Effects of ethanol alone or after pretreatment with 20% ethanol on phospholipid metabolism in rat gastric mucosa

312

Biochimica et Bioph_vsicu Acta 917 (1987) 372-380 Elsevier

BBA 52340

Effects of ethanol alone or after pretreatment with 20% ethanol on phospholipid metabolism in rat gastric mucosa Yukio Nishizawa

a, Hideki Sakurai a, Chiyuki Yamato and Motoyuki Moriga b

a

y Tsukuha Research Laboratories Eisni Co., Ltd., Tsukuba-gun, Ibaraki (Jopun) and ’ The 1st Deportment of Internal Medicine, Faculty of Medicine, University of Kyoto, Suk_vo-ku, Kyoto (Jopun) (Received

Key words:

Phospholipid

metabolism;

6 June 1986)

Ethanol;

Fatty acid; Glycerol;

Gastric

mucosa;

(Rat)

Changes in phospholipid metabolism in gastric mucosa caused by instillation of absolute ethanol (a cell-damaging agent) into the stomach of rats and the effects of pretreatment with 20% ethanol (a mild irritant) were investigated by using radioisotope-labeled fatty acids and glycerol. The labeled precursors were incorporated mainly into phosphatidylcholine and tiacylglycerol, and also to lesser extents into phosphatidylethanolamine and phosphatidylinositol + phosphatidylserine. The instillation of absolute ethanol reduced the incorporation of fatty acids and glycerol into phospholipids within 15 min, indicating the inhibition by ethanol of de novo synthesis of phospholipids. Pretreatment with 20% ethanol caused the incorporation of fatty acids into phospholipids to be maintained after absolute ethanol instillation. These results suggest that the pretreatment with 20% ethanol may protect the cellular synthetic activity of phospholipids against damage by absolute ethanol. The incorporation of fatty acids into the free fatty acid fraction, monoacylglycerol and diacylglycerol was increased by absolute ethanol instillation, suggesting damage to the blood vessels of the gastric mucosa, and these changes were inhibited to some extent by the pretreatment with 20% ethanol.

Introduction Phospholipids in cells are basic components of cellular membranes, acting as regulators of membrane-bound enzymes [1,2] and membrane transport of substances [3-51. Phospholipid turnover has been recognized as being involved in cellular responses to hormones and transmitters [6]. Phos-

Abbreviations; lethanolamine; dylserine.

PC, PI,

phosphatidylcholine; phosphatidylinositol;

PE, phosphatidyPS, phosphati-

Correspondence: Y. Nishizawa, Tsukuba Research Laboratories Eisai Co. Ltd., 1-3 Tokodai 5-Chome, Toyosato-machi, Tsukuba-gun, Ibaraki 300-26, Japan.

0005.2760/87/$03.50

0 1987 Elsevier Science Publishers

pholipids appear as pulmonary surfactants in lungs [7], and as detergents in micelle formation of fats present in foods [8]. Since these functions of phospholipids are related to their turnover (including synthesis, degradation and modifications), the regulation of turnover rates of phospholipids is an important factor in cellular functions. Gastric mucosal cells excrete HCl and pepsin to digest foods, and these cells are always exposed to cell-damaging agents such as HCl, bile acids and other chemicals. Although the mechanisms of excretion of HCl and gastric mucus have been studied in many laboratories, little is known about lipid metabolism in gastric mucosal cells and its relation to the cellular functions. Horowitz and his colleagues detected in rat stomach several kinds of

B.V. (Biomedical

Division)

