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Nonprotein sulfhydryls as possible components of the protective effect of rosaprostol on the rat gastric mucosa
PROSTAGLANDINS
NONPROTEIN SULFHYDRYLS AS POSSIBLE COMPONENTS OF THE PROTECTIVE EFFECT OF ROSAPROSTOL ON THE RAT GASTRIC MUCOSA R. Fumagalli', R. Caponi2, A. Eorsini', A. Bra$billa2, M. Palmira2, F. Bernini and U. Valcavi 1
Institute of Pharmacology and Pharmacognosy, 2
University of Milan, 20129 Milan, Italy Istituto Biochimico Italiano, Milan, Italy ABSTRACT
Experiments
were
designed to examine the possibility
that non-
protein sulfhydryl groups of the gastric mucosa could participate in the protection of rat gastric mucosa by rosaprostol (the Na salt of 9-hydroxy-8,12 trans-19,20-bis-nor-prostanoic acid). Gastric mucosal lesions and the content of nonprotein sulfhydryls were evaluated after orally administered absolute ethanol. Pretreatment with rosaprostol
by
gavage
prevented
gastric
lesions
and
reduced
or
prevented the decrease of mucosal nonprotein thiols. N-ethylmaleimide, a sulfhydryl blocker, worsened the ethanol-induced gastric lesions and lowered further the non protein thiols. Both variables were improved by the PG analogue and by PGE These results suggest a 2' possible role of endogenous nonprotein sulfhydryl groups in the gastric protective effect of rosaprostol. INTRODUCTION Prostaglandins in non-antisecretory doses can prevent acute hemorrhagic erosions of the rat gastric mucosa induced by a number of noxious substances such as absolute ethanol, concentrated acid and alkali, hypertonic NaCl solution and boiling water (1). Several protective mechanisms have been proposed including enhanced mucus formation (2,3). A new insight is the work of Szabo et al. (4), on mucosal nonprotein sulfhydryl groups (NPSH). A synthetic prostanoid, trans-19,20-bis-nor-prostanoate the sodium 9-hydroxy-8,12 (rosaprostol), is a gastric antisecretory and antiulcer compound in experimental animals and is reported to have no hemodynamic, uterine and intestinal activity (5). The present investigation was undertaken to evaluate the effect of this prostanoid on the content of mucosal NPSH in relation to its preventive effect on acute gastric lesions induced by ethanol.
MARCH
1985 VOL. 29 NO. 3
PROSTAGLANDINS
MATERIALS AND METHODS Reduced glutathione, N-ethylmaleimide (PGE ) were
obtained
from
Sigma
(St.
(NEM) and prostaglandin E2 Louis,
MO,
U.S.A.);
5,5'-
dithfo-2-nitrobenzoic acid was from Aldrich (Milwaukee, Wi, U.S.A.). Rosaprostol (Istituto Biochimico Italiano, Milan, Italy) and NEM were dissolved in distilled water; PGE2 was first dissolved in 70% ethanol and
stored at -2OOC; aliquots of this solution were diluted with distilled water immediately before use (ethanol final concentration: l-4%, v:v). N-ethylmaleimide was administered subcutaneously, whereas the other drugs were given by stomach intubation: in both cases the administered volumes were 5 ml/kg b.w., and control animals received equal
volumes of water. Absolute ethanol gavage as the stomach damaging agent.
