0022-3565/02/3013-969 –974$7.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics
JPET 301:969–974, 2002
Vol. 301, No. 3
4776/987467
Printed in U.S.A.
Role of ATP-Sensitive Potassium Channels in ProstaglandinMediated Gastroprotection in the Rat
BRIGITTA M. PESKAR, KARLHEINZ EHRLICH, and BERNHARD A. PESKAR
Department of Experimental Clinical Medicine, Ruhr University of Bochum, Bochum, Germany (B.M.P., K.E.); and Department of Experimental
and Clinical Pharmacology, University of Graz, Graz, Austria (B.A.P.)
Received November 26, 2001; accepted March 4, 2002
This article is available online at http://jpet.aspetjournals.org
dimercaprol. The blocker of KATP channels glibenclamide (5–10
mg/kg) significantly antagonized the protective effect of 16,16dimethyl-PGE2, 20% ethanol, sodium salicylate, lithium chloride, diethylmaleate, and dimercaprol. The inhibition of protection induced by glibenclamide was reversed by pretreatment
with the KATP channel activator cromakalim (0.3– 0.5 mg/kg). In
conclusion, our results indicate a role of KATP channels in the
gastroprotective effect of 16,16-dimethyl-PGE2 and of the
other agents tested. Since the protection afforded by these
agents is additionally indomethacin-sensitive, it is suggested
that under these conditions endogenous prostaglandins act as
activators of KATP channels, and this mechanism, at least in
part, mediates gastroprotection.
Various natural and synthetic prostaglandins (PGs)
have been shown to be protective against tissue injury in
animal models of myocardial infarction (Thiemermann and
Zacharowski, 2000). This effect is, at least in part, caused
by activation of ATP-sensitive potassium (KATP) channels
(Thiemermann and Zacharowski, 2000). Exogenous and
endogenous prostaglandins are potent protective agents in
the stomach that are effective against a variety of noxious
stimuli (Hawkey and Rampton, 1985). The importance of
endogenous prostaglandins for gastric mucosal integrity is
underlined by the observation that cyclooxygenase inhibitors such as indomethacin can damage the gastric mucosa
directly. In addition, cyclooxygenase inhibitors can inhibit
the protection exerted by various exogenous agents, e.g.,
20% ethanol (Robert et al., 1983). The direct damaging
effect of indomethacin in rat gastric mucosa has been
shown to be aggravated by the blocker of KATP channels,
glibenclamide, and inhibited by activators of these channels such as cromakalim or diazoxide (Akar et al., 1999;
Toroudi et al., 1999).
Intragastric instillation of concentrated ethanol induces
macroscopic and histologic mucosal injury within seconds,
associated with vascular stasis, increased vascular permeability, subepithelial hemorrhages, cellular exfoliation,
and enhanced leukocyte-endothelial cell interaction (Guth
et al., 1984; Szabo, 1987; Kvietys et al., 1990; Peskar,
1991). Rat gastric mucosal damage induced by high concentrations of ethanol has been widely used to investigate
gastroprotective phenomena (Robert et al., 1984; Hawkey
and Rampton, 1985; Peskar et al., 1988; Peskar, 1991;
Stroff et al., 1996; Gretzer et al., 1998; Araki et al., 2000).
Numerous agents in addition to prostaglandins, including
sodium salicylate, metals, thiols, and sulfhydryl blockers,
protect the gastric mucosa against damage induced by
ethanol. The effect of modulators of KATP channels on
these gastroprotective phenomena has so far not been investigated. We have now compared the effects of indomethacin with those of glibenclamide on the gastroprotective
activity of such agents. These investigations should elucidate the contribution of endogenous prostaglandins to the
activity of the protective agents as well as the importance
of KATP channels. Finally, it is already known that gastroprotection afforded by pretreatment with 20% ethanol
(adaptive gastroprotection) is inhibited by selective cyclooxygenase-2 inhibitors (Gretzer et al., 1998) as well as by
nonspecific inhibitors like indomethacin (Robert et al.,
1983; Gretzer et al., 1998). We have now investigated the
effect of glibenclamide and its modification by cromakalim
on adaptive gastroprotection induced by 20% ethanol.