313

enzyme activities involved in the metabolism of phospholipids [9-111. Hills and his colleagues have recently found phospholipids in the stomach surface [12], and they have proposed that this phospholipid layer acts as a defensive factor of the mucosal cells against injury by cell-damaging agents [13,14]. They also found that a prostaglandin E, derivative increases the phospholipid content in the surface layer of rat stomach [15]. Slomiany and his coworkers have proposed that the lipids associated with mucus in the stomach play a role in maintaining the viscosity of the mucus and have a barrier function against hydrogen ions present in the gastric lumen [16,17]. If the phospholipids and other lipids are present in the mucous layer of the stomach, these lipids may be excreted from the mucosal cells after synthesis, just as a lung surfactant, dipalmitoylphosphatidylcholine, is excreted from alveolar type II cells of lung [18]. The purposes of the present work were to elucidate the metabolic turnover of phospholipids and other lipids in rat gastric mucosa by using radioisotope-labeled fatty acids and glycerol as precursors, and to investigate the changes in lipid metabolism caused by a cell-damaging agent, absolute ethanol. We also evaluated the effects of pretreatment with 20% ethanol as a mild irritant on the changes in lipid metabolism induced by absolute ethanol, because this pretreatment protects mucosal cells against injury by subsequent treatment with absolute ethanol [19], through the stimulation of prostaglandin production in mucosal cells [20]. Methods Chemicals. [1-‘4C]Palmitic acid (58 mCi/ mmol), [9,10- 3H]oleic acid (4.3 Ci/mmol), and [5,6,8,9,11,12,14,15-3H]arachidonic acid (163 Ci/ mmol) were purchased from Amersham Inc., U.K. and [2-3H]glycerol (2 Ci/mmol) from ICN Radiochemicals (CA, U.S.A.). Bovine serum albumin, pancreatic phospholipase A, and indomethacin were obtained from Sigma Chemical Co., and other chemicals used were of reagent grade. Administration of labeled precursors. Labeled precursor was dissolved in 5% (w/v) bovine serum

albumin solution and 0.5 ml of the solution (40 PCi for fatty acids, 100 PCi for glycerol) was injected into the femoral vein of male SpragueDawley rats aged 7 to 8 weeks. Rats were fasted for 24 h before use. The labeled precursors were injected 20 s before or 15 min after the instillation of absolute ethanol (1.2 ml) into the stomach, and the rats were killed 15 min or 30 min after the injection, respectively. In some rats, 20% ethanol (1.2 ml) was administered orally as a mild irritant 15 min before the instillation of absolute ethanol. Rats treated with 20% ethanol alone, but without absolute ethanol injury, were injected with labeled precursors 15 min after the administration of 20% ethanol, and killed 30 min after the injection. In experiments to examine the effect of indomethatin, rats were treated with 20% ethanol and then [3H]arachidonic acid was injected 20 s before the instillation of absolute ethanol. The rats were killed 15 min after the injection. In some rats indomethacin (2.5 mg/kg) was administered orally to inhibit prostaglandin synthesis 60 min before the administration of 20% ethanol. The rats of saline control groups were given saline (1.2 ml) instead of absolute ethanol and 20% ethanol. Extraction of mucosal lipids. The stomach was excised, opened with scissors along the greater curvature, and washed in ice-cold saline. Gastric mucosa was then wiped with a filter paper and scraped with a metal spatula on an ice-cold glass plate. Mucosal cells were homogenized in 5 ml of ice-cold 0.5 mM EDTA with a Teflon homogenizer, and the volume of homogenate was adjusted to 7 ml. Total lipids were extracted from 5 ml of the homogenate by the method of Bligh and Dyer [21]. The chloroform layer obtained was evaporated to dryness, and the residue was dissolved in 5 ml of chloroform/methanol (2 : 1) and stored at -20°C until use. Determination of incorporated radioactivity. 1 ml of the extract was concentrated under nitrogen gas and applied to a precoated TLC plate (silica gel 60 F 254) ER Merck, F.R.G.). Neutral lipids were separated by the method of Ando et al. [22], and phospholipids by the method of Billah et al. [23]. Lipids were located by exposure of the plate to iodine vapor and the silica gel corresponding to each lipid fraction was scraped into counting vials. A scintillant (5 ml, ACS-II, Amersham Inc.) was

