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Modulation of rat gastric mucosal prostaglandin E2 release by dietary linoleic acid: Effects on gastric acid secretion and stress-induced mucosal damage
GASTROENTEROLOGY
1988;95:18-25
Modulation of Rat Gastric Mucosal Prostaglandin E2 Release by Dietary Linoleic Acid: Effects on Gastric Acid Secretion and Stress-Induced Mucosal Damage WOLFGANG
SCHEPP,
BEATRIX
STEFFEN,
HANS-JORG
RUOFF,
VOLKER SCHUSDZIARRA, and MEINHARD CLASSEN Department of Internal Medicine II, Technical University of Munich, Munich, and Department of Pharmacology, University of Tiibingen, Tiibingen, Federal Republic of Germany
We studied chronic intake of diets deficient in or supplemented with linoleic acid to determine whether it affects gastric acid secretion, release of lesions. For prostaglandin E 2, and stress-induced 8-10 wk rats were fed three dietary regimens supplying 3.5% (control group), 0.3%,and 10% of total calories as linoleic acid. We found that diets deficient in linoleic acid (0.3%) reduced release of prostaglandin E2 into the gastric lumen (- 77%) and increased basal (+ 133%) and pentagastrin-stimulated acid secretion (+93%) and the area of cold restraint-induced gastric mucosal lesions (+ 280%), when compared with the control group. Diets supplemented with linoleic acid (10%) increased prostaglandin Ez release into the gastric lumen (+106%) and reduced basal (-44%) and pentagastrin-stimulated acid secretion (- 78%) and the area of cold restraint-induced mucosal lesions (-80%). Prevention of these lesions by the 10% linoleic acid diet was confirmed by quantitative histology. Pretreatment with indomethacin (8 mg/kg intraperitoneally) abolished the effects of the 10% linoleic acid diet on prostaglandin formation, acid secretion, and mucosal injury. We conclude that in rats chronic intake of dietary linoleic acid reduces acid secretion and prevents cold restraint-induced mucosal lesions, possibly because of augmented synthesis of endogenous prostaglandins in the gastric mucosa.
xogenously administered prostaglandins have been studied extensively with respect to their antisecretory and protective effects on the gastric mucosa (1,Z). However, considerably less is known about the role of endogenous prostaglandins. Inhibi-
E
tion of cyclooxygenase by indomethacin has been used to demonstrate the effect of impaired endogenous prostaglandin production on gastric acid and bicarbonate secretion in rats (3), dogs (4), and humans (5,6). Oral administration of arachidonic acid (AA) or linoleic acid (LA) offers another opportunity to study the role of endogenous prostaglandins as these essential fatty acids are precursors for the biosynthesis of many prostaglandins (7). It has been reported that in rats acute intragastric administration of AA (8,9)or LA (10)is followed by a marked increase of gastric mucosal prostaglandin release and protection against alcohol-induced injury, which is supposed to be independent of gastric acid. In this study we chose a different approach: diets deficient in or supplemented with LA were supplied on a long-term basis, and the prevention of cold restraint-induced gastric mucosal lesions, which are supposed to be dependent on gastric acid secretion, was studied. Formula diets supplying 10% of the total dietary energy as LA have been shown to stimulate endogenous prostaglandin formation in rats (11,12). Modulation of endogenous prostaglandin production by dietary LA intake has been reported to influence physiologic functions in various tissues, e.g., platelets (13) and kidney (14). The objectives of our study were to examine the effect of chronic modulation of dietary LA intake (a) on basal and stimulated gastric acid output and (b) on prevention of stress-induced gastric mucosal lesions. Our hypothesis was that deprivation of dietary LA Abbreviations used in this paper: AA, arachidonic acid; LA, linoleic acid; PGE, prostaglandin E. 6 1988 by the American Gastroenterological Association 0016-5085/88/$3.50
LINOLEIC
July lY88
might reduce formation of endogenous prostaglandins, augment acid secretion, and increase the number of cold restraint-induced mucosal lesions; on the formaother hand, high dietary LA might augment tion
of endogenous
cretion,
and
should report form
prostaglandins,
prevent
be reversed of these
mucosal
reduce
lesions.
by indomethacin.
studies
has been
acid
These
se-
effects
A preliminary
published
in abstract
(15).
Materials and Methods Dietary
Regimens
Female Wistar rats (250-270 g) were fed three isocaloric, semisynthetic formula diets (Altromin, Lage, F.R.G.) that differed only by their content of LA. In a standard rat diet 3.5% of the total dietary energy is supplied as LA. Hence, in this study the control group was fed a 3.5% LA diet. Two test groups received diets containing 0.3% and 10% LA, respectively. The animals had free access to water and were housed in individual cages to control their daily dietary intake. Wire-bottom cages were used to prevent coprophagg. From these data LA intake per day was calculated for each dietary group (Table 1). Weight controls were performed weekly, demonstrating comparable weight gain in the three groups (Table 1). Experiments
in Fistula
Rats
To adapt the rats to semisynthetic formula diets 30 rats received the 3.5% LA control diet for 2 wk. A gastric fistula was implanted under hexobarbital anesthesia (60 mgikg intraperitoneally). Thereafter the animals were arbitrarily divided into three groups of 10 rats each. The control group continued receiving the 3.5% LA diet and the test groups were fed the 0.3% and loo/, LA diets for 10 wk. After 2. 4, 6, and 8 wk gastric secretion was collected through the fistulas. Before these experiments the animals were fasted for 24 h with free access to a 5% glucose solution that was removed 1 h before the experiment. All experiments started at 8 ‘~1 and were performed in the same laboratory (22” + 1°C humidity 60%-70%). Secretions from the first 30-min collection period were discarded. Then gastric juice was continuously collected for three 60-min periods. After centrifugation of the 60..min sam-
Table
I. Food Intake, Linoleic Acid Intake, and Body Weight of 120 Rats Before Diet and After 8 Weeks on the Diets’
Food intake Diet
3.5% LA 0.376LA 10.0% LA LA, linoleic
(g/day)
14.3t 1.8 14.7t 0.6 13.2t 1.4 acid.
” Mean
Linoleic acid intake (Wdoy)
501 k 47 40 k 6 1323 r 149 t SEM
Body weight before diet
Body weight after 8 wk
&I
(Sl
200 k 30 220 ? 35 210 r 25
270 2 35 280 i 30 275 t 25
ACID AND GASTRIC MUCOSAL DAMAGE
19
ples, volume was measured to the nearest 0.01 ml; a lOO-~1 sample was taken to determine acid output by automatic titration to pH 7 with 0.1 N sodium hydroxide. The remaining supernatant was stored at -20°C until assayed for prostaglandin E, (PGEJ within 2 wk. Basal acid output and PGE2 release into the gastric juice were measured every 3 h and were calculated as the sum of the three 60-min periods. After 8 wk, acid output and PGE, release were also determined during stimulation by pentagastrin (125 pglkg). After discarding the initial 30-min secretion, gastric juice was collected over a 60-min period for measurements of basal acid output and PGEZ release. Then the respective stimulant was intraperitoneally administered and gastric secretion was collected for two more 60-min periods. After 10 wk on the diet the animals were killed by cervical dislocation. The stomachs were removed and placed on ice; IO-15-mg specimens were immediately taken from the fundic mucosa. Each specimen was incubated for 60 min at 37°C in a shaking bath [I5 mg of mucosa per milliliter of buffer (mmol/L: NaH2P04, 0.5; Na2HP0,, 1.0;NaHC03, 20; NaCl, 70; KCl, 5; CaCIZ, 1.0; MgC12, 1.0; glucose, 11; 4-(2-hydroxyethyl)-l-piperazineethane sulfonic acid, 50; bovine serum albumin, 1 mg/ml, pH 7.8)]. At the end of the incubation PGE2 release into the medium was determined in 100-p] duplhates. Prostaglandin E, release (picograms per milligram of mucosa per 60 min) was calculated from the duplicates of each individual animal and the mean PGEz release of the dietary group was documented. Experiments
in Pylorus-Ligated
Rats
Another 30 rats were arbitrarily divided into three groups receiving the different dietary regimens. After 5 wk the animals were fasted for 24 h with free access to 5% glucose solution which was removed 1 h before the experiment. The pylorus was ligated under hexobarbital anesthesia (60 mgikg intraperitoneally). After 3 h the animals were killed by cervical dislocation. The stomachs were removed and the gastric juice was collected for measurements of acid output and PGEz release. Effect
of Cold-Restraint
Stress
A combination of cold and restraint was used to produce stress ulcers in rats (16).Three groups of 10 rats each were fed the different diets for 5 wk. After being fasted for 24 h with free access to 5% glucose solution, the animals were put into individual close-fitting restraint cages and were exposed to a temperature of 4°C for 3 h. Thereafter all rats were killed by cervical dislocation, and their stomachs were excised and opened along the greater curvature. The mucosa was examined by an observer who was unaware of the dietary pretreatment. The incidence of lesions per rat was recorded and the lesion area was determined using a 1 x l-mm calibrated grid under a dissecting microscope (X 20) (I 7). When assessing the size of petechiae, five such lesions were considered equivalent to a l-mm’ ulcer. The sum of the lesion area in each group was divided by the number of rats in that group. Mean
20
SCHEPP ET AL.
