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Ghrelin, an endogenous ligand for the growth hormone secretagogue receptor, is an important regulator of nitric oxide synthase (NOS) and cyclooxygenase (COX) enzyme systems, the products of which are of major significance to the processes of gastric mucosal defense and repair. Here, using primary culture of rat gastric mucosal cells, we report on the mechanism of ghrelin protection against ethanol cytotoxicity. We show that the protective effect of ghrelin was associated with the increase in NO and PGE2 production, and characterized by a marked up-regulation in cytosolic phospholipase A(2) (cPLA(2)) activity and arachidonic acid (AA) release. The loss in countering effect of ghrelin on the ethanol cytotoxicity was attained with constitutive NOS (cNOS) inhibitor, L-NAME, as well as indomethacin and a specific COX-1 inhibitor, SC-560, while specific COX-2 inhibitor, NS-398, and a selective inducible NOS (iNOS) inhibitor, 1400W, had no effect. The effect of L-NAME was reflected in the inhibition of ghrelin-induced mucosal cell capacity for NO production, cPLA(2) activation, and PGE2 generation, whereas indomethacin caused only the inhibition in PGE2 generation. Moreover, the ghrelin-induced up-regulation in AA release was reflected in the cPLA(2) enzyme protein phosphorylation and S-nitrosylation. Preincubation with L-NAME resulted in the inhibition of the ghrelin-induced S-nitrosylation, whereas the ERK inhibitor, PD98059, caused the blockage in cPLA(2) protein phosphorylation as well as S-nitrosylation. The findings demonstrate that ghrelin protection of gastric mucosa against ethanol cytotoxicity involves cNOS-derived NO induction of cPLA(2) activation for the increase in PGE2 synthesis. This activation process apparently includes the cPLA(2) phosphorylation followed by S-nitrosylation.

A pleiotropic hormone, leptin, secreted into saliva by the acinar cells of salivary glands is an important mediator of the processes of oral mucosal defense. Here, we report on the role of epidermal growth factor receptor (EGFR) transactivation in the signaling events that mediate leptin protection of sublingual salivary gland acinar cells against ethanol cytotoxicity. We show that the protective effect of leptin against ethanol cytotoxicity was associated with the increased EGFR protein tyrosine kinase and cytosolic phospholipase A(2) (cPLA(2)) activity, and characterized by a marked increase in matrix metalloproteinase MMP-9 and arachidonic acid (AA) release, and PGE(2) generation. The loss in countering capacity of leptin against ethanol cytotoxicity was attained with JAK inhibitor AG490, Src inhibitor PP2, and EGFR inhibitor AG1478, as well as ERK inhibitor PD98059. Moreover, the agents evoked also the inhibition in leptin-induced up-regulation in cPLA(2) activity, AA release, and PGE(2) generation. The changes caused by leptin in EGFR phosphorylation, MMP-9, and cPLA(2) activation were susceptible to suppression by metalloprotease inhibitor GM6001, but the production of MMP-9 was not affected by EGFR inhibitor AG1478 or PKC inhibitor Ro318220. These findings point to the involvement of MMP-9 in the event of leptin-induced EGFR transactivation that results in the signaling cascade leading to cPLA(2) activation and up-regulation in PGE(2) generation, thus providing new insights into the mechanism of oral mucosal protection against ethanol toxicity.

Advances in understanding the functional aspects of leptin in the processes affecting peripheral tissues have brought to the forefront the role of this pluripotent cytokine in the processes of gastric mucosal defense and repair. Here, we report that leptin protects the gastric mucosal cells against ethanol cytotoxicity. We show that ethanol cytotoxicity, characterized by a marked drop in the mucosal cells capacity for NO production, arachidonic acid release and prostaglandin generation, was subject to suppression by leptin. The loss in countering capacity of leptin on the ethanol-induced cytotoxicity was attained with cyclooxygenase inhibitor, indomethacin and nitric oxide synthase (cNOS) inhibitor, L-NAME, as well as PP2, an inhibitor of Src kinase. Indomethacin caused the inhibition in PGE(2) generation, pretreatment with L-NAME led to the inhibition in NO production, whereas PP2 exerted the inhibitory effect on leptin-induced changes in NO, arachidonic acid and PGE(2). The leptin-induced changes in arachidonic acid release and PGE(2) generation were blocked by ERK inhibitor, PD98059, but not by PI3K inhibitor, wortmannin. Moreover, the stimulatory effect of leptin on the mucosal cells cNOS activity was inhibited not only by PP2, but also by Akt inhibitor, SH-5. Our findings demonstrate that leptin protection of gastric mucosa against ethanol cytotoxicity involves Src kinase-mediated bifurcated activation of MAPK/ERK and Akt that leads to up-regulation of the respective prostaglandin and nitric oxide synthase pathways.

Leptin, a pleiotropic cytokine secreted by adipocytes but also identified in salivary glands and saliva, is recognized as an important element of oral mucosal defense. Here, we report that in sublingual salivary glands leptin protects the acinar cells of against ethanol cytotoxicity. We show that ethanol- induced cytotoxicity, characterized by a marked drop in the acinar cell capacity for NO production, arachidonic acid release and prostaglandin generation, was subject to suppression by leptin. The loss in countering capacity of leptin on the ethanol-induced cytotoxicity was attained with cyclooxygenase inhibitor, indomethacin and nitric oxide synthase (cNOS) inhibitor, L-NAME, as well as PP2, an inhibitor of Src kinase. Indomethacin, while not affecting leptin-induced arachidonic acid release, caused the inhibition in PGE2 generation, pretreatment with L-NAME led to the inhibition in NO production, whereas PP2 exerted the inhibitory effect on leptin-induced changes in NO, arachidonic acid, and PGE2. The leptin-induced changes in arachidonic acid release and PGE2 generation were blocked by ERK inhibitor, PD98059, but not by PI3K inhibitor, wortmannin. Further, leptin suppression of ethanol cytotoxicity was reflected in the increased Akt and cNOS phosphorylation that was sensitive to PP2. Moreover, the stimulatory effect of leptin on the acinar cell cNOS activity was inhibited not only by PP2, but also by Akt inhibitor, SH-5, while wortmannin had no effect. Our findings demonstrate that leptin protection of salivary gland acinar cells against ethanol cytotoxicity involves Src kinase-mediated parallel activation of MAPK/ERK and Akt that result in up-regulation of the respective prostaglandin and nitric oxide synthase pathways.