ABBREVIATIONS: PG, prostaglandin; CGRP, calcitonin gene-related peptide; KATP channels, ATP-sensitive potassium channels.
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ABSTRACT
This study compares the involvement of ATP-sensitive potassium (KATP) channels and prostaglandins in various forms of
gastroprotection in the rat. Instillation of 1 ml of 70% ethanol
induced severe gastric mucosal damage (lesion index 39 ⫾
0.8), which was substantially but not maximally reduced by oral
pretreatment with 16,16-dimethyl-prostaglandin (PG) E2 (75 ng/
kg), 20% ethanol (1 ml), sodium salicylate (15 mg/kg), the metal
salt lithium chloride (7 mg/kg), the sulfhydryl-blocking agent
diethylmaleate (5 mg/kg), and the thiol dimercaprol (10 mg/kg).
Administration of indomethacin (20 mg/kg) increased gastric
mucosal damage induced by 70% ethanol (lesion index 45 ⫾
0.8) and significantly reduced the protective effect of 20%
ethanol, sodium salicylate, lithium chloride, diethylmaleate, and
970
Peskar et al.
Materials and Methods
Results
Oral instillation of 1 ml of 70% ethanol induced severe
gastric mucosal damage (lesion index 39 ⫾ 0.8). Pretreatment with indomethacin (20 mg/kg, p.o., 60 min) augmented
the injurious effect of 70% ethanol (lesion index 45 ⫾ 0.8, p ⬍
0.001).
Neither the solvent for glibenclamide (lesion index 39 ⫾
0.9), the solvent for cromakalim (lesion index 39 ⫾ 1.8), nor
the solvent for indomethacin and the protective agents (lesion index 38 ⫾ 2) interfered with the damaging effect of 70%
ethanol. Furthermore, neither pretreatment with cromakalim (0.3 mg/kg and 0.5 mg/kg, p.o., 90 min before 70%
ethanol; lesion index 38 ⫾ 3.8 and 36 ⫾ 2.8, respectively) nor
the dose of glibenclamide used in a specific set of experiments
(5–10 mg/kg, p.o., 60 min before 70% ethanol) modified the
injury caused by 70% ethanol. However, cromakalim (0.5
mg/kg, p.o., 90 min before 70% ethanol) induced significant
(p ⬍ 0.001) gastroprotection (lesion index 16 ⫾ 3) in rats
treated additionally with indomethacin (5 mg/kg, p.o., 60 min
before ethanol) as compared with controls treated with cromakalim only (lesion index 39 ⫾ 2).
Gastric mucosal fragments obtained from vehicle-treated
rats released 425 ⫾ 45 pg/mg/10 min 6-keto-PGF1␣ during
incubation in vitro. Release of 6-keto-PGF1␣ was not significantly different when glibenclamide (final concentration 10
␮M) was added to the incubation medium (379 ⫾ 68 pg/mg/10
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Drugs. The prostaglandin analog 16,16-dimethyl-PGE2 was obtained from Paesel & Lorei (Frankfurt, Germany). Indomethacin,
sodium salicylate, lithium chloride, diethylmaleate, dimercaprol,
glibenclamide, cromakalim, and all other compounds were purchased from Sigma-Aldrich (St. Louis, MO).
Animals. Male Wistar rats (weighing 180 –220 g) were purchased
from Harlan-Winkelmann (Paderborn, Germany). They were deprived of food for 24 h with free access to tap water. Rats were kept
on a 12-h light/dark cycle and under conditions of controlled temperature (22 ⫾ 1°C). The studies reported in this article have been
carried out in accordance with the Guide for the Care and Use of
Laboratory Animals as adopted and promulgated by the U.S. National Institutes of Health. All experimental protocols were approved
by the Animal Care Committee of the Ruhr-University of Bochum.
Assessment of Gastric Mucosal Damage. Rats received 1 ml of
70% ethanol by oral intubation. After a further 5 min, rats were
killed by cervical dislocation. The stomach was removed and gross
mucosal damage was assessed in a blinded manner by calculation of
a lesion index by use of a 0 –3 scoring system based on the number
and severity factor of lesions as described previously (Stroff et al.,
1996). The severity factor was defined according to the length of the
lesions. Severity factor 0 ⫽ no lesions; I ⫽ lesions ⬍1 mm; II ⫽
lesions 2– 4 mm; III ⫽ lesions ⬎4 mm. The lesion index was calculated as the total number of lesions multiplied by their respective
severity factor.
