Acute exposure of small intestine to ethanol

Digestive Diseases and Sciences -     

Acute and chronic exposure to ethanol and the electrophysiology of the brush border membrane of rat small intestine.

In this study we have investigated the effects of (a) chronic ethanol intake on glucose and galactose absorption across the rat jejunum in vivo and on the potential difference across the isolated brush border membrane (Vm) and (b) acute exposure to ethanol (4% or 8%) and acetaldehyde (0.25%) on changes in Vm associated with Na(+)-dependent galactose absorption across the jejunum and ileum. Chronic ethanol intake was associated with hyperpolarization of Vm and an enhanced galactose but not glucose transport. Acute ethanol and acetaldehyde were without effect on Vm whether or not galactose was present. We conclude that while a greater electrochemical gradient across the brush border membrane is a likely explanation for the stimulation of galactose absorption induced by ethanol feeding, factors other than changes in Vm are responsible for the inhibitory effects of acute ethanol.     

Induced mucosal penetration and transfer to protal blood of luminal horseradish peroxidase after exposure of mucosa of guinea pig small intestine to ethanol and lysolecithin

The effect of luminal 150 mmol saline, 0.05–0.2% (w/v) lysolecithin, and 5–20% (v/v) ethanol was studied on the mucosal morphology of the proximal small intestine in conscious guinea pigs as well as on the mucosal penetration and transfer to portal venous blood of luminal horseradish peroxidase (HRP). No ultrastructural evidence of mucosal damage was identified in any of the lysolecithin-perfused animals compared with saline controls. Ten and 20% ethanol (v/v) resulted in the appearance of fluid-filled spaces between enterocytes and in cytoplasmic lipid deposits and an increased number of autophagic vesicles within the cells themselves. Tight junctions remained intact. These changes after luminal 5% ethanol (v/v) were much less conspicuous. In the presence of saline, luminal HRP was largely confined to the brush border. Both lysolecithin and ethanol (5% v/v) rapidly induced mucosal penetration of HRP which was seen in cytoplasmic vesicles within enterocytes, between enterocytes, and in the lamina propria. Peak portal venous blood levels of HRP studied in multiple samples over 3 hr were one log unit greater than saline controls. Absorption of HRP was proportional to the luminal concentration of lysolecithin in the range tested. These studies show that mucosal penetration and absorption of functional exogenous macromolecules may be induced, in the absence of morphological evidence of mucosal damage, by luminal constituents which may perturb the structure of enterocyte membranes.     

Acute ethanol dosage reduces the synthesis of smooth muscle contractile proteins in the small intestine of the rat.

The effects of an acute dose of ethanol (75 mmol/kg body weight; ip) on protein synthesis were investigated in the small intestine of the rat (n = 6). Control rats (n = 6) were injected with isovolumetric 0.15 mol/l NaCl, ip. After 2.5 h, fractional rates of protein synthesis (defined as the percentage of tissue protein renewed each day by synthesis and RNA efficiencies (defined as the amount of protein synthesised per unit RNA) were measured with a large flooding dose (0.3 Ci/mol; 150 mmol/l; 150 mumol/100 g body weight; iv) of [4(3)H]-phenylalanine. Rats were killed 10 minutes after injection of the isotope and portions of the small intestine were rapidly dissected and frozen. Tissues and plasma were processed for phenylalanine specific radioactivities to obtain fractional rates of protein synthesis or protein synthesis rates relative to RNA. Rates of protein synthesis in mixed tissue proteins fell approximately 15-25% (p ranged from less than 0.005 to greater than 0.05), in response to acute ethanol dosage. The decrease in the synthesis rates of the cytoplasmic protein fraction was similar (p less than 0.025). Proteins extracted from the smooth muscle contractile apparatus, however, showed a greater response to ethanol--that is, 40-50% inhibition in protein synthesis (p less than 0.001). It is therefore possible that the functional disturbances in the ethanol-exposed gut may be because of changes in smooth muscle protein turnover with decreased amounts of contractile apparatus.     