Earlier investigations on the effect of ethanol on synthesis and posttranlational glycosylation of gastric mucus glycoprotein (mucin) revealed quantitative changes in the apoprotein assembly, glycosylation, and mucin retention on the mucosal surface (Slomiany et al.., Alcoholism: Clin. Exp. Res. 21, 417-423, 1998). To assess whether metabolic consequences of ethanol ingestion, documented in the in vitro system are also occurring in vivo the rats were subjected to 8 weeks of ethanol containing liquid diet. The retention of mucin on the surface of gastric mucosa was quantitated by measuring the binding of gastric mucin to Mucin Binding Protein (MBP) of gastric mucosa. The results were compared with those obtained with the rats subjected to pair-feeding the isocaloric-control diet. Before alcohol administration, and in two weeks' intervals thereafter, the gastric contents from the animals was collected and mucin purified. After 8 weeks of the respective diet, the animals were sacrificed and their gastric mucosa used for MBP preparation. The binding of mucin to MBP before ethanol, and after 2, 4, 6, and 8 weeks of ethanol diet was quantitated with Enzyme Linked Lectin Assay (ELLA). The study with standard mucin revealed that binding of mucin to MBP differs substantially between individual animals. The same variability in binding was observed with the individual mucin preparations collected at the onset of the experiment. However, with the progression of ethanol feeding, the mucin samples besides displaying the variable and animal-specific binding to MBP at the initiation of the experiment, also showed a dramatic decrease in binding. In five animals, after two weeks of ethanol diet, mucin binding to MBP decreased by 50%; in two animals, the drastic decrease in binding was observed in mucin collected after four weeks of alcohol feeding; and in one animal a 20% decrease in binding persisted for six weeks, and then decreased to 50% in the last collection. Also, in two animals, the mucin collected after 8 weeks of ethanol feeding retained only 6-9% of the initial binding capacity. In contrast, in pair-fed controls, the mucin binding to MBP remained the same or increased up to 20%. Results of the studies, performed on mucin of the individual animals and matching preparations of MBP, showed that each animal expresses different degree of mucin binding. Moreover, in chronic ethanol ingestion, the individual variations are accompanied by a decrease in mucin binding to MBP. Since the observed decrease in binding occurred in samples containing the same preparation of MBP, the component affected by alcohol resides on mucin. Thus, considering the in vitro impact of ethanol on generation of carbohydrate chains in Golgi, and the finding on mucin oligosaccharides-dependent mucin-MBP complex formation, we conclude that ethanol impairs the synthesis of mucin oligosaccharide structures required for binding with MBP, and the retention on gastric mucosal surfaces.

Among the factors affecting the efficiency of soft oral tissue healing is endothelin-1 (ET-1), a potent vasoactive peptide produced from a biologically inactive big ET-1 by the action of endothelin-converting enzyme-1 (ECE-1). We investigated the expression of ECE-1 during buccal mucosal ulcer healing in rats maintained for 5 weeks on alcohol containing or control diet. The mucosal activity of ECE-1, characterized by sensitivity to phosphoramidon, was associated with microsomal fraction and showed an elevated (3.1-fold) level in the alcohol diet group. Moreover, the ulcer onset in the alcohol group was reflected in a 39% greater expression of ECE-1 activity, and was accompanied by a 1.4-fold greater increase in TNF-alpha and a 2.5-fold greater enhancement in epithelial cell apoptosis. While in both groups the ulcer healing was associated with a decrease in buccal mucosal expression of ECE-1, as well as a decline in TNF-alpha and apoptosis, the changes were significantly slower in the alcohol diet group and manifested by a 40% delay in healing. Thus, chronic alcohol ingestion leads to up-regulation of ECE-1 expression, induction of TNF-alpha, and triggering apoptotic events that delay the mucosal repair., (Copyright 2000 Academic Press.)

We investigated the effect of chronic alcohol ingestion on buccal mucosal ulcer healing by analyzing the interplay between mucosal expression of tumor necrosis factor-alpha (TNF-alpha), endothelin-1 (ET-1), and interleukin-4 (IL-4). Chronic ulceration was induced in rats maintained for 5 weeks on alcohol-containing or control liquid diet. In both groups, the ulcer onset was characterized by a massive increase (6.5-8.9-fold) in TNF-alpha and ET-1 (1.6-4.0-fold), and a decrease (1.4-1.5-fold) in IL-4. However, the group on the alcohol diet exhibited a 38.3% higher mucosal expression of TNF-alpha, a 26. 2% higher ET-1 level, and a 6.5% lower content of IL-4. While in both groups the ulcer healing was accompanied by an increase in buccal mucosal expression of IL-4, and a decline in ET-1 and TNF-alpha, the changes were significantly slower in the alcohol diet group and manifested by a 4 day delay in ulcer healing. The results suggest that chronic alcohol ingestion exerts detrimental effects on the buccal mucosal IL-4 expression, causing dysregulation of ET-1 production, induction of TNF-alpha, and triggering apoptotic events that delay the mucosal repair., (Copyright 1999 Academic Press.)

The outcome of chronic ethanol consumption recorded in liver by in situ staining of the genomic DNA in fragmented nuclei indicates the course of cellular events that has been coined as apoptosis or programmed cell death. Hence, we designed the study to determine which ethanol-induced modification of the cellular make-up is responsible for the hastening the cell damage. The in vitro assays were performed with cellular organelles and cytosol prepared from hepatocytes derived from rats subjected to 9 weeks of chronic alcohol consumption. The results were compared with the pair-fed controls receiving isocaloric liquid diet. In the initial phase of the studies, we established that the process of apoptosis was not triggered by the aberrant activity of neutral or acidic sphingomyelinase. The hepatocytes derived from alcohol and control diets manifested equal enzymatic activity. The de novo synthesis of sphingoid bases and ceramides in the alcohol-derived sample of endoplasmic reticulum was reduced, but the in situ apoptosis was up to 36-fold higher than in the control. Also, the isolated hepatocytes contained a 2- to 4-fold higher amount of nucleosomal fragments in the cytosolic extracts. The endosomes from liver hepatocytes of ethanol-consuming rats, in the presence of the cytosol and mitochondria from pair-fed controls, disclosed 2 to 3 times higher apoptotic potential than sample consisting of ethanol-derived fractions only, and 3 to 5 times higher than the control-derived fractions. The substantial increase in apoptosis, as recorded in the amount of DNA fragments released to the cytosol from the fresh nuclei, was also recorded when the microsomal membranes of endoplasmic reticulum and Golgi were incubated in the conditions with preserved intracellular transport. The maximal 20-fold increase of apoptotic activity was recorded in the incubation mixtures of ethanol-derived endoplasmic reticulum-Golgi membranes with control-derived cytosol in the presence of the ATP generating system. Results infer that the intracellular transport vesicles, generated from ethanol-modified membranes in the presence of the substrates that are available in the cytosol of the control hepatocytes, activate the apoptotic activity in the in vitro system. This interpretation is supported by the results of analysis of the clathrin-coated transport vesicles that, in contrast to nonclathrin transport vesicles, contain a sizable accumulation of ceramides that are known to induce apoptosis.