Assessment of Gastric Mucosal 6-Keto-PGF1␣ Formation.
Groups of six rats were treated with glibenclamide (10 mg/kg, p.o.) or
vehicle. Mucosal fragments were excised from the glandular part of
the stomach 60 min later, and 2 aliquots (40 mg) were incubated in
oxygenated Tyrode’s solution at 37°C for 10 min. In addition, glibenclamide (final concentration 10 ␮M) was added in vitro to the
incubation medium of a third aliquot of mucosal tissue obtained from
vehicle-treated control rats. It has been shown previously that under
identical experimental conditions, indomethacin causes dose-dependent inhibition of prostaglandin formation (Peskar et al., 1988;
Gretzer et al., 1998). The medium was analyzed for the content of
6-keto-PGF1␣ by radioimmunoassay (Peskar et al., 1988; Gretzer et
al., 1998). The radioimmunoassay used is highly specific for 6-ketoPGF1␣ with less than 0.1% cross-reaction by other eicosanoids or
related compounds.
Administration of Protective Agents. Groups of six to eight
rats were treated orally with the following protective agents: 16,16dimethyl-PGE2 (75 ng/kg), 1 ml of the mild irritant 20% ethanol,
sodium salicylate (15 mg/kg), the metal salt lithium chloride (7
mg/kg), the sulfhydryl-blocking agent diethylmaleate (5 mg/kg), and
the sulfhydryl-containing compound dimercaprol (10 mg/kg). None of
these agents affected gastric mucosal integrity in the absence of
ethanol. Ethanol was diluted in distilled water, and drugs were
administered in methylcellulose (0.25%, 2.5 ml/kg). Effects of indomethacin and glibenclamide described below could only be observed
when doses of the protective agents were used that did not cause
maximal protection (lesion index 0). At supratherapeutic doses that
completely prevented gross damage induced by 70% ethanol, the
effect of the protective agents was not modified by pretreatment with
either indomethacin or glibenclamide. The doses of protective agents
were selected from previous dose-range studies (Peskar et al., 1988;
Peskar, 1991). In addition, for confirmation, titration experiments
for the protective potency of each agent were performed in the
present study, and for each compound used, a dose was selected that
reduced ethanol-induced damage to a lesion index ranging from 7 to
14. Protective agents were administered 30 min before instillation of
1 ml of 70% ethanol. Controls received the vehicle 30 min before 70%
ethanol.
Pretreatment with Indomethacin. Indomethacin (20 mg/kg)
was administered orally in 2.5 ml/kg methylcellulose (0.25%) 30 min
before the protective agents (n ⫽ 6 per group). Controls received
indomethacin (20 mg/kg) 30 min before administration of 2.5 ml/kg
0.25% methylcellulose (vehicle for the protective agents) or 1 ml of
distilled water (diluent for 20% ethanol). The dose of indomethacin
used has been shown previously to inhibit rat gastric mucosal prostaglandin formation under identical experimental conditions by
more than 90% (Peskar et al., 1988; Gretzer et al., 1998). All rats
were challenged with 1 ml of 70% ethanol 30 min after administration of the protective agent or vehicle and were killed 5 min later.
Pretreatment with Glibenclamide and Cromakalim. Groups
of six to eight rats were treated with glibenclamide (5–10 mg/kg, p.o.)
30 min before administration of the protective agents. Glibenclamide
was administered in 2.5 ml/kg 0.02 N NaOH containing 4% glucose
to minimize hypoglycemia. Glibenclamide exhibited protective activity against 70% ethanol when the dose was increased above 5 to 10
mg/kg or the pretreatment period was prolonged over 60 min. Therefore, for each set of experiments a threshold dose of glibenclamide
without protective activity when given alone was determined, and
the doses finally used differed between 5 and 10 mg/kg. Additional
groups of six rats received cromakalim (0.3– 0.5 mg/kg, p.o.). Cromakalim was dissolved in 30% ethanol and further diluted with
distilled water. Cromakalim was administered in a volume of 2.5
ml/kg 30 min before glibenclamide, and the final concentration of
ethanol was 6%. All rats received either active agents or the corresponding vehicle so that the background of volumes and solvents was
identical in all groups. All rats were challenged with 70% ethanol 30
min after administration of the protective agent. Vehicles for all
drugs and their combinations were tested in groups of four to six rats
for a possible interference with the damaging effect of 70% ethanol.
