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Effect of 16,16-dimethyl prostaglandin E2 on ethanol-induced damage to canine oxyntic mucosa
00165085/78/7506.1061$02.00/0 GASTROENTEROLOGY 75:1061-1065, 1978 Copyright 0 1978 by the AmericanGastroenterologlcal Association
Vol. 75, No. 6 Printed
in U.S.A.
EFFECT OF 16,16-DIMETHYL PROSTAGLANDIN E, ON ETHANOLINDUCED DAMAGE TO CANINE OXYNTIC MUCOSA BARRY L. TEPPERMAN, JOHNSON,
PH.D.
Departments Texas
ofPhysiology
PH.D.,
THOMAS
and Surgery,
A.
MILLER,
M.D.,
AND LEONARD R.
The University of Texas Medical School at Houston, Houston,
Previous reports have demonstrated that the synthetic prostaglandin analogue, 16,16dimethyl prostaglandin E, (dm PGE,), protects the rat gastric mucosa against the damaging effects of a variety of ulcerogenic agents, including alcohol. The effects of dm PGE, on alcohol-induced gastric mucosal injury in the dog has not been investigated. Using the canine Heidenhain pouch, the present study examined the action of dm PGE, on gastric mucosal damage induced by 15% ethanol, both in the presence and absence of HCl. Ethanol by itself damaged the pouches as evidenced by large net fluxes of Na+ and K+ into the pouch. The combination of ethanol and 100 mN HCl produced the same effects on Nat and K+ and also increased pepsin secretion into the pouch and the loss of H+ from the bathing solution. Intravenous injection of dm PGE, in the doses 0.01, 0.1, and 1.0 pg per kg, one-half hour before administration of ethanol in acid solution, significantly reduced the net loss of H’ from the pouch and the gain of Na+, K+, and pepsin. The most effective doses for protection were 0.1 and 1 .O pg per kg. The increases in Na+ and K+ efflux elicited by.ethanol in the absence of HCl were also significantly reduced by dm PGE,. It is concluded that dm PGE, effectively protects the canine gastric mucosa against the damaging effects of alcohol and that this protection is doserelated. Although the mechanism underlying this protection is not apparent from this study, it appears to be independent of inhibition of acid secretion because equal protection against ethanol was observed both in the presence and absence of HCl. Robert and colleagues’-:’ have qualitatively demonstrated in the rat that the synthetic prostaglandin analogue 16,18dimethyl prostaglandin E, (dm PGE,) prevented or reduced the incidence of mucosal lesions which occurred after treating the stomach with various ulcerogenic agents. This antiulcer property has been termed “cytoprotection.“’ Robert’s visual findings, however, have given no indication as to the degree of disruption of the gastric mucosal barrier in terms of ionic fluxes. Only a short report exists which indicates that intravenous administration of dm PGE, reduces the disappearance of H+ associated with topical aspirin administration from the lumen of the stomach.4 Aspirin is known to inhibit prostaglandin synthetase,” the enzyme system responsible for endogenous prostaglandin production. Therefore the cytoprotective action of dm PGE, against aspirin-induced injury may be explained on the basis of replenishing gastric muReceived September 13, 1977. Accepted June 12, 1978. Address requests for reprints to: Thomas A. Miller, M.D., Department of Surgery, The University of Texas Medical School, 6431 Fannin, Houston, Texas 77030. This study was supported in part by Grants AM16505 and AM18164 from the National Institutes of Health, and a research grant from the Distilled Spirits Council of the United States, Inc. The authors are grateful to Dr. John Pike and the Upjohn Company (Kalamazoo, Michigan) for supplying the 16,16-dimethyl PGE,.
cosal prostaglandin. However, dm PGE, has also been shown to protect against gross mucosal ulcerations induced by ethanol,” bile,” and serotonin’ in the rat. Because these agents have not been shown to inhibit the prostaglandin synthetase system, mechanisms other than alterations in prostaglandin synthesis must be involved to confer this protection. The purpose of the present study was to assess quantitatively the possible cytoprotective effects of dm PGE, on the damaging effects of one of the above agents, namely ethanol, in terms of alterations produced in the ionic integrity of the gastric mucosal barrier. Materials
and Methods
Mongrel dogs of both sexes were surgically prepared with a gastric fistula drained by a Thomas cannulaX and a vagally denervated Heidenhain pouch drained with a Gregory cannula.!’ Experiments were begun 3 weeks postoperatively. The animals were fasted for 18 hr before an experiment but were allowed water ad libitum. At the start of each experiment, the dogs were placed on tables where they stood quietly, partially supported by slings. The gastric fistula was opened and the stomach rinsed with saline. The fistula was left open for the duration of the experiment to prevent gastric juice from entering the duodenum. The metal cannula draining the Heidenhain pouch was connected by rubber tubing to a reservoir containing 50 ml of a control solution which, depending on the experiment, contained either 50 mN NaCl plus 100 mN HCl or 150 mN NaCl (pH 7.0). The solution was allowed to
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1062
Vol.75,No. 6
TEPPERMANETAL.