In this study, we investigated the effect of chronic alcohol ingestion on the interplay between the receptor-bound basic fibroblast growth factor (bFGF-R) and the expression of cyclin-dependent kinase (Cdk2) in buccal mucosa during ulcer healing. Chronic ulceration was induced by a topical application of acetic acid to the buccal mucosa of rats maintained for 5 weeks on alcohol-containing or control liquid diet. In both groups, the ulcer healing was accompanied by an increase in buccal mucosal expression of bFGF and Cdk2. In the control group, the ulcer healed within 10 days and maximum induction in bFGF (2.6-fold) and Cdk2 (2.4-fold) occurred by the 2nd day of healing. In contrast, the alcohol diet group showed a marked delay in ulcer healing (14 days), associated with the shift in maximum of bFGF and Cdk2 expression to the 4-6th day, and the values were reduced by 35 to 38%. The findings show that chronic alcohol ingestion exerts detrimental effect on the signaling events initiated by bFGF-receptor activation and propagated by Cdk2 that propels the cell cycle progression essential for rapid mucosal repair.

Endothelin-1 (ET-1) and nitric oxide are emerging key mediators in the maintenance of mucosal homeostasis, but little is known about these substances in soft oral tissue with alcohol abuse. Hence, we examined the expression of ET-1 and activity of the constitutive nitric oxide synthase (NOS) in buccal mucosa of rats subjected to chronic ethanol diet. The immunometric assays revealed the buccal mucosal level of ET-1 in the controls at 40.2 pg/mg protein and showed a 4.1-fold increase with alcohol diet to 166.2 pg/mg protein. The NOS assays established that comparing to the controls, the alcohol diet group exhibited a 57% decrease in buccal mucosal NOS activity. Moreover, the expression of ET-1 showed an inverse correlation (r = -0.75) with the extent of the induced changes in NOS. The results suggest that an increase in vasoconstrictive ET-1 levels combined with a loss of compensatory action by the constitutive NOS may be responsible for the weakening of oral mucosal defenses in alcoholics.

In this study we investigated buccal mucosal cells apoptosis and the expression of regulatory cytokines, tumor necrosis factor-alpha (TNF-alpha) and interleukin-4(IL-4) with chronic ethanol ingestion. The buccal mucosa of rats maintained for 23 days on alcohol-containing and control liquid diet was assessed for IL-4 and TNF-alpha content, and the extent of epithelial cells apoptosis. While the expression of TNF-alpha in alcohol diet group showed a significant increase (1.9-fold) over that of the controls, less apparent differences between the two groups were observed in the content of IL-4 (141.8 +/- 28.2 vs. 119.8 +/- 7.3 pg/mg protein). The DNA fragmentation assays revealed that alcohol diet group also exhibited a 3.5-fold enhancement in buccal mucosal cells apoptosis. Moreover, the apoptotic index showed positive correlation (r = 0.53) with the extent of induced changes in TNF-alpha. These results demonstrate that ethanol-induced buccal mucosal cells apoptosis is triggered by the enhancement in TNF-alpha expression.

The study of ethanol effects on intracellular transport and membrane biogenesis in rat hepatocytes revealed that, during synthesis of transport vesicles, the cytosolic phosphatidylinositol 3-kinase incorporated into the membrane of Golgi transport vesicles and a portion of the vesicular phosphatidylinositol was phosphorylated to phosphatidylinositol 3-phosphate. Association of the enzyme with Golgi transport vesicles and the transport to the apical portion of the cell membrane was not affected by 0 to 120 mM ethanol, but was dependent on the presence of the p85 subunit of the phosphatidylinositol 3-kinase. In the presence of ATP-enriched cytosol and calcium ions, association of Golgi transport vesicles with the apical membrane was followed by phospholipase A2-specific hydrolysis of phosphatidylinositol 3-phosphate and incorporation of the transport vesicle membrane into the apical membrane. Association of Golgi transport vesicles with apical membranes was not affected by preincubation of the cell membrane or Golgi transport vesicles with 0 to 120 mM ethanol, but was inhibited when the p85 phosphatidylinositol 3-kinase was incorporated into the membrane before incubation with Golgi transport vesicles. The fusion of Golgi transport vesicles with the apical membrane and generation of lysophosphatidylinositol 3-phosphate and arachidonate was inhibited with EGTA or after depletion of ATP from cytosol. Results of these studies provide evidence that phosphatidylinositol 3-kinase and phospholipase A2 activities are crucial for the final step of exocytotic transport. The process consists of two stages. First, the p85 subunit of phosphatidylinositol 3-kinase is involved in the specific association of the vesicle with membrane receptor, and that is followed by phospholipase A2-specific lysophospholipid generation, perturbation of the membranes, and fusion of the transport vesicle membrane with the apical membrane. Addition of ethanol to the in vitro transport system decreased production of Golgi transport vesicles, but had no effect on their association with apical membrane or fusion with the membrane.

We investigated the extent of induction in sublingual salivary gland cells apoptosis and tumor necrosis factor-alpha (TNF-alpha) expression with chronic ethanol ingestion. The experiments were conducted on rats pair-fed for 8 weeks with alcohol-containing and control liquid diet. The animals were killed, their sublingual glands dissected, and the glandular tissue used for quantization of TNF-alpha expression and the assays of acinar cells apoptosis employing sandwich enzyme immunoassay for histone-associated DNA fragments. The mean value for TNF-alpha in sublingual gland of the control group was 22.3 pg/mg of protein and showed a 1.6-fold increase in the chronic ethanol diet group to 36.5 pg/mg of protein. In comparison with the controls, the sublingual gland of the chronic ethanol diet group also exhibited a 3.4-fold enhancement in acinar cell apoptosis. These findings suggest that chronic ethanol ingestion causes the enhancement in TNF-alpha expression and leads to the induction in salivary gland acinar cells apoptosis. Thus, the diminished secretion of saliva in alcoholics may be a direct result of increased salivary gland apoptosis.