(5 ml/kg)
was
given
by
Experimental protocols. Male Wistar rats of 200-220 g body weight were used after 18 hr fasting (water ad libitum until 30 min before treatments). All the experiments were performed between 2 and 4 p.m. because of the diurnal variation in the content of nonprotein sulfhydryl groups in the rat gastric mucosa (6). In a first experiment rosaprostol was given at various time-intervals before absolute ethanol; in all the other experiments this drug or PGE were 2 administered 30 min before the damaging agent. When used, N-ethylmaleimide ethanol, tively. damaging
was injected subcutaneously 60 or 20 min before absolute i.e. 30 min before and 10 min after rosaprostol, respecRats were killed by decapitation 5 to 60 min after the agent. The stomachs were removed, opened along the greater
curvature, and washed with saline. Lesion score. The gastric mucosa was examined with a 6.4 x binocular magnifier and the lesions were assessed independently by two observers unaware of the experimental protocols. Lesions were scored according to an arbitrary scale ranging from 0 to 10 and taking into account the presence, number, extension and confluency of erosions, petechiae'and hemorrhagic streaks. Nonprotein sulfhydryl groups in the gastric mucosa. After scoring, the glandular mucosa was scraped off with a blunt knife, and nonprotein sulfhydryl content of the gastric mucosa measured using the method of Elmann (7,8) as modified by Sedlack and Lindsay (9). The mucosa was homogenized in 40 volumes of 0.2 M acqueous EDTA. A 5 ml aliquot of the homogenate was mixed thoroughly with 4 ml distilled water and 1 ml 50% TCA, and then centrifuged at 3000 g for 15 min. Four ml of Tris buffer (0.4 M, pH 8.9) and 100 ul 5,5'-dithio-2-
468
MARCH
1985 VOL. 29 NO. 3
PROSTAGLANDINS
nitrobenzoic supernatant by measuring
acid
0.01
M,
and nonprotein the absorbance
were
added
to
a
2
ml
aliquot
of
the
sulfhydryls were determined in duplicate at 412 nm vs a standard curve obtained
with reduced glutathione. Protein was estimated according to Lowry et al. (10). Statistics. Data are expressed as mean + SEM, or as median (range). The comparison of gastric lesion scores between control and treated animals was based on non-parametric
analysis using Wilcoxon's test for unpaired data. The NPSH content of the gastric mucosa of the different experimental groups was analyzed by two-tailed Student's t-test for unpaired
data and by Duncan's test. Dose-dependency
was
assessed by regression analysis. RESULTS The protective effect of rosaprostol (DL by gavage: >, 5000 mg/kg) against acute gastric lesions induced aft:! 60 min exposure to absolute ethanol in rats was dependent upon the time-interval between the administration of the drug and the damaging agent. The effect was dose-dependent, reached a maximum when the drug was administered 30-15 min before‘. ethanol, and still occurred when the drug administration was only 0.5 min before that of ethanol (Figure 1). Absolute ethanol decreased the mucosal nonprotein sulfhydryl content, the highest effect being observed 15 min after alcohol administration. This reduction decreased by rosaprostol given by gavage 30 min before ethanol (Figure 2). The dose-related protective effect of the synthetic prostanoid on the acutely induced gastric lesions was paralleled by an increased content of gastric mucosa NPSH (Table 1). The administration of N-ethylmaleimide, a sulfhydryl blocking agent which decreases mucosal NPSH in normal animals (4), increased the gastric erosion Score elicited by ethanol in parallel with a further decrease :bf the mucosal NPSH (Table 2). Protection by rosaprostol was displayed when either preceded or followed the sulfhydryl blocker the drug administration (Table 2). Increasing doses of rbsaprostol or prostaglandin E given to rats killed 60 min after receiving ethanol progressively rsduced the stomach lesion score and the reduction of non protein sulfhydryls (Table 3).
MARCH
1985 VOL. 29 NO. 3
469
PROSTAGLANDINS
100
7
h1
A
B C
0 5oIL
60
120
T,ME
(MIN)
mg/
kg
B 200
mgl
kg
C 100
mglkg
0.5
5
15
30
A 400
BEFORE
ETHANOL
Fig. 1 Effect of rosaprostol on the rat stomach damaged by ethanol.
ETHANOL
+
ROSAPROSTOL
l
*
ETHANOL
5
15
30 TIME
pc
Fig.
470
2.