enter or leave the pouch influenced only by gravity and pouch contractions. The level of fluid in the reservoir was 20 cm above the pouch. At the end of each 15-min period, the reservoir and pouch were drained and the solution replaced. The first two 15-min periods (periods 1 and 2) were discarded to ensure thorough rinsing of the pouch. Collection of the control solution continued for two additional periods (periods 3 and 4). The pouches were then irrigated for four periods (periods 5 through 8) with a solution composed of either 15% ethanol (w/v) plus 50 mN NaCl and 100 mN HCl or 15% ethanol (w/v) plus 150 mN NaCl. A final hour of collections (periods 9 through 12) with the control solution ended the experiment. In another series of experiments the effects of various doses (0.01, 0.1, and 1.0 pg per kg) ofdm PGE, on pepsin output and the changes in net flux of Na’, K+, and H+ induced by 15% ethanol plus 50 mN NaCl and 100 mN HCl were assessed. Based on the results of these studies, a further series of experiments were conducted in which the effects of 1.0 Fg per kg of dm PGE, on Na’ and K+ flux induced by 15% ethanol plus 150 mN NaCl were evaluated. The design of these experiments was similar to those described above except that, after the control period, dm PGE, was injected intravenously as a single bolus in the appropriate test dose, after which the control period was extended for an additional 30 min. After each 15-min collection, the volume was measured and the acid concentration determined by titration to pH 7.0 with 0.2 N NaOH using an Autoburet andpH meter (Radiometer, Copenhagen, Denmark). The Na+ and K+ concentration of each sample was determined by flame photometry (Brinkmann Eppendorfmodel700, Westbury, N. Y.). Total amounts of Na’, K’, and H+ in the recovered fluid were calculated for each period. The net flux of each ion was determined by subtracting the amount put in the pouch from the amount recovered and expressed as microequivalents per 15 min. The pepsin concentration of each sample was determined using a modification of the Anson hemoglobin method.“’ Results were expressed as milligrams of pepsin per milliliter after reading the trichloroacetic acid supernatant fluid at 280 rnp and comparing it with solutions incubated with different concentrations of pepsin (hog pepsin, three times crystallized, Pentex Biochemicals, Kankakee, Ill.) Pepsin outputs were calculated and expressed as the percentage of the mean of the final two control periods when the pouches were irrigated with 100 mN HCl and 50 mN NaCl. This was done to eliminate some of the variability between individual animals which had consistent but different basal levels of pepsin secretion. Experiments were done on each dog no more than twice a week. All of the results are expressed as means and standard errors of the means. Control values in each experiment were obtained by averaging the final two 15-min periods of the initial control period. The effects of ethanol and dm PGE, were compared using the t-test for either unpaired or paired data. Differences were considered significant when P < 0.05.
Results During the two final 1Bmin control periods of the experiment in which 15% ethanol in acid or neutral solution alone was given, there was a small loss or net negative flux of H+ equal to about 4% of that in the pouch. Likewise there was a small positive gain of Na+ and K+ into the luminal bathing fluid. Irrigation of the pouch with 15% ethanol in combination with 100 mN HCI caused a significant increase in the net negative flux of H+ compared to control periods (fig. 1). The loss of H+ reached maximal levels during periods 7 and 8. Removal of ethanol and irrigating the pouches with 100
50 mN N&I
o-
g E z $
+ 100 mN I-ICI
15% Ethanol
Ethanol
+ 0.1 pglkg
Ed,.“.,,
+ 0.01 pglkg
dm PCEZ
200Edwnol + 1 .O yglkg
dm PGEZ dm PGEZ
400-
3 9 2 600f 5 = 6004\, 3
,
#
,
,
,
,
,
,
,
4
5
6
7
6
9
10
11
12
Time (15 min periods)
FIG. 1. Effect of 16,lgdimethyl prostaglandin E, (dm PGE,) on net H’ loss in response to topical application of 15% ethanol in 100 mN HCl. Control series (0-O; ethanol alone) represents the mean of eight experiments (*SEMI. Dm PGE, was given as an intravenous bolus injection after period 4. In this and subsequent figures the effect of intravenous dm PGE, in the doses 0.01 CO-. -01, 0.1 (A-- -A) and 1.0 (A---A) pg per kg are presented and represented by the means of 10, 8, and 9 experiments (~sEM). respectively. Asterisks, significant differences (P i 0.05) between corresponding points of each dm PGE, series and ethanol alone by the unpaired t-test.