Reversal of ethanol and indomethacin-induced suppression of hepatic DNA synthesis by 16,16-dimethyl prostaglandin E2

Investigations were undertaken to determine effectiveness of 16,16-dimethyl prostaglandin E2 (dmPGE2) in overcoming the suppressive effects of ethanol and/or indomethacin on hepatic DNA synthesis. Adult litter mate Sprague-Dawley rats were subjected to sham operation or partial hepatectomy. Immediately after partial hepatectomy, and at 8-hr intervals for 24 hr, the rats were given: (a) ethanol with and without dmPGE2 or (b) indomethacin with and without ethanol and/or dmPGE2. DmPGE2 produced a significant increase in DNA synthesis in sham-operated (p less than 0.001) and untreated partially hepatectomized animals (p less than 0.025). Ethanol and indomethacin caused a 6- and 18-fold reduction, respectively, in hepatic DNA synthesis following partial hepatectomy. DmPGE2 overcame the inhibitory effect of ethanol (p less than 0.005) and indomethacin (p less than 0.0005) in partially hepatectomized animals. Mitoses were decreased concomitantly with ethanol and/or indomethacin-induced reduction in DNA synthesis and increased with administration of dmPGE2. It is concluded that dmPGE2 increases hepatic DNA synthesis and regeneration in normal rat liver and overcomes their inhibition when ethanol and/or indomethacin is given after partial hepatectomy. Timing of dmPGE2 administration is crucial. When given 30 min before ethanol, it completely inhibits suppression of regenerative activity; omission of this "priming" dmPGE2 dose results in only 44% of DNA synthesis obtained in control animals.     

Acute exposure of rabbit jejunum to ethanol

The effect of acute exposure of rabbit jejunum to ethanol on the uptake of three hexoses was examinedin vitro. With ethanol present in the preincubation medium for 30 min, or directly in the incubation medium for 6 min, glucose uptake was reduced. Kinetic analysis demonstrated that ethanol in the preincubation medium was associated with a rise in the value of the apparent Michaelis constant (K _m ^*), whereas the inhibition of glucose uptake observed with ethanol present directly in the incubation medium was associated with a reduction in the apparent passive permeability coefficient (P _d ^*), a reduction in the maximal transport rate (J _d ^m ), and an increase inK _m ^*. When increasing concentrations of ethanol were added to the preincubation or to the incubation medium, there was a reduction in the uptake of both 1 mM and 40 mM glucose, galactose, and 3-O-methyl glucose. The addition of 40 mM galactose or 1 mM phloridzin to 40 mM glucose was associated with a 50% reduction in glucose uptake, but this uptake was not further inhibited by the addition of 6% ethanol (v/v). Similarly, the uptake of 3-O-methyl glucose was inhibited by the addition of 40 mM glucose or galactose but no further reduction in uptake was achieved by adding ethanol. Finally, galactose uptake was inhibited by adding 40 mM glucose or 40 mM 3-O-MG, but the addition of 6% ethanol was associated with no further decline in the uptake of galactose. Previous studies have shown that ethanol has no effect on the effective resistance of the intestinal unstirred water layer when the bulk phase is stirred; thus the ethanol-related changes inK _m ^* andP _d ^* are associated with qualitatively similar changes in the values of the true Michaelis constant,K _m , and the true permeability coefficient,P _d . In summary, ethanol has a complex effect on the intestinal uptake of hexoses, influencing theK _m ,J _d ^m andP _d , but the type and extent of inhibition depends upon whether the intestine has previously been exposed to ethanol, or whether the ethanol is presented to the intestine concurrently with the hexose.     

Human cholecystitis is associated with increased gallbladder prostaglandin I2 and prostaglandin E2 synthesis.