374

added to each vial and radioactivity was measured with an Aloka LSC-900 liquid scintillation spectrometer. Analysis of the positional distribution of incorporated fatty acids. Lipid extract (1 ml) was separated into phospholipid fractions by TLC as described above, and the regions of silica gel corresponding to phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were scraped into tubes. Methanol (2 ml) was added to each tube, and the mixture was sonicated for 10 min in a bath-type ultrasonicator (Branson Co.), then 4 ml of chloroform were added to the tube. Phospholipids were further extracted by shaking for 30 min, and the organic phase was transferred to another tube. The extract was dried under nitrogen gas, 0.5 ml of distrilled water was added, and the mixture was sonicated for 5 min. Phospholipids dispersed in water were digested by incubation with phospholipase A, for 30 min at 37°C by the method of Wassef et al. [9]. The reaction mixture was then extracted as described above [21]. Fatty acid fraction and lysophosphatide were separated by TLC, and the radioactivity of each fraction was measured. These conditions allowed complete hydrolysis of fatty acids incorporated into the sn-2 positions of phospholipids. Positional distribution of fatty acids in mucosal phospholipids. Phospholipids (PC and PE) were separated and digested with phospholipase A, as described above, and a part of the digest was hydrolyzed in 0.5 M KOH/ethanol at 50°C for 30 min. Fatty acids were recovered by extraction with diethyl ether. Fatty acid contents in the digests (sn-2) and in the hydrolysates with alkali (total fatty acid) were determined by HPLC with fluorescence detection (JASCO, Tokyo, Japan) after derivatization with 9-diazomethylanthracene 1241. Statistical analysis. Results are expressed mean values with S.E. The data were analyzed using the unpaired Student’s t-test.

vivo experiments using labeled precursors. However, no data are available on the incorporation of fatty acids and glycerol into phospholipids in gastric mucosa. Thus, mucosal lipids were doubly labeled with [ 3Hjoleic acid and [ l4 C]palmitic acid and the radioactivity incorporated into the lipids was chased up to 120 min after the injection of labeled fatty acids (Fig. 1). Palmitic acid was incorporated mainly to into PC and triacylglycerol within 15 min after the injection. The amount of label incorporated into PC decreased slowly after 30 min, whereas that in triacylglycerol declined quickly after 30 min. The incorporation of palmitic acid into PE and phosphatidylinositol + phosphatidylserine (PI -t PS) was low. Oleic acid was incorporated into PC and PE more efficiently than palmitic acid, while the incorporation into triacylglycerol was less. The time course curves of incorporated radioactivity of oleic acid showed patterns similar to those of palmitic acid, but there was a slight increase in the label present in PC during 15-60 min. The distributions of labeled precursors among mucosal lipids were compared 30 min after the injection. The incorporation of glycerol was the highest in PC, followed by triacylglycerol, PE and PI + PS. All fatty acids used were incorporated mainly into PC and triacylglycerol, but the distributions between the two fractions were different. Arachidonic acid was incorporated into PC most efficiently, whereas palmitic acid was best incorporated into tri-

as by

Results

Incorporation of labeled precursors into mucosal lipids Synthesis and modification of phospholipids in liver and lung have been well characterized in in

Time after administration

(min)

Fig. 1. Time-course curves of the label in mucosal lipids after intravenous injection of [‘4C]palmitic acid (A) and [ 3H]oleic acid (B). PC, 0; PE, A; PI+PS, 0; triacylglycerol, 0. Each value is the mean with SE. from five rats.

375

acylglycerol. Oleic acid was incorporated into PC and triacylglycerol to similar degrees. The incorporation of palmitic acid into PE was less than that of the other fatty acids. PI + PS fraction was labeled by the various fatty acids to almost the same extents. Other lipids such as sphingomyelin, diacylglycerol and monoacylglycerol were less labeled (less than 5% of total lipids) and virtually no radioactivity was found in lysophosphatidylcholine. A small amount of radioactivity (less than 7% of total lipids) was detected in free fatty acid fractions in the experiments with labeled fatty acids. Positional distribution of incorporated fatty acids The fatty acid composition of mucosal PC and PE was examined (Table I). It was noted, from the comparison of the composition of overall fatty acids with the composition of fatty acids in the m-2 position, that palmitic and stearic acid were concentrated in the sn-1 position of PC and PE, whereas arachidonic and linoleic acid were mainly found in the ~2-2 position. Oleic acid was rich in the sn-2 position of PC, but was distributed in the sn-1 and sn-2 positions of PE in similar amounts. The positional distribution of labeled fatty acids incorporated into PC and PE was next examined. Palmitic acid in PC and PE was predominantly in the sn-1 position (58% for PC, 74% for PE), while arachidonic acid was specifically incorporated into the sn-2 position. Oleic acid was incorporated into the sn-1 and sn-2 positions to similar extents. Incorporation of fatty acids into phospholipids after absolute ethanol instillation Mucosal lipids were quickly labeled after the