mN HCl and 50 mN NaCl alone reduced the loss of H’ , but even after the 1-hr recovery period, the net flux of H+ had not returned to pretreatment values. Intravenous injection of dm PGE, in the doses 0.01, 0.1, and 1.0 pg per kg in each case did not totally prevent the increase in H+ loss which occurred after ethanol administration. However, dm PGE, in the dose of 0.1 pg per kg significantly reduced at all points (periods 5 to 12) the degree of H+ loss when compared to the series of experiments in which ethanol alone was given (fig. 1). Dm PGE, in the dose of 1.0 pg per kg significantly reduced the net loss of H+ in all periods with the exception of periods 9 and 10. The lowest dose of prostaglandin (0.01 pug per kg) did not significantly reduce the degree of H+ back diffusion produced by topical ethanol administration, although the mean H+ loss in the final 45 min of the experiment after ethanol was withdrawn was significantly less than in the ethanol-treated series alone. The net positive flux of Na+ was greatly exaggerated upon ethanol administration in acidic solution (fig. 2) and this flux did not return to pretreatment values upon ethanol withdrawal. The net flux of Na+ also significantly increased in spite of prior intravenous injection of dm PGE, (fig. 2). However, the highest doses of dm PGE, used in this study (0.1 and 1.0 pg per kg) significantly reduced the amount of Nat appearing in the solution recovered from the Heidenhain pouch compared to ethanol alone. The lowest dose of dm PGE, (0.01 pg per kg) did not appreciably alter the effect of 15% ethanol on the gastric mucosal barrier. Administration of ethanol in neutral solution (fig. 3) also caused a significant efJlux of Nat which did not return to control
December
1978 1
ETHANOL 50 mN N&l
q
T
T
T
6
7
6
reduced this et&x at all time periods throughout the experiment. The output of pepsin increased after ethanol treatment (fig. 6). Removal of ethanol did not ameliorate the pepsin response with the output displaying a tendency to increase even further during the recovery period. Only one dose of dm PGE, (1.0 pg per kg) prevented the increase in pepsin output that occurred upon topical treatment with ethanol. The other doses of dm PGE, (0.01 and 0.1 pg per kg) did not prevent the increase in pepsin output, although the response returned to preethanol levels faster than it did without injection of dm PGE,.
5
I
,” 600 3
3 2
400
cl + LO 2
3
4
5
9
10
11
Discussion
12
Tlme (15 mln perlods)
FIG. 2. Net fluxes of Na- into the Heidenhain experiment described in figure 1.
150 mN
1063
AND PROSTAGLANDINS
1
+ 100 mN HCI
15% Ethanol
DAMAGE
pouch during the
N&l
Alcohol when given in 100 mN HCl topically resulted in significant damage to the canine gastric mucosa. These results confirm the findings of Davenport” who reported that ethanol concentrations greater than 14% disrupted the gastric mucosal barrier as evidenced by large increases in the net fluxes of H+, Na+, and K+. In
15% Ethanol
2 E
40
2 2
30
3 S z
Ethanol alone
20
+ Y
FIG. 3. Effect of 16,16-dimethyl prostaglandin E, (dm PGEJ on net Na’ efflux in response to topical application of 15% ethanol in 150 mN NaCl. Control series (ethanol alone) represents the mean -t SEM of nine experiments, whereas dm PGE, treated series represents the mean YXSEM of six experiments. Asterisks, significant differences (P < 0.05) between corresponding points of each dm PGE, series and ethanol alone b’y the unpaired t-test.
I
10
L
,
,
(
,
,
1
,
4
5
6
7
6
9
10
I
11
,
12
Time (15 min periods) FIG. 4. Net fluxes
experiment
levels until ethanol withdrawal. Prior administration of dm PGE, almost entirely eliminated this Na+ efIlux. Ethanol in acid solution resulted in a significant increase in the net flux of K+ in the canine Heidenhain pouch and this flux did not return to pretreatment values after ethanol was withdrawn (fig. 4). Dm PGE, produced the same apparent protective ability on this index of barrier integrity as was displayed against the net flux of Na’. The highest doses of prostaglandin resulted in significant reductions in K+ flux after ethanol. Although not affecting the amount of K+ appearing in the pouch upon ethanol instillation, 0.01 pg per kg of dm PGE, still significantly reduced the net flux of K+ which occurred in the final 45 min of the recovery period. Ethanol in neutral solution (fig. 5) also resulted in a significant increase in KC efflux which did not return to pretreatment values after ethanol withdrawal. Prior administration of dm PGE, significantly
I
3
of K’ into the Heidenhain described in figure 1.
pouch during the
150 mN NaCI 15% Ethanol
.__
=
I
_~.
I
*
\
L
3
4
5
6
7
8
9
10
11
12
Time (15 mh perk&)
FIG. 5. Net fluxes
experiment
of K+ into the Heidenhain described in figure 3.
pouch during the
1064
TEPPERMAN 50 mN NaCI + 100 mN WI 15% Ethanol
3
I
’
I
’
,
1
I
I
I
4
5
6
7
6
9
10
11
12
Time (15 min periods)
FIG. 6. Pepsin output as a percentage experiment described in figure 1.
of control
during
the
addition, Davenport” showed that the presence of acid was not essential for ethanol to produce damage to the gastric mucosa by finding that alcohol was no more damaging when given in 100 mN HCl than it was in a solution buffered to pH 7.5. We have confirmed these observations as well. Intravenous administration of dm PGE, resulted in significant reduction in the net fluxes of H+, Na+, and K+ associated with ethanol-induced damage to the gastric mucosal barrier in both an acidic and neutral solution. We are aware of no other report which demonstrates that any prostaglandin, natural or synthetic, reduces the degree of barrier disruption associated with topical application of this ulcerogenic agent. This protective action of prostaglandin was manifested in the present study by an amelioration in the net ionic fluxes. These results extend the finding in the rat that both natural and synthetic prostaglandin analogues prevent or reduce gross mucosal damage induced by damaging agents.‘, 3,ti,i, 12 The antiulcer ability of the prostaglandins may be related to their potent antisecretory properties. However, evidence exists which would militate against such a possibility. First, prostaglandins which lack antisecretory activity still possess cytoprotective properties.’ Furthermore, it has been demonstrated that effective doses for cytoprotection are often much lower than the median (ED:,,,) effective doses for antisecretion.2, :LI2 In the present study, however, the optimal protective doses were 0.1 and 1.0 pg of dm PGE, per kg, a dose range which has been shown by Robert et al.’ to inhibit most effectively histamine-stimulated acid secretion in the Heidenhain pouch dog (the ED~,, being 0.1 pg per kg). Thus it appears that doses below the ED,,,antisecretory dose are ineffective cytoprotective agents in the Heidenhain pouch dog, at least against ethanol-induced damage. Nevertheless, the cytoprotective ability of dm PGE, observed in the present study cannot be explained solely in terms of inhibition of acid secretion. The animals used in this study were not stimulated to secrete acid. Even if small amounts of acid were secreted, protection
Vol. 7ii. No. 6
ET AL.