0.05
(0);
0.02(-I;
0.01 (.*.I;
60 OF 0.001
SACRIFICE
(MINI
(****I
Rosaprostol (400 mg/kg) reduced the fall of non protein sulfhydryls in the rat stomach mucosa (100% = 23.9 + 2.0 nmoles/mg prot.).
MARCH
1985 VOL. 29 NO. 3
PROSTAGLANDINS
Table
1. Preventive
effect of rosaprostol
on the formation
of rat
gastric mucosal lesions caused by alcohol.
TREATMENT
DOSE mg/kg
Pa
LESION SCORE median (range)
MUCOSAL NPSHb nmoles/mg prot.
2
(l-3)
---
15.2 + 0.9
100
1
(O-4)
n.s.
16.6 + 0.5
9,
200
0.5 (O-5)
0.01
18.3 + 0.5
,t
400
0
0.001
19.3 + 0.8
ETHANOL ETHANOL + ROSAPROSTOL
(O-1)
Rats (9-12/group) were killed 15 min after ethanol administration. a, Wilcoxon's test; b, dose-dependency was linear, as assessed by regression analysis (P
Table
2.
Protective
effect
of
rosaprostol
(400 mg/kg)
on the rat
stomach. TREATMENT
LESION SCORE median (range)
1 2 3 4 5 6 7
NONE ETHANOL ETHANOL + ROSAPROSTOL NEM + ETHANOL (20 min) ROSAPROSTOL + NEM + ETHANOL NEM + ETHANOL (60 min) NEM + ROSAPROSTOL + ETHANOL
0 2 0 8.5 0 7 1
(O-1) (O-4) (o-l)a (l-10) (o-l)a (4-10) (o-3)a
MUCOSAL NPSH nmoles/mg prot. 24.9 16.1 19.8 13.1 17.5 9.6 15.9
+ ; + + 5 ; :
l.Ob 0.5 1.1 1.4 0.9 1.3 0.6
Rats (8-lo/group) were killed 15 min after ethanol administration. Times refer to the interval between NEM administration and ethanol gavage; rosaprostol was given 30 min before ethanol. Wilcoxon's test: a (p
MARCH 1985 VOL. 29 NO. 3
471
PROSTAGLANDINS
Table 3. Protective effect of rosaprostol and PGE_ on the rat stomach TREATMENT
LESION SCORE median (range)
MUCOSAL NPSH' nmoles/mg prot.
NONE
0
(0-l)
24.5 + 1.5
ETHANOL
2
(l-3)
16.7 + 1.5
NEM + ETHANOL
6
(2-7)
11.3 : 0.8
ROSAPROSTOL (100
mg/kg)
2
(1-3jb
15.8 + 0.8d
NEM + ETHANOL ROSAPROSTOL (200 mg/kg) + NEM + ETHANOL
1
(O-3Jb
18.8 + 1.2d
ROSAPROSTOL (400 mg/kg)
0
(O-ljb
23.4 + l.ld
+
+ NEM + ETHANOL PGE;! (50 u&kg)
+ NEM + ETHANOL
PGE7 (300 ug/kg) + NEM + ETHANOL
3.5 (2-loia 1 (O-3)
13.3 + 1.2 21.5 f. 1.4
Rats (9-lo/group) were killed 60 min after ethanol administration. Rosaprostol and NEM were given 30 and 20 min before ethanol, respectively. Wilcoxon's test: a (pcO.05); b (p
was
linear, as assessed by
DISCUSSION Rosaprostol protects rat gastric stomach from lesions caused by various necrotizing agents, including concentrated HCl (0.6 M) and absolute ethanol. Although the prostanoid is effective in these experimental models at doses that are antisecretory, its protective properties seem to be independent from interference with gastric acid secretion (5). The observation by Szabo et al. (4) that rat hemorrhagic gastric erosions induced by absolute ethanol accompany a dose-dependent decrease of gastric mucosal NPSH suggests a new approach to the mechanisms of cytoprotection. Sulfhydryl-containing drugs protected against alcohol-induced gastric damage, while sulfhydyl blocking . The reduction compounds counteracted the protective effect of PGF by rosaprostol of gastric damage by ethanol accom$?.nied a smaller decrease of NPSH material in the gastric mucosa. Surprisingly,