Microsomal prostanoid synthesis was compared in normal gallbladders removed during organ donation and inflamed gallbladders removed at cholecystectomy. Normal human gallbladder microsomes demonstrated low rates of conversion of [14C]arachidonic acid to total labeled prostanoids, which increased during 1 to 30 min of incubation. Normal human gallbladder microsomes converted labeled substrate to all primary prostaglandins without demonstration of a major product. Inflamed human gallbladder microsomes increased the rate of conversion of [14C]arachidonic acid to total labeled prostanoids two or three times over the levels demonstrated by normal gallbladder microsomes at all times of incubation (p < 0.01). The main prostanoids synthesized by the inflamed human gallbladder microsomes were prostaglandin E2 and 6-keto-prostaglandin F1 alpha, which were increased four times over the levels demonstrated by normal gallbladder microsomes (p < 0.01). These data showed that inflammation of the human gallbladder was associated with increased synthesis of gallbladder 6-keto-prostaglandin F1 alpha and prostaglandin E2.     

Effect of 16,16-dimethyl prostaglandin E2 on the gastric mucosal barrier.

16,16-dimethyl prostaglandin E2 (16DM) can protect the gastric mucosa from injury and yet apparently damages the gastric mucosal barrier. The effect on the gastric mucosal barrier of 16DM (26.2 micromol/l), a dose 50 times the ED50 for inhibition of acid secretion, was investigated in Heidenhain pouches in four dogs by measuring plasma shedding from the pouches after the topical application of histamine (2.7 mmol/l) and ionic fluxes. The results were compared with those using 30% ethanol, a known barrier breaker. The topical application of histamine after three hours' perfusion with 30% ethanol led to plasma shedding at a rate of 7.5 (+/-2.6) ml/h, which was significantly greater than the rate of 1.2 (+/-1.4) ml/h after three hours' perfusion with 16DM and of 1.5 (+/-1.7) ml/h in the control group. Ethanol also caused an increase in the flux of H+, Na+, and Cl-, indicating an increase in mucosal permeability, whereas 16DM increased the flux of Na+ and Cl- but not of H+. It is concluded that 16DM does not damage the gastric mucosal barrier but stimulates the secretion of fluid containing Na+ and Cl-.     

Patients with adenomatous polyps and carcinomas have increased colonic mucosal prostaglandin E2.

Colorectal carcinoma in humans and animal models is associated with increased synthesis of prostaglandin E2 (PGE2). PGE2 synthesis was measured in normal and neoplastic human colorectal mucosa to investigate its role in the adenoma-carcinoma sequence. Paired mucosal biopsy specimens for PGE2 synthesis and histological examination were obtained during 39 diagnostic colonoscopies. Twelve control patients in whom colonoscopies and histology were normal synthesised similar amounts of PGE2 at all sites. Their results were (mean (SD) pg PGE2/mg tissue) caecum 102.8 (15.9) (n = 6), ascending colon 110.8 (24.3) (n = 10), transverse colon 103.9 (19.5) (n = 11), descending colon 102.9 (23.2) (n = 12), sigmoid colon 96.4 (18.0) (n = 12), and rectum 107.1 (17.6) (n = 12). Nineteen patients had a total of 27 adenomatous polyps (rectum (1), sigmoid (22), descending (1), transverse (1), and ascending colon (1): histology-tubular (16), tubulo-villous (8), and villous adenomous (3)). The polyps (178.0 (55.0), n = 27) synthesised more PGE2 than controls (p < 0.001), but the values in polyp-associated mucosa (mean (SD) 115.4 (21.9), n = 15) were not different to control results. Eight patients had carcinomas (rectal (2), sigmoid (4), and caecal (2)) all of which were adenocarcinomas. The cancers (193.6 (40.2), n = 8) synthesised more PGE2 than control specimens (p < 0.001), but were not different to polyps. Cancer-associated mucosa (140.3 (27.7) n = 8) synthesised more PGE2 than control and polyp-associated mucosa. Colorectal neoplasia is associated with a progressive increase in PGE2 synthesis which may have a role in tumourigenesis and be a pathophysiological explanation for the beneficial effects of NSAIDs in animal models and human disease.     