attributable to an inhibition of acid secretion cannot be the explanation for the observed protection because inhibition of acid output would appear to produce a greater loss of H+ from the pouch. This, however, was not the case. Finally, the observation that dm PGE, protects against ethanol-induced damage in neutral solution lends further support to the hypothesis that mechanisms other than inhibition of acid secretion must be involved in offering this protection. The mechanism underlying protection against ethanol-induced gastric mucosal injury by dm PGE, is not apparent from the present study. Although aspirin inhibits the enzyme system responsible for the conversion of arachidonic acid to the natural prostaglandin compounds,” alcohol has not been shown to be a synthetase inhibitor. The ability of dm PGE, to protect canine gastric mucosa from both aspirin- and ethanol-induced damage indicates that the mechanism of cytoprotection is not, at least in total, caused by an involvement of prostaglandin synthetase. Johnson’:’ has demonstrated that 15% ethanol in 100 mN HCl results in a significant increase in pepsin output from the damaged canine Heidenhain pouch. Furthermore, it was found that H+ ion must be present to back diffuse through the mucosa in order for stimulation of pepsin secretion to occur. Once the barrier to diffusion of H+ is broken, a low concentration of acid is sufficient to stimulate pepsin secretion. In the present study, dm PGE, significantly reduced but did not prevent back diffusion of H.+. Thus it would be expected that pepsin secretion might still continue in the dm PGE, series. However, it would also be expected that the reduced H+ back diffusion would result in a reduction in the peptic response inasmuch as Johnson14 has demonstrated a dependency between acid concentration and pepsin output. Such was the case, for all doses of dm PGE, lowered pepsin output during the recovery period even though only the highest dose (1.0 pg per kg) prevented pepsin secretion during the presence of ethanol. In conclusion, we have demonstrated that the synthetic prostaglandin analogue, dm PGE, aids in the maintenance of gastric mucosal barrier integrity as measured by ionic fluxes. The ability of a prostaglandin to protect against the damaging agent, ethanol, which is not assumed to be an inhibitor of prostaglandin synthetase, suggests that this enzyme system is not necessarily involved in the mechanism of prostaglandin cytoprotection. Because this protective effect was also observed in neutral solution, it does not depend on the antisecretory property of dm PGE,. REFERENCES Robert A, Schultz JR, Nezamis JE, et al: Gastric antisecretory and antiulcer properties of PGE,, l&methyl PGE, and 16,16dimethyl PGE,. Gastroenterology 70:359-370, 1976 Robert A: Antisecretory, antiulcer, cytoprotective and diarrheogenie properties of prostaglandins. In Advances in Prostaglandin and Thromboxane Research, vol 2. Edited by B Samuelsson, R Paoletti. New York, Raven Press, 1976, p 507-520 Robert A, Nezamis JE, Lancaster C, et al: Gastric cytoprotective property of prostaglandins (abstr). Gastroenterology 72:1121, 1977
December 1978 4. Miller TA, Tepperman
5.
6.
7.
8.
ETHANOL BL: Protection
against
DAMAGE
AND
aspirin-induced
gastric mucosal injury by 16,16 dimethyl prostaglandin E, (abstr). Gastroenterology 72:1102, 1977 Vane JR: Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs. Nature [New Biol] 231:232-235, 1971 Mann NS: 13ile-induced acute erosive gastritis. Its prevention by antacid, chtolestyramine and prostaglandin E,. Am J Dig Dis 21:89-92, 1976 Ferguson WW, Edmonds AW, Starling JR, et al: Protective effect of prostaglandin E, (PGE,) on lysosomal enzyme release in serotonin-induced gastric ulceration. Ann Surg 177:648-654, 1973 Thomas JE: An improved cannula for gastric and intestinal
PROSTAGLANDINS fistulas. Proc Sot Exp Biol Med 46:260-261. 1941 9. Gregory RA: Gastric secretory responses after portal
1065
venous
ligation. J Physiol (Lend) 144:123-127, 1958 10. Anson ML: The estimation of pepsin, trypsin, papain and cathepsin with hemoglobin. J Gen Physiol 22:79-89, 1938 11. Davenport HW: Ethanol damage to canine oxyntic glandular mucosa. Proc Sot Exp Biol Med 126:657-662, 1967 12. Main IHM, Whittle BJR: Potency and selectivity of methyl analogues of prostaglandin E, on rat gastrointestinal functions. Br J Pharmacol 54:309-317, 1975 13. Johnson LR: Pepsin secretion during damage by ethanol and salicylic acid. Gastroenterology 62:412-416, 1972 14. Johnson LR: Regulation of pepsin secretion by topical acid in the stomach. Am J Physiol 223:847-850, 1972
Vol. 75, No. 6 Printed
in U.S.A.