GASTROENTEROLOGY Vol. 95.No. 1
lesion area per dietary group was expressed in square millimeters. Prostaglandin Ez release from macroscopitally intact fundic mucosa of the three groups was measured as described above. Experiments
in the Presence
Before being assayed gastric juice was adjusted to pH 7 by 0.1 N NaOH. In preliminary experiments we demonstrated that PGEz extraction by ethylacetate did not enhance sensitivity of measurements; therefore this procedure was not employed in the present study.
of Statistical
lndomethacin A cyclooxygenase inhibitor was used to determine if the effects of the 10% LA diet on PGE2 release and acid secretion were caused by increased endogenous prostaglandin production. In fistula rats receiving 10% LA, PGEz release and acid secretion were measured after 2, 4, 6, and 8 wk as described above; 3 h before the experiments, indomethacin (8 mgikg) was injected intraperitoneally. Histologic
Evaluation
In additional rats cold-restraint experiments were performed after 8 wk on the test diet as described above. Three hours before exposure to stress the animals received a &ml intraperitoneal injection of either vehicle (sodium bicarbonate solution) or indomethacin (8 mg/kg, prepared immediately before use). We studied five groups of rats (8 rats each) that were fed the following diets: 0.3% LA plus vehicle, 3.5% LA plus vehicle, 3.5% LA plus indomethacin, 10% LA plus vehicle, and 10% LA plus indomethacin. After 3 h of cold restraint the animals were killed by cervical dislocation. Standard mucosal strips of the gastric wall (0.5 cm in width, extending through the entire glandular oxyntic mucosa) were obtained from all animals. Specimens were fixed in 10% buffered formalin and stained with hematoxylin and eosin (H&E) and with Alcian blue (pH 2.5) and periodic acid-Schiff. Coded mucosal specimens were evaluated under light microscopy by two investigators unaware of the code. During histologic evaluation special attention was given to the integrity of the surface epithelial cells, presence of erosions, and submucosal edema. We also performed morphometric measurements of the extent of deep histologic necrosis (defined as necrotic lesions penetrating into the mucosa deeper than 0.2 mm). We measured the length of the mucosal strip and total length of necrotic lesions for each strip with the aid of a projecting lens (Nikon Inc., Garden City, N.Y.), which projected the image onto the stage of a Microplan II image analysis system. Radioimmunoassay
for Prostaglandin
E,
Prostaglandin Ez was determined by a specific radioimmunoassay using [3H]PGEz. Antibody against PGEz was raised in rabbits. The following cross-reactivities were found: prostaglandin AZ, 0%; thromboxane Bz, 0%; and PGE1, 13%. Lower and upper sensitivities were 10 and 1000 pg/lOO ~1 assay volume, respectively. Nonspecific binding was 4.1% k 0.3%. The recovery of 100 pg of PGEz added to gastric juice in vitro was 94% * 5% when measured immediately after addition and 81% 2 5% when determined after 1 h (n = 5 measurements). Duplicate samples (100 ~1) of gastric juice or incubation medium were assayed for PGEz within 2 wk after the experiments.
Analysis
The data concerning acid secretion and PGEz were analyzed by Student’s t-test for groups. Statistical comparisons of the extent of deep histologic necrosis were performed with the Kruskall-Wallis and Wilcoxon tests (18). Materials The following semisynthetic formula diets were purchased from Altromin: C 1000 (3.5% LA), C 1000 modified (10% LA), and C 1058 (0.3% LA). Hexobarbital was purchased from Bayer (Leverkusen, F.R.G.); Gastrodiagnost (pentagastrin) was purchased from Merck (Darmstadt, F.R.G.); bovine serum albumin, 4-(2-hydroxyethyl)l-piperazine-ethane sulfonic acid, and polyethylene glycol 6000 was purchased from Serva (Heidelberg, F.R.G.); and [3H]PGEz was purchased from DuPont (Montreal, Canada).
Results in Fistula
Experiments
Rats
Gastric juice was collected under basal condiafter 2, 4, 6, and 8 wk from fistula rats on the
tions three
dietary
regimens.
During
the
entire
period
secretion volumes did not significantly differ between the dietary groups. Prostaglandin E2 release into the gastric juice is shown in Figure 1, left. After 2 wk mean PGEz release was 25.9 2 3.7 ngl3 h in the control
group,
20.7
group,
and 50.1
t
During
the
entire
from the 0.3% controls,
*
4.8
ng/3 h in the
3.0 ng/3 h in the 10% observation
LA group
whereas
period
was lower
release
in
the
0.3%
LA
LA group.
PGE2
release
than
that in the
10%
LA
group
exceeded that in the controls. The differences between the 10% LA group and controls and the 10% LA group and 0.3%
LA group were significant
the
period
entire
difference
dietary between
was significant
(p <
the 0.3%
only
O.Ol),
LA group
during
whereas
the
and controls
after 4 wk (p < 0.01)
and 8 wk
(p < 0.05). The simultaneous measurements of gastric acid output in these experiments are shown in Figure 1, right. After 2 wk mean acid output was 147.8 2 7.9 pEql3
h in the control
group,
214.1
? 24.8 pEq in the
0.3% LA group (p CO.01 vs. controls and vs. 10% LA group), and 83.2 ? 11.4 PEq in the 10% LA group. The latter continued to produce less acid than controls (p < 0.05 after 6 and 8 wk), whereas in 0.3% LA group more acid was secreted than in controls. Maximal acid output occurred after 4 [317.7
t
24.0
pEq/3 h (p < 0.01
vs. control
the the the wk
and vs.
July 1988
++ 4 k_l **
-300
70-
l
*
++
XX
60-•+
*t i c7 5cxx xx ‘m xx c c LO- xx 0
o-,+, 2 L
+ +
l
*
l
9 100 s 2
-50 I 6
l171T0 2108
a weeks
Figure
Experiments
159 z
x2_
lo-
6
I. Gastric fistula rats: PGE, release (left) and acid secretion (right). For 8 wk 30 fistula rats (10 animals per group) were fed three isocaloric semisynthetic diets that differed only by LA content [3.5% of the total dietary energy, controls (m); 0.3% LA (A), and 10% LA (O)]. The animals were fasted for 24 h with free access to 5% glucose only. After discarding the initial 30-min sample, gastric juice was collected for three consecutive 60-min periods under basal conditions. The samples were adjusted to pH 7 and PGE, was radioimmunologically determined. Prostaglandin I$ release per animal was calculated as the sum of the three consecutive 60-min periods. Prostaglandin E2 release (mean + SEM) is plotted as nanograms per 3 h during 8 wk on the diets. Acid output was measured by automatic titration to pH 7 with 0.1 N sodium hydroxide in gastric juice samples of three consecutive 6o-min periods. Acid output (mean + SEM) is plotted as microequivalents per 3 h during 8 wk on the diets. Statistical analysis was performed by Student’s t-test for groups. *ix.‘+. p < 0.05; **lxx/++, p i 0.01. Comparisons: 10%) LA vs. control, t; loo/, LA vs. 0.3%1 LA, x: 0.3% LA vs. control, A.
in Pylorus-Ligated
kntacptnn
DAMAGE
21
Rats
l
1 *
**
Penkgast nn **
J
1Ol +
25 _
t
XX XX
_+I
20
xx
XX
+
-*
il rh xx
-60
l
LA)].
l
40
*
I
0
0.001).
After 8 wk PGEz release and acid output were measured during stimulation by pentagastrin (125 kg/kg intraperitoneally; Figure 2). Similar to the experiments under basal conditions, PGEz release differed between the three dietary groups. In the presence of pentagastrin, PGEz release tended to decrease from basal level, yet without attaining the level of statistical significance. One hour after stimulation, acid secretion in the 0.3% LA group was higher than in the controls (p < 0.05), whereas the 10% LA group revealed a much weaker response (p < 0.05 vs. controls; p < 0.01 vs. 10% LA group). To reexamine our in vivo results concerning PGEz release into the gastric juice, the fistula rats were killed after 10 wk on the test diets and PGE, release from fundic mucosal specimens was measured in vitro. Mean PGE, release was 762.7 + 98.5 pgimg
MUCOSAL
After ligation of the pylorus, the stomach becomes increasingly distended because of accumulation of gastric juice. We used this experimental model to study the effect of dietary LA on distentionstimulated acid secretion and PGEz release (Figure 3). Mean PGE, release was 12.6 ? 1.7 ng/3 h in the controls, 16.3 + 0.9 ngi3 h in the 10% LA group, and 7.5 2 0.7 ngl3 h in the 0.3% LA group (p < 0.05 vs. controls; p < 0.01 vs. 10% LA group). As expected
Acid secretion during the 8 wk on the test diet was inversely related to the release of PGE, Jr = 236.2 - 2.3x; r = - 0.75; p < (linear regression: 10%
ACID AND GASTRIC
mucosa per hour in the controls, 1498.7 IT 161.2 pg in the 10% LA group (p < 0.01 vs. controls), and 470.2 i 70.2 pg in the 0.3% LA group (p < 0.05 vs. controls; p < 0.01 vs. 10% LA group).
g +-200 f xx xx =
Pqf Y a
-250
++
LINOLEIC
10
I-73
l-73 h Figure
2. Gastric fistula rats: PGE, release [left) and acid secretion (right] during stimulation by pentagastrin (125 fig/kg). Thirty fistula rats (10 animals per group) were fed three isocaloric semisynthetic diets [3.5% LA, controls (W); 0.3% LA (A); 10% LA (e)]. After 8 wk the initial 30-min sample of gastric juice was discarded and basal PGE, release and acid secretion were determined in the following 60-min portion. Then pentagastrin was administered intraperitoneally and gastric juice was collected for two additional 60.min periods. Measurements of PGE, (radioimmunoassay) and acid (titration) were performed as described in the legend to Figure 1. Prostaglandin EZ release (ngih) and acid output (FEq/h) are shown for each dietary group (mean + SEMI. Statistical analysis was performed by Student’s t-test for groups. *lx/+, p < 0.05: **/xx/++, p < 0.01. Comparisons: 10% LA vs. control, * : 10% LA vs. 0.3% LA, x; 0.3% LA vs. control, +.
22
SCHEPP
ET AL.