In this study, we investigated gastric epithelial cells' apoptosis and tumor necrosis factor-alpha (TNF-alpha) expression with ethanol-induced mucosal injury, and the effect of antiulcer agents on this process. Rats received intragastric pretreatment with the agent or vehicle followed 1h later by ethanol, and after 30 min the gastric mucosa was assessed for TNF-alpha and apoptosis. In the absence of antiulcer agents, ethanol caused extensive mucosal lesions accompanied by a 9.5-fold enhancement in apoptosis and a 2.5-fold increase in TNF-alpha. Pretreatment with omeprazole evoked a 54% reduction in TNF-alpha, but had no effect on ethanol-induced mucosal damage or apoptosis, the sucralfate reduced the extent of mucosal damage by 95%, apoptosis by 39% and TNF-alpha by 52%, while ebrotidine not only prevented mucosal injury and rise in TNF-alpha, but also caused a 70% reduction in epithelial cells' apoptosis. The results demonstrate that ethanol-induced gastric epithelial cells apoptosis triggered by the enhancement in mucosal TNF-alpha is efficiently counteracted by ebrotidine.

The effect of ethanol on mucus glycoprotein synthesis, intracellular modification, transport, glycosylation, and secretion was studied in rat gastric mucous cells. Preincubation of the in vitro translation mixture containing gastric mucous cells mRNA for 60 min with 0 to 120 mM ethanol caused a decrease in the synthesis of mucus glycoprotein apopeptide by up to 40%. The reduction in translation was time- and ethanol concentration-dependent. After 60 min, translation in the presence of 30, 60, and 120 mM ethanol decreased to 83.3 +/- 2.3%, 75.5 +/- 0.4%, and 63.6 +/- 2.6%, respectively. The experiments conducted with endoplasmic reticulum microsomes, preincubated with ethanol, and used in the studies of cotranslational translocation of the apomucin showed a 20% decrease in the transfer of mucus glycoprotein apopeptide to the lumen of endoplasmic reticulum microsomes. In the presence of ethanol, processing of mucus glycoprotein apopeptide in Golgi was also inhibited. During the initial 30 min of incubation with 0 to 120 mM ethanol, glycosylation seemed to proceed at the same rate in the samples with and without ethanol. However, during consecutive 30 min of incubation, glycosylation in the presence of 60 mM ethanol decreased by 30 to 35%, and with 120 mM ethanol was completely inhibited. Measurements of the effect of ethanol on the discharge of mucus glycoprotein from the intracellular stores revealed that, on average, the secretory output of the rat gastric mucosa exposed to ethanol liquid diet for 8 weeks decreased by 77% or more, and adherence of the glycoprotein to the gastric epithelium was weakened. Results indicate that ethanol inhibits synthesis, transport, and processing of gastric mucus glycoprotein, and that the processes taking place in different intracellular compartments contribute in the additive fashion and, are reflected in a dramatic decrease in the delivery of mucus glycoprotein to the gastric epithelial surfaces.

The present study investigated the effect of acute ethanol (ETOH) treatment on the epidermal growth factor (EGF) receptor in rat gastric mucosa using two different experimental models. In the in vitro experiments, gastric mucosal cells were incubated with 0, 0.05, 0.1, 0.25, and 0.5% ethanol in Dulbecco's modified Eagle's medium (DMEM) for 30 min and then used for the membrane preparation. The EGF receptor binding assay indicated that cells incubated in the presence of ethanol displayed a concentration-dependent increase (r = 0.85) in the 125I-EGF binding. The Western blot analysis using anti-EGF-receptor antibody revealed that ethanol in vitro caused reduction in the immunoreactivity of the major 170-kDa protein. There were also alterations in the minor protein bands (140, 120, and 50 kDa). In the in vivo experiments, rats that fasted overnight were given 1.0 ml of saline or ethanol (5, 10, or 15%) by gastric intubation 30 min before sacrifice. In comparison with the saline controls, ethanol treatment caused a decrease of the EGF receptor binding to the gastric mucosal membrane (saline: 5%: 10%; 15% ETOH, 1.46 +/- 0.18: 1.13 +/- 0.17: 1.27 +/- 0.19: 0.84 +/- 0.14, p < 0.02; mean +/- SEM, n = 9). Furthermore, the immunoblot analysis revealed concentration-dependent decrease in the intensity of the major 170-kDa protein with ethanol. The reduction in the EGF receptor binding and the impairment of the receptor protein might be due to the nonspecific damage of the gastric mucosal membrane by ethanol.

To investigate the effect of ethanol on the intracellular transport in gastric epithelial cells, the in vitro system, generating transport vesicles which transfer mucus glycoprotein apopeptide (apomucin) from rough endoplasmic reticulum (RER) to Golgi, was assembled. The vesicles, generated from gastric mucous cell RER microsomes and labeled with [3H]palmitic acid, were isolated from the maternal RER and characterized. The electron microscopy revealed that these RER products consisted of 80 to 100 nm smooth membrane vesicles, while the immunochemical analyses showed that they contain apomucin but were devoid of the characteristic integral proteins of the RER membrane. Incubation of apomucin transporting vesicles with Golgi in the presence of UDP-[3H]galactose resulted in the formation of glycosylated mucin and fusion of the vesicles with Golgi. Formation of ER transport vesicles was dependent on the supply of lipid precursors, and the activity of phosphoglyceride and sphingolipid synthesizing enzymes. In the presence of 60 and 120 mM ethanol, the vesicles were formed, but their lipid composition was modified. The results suggest that ethanol-induced membrane alterations are initiated at the early stages of the membrane biogenesis and are first reflected in the lipid composition of the intracellular transport vesicles.

The dose-dependent effects of acute ethanol treatment on the prostaglandin E2 (PGE2) receptor in submandibular salivary glands was investigated. Rat submandibular-gland segments were preincubated with Dulbecco's modified Eagle's medium in the absence and the presence of various concentrations of ethanol (0.1-5%). After incubation, membranes were prepared and assayed for PGE2 receptor binding using [3H]-PGE2. Ethanol had no effect on receptor binding at concentrations below 1%, but binding was stimulated by higher concentrations. A 30% increase in binding occurred with 2.5% ethanol and 50% with 5% ethanol. This increase was due to an alteration of the binding characteristics. Scatchard analysis of the data from the control and 5% ethanol groups revealed that the ethanol-induced increase in PGE2 receptor binding was mainly due to a 26 and 58% decrease in the dissociation constant (Kd) for the high- and low-affinity binding sites, respectively. A 31% decrease of binding capacity (Bmax) was also observed in the low-affinity binding sites. The results demonstrate that PGE2 receptor binding in submandibular glands is not altered by acute treatment with ethanol at physiological doses. The receptor binding is also relatively stable on pretreatment with 1% ethanol. It is suggested that the increase of receptor binding induced by high concentrations of ethanol may be a spontaneous response to the decrease of PGE2 synthesis in the tissues.