EFFECT OF 16,16-DIMETHYL PROSTAGLANDIN E, ON ETHANOLINDUCED DAMAGE TO CANINE OXYNTIC MUCOSA BARRY L. TEPPERMAN, JOHNSON,
PH.D.
Departments Texas
ofPhysiology
PH.D.,
THOMAS
and Surgery,
A.
MILLER,
M.D.,
AND LEONARD R.
The University of Texas Medical School at Houston, Houston,
Previous reports have demonstrated that the synthetic prostaglandin analogue, 16,16dimethyl prostaglandin E, (dm PGE,), protects the rat gastric mucosa against the damaging effects of a variety of ulcerogenic agents, including alcohol. The effects of dm PGE, on alcohol-induced gastric mucosal injury in the dog has not been investigated. Using the canine Heidenhain pouch, the present study examined the action of dm PGE, on gastric mucosal damage induced by 15% ethanol, both in the presence and absence of HCl. Ethanol by itself damaged the pouches as evidenced by large net fluxes of Na+ and K+ into the pouch. The combination of ethanol and 100 mN HCl produced the same effects on Nat and K+ and also increased pepsin secretion into the pouch and the loss of H+ from the bathing solution. Intravenous injection of dm PGE, in the doses 0.01, 0.1, and 1.0 pg per kg, one-half hour before administration of ethanol in acid solution, significantly reduced the net loss of H’ from the pouch and the gain of Na+, K+, and pepsin. The most effective doses for protection were 0.1 and 1 .O pg per kg. The increases in Na+ and K+ efflux elicited by.ethanol in the absence of HCl were also significantly reduced by dm PGE,. It is concluded that dm PGE, effectively protects the canine gastric mucosa against the damaging effects of alcohol and that this protection is doserelated. Although the mechanism underlying this protection is not apparent from this study, it appears to be independent of inhibition of acid secretion because equal protection against ethanol was observed both in the presence and absence of HCl. Robert and colleagues’-:’ have qualitatively demonstrated in the rat that the synthetic prostaglandin analogue 16,18dimethyl prostaglandin E, (dm PGE,) prevented or reduced the incidence of mucosal lesions which occurred after treating the stomach with various ulcerogenic agents. This antiulcer property has been termed “cytoprotection.“’ Robert’s visual findings, however, have given no indication as to the degree of disruption of the gastric mucosal barrier in terms of ionic fluxes. Only a short report exists which indicates that intravenous administration of dm PGE, reduces the disappearance of H+ associated with topical aspirin administration from the lumen of the stomach.4 Aspirin is known to inhibit prostaglandin synthetase,” the enzyme system responsible for endogenous prostaglandin production. Therefore the cytoprotective action of dm PGE, against aspirin-induced injury may be explained on the basis of replenishing gastric muReceived September 13, 1977. Accepted June 12, 1978. Address requests for reprints to: Thomas A. Miller, M.D., Department of Surgery, The University of Texas Medical School, 6431 Fannin, Houston, Texas 77030. This study was supported in part by Grants AM16505 and AM18164 from the National Institutes of Health, and a research grant from the Distilled Spirits Council of the United States, Inc. The authors are grateful to Dr. John Pike and the Upjohn Company (Kalamazoo, Michigan) for supplying the 16,16-dimethyl PGE,.
cosal prostaglandin. However, dm PGE, has also been shown to protect against gross mucosal ulcerations induced by ethanol,” bile,” and serotonin’ in the rat. Because these agents have not been shown to inhibit the prostaglandin synthetase system, mechanisms other than alterations in prostaglandin synthesis must be involved to confer this protection. The purpose of the present study was to assess quantitatively the possible cytoprotective effects of dm PGE, on the damaging effects of one of the above agents, namely ethanol, in terms of alterations produced in the ionic integrity of the gastric mucosal barrier. Materials
and Methods
Mongrel dogs of both sexes were surgically prepared with a gastric fistula drained by a Thomas cannulaX and a vagally denervated Heidenhain pouch drained with a Gregory cannula.!’ Experiments were begun 3 weeks postoperatively. The animals were fasted for 18 hr before an experiment but were allowed water ad libitum. At the start of each experiment, the dogs were placed on tables where they stood quietly, partially supported by slings. The gastric fistula was opened and the stomach rinsed with saline. The fistula was left open for the duration of the experiment to prevent gastric juice from entering the duodenum. The metal cannula draining the Heidenhain pouch was connected by rubber tubing to a reservoir containing 50 ml of a control solution which, depending on the experiment, contained either 50 mN NaCl plus 100 mN HCl or 150 mN NaCl (pH 7.0). The solution was allowed to
1061
1062
Vol.75,No. 6
TEPPERMANETAL.