GASTROENTEROLOGY
*
from our experiments in fistula rats, the 0.3% LA group revealed a higher acid secretion (336.12 24.7 pEq/3 h) than the controls (171.3 + 20.6 pEq/3 h; p < O.Ol), and the 10% LA group (147.2 ? 16.4 pEqi3 h; p < 0.01). Effect of Cold-Restraint
Effect of lndomethacin The cyclooxygenase inhibitor indomethacin was used to examine whether reduced acid secretion **
** I
I
,
l *
* -
-
g20\
I
F
-330
I
-glS _
fl, WN 5_
05 0.3% Figure
3
3.5%
10%
- * n
Stress
Our studies hitherto demonstrated that dietary LA can modulate release of gastric mucosal PGEz and that this modulation is inversely related to the patterns of gastric acid secretion. Because, in addition, prostaglandins are supposed to prevent peptic lesions, we studied the incidence of coldrestraint ulcers during modulated LA intake (Figure 4). The lesion area in the control animals (4.0 + 1.1 mm”) was smaller than that in the 0.3 LA group (15.2 ? 2.6 mm’; p < 0.05), whereas the 10% LA group revealed a much smaller lesion area (0.8 ? 0.4 mm’) than the controls (p < 0.05) and the 0.3% LA group (p < 0.01). Prostaglandin E2 release from fundic mucosal specimens was 912.7 ? 152.3 pgimg mucosa per hour in the controls, 632.9 ? 64.9 pg in the 0.3% LA group (p < 0.05 vs. controls), and 1516.2 If: 219.8 pg in the 10% LA group (p < 0.05 vs. controls; p < 0.01 vs. 0.3% LA group). The inverse correlation between lesion area and PGE, release of the three dietary groups was corroborated by calculating the linear regression (y = 1289 - 43.3x; r = -0.63; p < 0.01).
t
20 1
l
0.3%
3.5%
10%
Prostaglandin Ez release and acid secretion in pylorusligated rats. After 5 wk on the diets [3.5% LA, controls (0); 0.3% LA (U); 10% LA (m); n = 10 rats per group] pylorus ligation was performed under hexobarbital anesthesia. Prostaglandin EZ release (left) and acid output into the gastric juice (right) was determined after 3 h. For further details see legends to Figure 1. *, p < 0.05; **, p < 0.01.
a3% 3.5%10% Figure
l
1 l
Vol. 95, No. 1
l
*
1
-II
1
03% 35% 10%
4. Cold restraint-induced gastric mucosal damage after 5 wk on the diets [3.5% LA, controls (0); 0.3% LA (BI); 10% LA (me); n = 10 rats per group]. Animals were fasted for 24 h with free access to 576 glucose solution only. After exposure to 4°C for 3 h in close-fitting restraint cages the animals were killed. Using a 1 x l-mm eye grid under the dissecting microscope (X ZO), the incidence and area of gastric mucosal lesions were determined. Mean lesion areas of the dietary groups are plotted as square millimeters (left). In addition, specimens from macroscopically intact fundic mucosa were used for duplicate measurements of PGE, release in vitro during 60 min of incubation at 37°C in a shaking bath. Mean PGEz release from the specimens of the three dietary groups is plotted as picograms per milligram of mucosa per 60 min (right).
and reduced occurrence of stress-induced lesions were caused by increased formation of endogenous prostaglandins in the 10% LA group. Simultaneous with the experiments shown in Figure 1, PGEz release and acid secretion were measured in fistula rats on the 10% LA diet receiving indomethacin (8 mg/kg intraperitoneally) 3 h before the experiments. Under these conditions PGE2 release into the gastric juice ranged between 7.7 ? 0.3 ngi3 h (2 wk) and 9.4 ? 0.4 ng/3 h (8 wk; Figure 5, left) and acid secretion was 194 + 19 pEq/3 h (2 wk) and 194 -+ 12 pEql3 h (8 wk; Figure 5, right). Comparison of these results with the data given in Figure 1 demonstrates that in animals on the 10% LA diet indomethacin reduces the PGEz release and increases acid secretion to the same levels simultaneously measured in the 0.3% LA group (p < 0.01 vs. 10% LA diet without indomethacin after 2, 4, 6, and 8 wk) (see Results, Experiments in Fistula Rats). After 8 wk on the 10% LA diet and 3 h after indomethacin (8 mg/kg intraperitoneally), PGEz release from fundic mucosal specimens in vitro was 85.1 r 11.8 pg/mg mucosa per hour (p < 0.01 vs. 10% LA without preceding indomethacin (see Results, Experiments in Fistula Rats).
LINL’OLEIC ACID AND GASTRIC
luly 1988
o’-
2
1
0
-
8
2
1
6
8
Lo
weeks Figure
5. Gastric fistula rats: PGE? release (left) and acid secretion (right] under basal conditions. Simultaneously with the experiments shown in Figure 1, 10 fistula rats were fed the 10% LA diet (01 for 8 wk. Three hours after administration of indomethacin (8 mgikg i.p.), PGE, release (radioimmunoassay) and acid output (titration] were measured as described in the legend to Figure 1. For better comparison the dashed line shows the results with lOoh, LA in the absence of indomethacin (Figure 11. +*, p < 0.01 (10% LA + indomethacin vs. 10% LA alone].
Histologic
Evaluation
In the animals on the 0.3% LA diet, cold restraint produced prominent histologic changes: exfoliation of the surface epithelium, deep necrotic lesions of focal character penetrating into half of the entire mucosal thickness, and edema of the lamina propria and submucosa. Deep necrotic lesions involved 1.87% ? 0.5% of the mucosal section length in the 0.3% LA group and 1.4% ? 0.4% in the controls. In contrast, in rats receiving the 10% LA diet deep necrotic lesions were markedly reduced to 0.04% ? O.OZ%, of the mucosal section length (p < 0.05 vs. 0.3% LA group). Prevention of cold restraintinduced mucosal lesions by the 10% LA diet was more than reversed by pretreatment with indomethacin: deep necrotic lesions involved 3.70%, 2 0.8% of the mucosal section length in this group (p < 0.01 vs. 10% LA diet without indomethacin). Thus, in rats on the 10% LA diet indomethacin increased the stress-induced damage to a lesser extent than in the control animals (5.7% f 0.7% of the mucosal section length; p < 0.05 vs. 10% LA diet plus indomethacin).
Discussion This study demonstrates that in rats deprivation of dietary LA is followed by reduced gastric mucosal PGE;! release, augmented gastric acid secretion, and increased occurrence of stress-induced lesions. On the other hand, animals fed an LA-rich diet revealed enhanced gastric mucosal PGE, re-
MLJCOSAL DAMAGE
23
lease, reduced acid output, and reduced occurrence of mucosal lesions. Indomethacin completely reversed these effects of the 10% LA diet, suggesting that they are due to increased formation of endogenous prostaglandins. These data support a role for endogenous prostaglandins in the regulation of gastric acid secretion and a potential role in the prevention of stress-induced mucosal lesions by additional activation of defense mechanisms. To date, however, experiments with indomethacin do not unequivocally prove such a role for prostaglandins: the agent increased stress-induced lesions in animals receiving the control diet, indicating that indomethacin might exert additional effects unrelated to inhibition of the cyclooxygenase. Linoleic acid, a polyunsaturated fatty acid, is an essential dietary constituent (19). Standard rat diets supply 3%-4%, of total dietary energy as LA, equivalent to 480 mg LA per day. Therefore, a 0.3% and a 10% LA diet should result in sufficiently different LA intake to study the effects of LA deprivation or enrichment, respectively. The enzyme delta-6desaturase transforms LA to AA (ZO), which is the precursor for the formation of various prostaglandins. Because PGEz is a main prostanoid formed in the gastric mucosa and its antisecretory and protective effects have been studied in detail, we decided to measure PGE, release from the rat stomach both in vivo and in vitro. This does not exclude the possibility that changes of membrane lipids or prostanoids other than PGE, might also be involved in mucosal protection or inhibition of acid secretion during modulation of dietary LA. As tissue levels offer limited information because of the immediate release of newly synthesized prostaglandins from their cells of origin (al), we measured PGE, release into the gastric juice. In addition, these in vivo results were confirmed by determining PGEz release from fundic mucosal specimens in vitro. To examine whether modulation of gastric mucosal PGE, release by dietary LA influences gastric functions that are partially dependent on PC&, we studied gastric acid secretion and occurrence of stress-induced lesions. Prostaglandins have been reported to reduce gastric acid secretion by inhibiting the histaminesensitive adenylate cyclase of the parietal cell (22, 23); recently it was reported that they interfere at the (24). In addition, inhibitory subunit of that enzyme prostaglandins have an overall protective effect on the gastric mucosa. This effect consists of mucus and bicarbonate secretion, maintenance of the microvascular integrity, and production of surface-active phospholipids providing a nonwettable hydrophobic surface barrier above the epithelium (1,2). Modulations of prostaglandin formation by rat
24
SCHEPP
ET AL.