1. The gastroprotective properties of a new H2-receptor antagonist, ebrotidine, against ethanol-induced mucosal injury was investigated. 2. Groups of rats, with and without indomethacin pretreatment, received intragastrically either a dose of ebrotidine or vehicle only, followed by ethanol given at various intervals up to 4 hr. The gastric mucosa, 30 min after the ethanol challenge, was then subjected to macroscopic and histologic examination, and physicochemical measurements. 3. Ebrotidine at doses of 50 mg and higher per kg body wt effectively prevented the alcohol-induced mucosal injury, even in the presence of indomethacin. The protective effect was demonstrable already at 30 min, reached maximum at 1 hr, and persisted up to 3 hr. 4. Physicochemical analyses established that ebrotidine elicited 30% increase in mucus gel dimension, caused 19-20% increase in glycolipids and phospholipids, and evoked 21% increase in sulfomucin and 18% in sialomucins. As a consequence, the mucus gel viscosity increased by 1.4-fold, H+ retardation capacity by 16%, and hydrophobicity by 65%. 5. The results demonstrate that ebrotidine is a unique H2-antagonist endowed with a remarkable mucosal strengthening capability.

1. Ethanol treatment caused a 47% decrease in [125I]EGF binding to the membrane preparations of buccal mucosa resulting from the decrease of binding sites rather than the affinity of high affinity receptor. 2. The immunoblot revealed a protein band of 170 kDa in the control sample, while a barely detectable 200 kDa protein was observed in the ethanol-treated sample. 3. Protein kinase assays using [32P]ATP as probe showed an EGF-stimulated phosphorylation band of 170 kDa in the control but not in ethanol-treated sample. 4. Ethanol did not change the basal incorporation of [3H]thymidine and [35S]methionine, but caused a 38% and 57% decrease of EGF-stimulated thymidine and methionine incorporation, respectively. 5. The data suggest that EtOH decreases EGF receptor binding through modification of the receptor molecule, thereby impairing receptor kinase activity and its physiological function.

The gastroprotective properties of GM1-ganglioside, an indigenous component of epithelial cell membrane, was investigated. The experiments were conducted with groups of rats with and without indomethacin pretreatment. The animals received intragastrically either a dose of GM1 as emulsion in 5% gum arabic or vehicle alone, followed by ethanol given at various time intervals up to 3 h after the GM1. The animals were sacrificed 30 min after the ethanol dose and their gastric mucosa subjected to macroscopic and histologic assessment, and physicochemical measurements. In the absence of GM1, ethanol caused extensive gastric hemorrhagic lesions which were significantly reduced by pretreatment with GM1 at dose as low as 70 micrograms/100 g body weight. Removal of sialic acid from GM1 led to the loss of gastroprotection. Furthermore, the effect of GM1 was not thwarted by indomethacin. The maximal protection was achieved 1 h following GM1 dose and this protective effect persisted at least 2.5 hr. The results of physicochemical measurements revealed that GM1 was capable of preventing the detrimental effect of indomethacin on the adherent mucus gel dimension, and on its content of sulfo- and sialomucins, protein, and phospholipids. The effects brought by GM1 were also accompanied by a significant (40-60%) increase in mucus gel viscosity, hydrogen ion retardation capacity (35-46%) and hydrophobicity (70-94%). The results indicate that the gastroprotective action of GM1 occurs through the enhancement of the physicochemical characteristics of the mucus layer, and does not appear to be mediated by endogenous prostaglandins.

The effect of ethanol on buccal mucosal epidermal growth factor receptors was investigated. The buccal mucosal cells from adult rats were incubated in DMEM at 4 degrees C and 37 degrees C in the presence of various concentrations (0-5%) of ethanol and then assayed for EGF receptor binding using [125I]-EGF. The results of binding analysis showed that the cells incubated in the presence of ethanol displayed diminished [125I]-EGF binding. The receptor binding capacity was dependent upon ethanol concentration and was more pronounced at 4 degrees C. A 30% decrease in receptor binding occurred with 1% ethanol, 34% with 2.5% ethanol, while 5% ethanol caused a 42% decrease in binding. Scatchard analysis suggested that the decrease of [125I]-EGF binding to the buccal mucosal cells pre-incubated with ethanol was due to a decrease of the high affinity binding site (Bmax). The association constant of the high affinity EGF receptor remained unchanged. The results suggest that ethanol due to its untoward effect on the receptor binding of salivary GF interferes with the process of epithelial renewal, thus leading to weakening of the mucosal defense perimeter of soft oral tissue and the onset of dysplasia.

Sublingual salivary acini and submandibular tissue were incubated in DMEM medium in the presence of various concentrations (0-5%) of ethanol and the content of the three major prostaglandins, PGE2, PGF2 alpha and 6-keto-PGF1 alpha, were determined by radioimmunoassay. In In the sublingual gland, ethanol caused a decrease in PGE2 and PGF2 alpha levels, but had no effect on 6-keto-PGF1 alpha, while all three prostaglandins were affected in the submandibular gland. At 2.5% ethanol, the production of PGF2 alpha and PGE2 in sublingual gland decreased by 10% and reached maximum inhibition at 5% ethanol, at which concentration there was a 20 and 30% decrease in their levels. In submandibular gland, 2.5% ethanol caused a 20% decrease in PGE2, 30% in PGF2 alpha and 50% in 6-keto-PGF1 alpha; 40% inhibition in PGE2, 57% in 6-keto-PGF1 alpha and 65% in PGF2 alpha occurred in the presence of 5% ethanol. These findings suggest that alcohol impairs the function of salivary glands by inhibiting prostaglandin production.

1. The mechanism of gastric mucosal protection by an anticular agent, nitecapone, against injury was investigated in rats with and without indomethacin pretreatment. 2. Animals received intragastrically either a dose of nitecapone or vehicle alone, followed by ethanol given at various intervals up to 5 hr, and their gastric mucosa subjected to histologic and physicochemical assessment. 3. Ethanol caused extensive gastric hemorrhagic lesions which were essentially prevented by nitecapone at doses of 30 mg and higher per kg body weight. The maximal protection was achieved by 1.5 hr which persisted up to 4 hr and was not thwarted by indomethacin. 4. Physicochemical measurements revealed that nitecapone evoked 78% increase in mucus gel dimension, and showed 21% increase in phospholipids, and the content of sulfo-(22%) and sialomucins (72%). This was accompanied by 1.6-fold increase in mucus viscosity, 31% increase in H+ retardation capacity and 2.2-fold increase in hydrophobicity. 5. The results suggest that the gastroprotective action of nitecapone occurs through the enhancement of the physicochemical characteristics of mucus layer.