472
MARCH
1985 VOL. 29 NO. 3
PROSTAGLANDINS
however,
rosaprostol
exerted
protection
and the mucosal
content of
NPSH remained higher despite the previous administration of the sulfhydryl b.Locker N-ethylmaleimide which, in the presence of absolute ethanol, further depleted gastric mucosal NPSH and worsened the Lesion scores. Prostaglandin E2 gave analogous results. The role played by endogenous NPSH in gastric mucosal protection is debated. The mediatory effect suggested by Szabo et al. (4) is at variance with results obtained by others using a different NPSH blocker (11,121. Our results do not rule out a possible role of NPSH in rat gastric mucosal protection. The failure of the sulfhydryl blocker to counteract the protective effect of rosaprostol is associated
with
a smaller
decrease
of mucosal NPSH levels. This is
difficult to interpret since it occurred when the sulfhydryl blocker was given either before or after the prostanoid. Rosaprostol alone, at variance with cysteamine, did not increase the mucosal NPSH content (unpublished data). Nonprotein thiols are highly concentrated (6), they bind reactive influence the structure
elnctrophilic of
gastric
in the stomach mucosa
radicals
mucus
(13), and they may
(14). Since
rosaprostol
stimulates mucus production in rats and humans (5,15) and enhances the mucoprotective index in humans (16), and since the mucus is considered one possible key to gastric cytoprotection (3,171, the capacity of rosaprostol to inhibit the fall of gastric NPSH elicited by alcohol could contribute to its protective properties. ACKNOWLEDGEMENTS The authors thank Prof. F. Berti (University of Milan, Italy) for his stimulating suggestions about this work. REFERENCES 1) Robert, A., J.E. Nezamis, C. Lancaster, and A.J. Hanchar. Cytoprotection by prostaglandins in rat. Prevention of gastric necrosis produced by alcohol, HCl, NaOH, hypertonic NaCl, and thermal injury. Gastroenterology 77~433. 1979. 2) Miller, T.A., and E.D. Jacobson. GastrointestinaL cytoprotection by prostaglandins. Gut 20:75. 1979. 3) Nezamis, J.E., and A. Robert. Gastric mucus may mediate cytoprotective effect of prostaglandins. Gastroenterology 1228. 1980. 4) Szabo, S., J.S. Trier and P.W. Frankel. Sulfhydryl compounds
MARCH
1985 VOL. 29 NO. 3
the 78: may
473
PROSTAGLANDINS
mediate gastric cytoprotection. Science 214:200. 1981. 5) Valcavi, U., R. Caponi, A. Brambilla, M. Palmira, F. Minoja, F. Bernini, R. Musanti and R. Fumagalli. Gastric antisecretory, antiulcer and cytoprotective properties of 9-hydroxy-19,20-norbis-prostanoid
acid
in experimental
animals.
Arzneim.
Forsch.
32:657. 1982. 6) Boyd, S.C., M.R. Sasame and M.R. Boyd. High concentration of glutathione in g.landular stomach: possible implication for carcinogenesis. Science 205:lOlO. 1979. 7) Ellman, G.L. A calorimetric method for determining low concentrations of mercaptans. Arch. Biochem. Biophys. 74:443. 1958. 8) Ellman, G.L. Tissue 82:70. 1959.
sulfhydryl
groups.
Arch. Biochem.
Biophys.