TABLE

injection of labeled fatty acids, and the labeling was almost completed within 15 min. Therefore, the short-term labeling of mucosal lipids for 15 min or 30 min reflects the synthesis or modification of lipids, rather than the degradation of lipids. Thus, the incorporations of [‘4C]palmitic acid and [3H]oleic acid into mucosal phospholipids were measured to study the effects of a cell-damaging agent, absolute ethanol, on the synthesis (or modification) of phospholipids. The effects of pretreatment with 20% ethanol on the absolute ethanol-induced changes were also evaluated. The values of incorporation are plotted at the median time points between the injection of fatty acids and the sacrifice of rats (Fig. 2). The treatment with 20% ethanol alone did not affect the incorporation of fatty acids, as compared with the saline control. The instillation of absolute ethanol reduced the incorporation of fatty acids into PC within 15 min (the first labeling period) and the values were decreased to 64-66% of the saline control within 15-45 min (the second labeling period). The pretreatment with 20% ethanol significantly maintained the incorporation of fatty acids into PC during the observation period. In the case of PE, palmitic acid incorporation was slightly increased within 15 min after the instillation of absolute ethanol, and then decreased to 72% of the saline control within 15-45 min, while the incorporation of oleic acid into PE was immediately decreased. The pretreatment with 20% ethanol prevented the reduction in labeling of PE induced by absolute ethanol. There was no significant increase of labeling in lysophosphatides, lysophosphatidylcholine or lysophosphatidylethanolamine, after the instillation of absolute ethanol. The incorporation of

I

POSITIONAL

DISTRIBUTION

OF FAW

ACIDS

IN MUCOSAL

PHOSPHOLIPIDS

Each value (mol% of total fatty acid) is the mean with SE. from five rats. PC

PE

overall

sn-2

overall

SW2

16:0 18:O

35.8+0.6 12.6~0.4

18:l 1x:2 20:4

12.s+o.3 23.1 i: 0.9 15.7kO.7

10.3 i 2.2 3.7kl.O 18.0?0.8 41.0 i 2.6 26.9kO.5

19.4i 1.3 15.7*0.4 23.2 + 0.4 10.3 io.2 31.4? 1.2

7.011.7 4.5 kO.9 25.5 i 0.6 16.3 k 0.8 46.9i_1.8

316

fatty acids into PI + PS decreased markedly compared to the case with PC and PE within 15 min, and although the pretreatment with 20% ethanol tended to maintain the incorporation of fatty acids, the effect was not statistically significant.

Time

t

alter absolute

-I-

O 0 30 Time after absolute

LULlI

00 30 Time after absolute

ethanol

0 0 ethanol

flo ethanol

treatment (mid

30 treatment (mid

30 treatment (min)

Incorporation of fatty acids into neutral lipids after absolute ethanol instillation The incorporation of fatty acids into neutral lipids and free fatty acid fraction was also determined (Fig. 3). The instillation of absolute ethanol increased the incorporation of labeled fatty acids into triacylglycerol within 15 min as compared with the saline control, but this increase did not continue from 15 to 45 min. The pretreatment with 20% ethanol enhanced the incorporations to about twice the values of the saline control. The radioactivity present in free fatty acid fraction was markedly increased (about 20-fold more than the saline control) by the instillation of absolute ethanol, and the pretreatment with 20% ethanol reduced the labeling to about lo-times the saline control. The incorporation of fatty acids into diacylglycerol and monoacylglycerol was also increased by the instillation of absolute ethanol. However, the pretreatment with 20% ethanol had no effect on the absolute ethanol-induced changes in diacylglycerol and monoacylglycerol. The pretreatment with 20% ethanol caused a marked increase in the fatty acid incorporation into triacylglycerol. It is well established that the treatment with a mild irritant stimulates prostaglandin production in the mucosal cells [20]. Therefore, the enhanced uptake of fatty acids into triacylglycerol may be due to the increased prostaglandin level in the cells. Thus, rats were treated with 20% ethanol and the incorporation of [ 3Hlarachidonic acid into triacylglycerol was determined. In some rats indomethacin was given to inhibit prostaglandin production 60 min before the administration of 20% ethanol. The incorporaFig. 2. Effects of pretreatment with 20% ethanol on the changes in the incorporation of labeled fatty acids into mucosal phospholipids induced by the instillation of absolute ethanol. Rats were injected with [r4C]palmitic acid and [ 3H]oleic acid intravenously and killed at the time points indicated in the Methods section. Diluted ethanol (20%) was administered orally 15 min before the instillation of absolute ethanol. Radioactivity incorporated into PC (A), PE (B), and PI + PS (C) was measured. Saline control, 0; absolute ethanol instillation, A; pretreatment with 20% ethanol, 0. The values indicated by arrows are from the rats treated with 20% ethanol alone or saline. Each value is the mean with SE. from five to ten rats. Significant differences: * P i 0.05, ** P < 0.01. *** P i 0.001 vs. saline control; @ P < 0.05, @@ P < 0.01, @@@ P < 0.001 vs. absolute ethanol instillation.