enter or leave the pouch influenced only by gravity and pouch contractions. The level of fluid in the reservoir was 20 cm above the pouch. At the end of each 15-min period, the reservoir and pouch were drained and the solution replaced. The first two 15-min periods (periods 1 and 2) were discarded to ensure thorough rinsing of the pouch. Collection of the control solution continued for two additional periods (periods 3 and 4). The pouches were then irrigated for four periods (periods 5 through 8) with a solution composed of either 15% ethanol (w/v) plus 50 mN NaCl and 100 mN HCl or 15% ethanol (w/v) plus 150 mN NaCl. A final hour of collections (periods 9 through 12) with the control solution ended the experiment. In another series of experiments the effects of various doses (0.01, 0.1, and 1.0 pg per kg) ofdm PGE, on pepsin output and the changes in net flux of Na’, K+, and H+ induced by 15% ethanol plus 50 mN NaCl and 100 mN HCl were assessed. Based on the results of these studies, a further series of experiments were conducted in which the effects of 1.0 Fg per kg of dm PGE, on Na’ and K+ flux induced by 15% ethanol plus 150 mN NaCl were evaluated. The design of these experiments was similar to those described above except that, after the control period, dm PGE, was injected intravenously as a single bolus in the appropriate test dose, after which the control period was extended for an additional 30 min. After each 15-min collection, the volume was measured and the acid concentration determined by titration to pH 7.0 with 0.2 N NaOH using an Autoburet andpH meter (Radiometer, Copenhagen, Denmark). The Na+ and K+ concentration of each sample was determined by flame photometry (Brinkmann Eppendorfmodel700, Westbury, N. Y.). Total amounts of Na’, K’, and H+ in the recovered fluid were calculated for each period. The net flux of each ion was determined by subtracting the amount put in the pouch from the amount recovered and expressed as microequivalents per 15 min. The pepsin concentration of each sample was determined using a modification of the Anson hemoglobin method.“’ Results were expressed as milligrams of pepsin per milliliter after reading the trichloroacetic acid supernatant fluid at 280 rnp and comparing it with solutions incubated with different concentrations of pepsin (hog pepsin, three times crystallized, Pentex Biochemicals, Kankakee, Ill.) Pepsin outputs were calculated and expressed as the percentage of the mean of the final two control periods when the pouches were irrigated with 100 mN HCl and 50 mN NaCl. This was done to eliminate some of the variability between individual animals which had consistent but different basal levels of pepsin secretion. Experiments were done on each dog no more than twice a week. All of the results are expressed as means and standard errors of the means. Control values in each experiment were obtained by averaging the final two 15-min periods of the initial control period. The effects of ethanol and dm PGE, were compared using the t-test for either unpaired or paired data. Differences were considered significant when P < 0.05.
Results During the two final 1Bmin control periods of the experiment in which 15% ethanol in acid or neutral solution alone was given, there was a small loss or net negative flux of H+ equal to about 4% of that in the pouch. Likewise there was a small positive gain of Na+ and K+ into the luminal bathing fluid. Irrigation of the pouch with 15% ethanol in combination with 100 mN HCI caused a significant increase in the net negative flux of H+ compared to control periods (fig. 1). The loss of H+ reached maximal levels during periods 7 and 8. Removal of ethanol and irrigating the pouches with 100
50 mN N&I
o-
g E z $
+ 100 mN I-ICI
15% Ethanol
Ethanol
+ 0.1 pglkg
Ed,.“.,,
+ 0.01 pglkg
dm PCEZ
200Edwnol + 1 .O yglkg
dm PGEZ dm PGEZ
400-
3 9 2 600f 5 = 6004\, 3
,
#
,
,
,
,
,
,
,
4
5
6
7
6
9
10
11
12
Time (15 min periods)
FIG. 1. Effect of 16,lgdimethyl prostaglandin E, (dm PGE,) on net H’ loss in response to topical application of 15% ethanol in 100 mN HCl. Control series (0-O; ethanol alone) represents the mean of eight experiments (*SEMI. Dm PGE, was given as an intravenous bolus injection after period 4. In this and subsequent figures the effect of intravenous dm PGE, in the doses 0.01 CO-. -01, 0.1 (A-- -A) and 1.0 (A---A) pg per kg are presented and represented by the means of 10, 8, and 9 experiments (~sEM). respectively. Asterisks, significant differences (P i 0.05) between corresponding points of each dm PGE, series and ethanol alone by the unpaired t-test.
mN HCl and 50 mN NaCl alone reduced the loss of H’ , but even after the 1-hr recovery period, the net flux of H+ had not returned to pretreatment values. Intravenous injection of dm PGE, in the doses 0.01, 0.1, and 1.0 pg per kg in each case did not totally prevent the increase in H+ loss which occurred after ethanol administration. However, dm PGE, in the dose of 0.1 pg per kg significantly reduced at all points (periods 5 to 12) the degree of H+ loss when compared to the series of experiments in which ethanol alone was given (fig. 1). Dm PGE, in the dose of 1.0 pg per kg significantly reduced the net loss of H+ in all periods with the exception of periods 9 and 10. The lowest dose of prostaglandin (0.01 pug per kg) did not significantly reduce the degree of H+ back diffusion produced by topical ethanol administration, although the mean H+ loss in the final 45 min of the experiment after ethanol was withdrawn was significantly less than in the ethanol-treated series alone. The net positive flux of Na+ was greatly exaggerated upon ethanol administration in acidic solution (fig. 2) and this flux did not return to pretreatment values upon ethanol withdrawal. The net flux of Na+ also significantly increased in spite of prior intravenous injection of dm PGE, (fig. 2). However, the highest doses of dm PGE, used in this study (0.1 and 1.0 pg per kg) significantly reduced the amount of Nat appearing in the solution recovered from the Heidenhain pouch compared to ethanol alone. The lowest dose of dm PGE, (0.01 pg per kg) did not appreciably alter the effect of 15% ethanol on the gastric mucosal barrier. Administration of ethanol in neutral solution (fig. 3) also caused a significant efJlux of Nat which did not return to control
December
1978 1
ETHANOL 50 mN N&l
q
T
T
T
6
7
6
reduced this et&x at all time periods throughout the experiment. The output of pepsin increased after ethanol treatment (fig. 6). Removal of ethanol did not ameliorate the pepsin response with the output displaying a tendency to increase even further during the recovery period. Only one dose of dm PGE, (1.0 pg per kg) prevented the increase in pepsin output that occurred upon topical treatment with ethanol. The other doses of dm PGE, (0.01 and 0.1 pg per kg) did not prevent the increase in pepsin output, although the response returned to preethanol levels faster than it did without injection of dm PGE,.