J.S. and R.H. Lindsay. Estimation of total, protein9) cdlak, bound and nonprotein sulfhydryl groups in tissue with Ellman's reagent. Anal. Biochem. 25:192. 1968. 10) Lowry, O.H., N.I. Rosebrough, A.L. Farr and R.J. Randall. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193:265. 1951. 11) Eberle, D., A. Robert and N. Kaplowitz. Glutathione (GSH) and gastric cytoprotection: an inverse relationship. Gastroenterology 12)
80:1140. 1981. Konturek, S.J., T. Brzozowski,
I. Piastucki,
T. Radecki and S.
Szabo. Role of endogenous prostaglandins in gastric cytoprotection induced by agents altering mucosal content of sulfhydryl compounds. V International Conference on Prostaglandins, Florence, Italy; Abstract book, 1982, p. 371. 13) Mead, J.F. In: Free radicals in biology (W.A. Pryor, ed.) Academic Press, New York, 1976, vol. 1, p. 51. 14) Allen, A. Structure of gastrointestinal mucus glycoprotein and the viscous and gel-forming properties of mucus. Br. Med. Bull. 34:28. 1978. Domschke, W., S. Domschke, S. Hornig and L. Demling. Prostaglan15) din-stimulated mucus secretion in man. Acta Hepato-gastroenterol. 25:292. 1978. 16) Guslandi, M., D. Foschi, F. Ferrante and V. Rovati. Changes in human gastric mucus following rosaprostol treatment. Curr. Ther. Res. 35:643. 1984. 17) Robert, A. Cytoprotection 77:761. 1979. Editor:
474
A.
by
prostaglandins.
Gastroenterology
Accepted:
Bennet
MARCH
12-17-84
1985 VOL. 29 NO. 3
NONPROTEIN SULFHYDRYLS AS POSSIBLE COMPONENTS OF THE PROTECTIVE EFFECT OF ROSAPROSTOL ON THE RAT GASTRIC MUCOSA R. Fumagalli', R. Caponi2, A. Eorsini', A. Bra$billa2, M. Palmira2, F. Bernini and U. Valcavi 1
Institute of Pharmacology and Pharmacognosy, 2
University of Milan, 20129 Milan, Italy Istituto Biochimico Italiano, Milan, Italy ABSTRACT
Experiments
were
designed to examine the possibility
that non-
protein sulfhydryl groups of the gastric mucosa could participate in the protection of rat gastric mucosa by rosaprostol (the Na salt of 9-hydroxy-8,12 trans-19,20-bis-nor-prostanoic acid). Gastric mucosal lesions and the content of nonprotein sulfhydryls were evaluated after orally administered absolute ethanol. Pretreatment with rosaprostol
by
gavage
prevented
gastric
lesions
and
reduced
or
prevented the decrease of mucosal nonprotein thiols. N-ethylmaleimide, a sulfhydryl blocker, worsened the ethanol-induced gastric lesions and lowered further the non protein thiols. Both variables were improved by the PG analogue and by PGE These results suggest a 2' possible role of endogenous nonprotein sulfhydryl groups in the gastric protective effect of rosaprostol. INTRODUCTION Prostaglandins in non-antisecretory doses can prevent acute hemorrhagic erosions of the rat gastric mucosa induced by a number of noxious substances such as absolute ethanol, concentrated acid and alkali, hypertonic NaCl solution and boiling water (1). Several protective mechanisms have been proposed including enhanced mucus formation (2,3). A new insight is the work of Szabo et al. (4), on mucosal nonprotein sulfhydryl groups (NPSH). A synthetic prostanoid, trans-19,20-bis-nor-prostanoate the sodium 9-hydroxy-8,12 (rosaprostol), is a gastric antisecretory and antiulcer compound in experimental animals and is reported to have no hemodynamic, uterine and intestinal activity (5). The present investigation was undertaken to evaluate the effect of this prostanoid on the content of mucosal NPSH in relation to its preventive effect on acute gastric lesions induced by ethanol.
MARCH
1985 VOL. 29 NO. 3
PROSTAGLANDINS
MATERIALS AND METHODS Reduced glutathione, N-ethylmaleimide (PGE ) were
obtained
from
Sigma
(St.
