16,16-dimethyl prostaglandin E 2 under in vivo conditions was 1.0 μg/ml. The diminished tolerance to ASA damage in vitro when compared with in vivo, and the inability of 16,16-dimethyl prostaglandin E 2 to prevent these damaging effects in vitro, underscores the probable crucial role for blood flow, and possibly neural innervation, in mediating the protective effects of prostaglandins.

Using a canine chambered stomach preparation, the effects of topical 16,16-dimethylprostaglandin E2 (dmPGE2) on gastric mucosal morphology, H+ loss and HCO3-secretion were assessed. Compared to control studies in which acid alone (100 mM HCl) bathed the epithelium, dmPGE2 (1 or 4 micrograms/ml), when applied to gastric mucosa in acid medium, significantly (P less than 0.01) enhanced H+ loss from the bathing solution in a dose-related fashion. These effects, however, were independent of any injury to the epithelium as confirmed by both light and electron microscopy. In other studies in which dmPGE2 was applied to gastric mucosa in neutral solution (150 mM NaCl, pH 7.0) using identical doses, a dose-related increase (P less than 0.01) in HCO3-secretion was observed. When the net HCO3-output (microEq/15 min) for each dose of dmPGE2 employed was compared with the net H+ loss (microEq/15 min) for each dose of PG used, no differences were noted. The correlation between H+ loss and HCO3-secretion was highly significant (r = 0.87; P less than 0.01). We conclude that with the doses studied, dmPGE2 is not damaging to gastric mucosa and that the apparent increase in H+ loss elicited by dmPGE2 in acid medium is due to acid neutralization secondary to HCO3-secretion.