5
I
,” 600 3
3 2
400
cl + LO 2
3
4
5
9
10
11
Discussion
12
Tlme (15 mln perlods)
FIG. 2. Net fluxes of Na- into the Heidenhain experiment described in figure 1.
150 mN
1063
AND PROSTAGLANDINS
1
+ 100 mN HCI
15% Ethanol
DAMAGE
pouch during the
N&l
Alcohol when given in 100 mN HCl topically resulted in significant damage to the canine gastric mucosa. These results confirm the findings of Davenport” who reported that ethanol concentrations greater than 14% disrupted the gastric mucosal barrier as evidenced by large increases in the net fluxes of H+, Na+, and K+. In
15% Ethanol
2 E
40
2 2
30
3 S z
Ethanol alone
20
+ Y
FIG. 3. Effect of 16,16-dimethyl prostaglandin E, (dm PGEJ on net Na’ efflux in response to topical application of 15% ethanol in 150 mN NaCl. Control series (ethanol alone) represents the mean -t SEM of nine experiments, whereas dm PGE, treated series represents the mean YXSEM of six experiments. Asterisks, significant differences (P < 0.05) between corresponding points of each dm PGE, series and ethanol alone b’y the unpaired t-test.
I
10
L
,
,
(
,
,
1
,
4
5
6
7
6
9
10
I
11
,
12
Time (15 min periods) FIG. 4. Net fluxes
experiment
levels until ethanol withdrawal. Prior administration of dm PGE, almost entirely eliminated this Na+ efIlux. Ethanol in acid solution resulted in a significant increase in the net flux of K+ in the canine Heidenhain pouch and this flux did not return to pretreatment values after ethanol was withdrawn (fig. 4). Dm PGE, produced the same apparent protective ability on this index of barrier integrity as was displayed against the net flux of Na’. The highest doses of prostaglandin resulted in significant reductions in K+ flux after ethanol. Although not affecting the amount of K+ appearing in the pouch upon ethanol instillation, 0.01 pg per kg of dm PGE, still significantly reduced the net flux of K+ which occurred in the final 45 min of the recovery period. Ethanol in neutral solution (fig. 5) also resulted in a significant increase in KC efflux which did not return to pretreatment values after ethanol withdrawal. Prior administration of dm PGE, significantly
I
3
of K’ into the Heidenhain described in figure 1.
pouch during the
150 mN NaCI 15% Ethanol
.__
=
I
_~.
I
*
\
L
3
4
5
6
7
8
9
10
11
12
Time (15 mh perk&)
FIG. 5. Net fluxes
experiment
of K+ into the Heidenhain described in figure 3.
pouch during the
1064
TEPPERMAN 50 mN NaCI + 100 mN WI 15% Ethanol
3
I
’
I
’
,
1
I
I
I
4
5
6
7
6
9
10
11
12
Time (15 min periods)
FIG. 6. Pepsin output as a percentage experiment described in figure 1.
of control
during
the
addition, Davenport” showed that the presence of acid was not essential for ethanol to produce damage to the gastric mucosa by finding that alcohol was no more damaging when given in 100 mN HCl than it was in a solution buffered to pH 7.5. We have confirmed these observations as well. Intravenous administration of dm PGE, resulted in significant reduction in the net fluxes of H+, Na+, and K+ associated with ethanol-induced damage to the gastric mucosal barrier in both an acidic and neutral solution. We are aware of no other report which demonstrates that any prostaglandin, natural or synthetic, reduces the degree of barrier disruption associated with topical application of this ulcerogenic agent. This protective action of prostaglandin was manifested in the present study by an amelioration in the net ionic fluxes. These results extend the finding in the rat that both natural and synthetic prostaglandin analogues prevent or reduce gross mucosal damage induced by damaging agents.‘, 3,ti,i, 12 The antiulcer ability of the prostaglandins may be related to their potent antisecretory properties. However, evidence exists which would militate against such a possibility. First, prostaglandins which lack antisecretory activity still possess cytoprotective properties.’ Furthermore, it has been demonstrated that effective doses for cytoprotection are often much lower than the median (ED:,,,) effective doses for antisecretion.2, :LI2 In the present study, however, the optimal protective doses were 0.1 and 1.0 pg of dm PGE, per kg, a dose range which has been shown by Robert et al.’ to inhibit most effectively histamine-stimulated acid secretion in the Heidenhain pouch dog (the ED~,, being 0.1 pg per kg). Thus it appears that doses below the ED,,,antisecretory dose are ineffective cytoprotective agents in the Heidenhain pouch dog, at least against ethanol-induced damage. Nevertheless, the cytoprotective ability of dm PGE, observed in the present study cannot be explained solely in terms of inhibition of acid secretion. The animals used in this study were not stimulated to secrete acid. Even if small amounts of acid were secreted, protection
Vol. 7ii. No. 6
ET AL.