(NEM) and prostaglandin E2 Louis,
MO,
U.S.A.);
5,5'-
dithfo-2-nitrobenzoic acid was from Aldrich (Milwaukee, Wi, U.S.A.). Rosaprostol (Istituto Biochimico Italiano, Milan, Italy) and NEM were dissolved in distilled water; PGE2 was first dissolved in 70% ethanol and
stored at -2OOC; aliquots of this solution were diluted with distilled water immediately before use (ethanol final concentration: l-4%, v:v). N-ethylmaleimide was administered subcutaneously, whereas the other drugs were given by stomach intubation: in both cases the administered volumes were 5 ml/kg b.w., and control animals received equal
volumes of water. Absolute ethanol gavage as the stomach damaging agent.
(5 ml/kg)
was
given
by
Experimental protocols. Male Wistar rats of 200-220 g body weight were used after 18 hr fasting (water ad libitum until 30 min before treatments). All the experiments were performed between 2 and 4 p.m. because of the diurnal variation in the content of nonprotein sulfhydryl groups in the rat gastric mucosa (6). In a first experiment rosaprostol was given at various time-intervals before absolute ethanol; in all the other experiments this drug or PGE were 2 administered 30 min before the damaging agent. When used, N-ethylmaleimide ethanol, tively. damaging
was injected subcutaneously 60 or 20 min before absolute i.e. 30 min before and 10 min after rosaprostol, respecRats were killed by decapitation 5 to 60 min after the agent. The stomachs were removed, opened along the greater
curvature, and washed with saline. Lesion score. The gastric mucosa was examined with a 6.4 x binocular magnifier and the lesions were assessed independently by two observers unaware of the experimental protocols. Lesions were scored according to an arbitrary scale ranging from 0 to 10 and taking into account the presence, number, extension and confluency of erosions, petechiae'and hemorrhagic streaks. Nonprotein sulfhydryl groups in the gastric mucosa. After scoring, the glandular mucosa was scraped off with a blunt knife, and nonprotein sulfhydryl content of the gastric mucosa measured using the method of Elmann (7,8) as modified by Sedlack and Lindsay (9). The mucosa was homogenized in 40 volumes of 0.2 M acqueous EDTA. A 5 ml aliquot of the homogenate was mixed thoroughly with 4 ml distilled water and 1 ml 50% TCA, and then centrifuged at 3000 g for 15 min. Four ml of Tris buffer (0.4 M, pH 8.9) and 100 ul 5,5'-dithio-2-
468
MARCH
1985 VOL. 29 NO. 3
PROSTAGLANDINS
nitrobenzoic supernatant by measuring
acid
0.01
M,
and nonprotein the absorbance
were
added
to
a
2
ml
aliquot
of
the
sulfhydryls were determined in duplicate at 412 nm vs a standard curve obtained
with reduced glutathione. Protein was estimated according to Lowry et al. (10). Statistics. Data are expressed as mean + SEM, or as median (range). The comparison of gastric lesion scores between control and treated animals was based on non-parametric
analysis using Wilcoxon's test for unpaired data. The NPSH content of the gastric mucosa of the different experimental groups was analyzed by two-tailed Student's t-test for unpaired
data and by Duncan's test. Dose-dependency
was
assessed by regression analysis. RESULTS The protective effect of rosaprostol (DL by gavage: >, 5000 mg/kg) against acute gastric lesions induced aft:! 60 min exposure to absolute ethanol in rats was dependent upon the time-interval between the administration of the drug and the damaging agent. The effect was dose-dependent, reached a maximum when the drug was administered 30-15 min before‘. ethanol, and still occurred when the drug administration was only 0.5 min before that of ethanol (Figure 1). Absolute ethanol decreased the mucosal nonprotein sulfhydryl content, the highest effect being observed 15 min after alcohol administration. This reduction decreased by rosaprostol given by gavage 30 min before ethanol (Figure 2). The dose-related protective effect of the synthetic prostanoid on the acutely induced gastric lesions was paralleled by an increased content of gastric mucosa NPSH (Table 1). The administration of N-ethylmaleimide, a sulfhydryl blocking agent which decreases mucosal NPSH in normal animals (4), increased the gastric erosion Score elicited by ethanol in parallel with a further decrease :bf the mucosal NPSH (Table 2). Protection by rosaprostol was displayed when either preceded or followed the sulfhydryl blocker the drug administration (Table 2). Increasing doses of rbsaprostol or prostaglandin E given to rats killed 60 min after receiving ethanol progressively rsduced the stomach lesion score and the reduction of non protein sulfhydryls (Table 3).