Gastric mucosal prostaglandin E2 levels in cirrhosis and portal hypertension

Prostaglandin E2 (PGE2) tissue levels were measured, using radioimmunoassay, in specimens from the gastric antral mucosa in (a) healthy subjects, (b) patients without liver diseases in the absence or presence of gastric ulcers, and (c) patients with alcoholic cirrhosis in the absence or presence of gastric ulcers. These groups were divided further into subgroups with or without portal hypertension. PGE2 tissue levels in subjects without liver diseases were not significantly different from those of cirrhotic patients without portal hypertension. However, in cirrhotic patients with portal hypertension, the PGE2 content was significantly lower, especially in the presence of congestion in the mucosa (about 70-80%). When a gastric antral ulcer was present, a further statistically significant decrease of the PGE2 levels was observed. It was concluded that a decrease in the PGE2 tissue levels in the gastric mucosa of cirrhotic patients is related to the presence of portal hypertension. Congestion in the mucosa, as a pathogenetically relevant factor for mucosal PGE2 deficiency, may play a role.     

Effect of chronic ethanol consumption on mucosal morphology and mitotic index in the rat small intestine

Rats were maintained 16 weeks on a well-balanced semisynthetic solid diet supplemented with ethanol which comprised 35% of total calories. A control group was pair-fed the same basic diet with sucrose replacing ethanol isocalorically. Striking changes in mucosal morphology and mitotic index were observed in the jejunum and ileum of ethanol-fed rats in comparison to pair-fed controls. Furthermore, it is significant that these changes were more pronounced in the ileum than in the jejunum. Since ethanol is almost completely absorbed in the stomach and upper intestine, under the conditions of this study, we propose that, apart from a possible topical toxic effect of ethanol, there appear to be other separate possible causes of the extensive small intestinal changes found in ethanol-fed rats. The first is that the changes are due to injurious effects of blood-borne ethanol; secondly, the changes could be a functional adaptation due to altered luminal nutrition as a consequence of the introduction of ethanol in the diet.     

Colonic and small intestinal response to intravenous prostaglandin F2 alpha and E2 in man.

The effects of intravenous infusions of prostaglandins (PGs) F2 alpha(0-4 or 0-8 mug kg-1 min-1) or E2 (0-08 or 0-1 mug kg-1 min equals1) on net colonic movement of water and electrolytes and on ileal flow were measured in eight healthy males by simultaneous ileal and colonic perfusion. Ileal flow was increased by PGF2 alpha (six subjects) from a mean of 1-69 ml min-1 to 4-63 ml min-1 (P smaller than 0-01); it also increased in the two subjects given PGE2. Colonic absorptive function was not significantly diminished by either prostaglandin. These results suggest that diarrhoea due to prostaglandins originates in the small intestine.     

Proliferation of rat and human lung fibroblasts following exposure to prostaglandin E2

Numerous investigators have reported that exogenous prostaglandin E2 (PGE2) can inhibit human lung fibroblast proliferation in vitro. Yet various lines of evidence derived from clinical and experimental studies suggest that PGE2 may not be of major importance in inhibiting fibroblast proliferation in vivo. We examined the effects of exogenously-supplied PGE2 on the in vitro proliferation of HFL-1 human lung fibroblasts and rat lung fibroblasts derived from Fischer 344 rats using a multisample assay system that provided a detailed kinetic picture of PGE2 effects on fibroblast proliferation. Exogenously supplied PGE2 (5-5000 ng/ml) had no effect on the proliferation of actively cycling or initially quiescent subconfluent populations of rat lung fibroblasts. In contrast, initially quiescent subconfluent or confluent cultures of HFL-1 cells that were treated with 50-5000 ng/ml PGE2 exhibited a dose-dependent, transitory inhibition of division when stimulated to return to a state of active proliferation. Once division resumed, the cells divided at the rate of the PGE2-free control condition, even in the continued presence of the prostaglandin. This initial postinhibitory resumption of division was not attributable to the emergence of a PGE2-resistant subpopulation. Thus, although exogenously supplied PGE2 indeed inhibits proliferation of human pulmonary fibroblasts in vitro, the duration of the inhibition appears to be much shorter than previously suspected.     