The mechanism of gastric mucosal protection by an antiulcer agent, colloidal bismuth subcitrate (CBS), against ethanol-induced injury was investigated using in vivo and in vitro systems. The experiments in vivo were conducted with groups of rats with and without indomethacin pretreatment, and the animals received either a dose of CBS (100 mg/kg) or a vehicle (saline), followed 30 min later by ethanol. In the in vitro studies, gastric mucosa segments were cultured in the presence of CBS, ethanol, or both. The results of in vivo experiments revealed that in the absence of CBS, ethanol caused extensive gastric hemorrhagic lesions which were significantly reduced following CBS pretreatment and this effect of CBS was not prevented by indomethacin. The data obtained with gastric mucosal culture established that in comparison to the controls, ethanol caused a 27% decrease in mucin synthesis, while mucin synthesis in the presence of CBS increased by 48%. The increase in mucin synthesis evoked by CBS was accompanied by the enhanced metabolism of mucosal phosphoinositides, as reflected by a decrease in PI (15%) and PIP2 (30%), and an increase in IP1 (26%) and IP3 (67%). In contrast, ethanol, which exhibited detrimental effect on mucin synthesis, caused a decrease in PIP (35%), IP2 (47%) and IP3 (38%), and an increase in PIP2 (80%), and IP1 (51%). However, when the mucosal culture was carried out in the presence of both CBS and ethanol, the detrimental changes evoked by ethanol on mucin synthesis were prevented, and the phosphoinositide and inositide phosphate distribution patterns were quite similar to those in the mucosa cultured in the presence of CBS only.(ABSTRACT TRUNCATED AT 250 WORDS)

The mechanism of protection by sucralfate against gastric mucosal injury induced by ethanol was investigated. The experiments in vivo were conducted with groups of rats with and without indomethacin pretreatment, and the animals received sucralfate followed by ethanol. In the in vitro experiments, gastric mucosa was cultured in the presence of sucralfate, ethanol, or both. The in vivo experiments revealed that ethanol caused extensive gastric hemorrhagic lesions which were significantly reduced following sucralfate pretreatment and that this effect of sucralfate was not prevented by indomethacin. The data with gastric mucosal culture demonstrated that ethanol caused a 24% decrease in mucin synthesis, while mucin synthesis in the presence of sucralfate increased by 32%. This increase was accompanied by the enhanced metabolism of mucosal phosphoinositides, as reflected by a 22% decrease in PI, 1,2-fold increase in IP1 and 3.4-fold increase in IP3. In contrast, ethanol, caused 1.5-fold increase in IP1 and PIP2, and 35% decrease in PIP, 47% decrease in IP2 and 38% decrease in IP3. However, when the mucosal culture was carried out in the presence of both sucralfate and ethanol, the detrimental changes evoked by ethanol in mucin synthesis were prevented. The results suggest that the mucosal protective action of sucralfate involves the metabolism of phosphoinositide-derived messenger molecules.

The effect of ethanol, 16,16-dimethyl prostaglandin E2 (DMPGE2), and ethanol after DMPGE2 pretreatment on the solubilization of protein, glycoprotein, and glycolipid constituents of gastric mucus was investigated. The Lucite chamber stomach-flap preparation was used in dogs whose basal H+ secretion was inhibited by intravenous cimetidine. Graded concentrations (5-80%) of ethanol produced a dose-dependent decrease in potential difference (PD) which was accompanied by an increase in the content of proteins, glycoproteins, and glycolipids of the instillates. This effect was most pronounced at 40% ethanol. Exposure of gastric mucosa to DMPGE2 at 0.01 microgram/ml had no effect on the transmucosal PD, and the content of investigated components in the instillates increased only slightly over the saline control levels. DMPGE2 at 0.1 microgram/ml, although it did not induce an evident fall of PD, evoked a moderate increase in glycoprotein and glycolipid liberation. Higher doses (above 1.0 microgram/ml) of DMPGE2 were associated with the fall of PD and the increased solubilization of proteins, glycoproteins, and glycolipids. Pretreatment of the mucosa with DMPGE2 in a dose of 1.0 microgram/ml diminished the liberation of the investigated components from the gastric mucosa by 5 and 10% ethanol, but did not prevent the changes evoked by higher concentrations (20-80%) of ethanol. These data indicate that DMPGE2 applied topically protects in part the gastric mucosa against the ethanol-induced solubilization of mucus constituents.

The effect of ethanol on the proteolytic activity of pepsin towards gastric mucus glycoprotein was investigated. Undegraded mucus glycoprotein isolated from pig stomach was incubated with pepsin in the presence and absence of ethanol, and the released alpha-amino residues were quantitated by the trinitrophenylation procedure. In the absence of ethanol the rate of proteolysis was proportional to mucus glycoprotein concentration up to 450 micrograms and remained constant with time of incubation for at least 30 min. The apparent Km value of pepsin for pig gastric mucus glycoprotein was 8.7 X 10(-7)M. The proteolytic activity of pepsin towards mucus glycoprotein was inhibited by ethanol. The rate of inhibition was proportional to the ethanol concentration up to 0.8 M and was of competitive type. The apparent K1 value calculated from the double-reciprocal plots for ethanol was 2.2 X 10(-6)M. The inhibitory effect of ethanol on the proteolysis of gastric mucus glycoprotein may be of physiological significance.

The resistance to proteolysis by pepsin of gastric mucus glycoprotein synthesized by tissue culture in the presence and absence of 0.1 M ethanol was investigated. The glycoprotein product of ethanol-supplemented culture was found to contain 68% less associated lipids and 81% less covalently bound fatty acids, but exhibited unaltered content of carbohydrate and protein. The lipid and fatty acyl deficient glycoprotein was 5-times more rapidly and 2-3-times more extensively degraded by pepsin than the glycoprotein synthesized in the absence of ethanol. Following delipidation with organic solvents and deacylation with hydroxylamine both glycoproteins were digested at the same rate and degraded to the same extent. The lower content of fatty acyl residues markedly affected the overall pattern of the proteolytic fragments identified by SDS gel electrophoresis. The peptides corresponding to the acylated fragments of control were degraded and an increase in the amount of smaller peptides was observed. The in vitro assays of the fatty acyltransferase activity towards the substrates obtained from control and alcohol-containing cultures revealed that the enzyme activity was similar and increased proportionally with increased concentration of both glycoprotein substrates and enzyme. However, addition of 0.1 M ethanol to the assay tubes containing complete incubation mixture decreased the acylation of either glycoprotein by 40%. Based on the results presented here, and on previous studies of mucus glycoprotein synthesis in the presence of ethanol, we conclude that ethanol interferes with the process of acylation of mucus glycoprotein with fatty acids.