MARCH
1985 VOL. 29 NO. 3
469
PROSTAGLANDINS
100
7
h1
A
B C
0 5oIL
60
120
T,ME
(MIN)
mg/
kg
B 200
mgl
kg
C 100
mglkg
0.5
5
15
30
A 400
BEFORE
ETHANOL
Fig. 1 Effect of rosaprostol on the rat stomach damaged by ethanol.
ETHANOL
+
ROSAPROSTOL
l
*
ETHANOL
5
15
30 TIME
pc
Fig.
470
2.
0.05
(0);
0.02(-I;
0.01 (.*.I;
60 OF 0.001
SACRIFICE
(MINI
(****I
Rosaprostol (400 mg/kg) reduced the fall of non protein sulfhydryls in the rat stomach mucosa (100% = 23.9 + 2.0 nmoles/mg prot.).
MARCH
1985 VOL. 29 NO. 3
PROSTAGLANDINS
Table
1. Preventive
effect of rosaprostol
on the formation
of rat
gastric mucosal lesions caused by alcohol.
TREATMENT
DOSE mg/kg
Pa
LESION SCORE median (range)
MUCOSAL NPSHb nmoles/mg prot.
2
(l-3)
---
15.2 + 0.9
100
1
(O-4)
n.s.
16.6 + 0.5
9,
200
0.5 (O-5)
0.01
18.3 + 0.5
,t
400
0
0.001
19.3 + 0.8
ETHANOL ETHANOL + ROSAPROSTOL
(O-1)
Rats (9-12/group) were killed 15 min after ethanol administration. a, Wilcoxon's test; b, dose-dependency was linear, as assessed by regression analysis (P
Table
2.
Protective
effect
of
rosaprostol
(400 mg/kg)
on the rat
stomach. TREATMENT
LESION SCORE median (range)
1 2 3 4 5 6 7
NONE ETHANOL ETHANOL + ROSAPROSTOL NEM + ETHANOL (20 min) ROSAPROSTOL + NEM + ETHANOL NEM + ETHANOL (60 min) NEM + ROSAPROSTOL + ETHANOL
0 2 0 8.5 0 7 1
(O-1) (O-4) (o-l)a (l-10) (o-l)a (4-10) (o-3)a
MUCOSAL NPSH nmoles/mg prot. 24.9 16.1 19.8 13.1 17.5 9.6 15.9
+ ; + + 5 ; :
l.Ob 0.5 1.1 1.4 0.9 1.3 0.6
Rats (8-lo/group) were killed 15 min after ethanol administration. Times refer to the interval between NEM administration and ethanol gavage; rosaprostol was given 30 min before ethanol. Wilcoxon's test: a (p
MARCH 1985 VOL. 29 NO. 3
471
PROSTAGLANDINS
Table 3. Protective effect of rosaprostol and PGE_ on the rat stomach TREATMENT
LESION SCORE median (range)
MUCOSAL NPSH' nmoles/mg prot.