Leishmania donovani-induced macrophages cyclooxygenase-2 and prostaglandin E2 synthesis

Prostaglandin E2 (PGE2) secretion during Leishmania infection has been reported. However, the signalling mechanisms mediating this response are not well understood. Since cyclooxygenase-2 (COX-2) and cytosolic phospholipase A2 (cPLA2) are involved in PGE2 synthesis in response to various stimuli, the implication of these enzymes was evaluated in Leishmania-infected phorbol myristate acetate-differentiated U937 human monocytic cell line. Time-course experiments showed that PGE2 synthesis increased significantly in parallel with COX-2 expression when cells were incubated in the presence of Leishmania donovani promastigotes or lipopolysaccharides (LPS). Increase in cPLA2 mRNA expression was only detected when cells were stimulated with LPS. Indomethacin, genistein, and H7, which are antagonists of COX-2, protein tyrosine kinase (PTK) and protein kinase C (PKC), respectively, inhibited PGE2 production induced by L. donovani and LPS. However, only H7 inhibited COX-2 mRNA synthesis, and there was a significant correlation between PGE2 inhibition and reduced COX-2 expression. Collectively, our results indicate that infection of U937 by L. donovani leads to the generation of PGE2 in part through a PKC-dependent signalling pathway involving COX-2 expression. They further reveal that PTK-dependent events are necessary for Leishmania-induced PGE2 generation, but not for COX-2 expression. A better understanding of the mechanisms by which Leishmania can induce PGE2 production could provide insight into the pathophysiology of leishmaniasis and may help to improve therapeutic approaches.     

Effect of acute and chronic alcohol feeding on prostaglandin E2 biosynthesis in the small intestine of the rat

The effect of acute and chronic alcohol ingestion on the PGE2 synthesis and PGE2 content in the duodenum and ileum was studied in rats. Following a single oral load of 20% alcohol (v/v; 4 g/kg body weight) the synthesis of PGE2 in isolated microsomes from both parts of the small intestine did not differ from that in control rats during the first 8 hours, but was increased significantly after 24 hours. Feeding a liquid diet containing alcohol (37% of total calories) for 1 week led to enhanced PGE2 synthesis in the duodenum. After feeding the diet for 6 and 12 weeks the rate of PGE2 synthesis was significantly reduced in the duodenum (-73% and -55%) and ileum (-34% and -45%) as compared with the control group. The PGE2 content of the tissue was not significantly changed 24 hours after the single alcohol load and after 1 week of feeding the alcohol-containing diet. However, after 6 and 12 weeks' feeding, the PGE2 content was reduced in both parts of the small intestine. The results suggest a biphasic response to alcohol of PGE2 synthesis in the small intestine. The increased rate of PGE2 synthesis in the initial phase might reflect an adaptive response to the injurious effect of alcohol which cannot be maintained after long-term ingestion of alcohol.     

An EP2 receptor-selective prostaglandin E2 agonist induces bone healing

The morbidity and mortality associated with impaired/delayed fracture healing remain high. Our objective was to identify a small nonpeptidyl molecule with the ability to promote fracture healing and prevent malunions. Prostaglandin E2 (PGE2) causes significant increases in bone mass and bone strength when administered systemically or locally to the skeleton. However, due to side effects, PGE2 is an unacceptable therapeutic option for fracture healing. PGE2 mediates its tissue-specific pharmacological activity via four different G protein-coupled receptor subtypes, EP1, -2, -3, and -4. The anabolic action of PGE2 in bone has been linked to an elevated level of cAMP, thereby implicating the EP2 and/or EP4 receptor subtypes in bone formation. We identified an EP2 selective agonist, CP-533,536, which has the ability to heal canine long bone segmental and fracture model defects without the objectionable side effects of PGE2, suggesting that the EP2 receptor subtype is a major contributor to PGE2's local bone anabolic activity. The potent bone anabolic activity of CP-533,536 offers a therapeutic alternative for the treatment of fractures and bone defects in patients.     

Moraxella catarrhalis induces ERK- and NF-kappaB-dependent COX-2 and prostaglandin E2 in lung epithelium.