317

0 0 30 Time after absolute

0 0 ethanol

00

30 treatment (min)

C 14C

Time

30 after absolute

00 ethanol

30 treatment (mid

D

500

T

3H

uo ethanol

30 treatment (mid

t

L If?o 30 Time after absolute

00 ethanol

ok. -uo Time

30 treatment (mid

30 after absolute

Fig. 3. Effects of pretreatment with 20% ethanol on the changes in the incorporation of labeled fatty acids into neutral lipids and free fatty acid fraction induced by the instillation of absolute ethanol. Rats were injected with [‘4C]palmitic acid and [ ‘H]oleic acid intravenously and killed at the time points indicated in the Methods section. Diluted ethanol (20%) was administered orally 15 min before the instillation of absolute ethanol. Radioactivity incorporated into triacylglycerol (A), free fatty acid (B), diacylglycerol (C). and monoacylglycerol (D) was measured. Saline control, 0; absolute ethanol instillation, a; pretreatment with 20% ethanol, 0. The values indicated by arrows are from the rats treated with 20% ethanol alone or saline. Each value is the mean with SE. of five rats. Significant differences: * P < 0.05, ** P < 0.01, *** P < 0.001 vs. saline control; @ P < 0.05, @@ P < 0.01, @@I@ < 0.001 vs. absolute ethanol instillation.

tion of arachidonic acid into triacylglycerol was increased in the rats pretreated with 20% ethanol, and the labeling was 178% + 21 (n = 4) of the saline control. Arachidonic acid was incorporated into triacylglycerol to almost the same degree as for palmitic acid and oleic acid after the pretreatment with 20% ethanol. The labeling in triacylglycerol in the rats, given indomethacin prior to the administration of 20% ethanol, was 168% k 19 (n = 5), indicating that indomethacin did not have any significant effect. Incorporation of glycerol The cell-damaging agent

absolute

ethanol

elicited marked changes in the metabolism of exogenously injected fatty acids. We examined further whether these changes were due to alterations of de novo synthesis of the lipids by using [ 3H]glycerol and [‘4C]palmitic acid (Table II). The incorporation of glycerol into PC, PE and sphingomyelin was significantly decreased by the instillation of absolute ethanol, and the *4C/3H ratios of incorporated radioactivity in the ethanol-injured rats were not significantly different from the values of the saline control rats. The incorporation of palmitic acid into PI + PS was also decreased, while the incorporation of glycerol into this fraction was slightly reduced; conse-

378 TABLE

II

INCORPORATION

OF [3H]GLYCEROL

AND [‘4C]PALMITIC

ACID

AFTER

ABSOLUTE

ETHANOL

INSTILLATION

[3H]Glycerol and [14C]palmitic acid were injected intravenously 15 min after the absolut ethanol instillation and the rats were killed 30 min after the injection. Saline was instilled instead of absolute ethanol in the rats of the saline control group. Each value is the mean with SE. from five rats. Incorporation [ 3HIglycerol PC PE PI+Ps Sphingomyelin Triacylglycerol Diacylglycerol ’ Percent

of saline control.