NONE
0
(0-l)
24.5 + 1.5
ETHANOL
2
(l-3)
16.7 + 1.5
NEM + ETHANOL
6
(2-7)
11.3 : 0.8
ROSAPROSTOL (100
mg/kg)
2
(1-3jb
15.8 + 0.8d
NEM + ETHANOL ROSAPROSTOL (200 mg/kg) + NEM + ETHANOL
1
(O-3Jb
18.8 + 1.2d
ROSAPROSTOL (400 mg/kg)
0
(O-ljb
23.4 + l.ld
+
+ NEM + ETHANOL PGE;! (50 u&kg)
+ NEM + ETHANOL
PGE7 (300 ug/kg) + NEM + ETHANOL
3.5 (2-loia 1 (O-3)
13.3 + 1.2 21.5 f. 1.4
Rats (9-lo/group) were killed 60 min after ethanol administration. Rosaprostol and NEM were given 30 and 20 min before ethanol, respectively. Wilcoxon's test: a (pcO.05); b (p
was
linear, as assessed by
DISCUSSION Rosaprostol protects rat gastric stomach from lesions caused by various necrotizing agents, including concentrated HCl (0.6 M) and absolute ethanol. Although the prostanoid is effective in these experimental models at doses that are antisecretory, its protective properties seem to be independent from interference with gastric acid secretion (5). The observation by Szabo et al. (4) that rat hemorrhagic gastric erosions induced by absolute ethanol accompany a dose-dependent decrease of gastric mucosal NPSH suggests a new approach to the mechanisms of cytoprotection. Sulfhydryl-containing drugs protected against alcohol-induced gastric damage, while sulfhydyl blocking . The reduction compounds counteracted the protective effect of PGF by rosaprostol of gastric damage by ethanol accom$?.nied a smaller decrease of NPSH material in the gastric mucosa. Surprisingly,
472
MARCH
1985 VOL. 29 NO. 3
PROSTAGLANDINS
however,
rosaprostol
exerted
protection
and the mucosal
content of
NPSH remained higher despite the previous administration of the sulfhydryl b.Locker N-ethylmaleimide which, in the presence of absolute ethanol, further depleted gastric mucosal NPSH and worsened the Lesion scores. Prostaglandin E2 gave analogous results. The role played by endogenous NPSH in gastric mucosal protection is debated. The mediatory effect suggested by Szabo et al. (4) is at variance with results obtained by others using a different NPSH blocker (11,121. Our results do not rule out a possible role of NPSH in rat gastric mucosal protection. The failure of the sulfhydryl blocker to counteract the protective effect of rosaprostol is associated
with
a smaller
decrease
of mucosal NPSH levels. This is
difficult to interpret since it occurred when the sulfhydryl blocker was given either before or after the prostanoid. Rosaprostol alone, at variance with cysteamine, did not increase the mucosal NPSH content (unpublished data). Nonprotein thiols are highly concentrated (6), they bind reactive influence the structure
elnctrophilic of
gastric
in the stomach mucosa
radicals
mucus
(13), and they may
(14). Since
rosaprostol
stimulates mucus production in rats and humans (5,15) and enhances the mucoprotective index in humans (16), and since the mucus is considered one possible key to gastric cytoprotection (3,171, the capacity of rosaprostol to inhibit the fall of gastric NPSH elicited by alcohol could contribute to its protective properties. ACKNOWLEDGEMENTS The authors thank Prof. F. Berti (University of Milan, Italy) for his stimulating suggestions about this work. REFERENCES 1) Robert, A., J.E. Nezamis, C. Lancaster, and A.J. Hanchar. Cytoprotection by prostaglandins in rat. Prevention of gastric necrosis produced by alcohol, HCl, NaOH, hypertonic NaCl, and thermal injury. Gastroenterology 77~433. 1979. 2) Miller, T.A., and E.D. Jacobson. GastrointestinaL cytoprotection by prostaglandins. Gut 20:75. 1979. 3) Nezamis, J.E., and A. Robert. Gastric mucus may mediate cytoprotective effect of prostaglandins. Gastroenterology 1228. 1980. 4) Szabo, S., J.S. Trier and P.W. Frankel. Sulfhydryl compounds
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Gastroenterology
Accepted:
Bennet
MARCH
12-17-84
1985 VOL. 29 NO. 3