heart, aorta, and platelets have been achieved by dietary LA enrichment and deprivation, respectively (13).Similarly, our results demonstrate that PGE2 release from the rat gastric mucosa can be modulated by comparable LA diets. Several lines of evidence indicate that endogenous prostaglandins are involved in the regulation of gastric acid secretion: acid secretion was increased during LA deprivation and reduced during high LA intake; this reduction was prevented by indomethacin. Similarly, Konturek and colleagues (25) observed inhibition of gastric acid secretion during intraarterial or topical application of AA in the dog. Using different approaches, our diet experiments and Konturek’s acute application of prostaglandin precursors are in line with observations of increased basal or stimulated acid secretion during pharmacologic inhibition of endogenous prostaglandin formation in rats (3), dogs (41, and humans (5,6). As evidenced by quantitative histology, our 10% LA group revealed significant reduction of stressinduced lesions. Furthermore, we found a significant correlation between the reduced mucosal lesion area and PGEz release in the dietary groups, but the 10% LA diet did not prevent mucosal lesions after pretreatment with indomethacin. Thus our data suggest that the dietary precursor LA prevents stressinduced gastric mucosal lesions by induction of endogenous prostaglandin formation. The results of our dietary approach are in accordance with reports on rat gastric mucosal damage during indomethacininduced pharmacologic inhibition of endogenous prostaglandin formation (26). In our 10% LA group, acid secretion was reduced; on the other hand, cold restraint-induced lesions are known to depend on the presence of acid. Therefore, it cannot be decided from this study how much “protection” (27) contributed to the observed benefit of our LA-rich diet and how much was brought about by the inhibition of acid output. As acute administration of LA has been demonstrated to prevent alcohol-induced lesions (10) (which are known to be independent of gastric acid], it must be taken into consideration that LA not only inhibits acid secretion but, in addition, might also activate mucosal defense mechanisms. Protection of the rat gastric mucosa against alcohol-induced injury has been accomplished by acute intragastric administration of AA (8,9). Yet, there are differences between these experiments and our approach. Administering dietary LA on a long-term basis, we studied the effects of providing higher substrate concentrations for the synthesis of endogenous prostaglandins rather than the acute effect of a single excessive bolus of the ultimate precursor AA. According to physiologic needs, LA is transformed to AA and converted into prostaglandins, thereby
GASTROENTEROLOGY
Vol. 95, No. 1
resembling more closely the natural stimulation of prostaglandin formation. The delayed conversion of LA into prostaglandins might be reflected by the observation that PGE, concentration in the gastric juice of our LA-rich fed rats was lower than that reported after intragastric application of AA (8). Another explanation would be that-in contrast to Hollander and colleagues (8)-we did not include detergents to solubilize and enhance absorption of LA. In the study of Hollander and colleagues (8) intrajejunally administered AA failed to confer protection comparable with that after intragastric AA, indicating that direct contact of AA with the gastric mucosa is required for its acute action. An explanation for the failure of intrajejunal AA would be that immediately upon intestinal absorption AA is converted into prostaglandins, which are deactivated during the first pulmonary passage. From our data it cannot be excluded that a minor portion of dietary LA was directly converted into prostaglandins by the gastric mucosal cells before LA had reached the small intestine. However, the greater proportion of LA is absorbed in the small intestine (28) and transported by chylomicrons to peripheral cells (29), where enzymatic transformation into prostaglandins is performed. Therefore, in all likelihood LA does not require direct contact with the gastric mucosa for its chronic protective action.
References 1. Miller TA. Protective effects of prostaglandins against gastric mucosal damage: current knowledge and proposed mechanisms. Am J Physiol 1983;245:G601-23. 2. Hawkey CJ, Rampton DS. Prostaglandins and the gastrointestinal mucosa: are they important in its function, disease, or treatment? Gastroenterology 1985;89:1162-88, 3. Ligumsky M, Goto Y, Debas H, Yamada T. Prostaglandins mediate inhibition of gastric acid secretion by somatostatin in the rat. Science 1983;219:301-3. 4. Miller TA, Henagan JM, Kuo YJ, Shanbour LL. The role of sodium transport in prostaglandin cytoprotection. In: Allen A, Flemstrom G, Garner A, Silen W, Turnberg L, eds. Mechanisms of mucosal protection in the upper gastrointestinal tract. New York: Raven, 1984:357-61. EH. Effects of indomethacin on basal 5. Levine RA, Schwartzel and histamine stimulated human gastric acid secretion. Gut 1984;25:718-22. on gastric acid 6. Feldman M, Colturi TJ. Effect of indomethacin and bicarbonate secretion in humans. Gastroenterology 1984;87:1339-43. Lands WEM. Fatty acid-prostaglandin (autacoid) relationship. In: Boers RF, Bassett EG, eds. Nutritional factors: modulating effects on metabolic processes. New York: Raven, 1981:485-8. Hollander D, Tarnawski A, Ivey KJ, et al. Arachidonic acid protection of rat gastric mucosa against ethanol injury. J Lab Clin Med 1982;100:296-308. Tarnawski A, Hollander D, Stachura J, Krause WJ. Arachidonic acid protection of gastric mucosa against alcohol inju-
LINOLEIC
July 1988
10.
11,
12.
13.
14.
15.
16.
17.
18. 19.
20.
21.
22.
ry: sequential analysis of morphologic and functional changes. J Lab Clin Med 1983:102:340-51. Tarnawski A, Hollander D, Krause WJ, et al. Is linoleic acid (dietary essential fatty aciti-EFA) cytoprotective for the gastric mucosa (abstr)? Gastroenteroiogy 1985;88:1610. JMathias MM, Fine K, Lipinski B, DuPont J. Dietary linoleate effect upon prostaglandin and thromboxane synthesis. Prostaglandins 1978;15:706-10. Nugteren DH, van Evert WC, Soeting WH, Spuy JH. Effects of different amounts of linoleic acid in the diet on the excretion of urinary prostaglandin metabolites in the rat. In: Samuelsson B, Ramwell P, Paoletti R. eds. Advances in prostaglandin and thromboxane research. Volumes 6-8. New York: Raven, 1980:1793-6. tenHor F, deDeckere EAM. Haddeman E, Hornstra G, Quadt JFA. Dietary manipulation of prostaglandin and thromboxane synthesis in heart, aorta and blood platelets of the rat. In: Samuelsson B, Ramwell P. Paoletti R, eds. Advances in prostaglandin and thromboxane research. Volume 8. New York: Ralren, 1980:1771-81. Adam 0. Wolfram G, Zollner N. Effect of different linoleic acid intake on prostaglandin biosynthesis and kidney function in man. Am J Clin Nutr 1984;40:763-70. Schepp W. Steffen B, Ruoff H-J. Classen M. Effect of dietary linoleic acid on acid secretion and cold restraint ulcers in rats (abstr). Gastroenterology 1986;90:1617. Senay EC, Levine RJ. Synergism between cold and restraint for rapid production of stress ulcers in rats. Proc Sot Exp Biol Med 19f~7;124:1221-5. Waisman Y, Dinari G, Marcus H, et al. Naloxone is protective against indomethacin-induced intestinal ulceration in the rat. Gastroenterology 1985;89:86-91. Cono\er WJ. Practical nonparametric statistics. New York: John Wiley & Sons, 1971:39. FXO/WHO. Dietary fats and oils in human nutrition. A joint FAOWHO report. Rome: Food and Agriculture Organization of the United Nations, 1977. Brenner RR. Regulatory function of delta-6-desaturase, key enzyme of polyunsaturated fatty acid synthesis. Function and biosynthesis of lipids. In: Bazan NG, Brenner RR, Giusto NM, eds. Advances in experimental medicine and biology. Volume 83. New York: Plenum, 197785-101. Schepp W, Steffen B, Schusdziarra V, Classen M. Calcium, calmodulin. and cyclic adenosine monophosphate modulate prostaglandin E,-release from isolated human gastric rnucosal cells. J Clin Endocrinol Metab 1986;63:886-91. Sol1 AH. Specific inhibition by prostaglandins E, and I, of
ACID AND GASTRIC
histamine-stimulated
23.
24.
25.
26.
27.
28.
29.
MIJCOSAI,
[‘“Cl aminopyrine
DAMAGE
ancl cyclic
25
adenosine
monophosphate generation by isolated canine parietal cells. J Clin Invest 1980;65:1222-9. Schepp W, Ruoff H, Maslinski S. Aminopyrine accumulation of isolated parietal cells from the rat stomach. Effect of histamine and interaction with endogenous inhibitors. Arch lnt Pharmacodyn Ther 1983;265:293-308. Sol1 AH, Berglindh Th. Physiology of isolated gastric glands and parietal cells: receptors and effecters regulating function. In: Johnson LK, ed. Physiology of the gastrointestinal tract. New York: Raven, 1987:883-909. Konturek SJ, Mikos E, Pawlik W, Walus K. Direct inhibition of gastric secretion and mucosal blood Bow by arachidonic acid J Physiol 1979;286:15-28. Whittle BJR. Temporal relationship between cyclooxygenase inhibition as measured by prostacyclin biosynthesis and the gastrointestinal damage induced by indomethacin in the rat. Gastroenterology 1981;80:94-8. Robert A. Antisecretory. antiulcer, cytoprotective and diarrheogenic properties of prostaglandins. In: Samuelsson B. Kamwell PW, Paoletti R. eds. Advances in prostaglandin and thromboxane research. Volume 2. New York: Raven, 197fi: 507-20. Chow SL, Hollander 1). Linoleic absorption in the unanesthetized rat: mechanisms of transport and inlluence of luminal factors on absorption. Lipids 1978:13:378-85. Lewis B. The LDL theory and HDL hypothesis. In: Noseda C. Lewis B, Paoletti R. eds. Diets arrd drugs in atherosclerosis. New York: Raven, 1980:1-8.
Received May 5. 1986. Accepted November 30, 1987. Address requests for reprints to: Wolfgang Schepp, M.D., Medizinische Klinik II, Technischr Ilniversitat, Klinikum rechts der Isar, Ismaninger Strasse 22, LD 8000 Muncher1 80, Federal Republic of Germany This study was supported by grant Sche 22912-l from the Deutsche Forschungsgemeinschaft. This work was presented in part at the meeting of the American Gastroenterological Association, May 18-21, 1986, in San Francisco, California. The authors thank Heumann Pharmaceutics, Niirnberg, Germany for generous support: Mrs. C. Tatge for ter hnical assistance; and Professor Andrzej S. Tarnawski of the University of California at Irvine for performing quantitative histologic studies of rat gastric mucosal specimens.