of a

64.3+ 6.2’ 66.25 6.2 ’ 85.4+ 8.9 70.7i 8.1 b 100.4* 15.9 106.1k 6.9

l4 C/ 3H Ratio saline 0.738 0.687 1.03 1.29 2.15 1.56

b P i 0.05. ’ P < 0.01, d P < 0.001, significantly

quently the 14C/3H ratio was less in the ethanolinjured rats than in the saline control. Conversley, the incorporation of glycerol into diacylglycerol was not affected by the absolute ethanol instillation, while the incorporation of palmitic acid was greatly increased. These results indicated that de novo syntheses of PC, PE and sphingomyelin were inhibited by the absolute ethanol instillation, but the synthesis of PI + PS was less inhibited, and that the enhanced incorporation of fatty acids into diacylglycerol was not accompanied by the synthesis of diacylglycerol. Discussion Metabolic turnover in the gastric mucosa of labeled fatty acids exogenously injected into rats was investigated. The incorporation of fatty acids into triacylglycerol was completed within 15 min, and the label in triacylglycerol decreased quickly after 30 min, while the amount of labeled fatty acids incorporated into phospholipids slightly decreased during the observation period. A small portion of fatty acids in triacylglycerol may be transferred to phospholipids, because labeled oleic acid in phospholipids, especially in PC and PE, was slightly increased 15-60 min after the injection, but most of the fatty acids contained in triacylglycerol should be transferred quickly to the blood or oxidized in the cells. Therefore, triacylglycerol in mucosal cells should be a temporary storage pool for extracellularly supplied

ethanol iO.010 k 0.016 k 0.024 k 0.030 kO.072 +0.044 different

0.707 0.728 0.772 1.25 2.04 3.00

f 0.013 + 0.018 zk0.040 d k 0.044 kO.115 i 0.093 d

from saline control

fatty acids. Fatty acids contained in triacylglycerol are transferred to phospholipids in L fibroblasts and T-lymphocytes [25,26], and also released into the culture medium of endothelial cells as free fatty acid [27]. Palmitic acid was incorporated predominantly into the sn-1 position of PC and PE, whereas arachidonic acid was specifically incorporated into the ~2-2 position. It was found in rat liver and brain that the labeled palmitic acid are predominantly incorporated into the sn-1 position of phospholipids [28,29], and that arachidonic acid is specifically incorporated into the m-2 position by acyltransferase [30]. Our results are consistent with such observations. There is a disagreement as regards the fatty acid composition of PC between the present data and the results obtained by Wassef et al. [31], who found higher contents of palmitic acid than those shown in Table I. The difference may be due to the different methods used to prepare gastric mucosa, since we scraped the total stomach mucosa and they used ‘light scrapings’ of the stomach for analysis. In. addition, our results on the fatty acid composition of PC and PE showed similarly with those in microsomes and mitochondria of pig gastric mucosa [32,33], except for the arachidonic acid contents, which are less in pigs; therefore the fatty acid composition in the present studies should reflect mainly those in cellular membranes. Morphological and histological characterizations of ethanol-induced damage to gastric mucosa

319

have been carried out by many authors [34-371, although the intracellular metabolism of lipids has not previously been studied. The incorporation of fatty acids into phospholipids, PC, PE, sphingomyelin and PI + PS decreased rapidly after the instillation of absolute ethanol. Furthermore, there was no significant increase of labeling in lysophosphatides. The experiment using [3H]glycerol, together with the above results, indicated that de novo synthesis of PC, PE and sphingomyelin was decreased by the absolute ethanol instillation. The pretreatment with 20% ethanol (a mild irritant) maintained the incorporation of fatty acids into phospholipids such as PC and PE. Robert et al. found that the exposure of the gastric mucosa to a mildly damaging concentration of an agent such as 20% ethanol increases mucosal resistance to subsequent exposure to more damaging concentrations of the same agent such as absolute ethanol [19], and they proposed that these protective effects by the mild irritant are mediated by stimulating the release of prostaglandins by the stomach [20]. Ethanol can penetrate into mucosal cells when it is administered orally, and oral administration of prostaglandins does not prevent the entry of ethanol [34]. It is therefore likely that the pretreatment with 20% ethanol protects the cellular synthetic activity of phospholipids against damage by absolute ethanol. The instillation of absolute ethanol increased labeled fatty acids present in the free fatty acid fraction. This change seems due to the leakage of fatty acids from blood vessels to the gastric lumen and the penetration of extracellular fatty acids into the mucosal cells. This view is consistent with the histological evidence that the damage to gastric blood vessels is an early event in the development of gastric hemorrhagic lesions after the instillation of absolute ethanol, and the vascular permeability increases within l-3 min after the instillation [37]. The pretreatment with 20% ethanol reduced the increase of labeling in free fatty acid fraction suggesting that the pretreatment with 20% ethanol can protect the gastric blood vessels to some extent. The labeled fatty acids present in diacylglycerol and monoacylglycerol also increased rapidly after the instillation of absolute ethanol, implying that the inhibition of phospholipid