In an additional set of experiments, rats (n ⫽ 6) received cromakalim (0.5 mg/kg, p.o.) 90 min and indomethacin (5 mg/kg, p.o.) 60
min before 70% ethanol. A control group (n ⫽ 6) received cromakalim
(0.5 mg/kg, p.o.) followed by methylcellulose (0.25%, 2.5 ml/kg, p.o.)
instead of indomethacin.
Statistical Analysis. All data are expressed as mean ⫾ S.E.M. of
n values. Comparisons between groups were made by use of the
Wilcoxon rank test for nonparametric data. A p value of ⬍ 0.05 was
considered significant.
KATP Channels and Gastroprotection
Fig. 1. Gastroprotection by 16,16-dimethyl-PGE2 and effect of glibenclamide and cromakalim. Administration of 16,16-dimethyl-PGE2 (16,16DMPGE2, 75 ng/kg, p.o.) reduced gastric damage induced by 70% ethanol.
Pretreatment with glibenclamide (Glib, 10 mg/kg, p.o.) inhibited the
protection conferred by 16,16-dimethyl-PGE2. Administration of cromakalim (Crom, 0.3 mg/kg, p.o.) before glibenclamide restored the protective effect of 16,16-dimethyl-PGE2. Rats treated with 16,16-dimethylPGE2 alone (Veh) were pretreated with the vehicles for cromakalim and
glibenclamide. Values are the mean ⫾ S.E.M. of 5–7 rats. 多多多, p ⬍ 0.001
versus controls (Co); F F F, p ⬍ 0.001 versus 16,16-dimethyl-PGE2 alone;
# # #, p ⬍ 0.001 versus glibenclamide before 16,16-dimethyl-PGE2.
Fig. 2. Gastroprotection by the mild irritant 20% ethanol and effect of
glibenclamide, cromakalim, and indomethacin. Instillation of 20% ethanol (1 ml, p.o.) reduced gastric damage induced by 70% ethanol. Pretreatment with glibenclamide (Glib, 10 mg/kg, p.o.) inhibited the protection
conferred by the mild irritant; administration of cromakalim (Crom, 0.3
mg/kg, p.o.) before glibenclamide restored the protective effect of 20%
ethanol. Rats treated with 20% ethanol alone (Veh) were pretreated with
the vehicles for cromakalim and glibenclamide. Pretreatment with indomethacin (Indo, 20 mg/kg, p.o.) increased damage induced by 70% ethanol
and abolished the protection by 20% ethanol. Values are the mean ⫾
S.E.M. of 5– 6 rats. 多多多, p ⬍ 0.001 versus controls (Co); F F F, p ⬍ 0.001
versus 20% ethanol alone; # # #, p ⬍ 0.001 versus glibenclamide before
20% ethanol.
Fig. 3. Gastroprotection by sodium salicylate and effect of glibenclamide,
cromakalim, and indomethacin. Administration of sodium salicylate (15
mg/kg, p.o.) reduced gastric damage induced by 70% ethanol. Pretreatment with glibenclamide (Glib, 7.5 mg/kg, p.o.) inhibited the protection
conferred by sodium salicylate; administration of cromakalim (Crom, 0.3
mg/kg, p.o.) before glibenclamide restored the protective effect of sodium
salicylate. Rats treated with sodium salicylate alone (Veh) were pretreated with the vehicles for cromakalim and glibenclamide. Pretreatment with indomethacin (Indo, 20 mg/kg, p.o.) increased damage induced
by 70% ethanol and abolished the protection by sodium salicylate. Values
are the mean ⫾ S.E.M. of 5–7 rats. 多多多, p ⬍ 0.001 versus controls (Co);
F F F, p ⬍ 0.001 versus sodium salicylate alone; # # #, p ⬍ 0.001 versus
glibenclamide before sodium salicylate.
cantly inhibited by pretreatment with glibenclamide (10 mg/
kg, p ⬍ 0.001 versus lithium chloride in the absence of
glibenclamide). Cromakalim (0.3 mg/kg) reversed the effect
of glibenclamide (p ⬍ 0.001 versus pretreatment with glibenclamide). Similarly, indomethacin (20 mg/kg) attenuated
the protective effect of lithium chloride (p ⬍ 0.05 versus
lithium chloride in the absence of indomethacin). Results are
shown in Fig. 4.