1988;95:18-25
Modulation of Rat Gastric Mucosal Prostaglandin E2 Release by Dietary Linoleic Acid: Effects on Gastric Acid Secretion and Stress-Induced Mucosal Damage WOLFGANG
SCHEPP,
BEATRIX
STEFFEN,
HANS-JORG
RUOFF,
VOLKER SCHUSDZIARRA, and MEINHARD CLASSEN Department of Internal Medicine II, Technical University of Munich, Munich, and Department of Pharmacology, University of Tiibingen, Tiibingen, Federal Republic of Germany
We studied chronic intake of diets deficient in or supplemented with linoleic acid to determine whether it affects gastric acid secretion, release of lesions. For prostaglandin E 2, and stress-induced 8-10 wk rats were fed three dietary regimens supplying 3.5% (control group), 0.3%,and 10% of total calories as linoleic acid. We found that diets deficient in linoleic acid (0.3%) reduced release of prostaglandin E2 into the gastric lumen (- 77%) and increased basal (+ 133%) and pentagastrin-stimulated acid secretion (+93%) and the area of cold restraint-induced gastric mucosal lesions (+ 280%), when compared with the control group. Diets supplemented with linoleic acid (10%) increased prostaglandin Ez release into the gastric lumen (+106%) and reduced basal (-44%) and pentagastrin-stimulated acid secretion (- 78%) and the area of cold restraint-induced mucosal lesions (-80%). Prevention of these lesions by the 10% linoleic acid diet was confirmed by quantitative histology. Pretreatment with indomethacin (8 mg/kg intraperitoneally) abolished the effects of the 10% linoleic acid diet on prostaglandin formation, acid secretion, and mucosal injury. We conclude that in rats chronic intake of dietary linoleic acid reduces acid secretion and prevents cold restraint-induced mucosal lesions, possibly because of augmented synthesis of endogenous prostaglandins in the gastric mucosa.
xogenously administered prostaglandins have been studied extensively with respect to their antisecretory and protective effects on the gastric mucosa (1,Z). However, considerably less is known about the role of endogenous prostaglandins. Inhibi-
E
tion of cyclooxygenase by indomethacin has been used to demonstrate the effect of impaired endogenous prostaglandin production on gastric acid and bicarbonate secretion in rats (3), dogs (4), and humans (5,6). Oral administration of arachidonic acid (AA) or linoleic acid (LA) offers another opportunity to study the role of endogenous prostaglandins as these essential fatty acids are precursors for the biosynthesis of many prostaglandins (7). It has been reported that in rats acute intragastric administration of AA (8,9)or LA (10)is followed by a marked increase of gastric mucosal prostaglandin release and protection against alcohol-induced injury, which is supposed to be independent of gastric acid. In this study we chose a different approach: diets deficient in or supplemented with LA were supplied on a long-term basis, and the prevention of cold restraint-induced gastric mucosal lesions, which are supposed to be dependent on gastric acid secretion, was studied. Formula diets supplying 10% of the total dietary energy as LA have been shown to stimulate endogenous prostaglandin formation in rats (11,12). Modulation of endogenous prostaglandin production by dietary LA intake has been reported to influence physiologic functions in various tissues, e.g., platelets (13) and kidney (14). The objectives of our study were to examine the effect of chronic modulation of dietary LA intake (a) on basal and stimulated gastric acid output and (b) on prevention of stress-induced gastric mucosal lesions. Our hypothesis was that deprivation of dietary LA Abbreviations used in this paper: AA, arachidonic acid; LA, linoleic acid; PGE, prostaglandin E. 6 1988 by the American Gastroenterological Association 0016-5085/88/$3.50
LINOLEIC
July lY88
might reduce formation of endogenous prostaglandins, augment acid secretion, and increase the number of cold restraint-induced mucosal lesions; on the formaother hand, high dietary LA might augment tion
of endogenous
cretion,
and
should report form
prostaglandins,
prevent
be reversed of these
mucosal
reduce
lesions.
by indomethacin.
studies
has been
acid
These
se-
effects
A preliminary
published
in abstract
(15).
Materials and Methods Dietary
Regimens
Female Wistar rats (250-270 g) were fed three isocaloric, semisynthetic formula diets (Altromin, Lage, F.R.G.) that differed only by their content of LA. In a standard rat diet 3.5% of the total dietary energy is supplied as LA. Hence, in this study the control group was fed a 3.5% LA diet. Two test groups received diets containing 0.3% and 10% LA, respectively. The animals had free access to water and were housed in individual cages to control their daily dietary intake. Wire-bottom cages were used to prevent coprophagg. From these data LA intake per day was calculated for each dietary group (Table 1). Weight controls were performed weekly, demonstrating comparable weight gain in the three groups (Table 1). Experiments
in Fistula
Rats
To adapt the rats to semisynthetic formula diets 30 rats received the 3.5% LA control diet for 2 wk. A gastric fistula was implanted under hexobarbital anesthesia (60 mgikg intraperitoneally). Thereafter the animals were arbitrarily divided into three groups of 10 rats each. The control group continued receiving the 3.5% LA diet and the test groups were fed the 0.3% and loo/, LA diets for 10 wk. After 2. 4, 6, and 8 wk gastric secretion was collected through the fistulas. Before these experiments the animals were fasted for 24 h with free access to a 5% glucose solution that was removed 1 h before the experiment. All experiments started at 8 ‘~1 and were performed in the same laboratory (22” + 1°C humidity 60%-70%). Secretions from the first 30-min collection period were discarded. Then gastric juice was continuously collected for three 60-min periods. After centrifugation of the 60..min sam-
Table
I. Food Intake, Linoleic Acid Intake, and Body Weight of 120 Rats Before Diet and After 8 Weeks on the Diets’
Food intake Diet
3.5% LA 0.376LA 10.0% LA LA, linoleic
(g/day)
14.3t 1.8 14.7t 0.6 13.2t 1.4 acid.
” Mean
Linoleic acid intake (Wdoy)
501 k 47 40 k 6 1323 r 149 t SEM
Body weight before diet
Body weight after 8 wk
&I
(Sl
200 k 30 220 ? 35 210 r 25
270 2 35 280 i 30 275 t 25
ACID AND GASTRIC MUCOSAL DAMAGE
19
ples, volume was measured to the nearest 0.01 ml; a lOO-~1 sample was taken to determine acid output by automatic titration to pH 7 with 0.1 N sodium hydroxide. The remaining supernatant was stored at -20°C until assayed for prostaglandin E, (PGEJ within 2 wk. Basal acid output and PGE2 release into the gastric juice were measured every 3 h and were calculated as the sum of the three 60-min periods. After 8 wk, acid output and PGE, release were also determined during stimulation by pentagastrin (125 pglkg). After discarding the initial 30-min secretion, gastric juice was collected over a 60-min period for measurements of basal acid output and PGEZ release. Then the respective stimulant was intraperitoneally administered and gastric secretion was collected for two more 60-min periods. After 10 wk on the diet the animals were killed by cervical dislocation. The stomachs were removed and placed on ice; IO-15-mg specimens were immediately taken from the fundic mucosa. Each specimen was incubated for 60 min at 37°C in a shaking bath [I5 mg of mucosa per milliliter of buffer (mmol/L: NaH2P04, 0.5; Na2HP0,, 1.0;NaHC03, 20; NaCl, 70; KCl, 5; CaCIZ, 1.0; MgC12, 1.0; glucose, 11; 4-(2-hydroxyethyl)-l-piperazineethane sulfonic acid, 50; bovine serum albumin, 1 mg/ml, pH 7.8)]. At the end of the incubation PGE2 release into the medium was determined in 100-p] duplhates. Prostaglandin E, release (picograms per milligram of mucosa per 60 min) was calculated from the duplicates of each individual animal and the mean PGEz release of the dietary group was documented. Experiments
in Pylorus-Ligated
Rats
Another 30 rats were arbitrarily divided into three groups receiving the different dietary regimens. After 5 wk the animals were fasted for 24 h with free access to 5% glucose solution which was removed 1 h before the experiment. The pylorus was ligated under hexobarbital anesthesia (60 mgikg intraperitoneally). After 3 h the animals were killed by cervical dislocation. The stomachs were removed and the gastric juice was collected for measurements of acid output and PGEz release. Effect
of Cold-Restraint
Stress
A combination of cold and restraint was used to produce stress ulcers in rats (16).Three groups of 10 rats each were fed the different diets for 5 wk. After being fasted for 24 h with free access to 5% glucose solution, the animals were put into individual close-fitting restraint cages and were exposed to a temperature of 4°C for 3 h. Thereafter all rats were killed by cervical dislocation, and their stomachs were excised and opened along the greater curvature. The mucosa was examined by an observer who was unaware of the dietary pretreatment. The incidence of lesions per rat was recorded and the lesion area was determined using a 1 x l-mm calibrated grid under a dissecting microscope (X 20) (I 7). When assessing the size of petechiae, five such lesions were considered equivalent to a l-mm’ ulcer. The sum of the lesion area in each group was divided by the number of rats in that group. Mean
20
SCHEPP ET AL.