attributable to an inhibition of acid secretion cannot be the explanation for the observed protection because inhibition of acid output would appear to produce a greater loss of H+ from the pouch. This, however, was not the case. Finally, the observation that dm PGE, protects against ethanol-induced damage in neutral solution lends further support to the hypothesis that mechanisms other than inhibition of acid secretion must be involved in offering this protection. The mechanism underlying protection against ethanol-induced gastric mucosal injury by dm PGE, is not apparent from the present study. Although aspirin inhibits the enzyme system responsible for the conversion of arachidonic acid to the natural prostaglandin compounds,” alcohol has not been shown to be a synthetase inhibitor. The ability of dm PGE, to protect canine gastric mucosa from both aspirin- and ethanol-induced damage indicates that the mechanism of cytoprotection is not, at least in total, caused by an involvement of prostaglandin synthetase. Johnson’:’ has demonstrated that 15% ethanol in 100 mN HCl results in a significant increase in pepsin output from the damaged canine Heidenhain pouch. Furthermore, it was found that H+ ion must be present to back diffuse through the mucosa in order for stimulation of pepsin secretion to occur. Once the barrier to diffusion of H+ is broken, a low concentration of acid is sufficient to stimulate pepsin secretion. In the present study, dm PGE, significantly reduced but did not prevent back diffusion of H.+. Thus it would be expected that pepsin secretion might still continue in the dm PGE, series. However, it would also be expected that the reduced H+ back diffusion would result in a reduction in the peptic response inasmuch as Johnson14 has demonstrated a dependency between acid concentration and pepsin output. Such was the case, for all doses of dm PGE, lowered pepsin output during the recovery period even though only the highest dose (1.0 pg per kg) prevented pepsin secretion during the presence of ethanol. In conclusion, we have demonstrated that the synthetic prostaglandin analogue, dm PGE, aids in the maintenance of gastric mucosal barrier integrity as measured by ionic fluxes. The ability of a prostaglandin to protect against the damaging agent, ethanol, which is not assumed to be an inhibitor of prostaglandin synthetase, suggests that this enzyme system is not necessarily involved in the mechanism of prostaglandin cytoprotection. Because this protective effect was also observed in neutral solution, it does not depend on the antisecretory property of dm PGE,. REFERENCES Robert A, Schultz JR, Nezamis JE, et al: Gastric antisecretory and antiulcer properties of PGE,, l&methyl PGE, and 16,16dimethyl PGE,. Gastroenterology 70:359-370, 1976 Robert A: Antisecretory, antiulcer, cytoprotective and diarrheogenie properties of prostaglandins. In Advances in Prostaglandin and Thromboxane Research, vol 2. Edited by B Samuelsson, R Paoletti. New York, Raven Press, 1976, p 507-520 Robert A, Nezamis JE, Lancaster C, et al: Gastric cytoprotective property of prostaglandins (abstr). Gastroenterology 72:1121, 1977
December 1978 4. Miller TA, Tepperman
5.
6.
7.
8.
ETHANOL BL: Protection
against
DAMAGE
AND
aspirin-induced
gastric mucosal injury by 16,16 dimethyl prostaglandin E, (abstr). Gastroenterology 72:1102, 1977 Vane JR: Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs. Nature [New Biol] 231:232-235, 1971 Mann NS: 13ile-induced acute erosive gastritis. Its prevention by antacid, chtolestyramine and prostaglandin E,. Am J Dig Dis 21:89-92, 1976 Ferguson WW, Edmonds AW, Starling JR, et al: Protective effect of prostaglandin E, (PGE,) on lysosomal enzyme release in serotonin-induced gastric ulceration. Ann Surg 177:648-654, 1973 Thomas JE: An improved cannula for gastric and intestinal
PROSTAGLANDINS fistulas. Proc Sot Exp Biol Med 46:260-261. 1941 9. Gregory RA: Gastric secretory responses after portal
1065
venous
ligation. J Physiol (Lend) 144:123-127, 1958 10. Anson ML: The estimation of pepsin, trypsin, papain and cathepsin with hemoglobin. J Gen Physiol 22:79-89, 1938 11. Davenport HW: Ethanol damage to canine oxyntic glandular mucosa. Proc Sot Exp Biol Med 126:657-662, 1967 12. Main IHM, Whittle BJR: Potency and selectivity of methyl analogues of prostaglandin E, on rat gastrointestinal functions. Br J Pharmacol 54:309-317, 1975 13. Johnson LR: Pepsin secretion during damage by ethanol and salicylic acid. Gastroenterology 62:412-416, 1972 14. Johnson LR: Regulation of pepsin secretion by topical acid in the stomach. Am J Physiol 223:847-850, 1972