synthesis causes the increase in the labeling of these precursors. The uptake of fatty acids into triacylglycerol was increased by the pretreatment with 20% ethanol. Indomethacin failed to affect the enhanced uptake of arachidonic acid into triacylglycerol induced by the pretreatment with 20% ethanol; therefore, it is unlikely that prostaglandin itself enhanced the uptake of fatty acids into triacylglycerol. Although the mechanism of enhanced uptake of fatty acids into triacylglycerol remains to be resolved, it may be correlated with the stimulation of prostaglandin generation by the mild irritant. Several investigators have observed in other cells that the stimulation of prostaglandin synthesis by hormones and other agents enhances the incorporation of fatty acids into triacylglycerol [38-411. References 1 Coleman,

380

20 Robert, A., Nezamis, J.E., Lancaster, C., Davis, J.P., Field, S.O. and Hanchar, A.J. (1983) Am. J. Physiol. 245, G113-121 21 Bligh, E.G. and Dyer, W.J. (1959) Can. J. B&hem. Physiol. 37, 911-919 22 Ando, S., Kon, K., Tanaka, Y., Nagase, S. and Nagai, Y. (1980) J. Biochem. (Tokyo) 87, 1859-1862 23 Billah, M.M., Lapentina, E.G. and Cuuatrecasas, P. (1980) I. Biol. Chem. 255, 10227-10231 24 Barker, S.A., Monti, J.A., Christian, ST., Benington, F. and Morin, R.D. (1980) Anal. Biochem. 107,116-123 25 Tsai, Pi-Y. and Geyer, R.P. (1978) Biochim. Biophys. Acta 528, 344-354 26 Goppelt, M., Kohler, L. and Resch, K. (1985) B&him. Biophys. Acta 833, 463-472 27 Den&g, G.M., Figard, P.H., Kaduce, T.L. and Spector, A.A. (1983) J. Lipid Res. 24, 993-1001 28 Akesson, B., Elovson, J. and Arvidson, G. (1970) Biochim. Biophys. Acta 218, 44-56 29 Baker, R.R. and Thompson, W. (1972) B&him. Biophys. Acta 270, 489-503 30 Holub, B.J. and Kuksis, A. (1978) Adv. Lipid Res. 16, 1-111

31 Wassef, M.K., Lin, Y.N. and Horowitz, M.I. (1979) Biochim. Biophys. Acta 573,222-226 32 Sen, P.C. and Ray, T.K. (1979) Arch. B&hem. Biophys. 198, 548-555 33 Sen, P.C. and Ray, T.K. (1980) B&him. Biophys. Acta 618, 300-307 34 Robert, A., Lancaster, C., Davis, J.P., Field, S.O., Sinha, A.J.W. and Thomburgh, B.A. (1985) Gastroenterology 88, 328-333 35 Tarnawski, A., Hollander, D., Stachura, J., Krause, W.J. and Gergely, H. (1985) Gastroenterology 88, 334-352 36 Lacy, E.R. and Ito, S. (1985) Gastroenterology 88,619-625 37 Szabo, S., Trier, J.S., Brown, A. and Schnoor, J. (1985) Gastroenterology 88, 228-236 38 Raz, A. and Schwartzman, M. (1983) Biochem. Pharmacol. 32, 2843-2846 39 Downing, I., Hutchon, D.J.R. and Poyser, N.L. (1983) Prostaglandins 26, 55-69 40 Sivarajan, M., Hall, E.R., Wu, K.K,, Rafelson, M.E. and Manner, C. (1984) Biochim. Biophys. Acta 795, 271-276 41 Erman, A., Baer, P.G. and Nasjletti, A. (1985) J. Biol. Chem. 260,4679-4683