GASTROENTEROLOGY Vol. 95.No. 1
lesion area per dietary group was expressed in square millimeters. Prostaglandin Ez release from macroscopitally intact fundic mucosa of the three groups was measured as described above. Experiments
in the Presence
Before being assayed gastric juice was adjusted to pH 7 by 0.1 N NaOH. In preliminary experiments we demonstrated that PGEz extraction by ethylacetate did not enhance sensitivity of measurements; therefore this procedure was not employed in the present study.
of Statistical
lndomethacin A cyclooxygenase inhibitor was used to determine if the effects of the 10% LA diet on PGE2 release and acid secretion were caused by increased endogenous prostaglandin production. In fistula rats receiving 10% LA, PGEz release and acid secretion were measured after 2, 4, 6, and 8 wk as described above; 3 h before the experiments, indomethacin (8 mgikg) was injected intraperitoneally. Histologic
Evaluation
In additional rats cold-restraint experiments were performed after 8 wk on the test diet as described above. Three hours before exposure to stress the animals received a &ml intraperitoneal injection of either vehicle (sodium bicarbonate solution) or indomethacin (8 mg/kg, prepared immediately before use). We studied five groups of rats (8 rats each) that were fed the following diets: 0.3% LA plus vehicle, 3.5% LA plus vehicle, 3.5% LA plus indomethacin, 10% LA plus vehicle, and 10% LA plus indomethacin. After 3 h of cold restraint the animals were killed by cervical dislocation. Standard mucosal strips of the gastric wall (0.5 cm in width, extending through the entire glandular oxyntic mucosa) were obtained from all animals. Specimens were fixed in 10% buffered formalin and stained with hematoxylin and eosin (H&E) and with Alcian blue (pH 2.5) and periodic acid-Schiff. Coded mucosal specimens were evaluated under light microscopy by two investigators unaware of the code. During histologic evaluation special attention was given to the integrity of the surface epithelial cells, presence of erosions, and submucosal edema. We also performed morphometric measurements of the extent of deep histologic necrosis (defined as necrotic lesions penetrating into the mucosa deeper than 0.2 mm). We measured the length of the mucosal strip and total length of necrotic lesions for each strip with the aid of a projecting lens (Nikon Inc., Garden City, N.Y.), which projected the image onto the stage of a Microplan II image analysis system. Radioimmunoassay
for Prostaglandin
E,
Prostaglandin Ez was determined by a specific radioimmunoassay using [3H]PGEz. Antibody against PGEz was raised in rabbits. The following cross-reactivities were found: prostaglandin AZ, 0%; thromboxane Bz, 0%; and PGE1, 13%. Lower and upper sensitivities were 10 and 1000 pg/lOO ~1 assay volume, respectively. Nonspecific binding was 4.1% k 0.3%. The recovery of 100 pg of PGEz added to gastric juice in vitro was 94% * 5% when measured immediately after addition and 81% 2 5% when determined after 1 h (n = 5 measurements). Duplicate samples (100 ~1) of gastric juice or incubation medium were assayed for PGEz within 2 wk after the experiments.
Analysis
The data concerning acid secretion and PGEz were analyzed by Student’s t-test for groups. Statistical comparisons of the extent of deep histologic necrosis were performed with the Kruskall-Wallis and Wilcoxon tests (18). Materials The following semisynthetic formula diets were purchased from Altromin: C 1000 (3.5% LA), C 1000 modified (10% LA), and C 1058 (0.3% LA). Hexobarbital was purchased from Bayer (Leverkusen, F.R.G.); Gastrodiagnost (pentagastrin) was purchased from Merck (Darmstadt, F.R.G.); bovine serum albumin, 4-(2-hydroxyethyl)l-piperazine-ethane sulfonic acid, and polyethylene glycol 6000 was purchased from Serva (Heidelberg, F.R.G.); and [3H]PGEz was purchased from DuPont (Montreal, Canada).
Results in Fistula
Experiments
Rats
Gastric juice was collected under basal condiafter 2, 4, 6, and 8 wk from fistula rats on the
tions three
dietary
regimens.
During
the
entire
period
secretion volumes did not significantly differ between the dietary groups. Prostaglandin E2 release into the gastric juice is shown in Figure 1, left. After 2 wk mean PGEz release was 25.9 2 3.7 ngl3 h in the control
group,
20.7
group,
and 50.1
t
During
the
entire
from the 0.3% controls,
*
4.8
ng/3 h in the
3.0 ng/3 h in the 10% observation
LA group
whereas
period
was lower
release
in
the
0.3%
LA
LA group.
PGE2
release
than
that in the
10%
LA
group
exceeded that in the controls. The differences between the 10% LA group and controls and the 10% LA group and 0.3%
LA group were significant
the
period
entire
difference
dietary between
was significant
(p <
the 0.3%
only
O.Ol),
LA group
during
whereas
the
and controls
after 4 wk (p < 0.01)
and 8 wk
(p < 0.05). The simultaneous measurements of gastric acid output in these experiments are shown in Figure 1, right. After 2 wk mean acid output was 147.8 2 7.9 pEql3
h in the control
group,
214.1
? 24.8 pEq in the
0.3% LA group (p CO.01 vs. controls and vs. 10% LA group), and 83.2 ? 11.4 PEq in the 10% LA group. The latter continued to produce less acid than controls (p < 0.05 after 6 and 8 wk), whereas in 0.3% LA group more acid was secreted than in controls. Maximal acid output occurred after 4 [317.7
t
24.0
pEq/3 h (p < 0.01
vs. control
the the the wk
and vs.
July 1988
++ 4 k_l **
-300
70-
l
*
++
XX
60-•+
*t i c7 5cxx xx ‘m xx c c LO- xx 0
o-,+, 2 L
+ +
l
*
l
9 100 s 2
-50 I 6
l171T0 2108
a weeks
Figure
Experiments
159 z
x2_
lo-
6
I. Gastric fistula rats: PGE, release (left) and acid secretion (right). For 8 wk 30 fistula rats (10 animals per group) were fed three isocaloric semisynthetic diets that differed only by LA content [3.5% of the total dietary energy, controls (m); 0.3% LA (A), and 10% LA (O)]. The animals were fasted for 24 h with free access to 5% glucose only. After discarding the initial 30-min sample, gastric juice was collected for three consecutive 60-min periods under basal conditions. The samples were adjusted to pH 7 and PGE, was radioimmunologically determined. Prostaglandin I$ release per animal was calculated as the sum of the three consecutive 60-min periods. Prostaglandin E2 release (mean + SEM) is plotted as nanograms per 3 h during 8 wk on the diets. Acid output was measured by automatic titration to pH 7 with 0.1 N sodium hydroxide in gastric juice samples of three consecutive 6o-min periods. Acid output (mean + SEM) is plotted as microequivalents per 3 h during 8 wk on the diets. Statistical analysis was performed by Student’s t-test for groups. *ix.‘+. p < 0.05; **lxx/++, p i 0.01. Comparisons: 10%) LA vs. control, t; loo/, LA vs. 0.3%1 LA, x: 0.3% LA vs. control, A.
in Pylorus-Ligated
kntacptnn
DAMAGE
21
Rats
l
1 *
**
Penkgast nn **
J
1Ol +
25 _
t
XX XX
_+I
20
xx
XX
+
-*
il rh xx
-60
l
LA)].
l
40
*
I
0
0.001).
After 8 wk PGEz release and acid output were measured during stimulation by pentagastrin (125 kg/kg intraperitoneally; Figure 2). Similar to the experiments under basal conditions, PGEz release differed between the three dietary groups. In the presence of pentagastrin, PGEz release tended to decrease from basal level, yet without attaining the level of statistical significance. One hour after stimulation, acid secretion in the 0.3% LA group was higher than in the controls (p < 0.05), whereas the 10% LA group revealed a much weaker response (p < 0.05 vs. controls; p < 0.01 vs. 10% LA group). To reexamine our in vivo results concerning PGEz release into the gastric juice, the fistula rats were killed after 10 wk on the test diets and PGE, release from fundic mucosal specimens was measured in vitro. Mean PGE, release was 762.7 + 98.5 pgimg
MUCOSAL
After ligation of the pylorus, the stomach becomes increasingly distended because of accumulation of gastric juice. We used this experimental model to study the effect of dietary LA on distentionstimulated acid secretion and PGEz release (Figure 3). Mean PGE, release was 12.6 ? 1.7 ng/3 h in the controls, 16.3 + 0.9 ngi3 h in the 10% LA group, and 7.5 2 0.7 ngl3 h in the 0.3% LA group (p < 0.05 vs. controls; p < 0.01 vs. 10% LA group). As expected
Acid secretion during the 8 wk on the test diet was inversely related to the release of PGE, Jr = 236.2 - 2.3x; r = - 0.75; p < (linear regression: 10%
ACID AND GASTRIC
mucosa per hour in the controls, 1498.7 IT 161.2 pg in the 10% LA group (p < 0.01 vs. controls), and 470.2 i 70.2 pg in the 0.3% LA group (p < 0.05 vs. controls; p < 0.01 vs. 10% LA group).
g +-200 f xx xx =
Pqf Y a
-250
++
LINOLEIC
10
I-73
l-73 h Figure
2. Gastric fistula rats: PGE, release [left) and acid secretion (right] during stimulation by pentagastrin (125 fig/kg). Thirty fistula rats (10 animals per group) were fed three isocaloric semisynthetic diets [3.5% LA, controls (W); 0.3% LA (A); 10% LA (e)]. After 8 wk the initial 30-min sample of gastric juice was discarded and basal PGE, release and acid secretion were determined in the following 60-min portion. Then pentagastrin was administered intraperitoneally and gastric juice was collected for two additional 60.min periods. Measurements of PGE, (radioimmunoassay) and acid (titration) were performed as described in the legend to Figure 1. Prostaglandin EZ release (ngih) and acid output (FEq/h) are shown for each dietary group (mean + SEMI. Statistical analysis was performed by Student’s t-test for groups. *lx/+, p < 0.05: **/xx/++, p < 0.01. Comparisons: 10% LA vs. control, * : 10% LA vs. 0.3% LA, x; 0.3% LA vs. control, +.
22
SCHEPP
ET AL.