The effect of ethanol on the synthesis and secretion of mucus glycoprotein in gastric mucosal cells was investigated. The mucosal cell suspensions were subjected to a short-term (4 h) culture in the presence of 0-1.5 M ethanol, with [3H]proline and [3H]palmitic acid as markers for glycoprotein synthesis and acylation. The synthesized labeled mucus glycoprotein was isolated from the incubation medium (extracellular glycoprotein) and from the mucosal cells (intracellular glycoprotein), and analyzed. Depending upon the ethanol concentration in the cell culture medium, two distinct effects on the synthesis and secretion of mucus glycoprotein were observed. The cells cultured in the presence of 0.02-0.1 M ethanol showed increased ability for the incorporation of [3H]proline and [3H]palmitic acid, and for the secretion of the newly assembled mucus glycoprotein. The synthesis of the glycoprotein increased 18-fold, acylation 5-fold, and secretion 10-fold. The synthesized glycoprotein, however, contained four to five times less of acyl-bound fatty acids. Ethanol at 0.1-1.5 M caused a marked reduction (62-64%) in the mucus glycoprotein synthesis, but the amount of glycoprotein released to the medium remained constant. This indicated that higher concentrations of ethanol caused the release of the preformed intracellular mucus glycoprotein reserves. The results demonstrate that gastric mucosal cells incubated in the presence of ethanol exhibit impaired synthesis and secretion of mucus glycoprotein, and that the severity of impairment depends upon the ethanol concentration.

Effects of ethanol and prostaglandin on the metabolic processes of gastric mucosal cells have been evaluated by studying the biosynthesis and intra- and extracellular distribution of mucus glycoprotein. The rat gastric mucosal cell suspensions were subjected to a short-term tissue culture in the presence of 0-1.5 M ethanol and 0-100 ng/ml 16,16-dimethyl prostaglandin E2 (DMPGE2), using [3H]proline and [3H]palmitic acid as markers of mucin apoprotein synthesis and modification. Ethanol at concentrations of 0.02-0.1 M stimulated the glycoprotein synthesis, but the product was 2-3 times more extensively and rapidly degraded by pepsin than the glycoprotein synthesized in the absence of ethanol. Higher concentrations of ethanol (0.1-1.5 M) caused a marked reduction in the glycoprotein synthesis and a depletion of the intracellular mucus glycoprotein stores, and at 1.5 M ethanol the synthetic processes ceased to function. When the incubated tissue cultures were challenged with DMPGE2, the highest synthetic and secretory stimulation was achieved at 10 ng/ml. The peptic susceptibility of the glycoprotein synthesized in the prostaglandin-enriched culture was somewhat lower than that of controls, and the proportion of the undegraded glycoprotein remaining after 22 h of digestion was significantly higher. Furthermore, the intracellular compartments were not depleted of mucus glycoprotein and the amount of the mucin apoprotein precursor increased to about 30%. Addition of DMPGE2 prior to ethanol treatment resulted in the stabilization of cellular processes and the cells responded as if ethanol was not present in the medium. Moreover, the intracellular stores of mucus glycoprotein were not depleted and the secretory rate was moderately elevated. The regulatory effect of DMPGE2 on the glycoprotein synthesis and distribution was significant, but diminished with increasing amounts of ethanol in the medium. The results suggest that DMPGE2 imposes a stabilizing effect on the secretory processes and controls the mucus glycoprotein synthesis, but the extent of this protective action against ethanol is limited and depends upon the concentration of ethanol penetrating the mucosal cells.

In vitro sulfation of mucus glycoprotein by sulfotransferase from rat submandibular salivary gland and the effect of ethanol on this enzyme activity was investigated. Subcellular fractionation studies revealed that the enzyme which catalyzes the transfer of sulfate ester group from 3'-phosphoadenosine-5'-phosphosulfate to submandibular gland mucus glycoprotein is associated with Golgi-rich membrane fraction. The sulfotransferase enzyme exhibited optimum activity at pH 6.8 in the presence of 0.5% Triton X-100, 4 mM MgCl2, and 25 mM NaF. The enzyme was equally capable of sulfation of the desulfated intact as well as proteolytically degraded desulfated glycoprotein preparations, whereas the acceptor capacity of the intact mucus glycoprotein was 70% lower. The submandibular gland sulfotransferase activity was inhibited by ethanol. The rate of inhibition of mucus glycoprotein sulfation was proportional to the concentration of ethanol up to 0.4 M, at which concentration a 39% reduction in the sulfotransferase activity occurred. The apparent Km value of the enzyme for salivary mucus glycoprotein was 11.1 microM, and the Kl in the presence of ethanol was 0.93 M. The synthesized 35S-labeled glycoprotein gave on CsCl equilibrium density gradient centrifugation 35S-labeled peak which coincided with that of the glycoprotein. Alkaline borohydride treatment of this glycoprotein led to the liberation of the label into the acidic oligosaccharide alditol fraction. As the inhibition by ethanol of sulfotransferase enzyme occurred below its isosmotic concentration to plasma, the observed effect could also be detrimental to salivary mucus glycoprotein sulfation in vivo.

The enzyme activity which catalyzes the transfer of sulfate group from 3'-phosphoadenosine 5'-phosphosulfate to C-3 of the galactose residue of galactosylceramide and lactosylceramide has been demonstrated in the Triton X-100 extracts of the microsomal fraction of rat gastric mucosa. The sulfotransferase activity of this fraction in antral mucosa was 1.2-1.3 times greater than that of the body and 19-22 times greater than that of the forestomach. The enzyme did not catalyze the transfer of sulfate to glucosylceramide, trihexosylceramide, and triglucosyl monoalkylmonoacylglycerol. Optimum enzymatic activity was obtained using 0.4% Triton X-100, 33 mM NaF, and 15 mM MgCl2 at a pH of 6.8. The sulfotransferase activity was inhibited by ethanol. With both glycolipid substrates, little inhibition of enzyme activity was obtained up to 0.5 M ethanol. However, higher concentrations of ethanol produced severe inhibitory effect. This inhibition of sulfation of galactosylceramide and lactosylceramide by ethanol was of the competitive type. The apparent KI values were 8.3 X 10(-5) for galactosylceramide and 5.6 X 10(-5) for lactosylceramide.