Effect of Diethylmaleate. The sulfhydryl-blocking compound diethylmaleate (15 mg/kg) inhibited gastric mucosal
injury induced by 70% ethanol by 66%. Pretreatment with
glibenclamide (5 mg/kg) reduced the protective effect of diethylmaleate (p ⬍ 0.001 versus diethylmaleate alone). Pro-
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min). Furthermore, oral treatment with glibenclamide (10
mg/kg) did not inhibit gastric mucosal release of 6-ketoPGF1␣ ex vivo (411 ⫾ 48 pg/mg/10 min) as compared with
controls.
Effect of 16,16-Dimethyl-PGE2. Ethanol-induced gastric
mucosal damage was significantly (by 74%) reduced by pretreatment with 16,16-dimethyl-PGE2 (75 ng/kg). The protective effect of the prostaglandin was attenuated by pretreatment with glibenclamide (10 mg/kg, p ⬍ 0.001 versus 16,16dimethyl-PGE2 alone). Administration of cromakalim (0.3
mg/kg) 30 min before glibenclamide (10 mg/kg) fully restored
the protective effect of 16,16-dimethyl-PGE2. Results are
shown in Fig. 1.
Adaptive Gastroprotection. Oral instillation of 1 ml of
the mild irritant 20% ethanol reduced gastric mucosal damage induced by a subsequent instillation of 1 ml of 70%
ethanol by 83% (p ⬍ 0.001). Pretreatment with indomethacin
(20 mg/kg, 30 min) abolished the protective effect of 20%
ethanol. Pretreatment with glibenclamide (10 mg/kg) 30 min
before instillation of 20% ethanol near-maximally inhibited
the protection induced by the mild irritant (p ⬍ 0.001 versus
20% ethanol alone). Cromakalim (0.3 mg/kg) counteracted
the effect of glibenclamide and restored the protective activity of the mild irritant (p ⬍ 0.001 versus glibenclamide before
20% ethanol). Results are shown in Fig. 2.
Effect of Sodium Salicylate. Sodium salicylate (15 mg/
kg) reduced the injurious effect of 70% ethanol by 80% (p ⬍
0.001 versus controls treated with vehicle instead of sodium
salicylate). The protective effect of sodium salicylate was
significantly diminished by pretreatment with glibenclamide
(7.5 mg/kg, p ⬍ 0.001 versus sodium salicylate alone) and
was restored after combined treatment with cromakalim (0.3
mg/kg) and glibenclamide (p ⬍ 0.001 versus sodium salicylate and glibenclamide). Indomethacin (20 mg/kg) abolished
the protection conferred by sodium salicylate. Results are
shown in Fig. 3.
Effect of Lithium Chloride. Administration of lithium
chloride (7 mg/kg) reduced gastric mucosal damage induced
by 70% ethanol by 67%. The protective effect was signifi-
971
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Peskar et al.
fect of dimercaprol (p ⬍ 0.01 versus dimercaprol alone). Results are shown in Fig. 6.
Discussion
Fig. 5. Gastroprotection by the sulfhydryl-blocking compound diethylmaleate and effect of glibenclamide, cromakalim, and indomethacin. Administration of diethylmaleate (15 mg/kg, p.o.) reduced gastric damage
induced by 70% ethanol. Pretreatment with glibenclamide (Glib, 5 mg/kg,
p.o.) inhibited the protection conferred by diethylmaleate; administration
of cromakalim (Crom, 0.5 mg/kg, p.o.) before glibenclamide restored the
protective effect of diethylmaleate. Rats treated with diethylmaleate
alone (Veh) were pretreated with the vehicles for cromakalim and glibenclamide. Pretreatment with indomethacin (20 mg/kg, p.o.) increased
damage induced by 70% ethanol and inhibited the protective effect of
diethylmaleate. Values are the mean ⫾ S.E.M. of 5– 6 rats. 多多多, p ⬍ 0.001
versus controls (Co); F F F, p ⬍ 0.001 versus diethylmaleate alone; # # #,
p ⬍ 0.001 versus glibenclamide before diethylmaleate.