GASTROENTEROLOGY
*
from our experiments in fistula rats, the 0.3% LA group revealed a higher acid secretion (336.12 24.7 pEq/3 h) than the controls (171.3 + 20.6 pEq/3 h; p < O.Ol), and the 10% LA group (147.2 ? 16.4 pEqi3 h; p < 0.01). Effect of Cold-Restraint
Effect of lndomethacin The cyclooxygenase inhibitor indomethacin was used to examine whether reduced acid secretion **
** I
I
,
l *
* -
-
g20\
I
F
-330
I
-glS _
fl, WN 5_
05 0.3% Figure
3
3.5%
10%
- * n
Stress
Our studies hitherto demonstrated that dietary LA can modulate release of gastric mucosal PGEz and that this modulation is inversely related to the patterns of gastric acid secretion. Because, in addition, prostaglandins are supposed to prevent peptic lesions, we studied the incidence of coldrestraint ulcers during modulated LA intake (Figure 4). The lesion area in the control animals (4.0 + 1.1 mm”) was smaller than that in the 0.3 LA group (15.2 ? 2.6 mm’; p < 0.05), whereas the 10% LA group revealed a much smaller lesion area (0.8 ? 0.4 mm’) than the controls (p < 0.05) and the 0.3% LA group (p < 0.01). Prostaglandin E2 release from fundic mucosal specimens was 912.7 ? 152.3 pgimg mucosa per hour in the controls, 632.9 ? 64.9 pg in the 0.3% LA group (p < 0.05 vs. controls), and 1516.2 If: 219.8 pg in the 10% LA group (p < 0.05 vs. controls; p < 0.01 vs. 0.3% LA group). The inverse correlation between lesion area and PGE, release of the three dietary groups was corroborated by calculating the linear regression (y = 1289 - 43.3x; r = -0.63; p < 0.01).
t
20 1
l
0.3%
3.5%
10%
Prostaglandin Ez release and acid secretion in pylorusligated rats. After 5 wk on the diets [3.5% LA, controls (0); 0.3% LA (U); 10% LA (m); n = 10 rats per group] pylorus ligation was performed under hexobarbital anesthesia. Prostaglandin EZ release (left) and acid output into the gastric juice (right) was determined after 3 h. For further details see legends to Figure 1. *, p < 0.05; **, p < 0.01.
a3% 3.5%10% Figure
l
1 l
Vol. 95, No. 1
l
*
1
-II
1
03% 35% 10%
4. Cold restraint-induced gastric mucosal damage after 5 wk on the diets [3.5% LA, controls (0); 0.3% LA (BI); 10% LA (me); n = 10 rats per group]. Animals were fasted for 24 h with free access to 576 glucose solution only. After exposure to 4°C for 3 h in close-fitting restraint cages the animals were killed. Using a 1 x l-mm eye grid under the dissecting microscope (X ZO), the incidence and area of gastric mucosal lesions were determined. Mean lesion areas of the dietary groups are plotted as square millimeters (left). In addition, specimens from macroscopically intact fundic mucosa were used for duplicate measurements of PGE, release in vitro during 60 min of incubation at 37°C in a shaking bath. Mean PGEz release from the specimens of the three dietary groups is plotted as picograms per milligram of mucosa per 60 min (right).
and reduced occurrence of stress-induced lesions were caused by increased formation of endogenous prostaglandins in the 10% LA group. Simultaneous with the experiments shown in Figure 1, PGEz release and acid secretion were measured in fistula rats on the 10% LA diet receiving indomethacin (8 mg/kg intraperitoneally) 3 h before the experiments. Under these conditions PGE2 release into the gastric juice ranged between 7.7 ? 0.3 ngi3 h (2 wk) and 9.4 ? 0.4 ng/3 h (8 wk; Figure 5, left) and acid secretion was 194 + 19 pEq/3 h (2 wk) and 194 -+ 12 pEql3 h (8 wk; Figure 5, right). Comparison of these results with the data given in Figure 1 demonstrates that in animals on the 10% LA diet indomethacin reduces the PGEz release and increases acid secretion to the same levels simultaneously measured in the 0.3% LA group (p < 0.01 vs. 10% LA diet without indomethacin after 2, 4, 6, and 8 wk) (see Results, Experiments in Fistula Rats). After 8 wk on the 10% LA diet and 3 h after indomethacin (8 mg/kg intraperitoneally), PGEz release from fundic mucosal specimens in vitro was 85.1 r 11.8 pg/mg mucosa per hour (p < 0.01 vs. 10% LA without preceding indomethacin (see Results, Experiments in Fistula Rats).
LINL’OLEIC ACID AND GASTRIC
luly 1988
o’-
2
1
0
-
8
2
1
6
8
Lo
weeks Figure
5. Gastric fistula rats: PGE? release (left) and acid secretion (right] under basal conditions. Simultaneously with the experiments shown in Figure 1, 10 fistula rats were fed the 10% LA diet (01 for 8 wk. Three hours after administration of indomethacin (8 mgikg i.p.), PGE, release (radioimmunoassay) and acid output (titration] were measured as described in the legend to Figure 1. For better comparison the dashed line shows the results with lOoh, LA in the absence of indomethacin (Figure 11. +*, p < 0.01 (10% LA + indomethacin vs. 10% LA alone].
Histologic
Evaluation
In the animals on the 0.3% LA diet, cold restraint produced prominent histologic changes: exfoliation of the surface epithelium, deep necrotic lesions of focal character penetrating into half of the entire mucosal thickness, and edema of the lamina propria and submucosa. Deep necrotic lesions involved 1.87% ? 0.5% of the mucosal section length in the 0.3% LA group and 1.4% ? 0.4% in the controls. In contrast, in rats receiving the 10% LA diet deep necrotic lesions were markedly reduced to 0.04% ? O.OZ%, of the mucosal section length (p < 0.05 vs. 0.3% LA group). Prevention of cold restraintinduced mucosal lesions by the 10% LA diet was more than reversed by pretreatment with indomethacin: deep necrotic lesions involved 3.70%, 2 0.8% of the mucosal section length in this group (p < 0.01 vs. 10% LA diet without indomethacin). Thus, in rats on the 10% LA diet indomethacin increased the stress-induced damage to a lesser extent than in the control animals (5.7% f 0.7% of the mucosal section length; p < 0.05 vs. 10% LA diet plus indomethacin).
Discussion This study demonstrates that in rats deprivation of dietary LA is followed by reduced gastric mucosal PGE;! release, augmented gastric acid secretion, and increased occurrence of stress-induced lesions. On the other hand, animals fed an LA-rich diet revealed enhanced gastric mucosal PGE, re-
MLJCOSAL DAMAGE
23
lease, reduced acid output, and reduced occurrence of mucosal lesions. Indomethacin completely reversed these effects of the 10% LA diet, suggesting that they are due to increased formation of endogenous prostaglandins. These data support a role for endogenous prostaglandins in the regulation of gastric acid secretion and a potential role in the prevention of stress-induced mucosal lesions by additional activation of defense mechanisms. To date, however, experiments with indomethacin do not unequivocally prove such a role for prostaglandins: the agent increased stress-induced lesions in animals receiving the control diet, indicating that indomethacin might exert additional effects unrelated to inhibition of the cyclooxygenase. Linoleic acid, a polyunsaturated fatty acid, is an essential dietary constituent (19). Standard rat diets supply 3%-4%, of total dietary energy as LA, equivalent to 480 mg LA per day. Therefore, a 0.3% and a 10% LA diet should result in sufficiently different LA intake to study the effects of LA deprivation or enrichment, respectively. The enzyme delta-6desaturase transforms LA to AA (ZO), which is the precursor for the formation of various prostaglandins. Because PGEz is a main prostanoid formed in the gastric mucosa and its antisecretory and protective effects have been studied in detail, we decided to measure PGE, release from the rat stomach both in vivo and in vitro. This does not exclude the possibility that changes of membrane lipids or prostanoids other than PGE, might also be involved in mucosal protection or inhibition of acid secretion during modulation of dietary LA. As tissue levels offer limited information because of the immediate release of newly synthesized prostaglandins from their cells of origin (al), we measured PGE, release into the gastric juice. In addition, these in vivo results were confirmed by determining PGEz release from fundic mucosal specimens in vitro. To examine whether modulation of gastric mucosal PGE, release by dietary LA influences gastric functions that are partially dependent on PC&, we studied gastric acid secretion and occurrence of stress-induced lesions. Prostaglandins have been reported to reduce gastric acid secretion by inhibiting the histaminesensitive adenylate cyclase of the parietal cell (22, 23); recently it was reported that they interfere at the (24). In addition, inhibitory subunit of that enzyme prostaglandins have an overall protective effect on the gastric mucosa. This effect consists of mucus and bicarbonate secretion, maintenance of the microvascular integrity, and production of surface-active phospholipids providing a nonwettable hydrophobic surface barrier above the epithelium (1,2). Modulations of prostaglandin formation by rat
24
SCHEPP
ET AL.