The mechanism of gastric mucosal protection by an antiulcer agent, geranylgeranylacetone (GGA), against ethanol-induced injury was investigated. The experiments were conducted with groups of rats with and without intraperitoneal indomethacin pretreatment. Animals received intragastrically either a dose of GGA (200 mg/kg) or a vehicle, followed 30 min later by 1 ml of absolute ethanol. The rats were sacrificed after 30 min and the gastric mucosa was subjected to macroscopic and histologic assessment and the measurements of adherent mucus, its dimension and chemical composition. In the absence of GGA, ethanol produced advanced macroscopic necrosis (greater than 38%) and the extensive necrotic lesions were visible upon histologic examination. Pretreatment with GGA significantly reduced (p less than 0.001) the extent and depth of mucosal necrotic lesions caused by ethanol, and this protection was not thwarted by indomethacin. Evaluation of the adherent mucus and its dimension by Alcian blue uptake and inverted microscope technique revealed that GGA was also capable of preventing the untoward effect of indomethacin on the adherent gastric mucus gel and its thickness. Results of chemical analyses established that in the absence of GGA indomethacin caused an increase in mucus protein (15%) and a decrease in its covalently bound fatty acids (67%) and lipids (36%). The decrease in lipids was particularly reflected in the content of phospholipids. Indomethacin, however, had no apparent effect on the composition of gastric mucus elaborated in the presence of GGA. The results suggest that gastric mucosal protective action of GGA is not mediated by endogenous prostaglandins but rather appears to involve the metabolism of mucosal lipids.

The magnitude of prostaglandin [16,16-dimethylprostaglandin E2 (DMPGE2)] protection against ethanol action has been evaluated by studying the intracellular events leading to synthesis, modification, intracellular retention, and secretion of mucus glycoprotein in the rat gastric mucosal cell culture. When metabolic activity of the cultured cells was expressed as the amount of radioactive tracer in purified mucus glycoprotein, it was found that ethanol at low concentration (0.1 M) caused an 8- to 9-fold increase in proline and a 5- to 6-fold increase in palmitic acid incorporation into mucus glycoprotein; however, at 1.5 M ethanol, the synthetic processes ceased to function. In the presence of DMPGE2 (10 ng/ml), a 2-fold increase in proline and a 3-fold increase in palmitic acid incorporation into mucus glycoprotein were observed. A simultaneous addition of DMPGE2 (10 ng/ml) and ethanol (0.1-1.5 M), or pretreatment with DMPGE2 (10 ng/ml) for 20 min followed by the addition of ethanol (0.1-1.5 M), resulted in the stabilization of glycoprotein synthesis and secretion, and in restoration of the function at the level observed with DMPGE2 alone. The addition of 10 ng/ml of DMPGE2 caused a 32% increase in mucus glycoprotein secretion and an 11% increase in the intracellular content of the glycoprotein. The amount of mucus apoprotein precursor rose by 18%, and the fatty acylation of the synthesized peptides was up by 38%. Addition of DMPGE2, together with ethanol, prevented depletion of the intracellular glycoprotein stores. As observed with ethanol alone, the secretion was elevated by 16-27%, whereas the intracellular glycoprotein stores remained similar to those of controls. The synthesis of the mucus apoprotein precursor was highly sensitive to ethanol, and addition of DMPGE2 only partially prevented its inhibitory action. Pretreatment with DMPGE2, however, eliminated ethanol toxicity, and the precursor pool level remained elevated (28-30%) at all concentrations of ethanol tested. The fatty acylation, although positively affected by DMPGE2, decreased steadily with increments of ethanol concentration. The results show that DMPGE2 stabilizes the cellular membrane processes and thus imposes control over mucus glycoprotein synthesis, secretion, and its intracellular retention.

A sulfotransferase activity that catalyzes the transfer of sulfate ester group from 3'-phosphoadenosine 5'-phosphosulfate to carbohydrate chains of gastric mucus glycoprotein has been demonstrated in the antral and body mucosa of rat stomach. Subcellular fractionation studies revealed that the enzyme is associated with Golgi-rich membrane fraction. The sulfotransferase activity of this fraction in antral mucosa was about 35% lower than that in the body. Optimum enzyme activity was obtained with 0.5% Triton X-100 and 30 mM NaF at a pH of 6.8 using desulfated mucus glycoprotein substrate. The enzyme was equally capable of sulfation of the proteolytically degraded and reduced forms of the desulfated glycoprotein, but the acceptor capacity of the intact mucus glycoprotein was about 60% lower than that of the desulfated preparation. The enzyme preparation also catalyzed the transfer of sulfate to galactosylceramide. The sulfation of mucus glycoprotein, however, was not affected by the presence of this glycolipid, suggesting that the sulfotransferase involved in mucus glycoprotein sulfation is different from that responsible for the synthesis of sulfatoglycosphingolipid. The mucus glycoprotein sulfotransferase activity was inhibited by ethanol. The rate of inhibition was proportional to the concentration of ethanol up to 0.3 M and was of the competitive type. The apparent Km value of the enzyme for mucus glycoprotein was 10.5 X 10(-6) M (21 mg/ml), and the KI in the presence of ethanol was 4.7 x 10(-1) M. The 35S-labeled mucus glycoprotein product of the enzyme reaction gave in CsCl density gradient a band in which the 35S label coincided with the glycoprotein. Alkaline borohydride reductive cleavage of this glycoprotein led to the liberation of the label into reduced acidic oligo-saccharide fraction. Most of the label was found incorporated in three oligosaccharides. These were identified as tri-, tetra-, and pentasaccharides, each carrying a labeled sulfate ester group on the terminal N-acetyl-glucosamine residue. Based on the results of structural analyses, the most abundant oligosaccharide was characterized as SO3H----6GlcNAc beta 1----3Gal beta 1----3GalNAc-ol.

The enzyme activity that catalyzes the transfer of palmitic acid from palmitoyl coenzyme A to the deacylated intact or deglycosylated gastric mucus glycoprotein was demonstrated in the detergent extracts of the microsomal fraction of antral and body mucosa of the rat stomach. Both types of mucosa exhibited similar acyltransferase activities and acceptor specificities. A 10-14% decrease in the fatty acyltransferase activity was observed with the reduced and S-carboxymethylated mucus glycoprotein, but the proteolytically degraded glycoprotein showed no acceptor capacity. This indicated that the acylation of mucus glycoprotein with fatty acids occurs at its nonglycosylated polypeptide regions and that some of the fatty acids may be linked via thiol esters. Optimum enzyme activity was obtained at pH 7.4 with the detergent Triton X-100, NaF, and dithiothreitol. The apparent Km values for the intact and deglycosylated mucus glycoproteins were 0.45 and 0.89 microM, respectively. The acyltransferase activity of the microsomal enzyme was inhibited by ethanol. With both intact and deglycosylated glycoprotein substrates, the rate of inhibition was proportional to the ethanol concentration up to 0.4 M and was of the competitive type. The K1 values were 0.80 microM for the intact mucus glycoprotein and 1.82 microM for the deglycosylated glycoprotein. Preincubation of the microsomal enzyme with low concentrations of ethanol (up to 0.5 M) did not seem to exert any additional deterrent effect on acyltransferase activity. Higher concentrations of ethanol (1.0 M and above), however, caused substantial reduction in the transferase activity due to denaturation of the enzyme.