Moraxella catarrhalis is a major cause of infectious exacerbations of chronic obstructive lung disease. Cyclooxygenase (COX)-derived prostaglandins, such as prostaglandin E(2) (PGE(2)), are considered to be important regulators of lung function. The present authors tested the hypothesis that M. catarrhalis induces COX-2-dependent PGE(2) production in pulmonary epithelial cells. In the present study, the authors demonstrate that M. catarrhalis specifically induces COX-2 expression and subsequent PGE(2) release in pulmonary epithelial cells. Furthermore, the prostanoid receptor subtypes EP2 and EP4 were also upregulated in these cells. The M. catarrhalis-specific ubiquitous cell surface protein A1 was important for the induction of COX-2 and PGE(2). Moreover, M. catarrhalis-induced COX-2 and PGE(2) expression was dependent on extracellular signal-regulated kinase 1/2-driven activation of nuclear factor-kappaB, but not on the activation of p38 mitogen-activated protein kinase. In conclusion, the present data suggest that ubiquitous cell surface protein A1 of Moraxella catarrhalis, extracellular signal-regulated kinase 1/2 and nuclear factor-kappaB control cyclooxygenase-2 expression and subsequent prostaglandin E(2) release by lung epithelial cells. Moraxella catarrhalis-induced prostaglandin E(2) expression might counteract lung inflammation promoting colonisation of the respiratory tract in chronic obstructive pulmonary disease patients.     

Microparticles stimulate the synthesis of prostaglandin E2 via induction of cyclooxygenase 2 and microsomal prostaglandin E synthase 1

Objective

Microparticles are small vesicles that are released from activated or dying cells and that occur abundantly in the synovial fluid of patients with rheumatoid arthritis (RA). The goal of these studies was to elucidate the mechanisms by which microparticles activate synovial fibroblasts to express a proinflammatory phenotype.

Methods

Microparticles from monocytes and T cells were isolated by differential centrifugation. Synovial fibroblasts were cocultured with increasing numbers of microparticles. Gene expression was analyzed by real‐time polymerase chain reaction and confirmed by Western blotting and enzyme immunoassay. Arachidonic acid labeled with tritium was used to study the transport of biologically active lipids by microparticles. The roles of NF‐κB and activator protein 1 (AP‐1) signaling were analyzed with electrophoretic mobility shift assay and transfection with small interfering RNA and IκB expression vectors.

Results

Microparticles strongly induced the synthesis of cyclooxygenase 2 (COX‐2), microsomal prostaglandin E synthase 1 (mPGES‐1), and prostaglandin E₂ (PGE₂). In contrast, no up‐regulation of COX‐1, mPGES‐2, cytosolic PGES, or phospholipase A₂ was observed. The induction of PGE₂ was blocked by selective inhibition of COX‐2. Microparticles activated NF‐κB, AP‐1, p38, and JNK signaling in synovial fibroblasts. Inhibition of NF‐κB, AP‐1, and JNK signaling reduced the stimulatory effects. Arachidonic acid was transported from leukocytes to fibroblasts by microparticles. Arachidonic acid derived from microparticles was converted to PGE₂ by synovial fibroblasts.

Conclusion

These results demonstrate that microparticles up‐regulate the production of PGE₂ in synovial fibroblasts by inducing COX‐2 and mPGES‐1. These data provide evidence for a novel mechanism by which microparticles may contribute to inflammation and pain in RA.

     

Modulation of prostaglandin E2 synthesis in rabbit synoviocytes

Prostaglandin E2 (PGE2) production by rabbit synoviocytes was markedly stimulated by hydroxyapatite only after a 60-minute delay. Release of 3H-arachidonic acid (C20:4) and 3H-PGE2 from cells with phospholipids that were prelabeled with 3H-C20:4 did not occur in parallel. At 240 minutes, phospholipase activity in sonicated cell suspensions had increased only 38%, while cyclooxygenase activity had doubled (109%). This doubling, as well as the production of synoviocyte PGE2, was prevented by inhibiting the synthesis of protein. Cyclooxygenase is an inducible enzyme, and as such, it is a rate-controlling step in PGE2 production.