heart, aorta, and platelets have been achieved by dietary LA enrichment and deprivation, respectively (13).Similarly, our results demonstrate that PGE2 release from the rat gastric mucosa can be modulated by comparable LA diets. Several lines of evidence indicate that endogenous prostaglandins are involved in the regulation of gastric acid secretion: acid secretion was increased during LA deprivation and reduced during high LA intake; this reduction was prevented by indomethacin. Similarly, Konturek and colleagues (25) observed inhibition of gastric acid secretion during intraarterial or topical application of AA in the dog. Using different approaches, our diet experiments and Konturek’s acute application of prostaglandin precursors are in line with observations of increased basal or stimulated acid secretion during pharmacologic inhibition of endogenous prostaglandin formation in rats (3), dogs (41, and humans (5,6). As evidenced by quantitative histology, our 10% LA group revealed significant reduction of stressinduced lesions. Furthermore, we found a significant correlation between the reduced mucosal lesion area and PGEz release in the dietary groups, but the 10% LA diet did not prevent mucosal lesions after pretreatment with indomethacin. Thus our data suggest that the dietary precursor LA prevents stressinduced gastric mucosal lesions by induction of endogenous prostaglandin formation. The results of our dietary approach are in accordance with reports on rat gastric mucosal damage during indomethacininduced pharmacologic inhibition of endogenous prostaglandin formation (26). In our 10% LA group, acid secretion was reduced; on the other hand, cold restraint-induced lesions are known to depend on the presence of acid. Therefore, it cannot be decided from this study how much “protection” (27) contributed to the observed benefit of our LA-rich diet and how much was brought about by the inhibition of acid output. As acute administration of LA has been demonstrated to prevent alcohol-induced lesions (10) (which are known to be independent of gastric acid], it must be taken into consideration that LA not only inhibits acid secretion but, in addition, might also activate mucosal defense mechanisms. Protection of the rat gastric mucosa against alcohol-induced injury has been accomplished by acute intragastric administration of AA (8,9). Yet, there are differences between these experiments and our approach. Administering dietary LA on a long-term basis, we studied the effects of providing higher substrate concentrations for the synthesis of endogenous prostaglandins rather than the acute effect of a single excessive bolus of the ultimate precursor AA. According to physiologic needs, LA is transformed to AA and converted into prostaglandins, thereby
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resembling more closely the natural stimulation of prostaglandin formation. The delayed conversion of LA into prostaglandins might be reflected by the observation that PGE, concentration in the gastric juice of our LA-rich fed rats was lower than that reported after intragastric application of AA (8). Another explanation would be that-in contrast to Hollander and colleagues (8)-we did not include detergents to solubilize and enhance absorption of LA. In the study of Hollander and colleagues (8) intrajejunally administered AA failed to confer protection comparable with that after intragastric AA, indicating that direct contact of AA with the gastric mucosa is required for its acute action. An explanation for the failure of intrajejunal AA would be that immediately upon intestinal absorption AA is converted into prostaglandins, which are deactivated during the first pulmonary passage. From our data it cannot be excluded that a minor portion of dietary LA was directly converted into prostaglandins by the gastric mucosal cells before LA had reached the small intestine. However, the greater proportion of LA is absorbed in the small intestine (28) and transported by chylomicrons to peripheral cells (29), where enzymatic transformation into prostaglandins is performed. Therefore, in all likelihood LA does not require direct contact with the gastric mucosa for its chronic protective action.
References 1. Miller TA. Protective effects of prostaglandins against gastric mucosal damage: current knowledge and proposed mechanisms. Am J Physiol 1983;245:G601-23. 2. Hawkey CJ, Rampton DS. Prostaglandins and the gastrointestinal mucosa: are they important in its function, disease, or treatment? Gastroenterology 1985;89:1162-88, 3. Ligumsky M, Goto Y, Debas H, Yamada T. Prostaglandins mediate inhibition of gastric acid secretion by somatostatin in the rat. Science 1983;219:301-3. 4. Miller TA, Henagan JM, Kuo YJ, Shanbour LL. The role of sodium transport in prostaglandin cytoprotection. In: Allen A, Flemstrom G, Garner A, Silen W, Turnberg L, eds. Mechanisms of mucosal protection in the upper gastrointestinal tract. New York: Raven, 1984:357-61. EH. Effects of indomethacin on basal 5. Levine RA, Schwartzel and histamine stimulated human gastric acid secretion. Gut 1984;25:718-22. on gastric acid 6. Feldman M, Colturi TJ. Effect of indomethacin and bicarbonate secretion in humans. Gastroenterology 1984;87:1339-43. Lands WEM. Fatty acid-prostaglandin (autacoid) relationship. In: Boers RF, Bassett EG, eds. Nutritional factors: modulating effects on metabolic processes. New York: Raven, 1981:485-8. Hollander D, Tarnawski A, Ivey KJ, et al. Arachidonic acid protection of rat gastric mucosa against ethanol injury. J Lab Clin Med 1982;100:296-308. Tarnawski A, Hollander D, Stachura J, Krause WJ. Arachidonic acid protection of gastric mucosa against alcohol inju-
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10.
11,
12.
13.
14.
15.
16.
17.
18. 19.
20.
21.
22.
ry: sequential analysis of morphologic and functional changes. J Lab Clin Med 1983:102:340-51. Tarnawski A, Hollander D, Krause WJ, et al. Is linoleic acid (dietary essential fatty aciti-EFA) cytoprotective for the gastric mucosa (abstr)? Gastroenteroiogy 1985;88:1610. JMathias MM, Fine K, Lipinski B, DuPont J. Dietary linoleate effect upon prostaglandin and thromboxane synthesis. Prostaglandins 1978;15:706-10. Nugteren DH, van Evert WC, Soeting WH, Spuy JH. Effects of different amounts of linoleic acid in the diet on the excretion of urinary prostaglandin metabolites in the rat. In: Samuelsson B, Ramwell P, Paoletti R. eds. Advances in prostaglandin and thromboxane research. Volumes 6-8. New York: Raven, 1980:1793-6. tenHor F, deDeckere EAM. Haddeman E, Hornstra G, Quadt JFA. Dietary manipulation of prostaglandin and thromboxane synthesis in heart, aorta and blood platelets of the rat. In: Samuelsson B, Ramwell P. Paoletti R, eds. Advances in prostaglandin and thromboxane research. Volume 8. New York: Ralren, 1980:1771-81. Adam 0. Wolfram G, Zollner N. Effect of different linoleic acid intake on prostaglandin biosynthesis and kidney function in man. Am J Clin Nutr 1984;40:763-70. Schepp W. Steffen B, Ruoff H-J. Classen M. Effect of dietary linoleic acid on acid secretion and cold restraint ulcers in rats (abstr). Gastroenterology 1986;90:1617. Senay EC, Levine RJ. Synergism between cold and restraint for rapid production of stress ulcers in rats. Proc Sot Exp Biol Med 19f~7;124:1221-5. Waisman Y, Dinari G, Marcus H, et al. Naloxone is protective against indomethacin-induced intestinal ulceration in the rat. Gastroenterology 1985;89:86-91. Cono\er WJ. Practical nonparametric statistics. New York: John Wiley & Sons, 1971:39. FXO/WHO. Dietary fats and oils in human nutrition. A joint FAOWHO report. Rome: Food and Agriculture Organization of the United Nations, 1977. Brenner RR. Regulatory function of delta-6-desaturase, key enzyme of polyunsaturated fatty acid synthesis. Function and biosynthesis of lipids. In: Bazan NG, Brenner RR, Giusto NM, eds. Advances in experimental medicine and biology. Volume 83. New York: Plenum, 197785-101. Schepp W, Steffen B, Schusdziarra V, Classen M. Calcium, calmodulin. and cyclic adenosine monophosphate modulate prostaglandin E,-release from isolated human gastric rnucosal cells. J Clin Endocrinol Metab 1986;63:886-91. Sol1 AH. Specific inhibition by prostaglandins E, and I, of
ACID AND GASTRIC
histamine-stimulated
23.
24.
25.
26.
27.
28.
29.
MIJCOSAI,
[‘“Cl aminopyrine
DAMAGE
ancl cyclic
25
adenosine
monophosphate generation by isolated canine parietal cells. J Clin Invest 1980;65:1222-9. Schepp W, Ruoff H, Maslinski S. Aminopyrine accumulation of isolated parietal cells from the rat stomach. Effect of histamine and interaction with endogenous inhibitors. Arch lnt Pharmacodyn Ther 1983;265:293-308. Sol1 AH, Berglindh Th. Physiology of isolated gastric glands and parietal cells: receptors and effecters regulating function. In: Johnson LK, ed. Physiology of the gastrointestinal tract. New York: Raven, 1987:883-909. Konturek SJ, Mikos E, Pawlik W, Walus K. Direct inhibition of gastric secretion and mucosal blood Bow by arachidonic acid J Physiol 1979;286:15-28. Whittle BJR. Temporal relationship between cyclooxygenase inhibition as measured by prostacyclin biosynthesis and the gastrointestinal damage induced by indomethacin in the rat. Gastroenterology 1981;80:94-8. Robert A. Antisecretory. antiulcer, cytoprotective and diarrheogenic properties of prostaglandins. In: Samuelsson B. Kamwell PW, Paoletti R. eds. Advances in prostaglandin and thromboxane research. Volume 2. New York: Raven, 197fi: 507-20. Chow SL, Hollander 1). Linoleic absorption in the unanesthetized rat: mechanisms of transport and inlluence of luminal factors on absorption. Lipids 1978:13:378-85. Lewis B. The LDL theory and HDL hypothesis. In: Noseda C. Lewis B, Paoletti R. eds. Diets arrd drugs in atherosclerosis. New York: Raven, 1980:1-8.
Received May 5. 1986. Accepted November 30, 1987. Address requests for reprints to: Wolfgang Schepp, M.D., Medizinische Klinik II, Technischr Ilniversitat, Klinikum rechts der Isar, Ismaninger Strasse 22, LD 8000 Muncher1 80, Federal Republic of Germany This study was supported by grant Sche 22912-l from the Deutsche Forschungsgemeinschaft. This work was presented in part at the meeting of the American Gastroenterological Association, May 18-21, 1986, in San Francisco, California. The authors thank Heumann Pharmaceutics, Niirnberg, Germany for generous support: Mrs. C. Tatge for ter hnical assistance; and Professor Andrzej S. Tarnawski of the University of California at Irvine for performing quantitative histologic studies of rat gastric mucosal specimens.