Fig. 6. Gastroprotection by the sulfhydryl-containing agent dimercaprol
and effect of glibenclamide and indomethacin. Administration of dimercaprol (10 mg/kg, p.o.) inhibited gastric damage induced by 70% ethanol.
Pretreatment with glibenclamide (Glib, 10 mg/kg, p.o.) inhibited the
protection conferred by dimercaprol; administration of cromakalim
(Crom, 0.4 mg/kg, p.o.) before glibenclamide restored the protective effect
of dimercaprol. Rats treated with dimercaprol alone (Veh) were pretreated with the vehicles for cromakalim and glibenclamide. Pretreatment with indomethacin (Indo, 20 mg/kg, p.o.) increased damage induced
by 70% ethanol and attenuated the protective effect of diethylmaleate.
Values are the mean ⫾ S.E.M. of 5– 6 rats. 多多多, p ⬍ 0.001 versus controls
(Co); F F, p ⬍ 0.01 versus dimercaprol alone; # # #, p ⬍ 0.001 versus
glibenclamide before dimercaprol.
Fig. 4. Gastroprotection by lithium chloride and effect of glibenclamide,
cromakalim, and indomethacin. Administration of lithium chloride (7
mg/kg, p.o.) reduced gastric damage induced by 70% ethanol. Pretreatment with glibenclamide (Glib, 10 mg/kg, p.o.) inhibited the protection
conferred by lithium chloride; administration of cromakalim (Crom, 0.3
mg/kg, p.o.) before glibenclamide restored the protective effect of lithium
chloride. Rats treated with lithium chloride alone (Veh) were pretreated
with the vehicles for cromakalim and glibenclamide. Pretreatment with
indomethacin (Indo, 20 mg/kg, p.o.) increased damage induced by 70%
ethanol and inhibited the protective effect of lithium chloride. Values are
the mean ⫾ S.E.M. of 5–7 rats. 多多多, p ⬍ 0.001 versus controls (Co); F, p ⬍
0.05, F F F, p ⬍ 0.001 versus lithium chloride alone; # # #, p ⬍ 0.001
versus glibenclamide before lithium chloride.
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tection was restored when rats received combined treatment
with cromakalim (0.5 mg/kg) and glibenclamide (p ⬍ 0.001
versus glibenclamide pretreatment). Likewise, pretreatment
with indomethacin (20 mg/kg) attenuated the protective effect of diethylmaleate (p ⬍ 0.001 versus diethylmaleate
alone). Results are shown in Fig. 5.
Effect of Dimercaprol. The sulfhydryl-containing agent
dimercaprol (10 mg/kg) exerted 67% protection against gastric damage induced by 70% ethanol. Pretreatment with glibenclamide (10 mg/kg) reduced the protective effect of dimercaprol (p ⬍ 0.01 versus dimercaprol alone). Cromakalim (0.4
mg/kg) reversed the effect of glibenclamide (p ⬍ 0.001 versus
pretreatment with glibenclamide). Similarly, pretreatment
with indomethacin (20 mg/kg) attenuated the protective ef-
The main new findings of the present work are: 1) the
gastroprotection of various agents of different chemical
classes in submaximal doses depends on an intact prostaglandin system; and 2) the prostaglandins mediating the
protective effect act, at least in part, by opening KATP channels. A scheme summarizing the proposed mechanism of
gastroprotection and the targets of drug actions is shown in
Fig. 7. The conclusion that the gastroprotection conferred by
submaximally effective doses of 16,16-dimethyl-PGE2 as well
as of sodium salicylate, 20% ethanol, lithium chloride, diethylmaleate, and dimercaprol is, at least partially, mediated by
activation of KATP channels is supported by the inhibition of
protection by glibenclamide, a blocker of KATP channels
(Sturgess et al., 1985; Quast, 1993). Furthermore, cromakalim, an opener of such channels (Quast and Cook, 1989;
Quast 1993; Quayle et al., 1995) prevents the glibenclamide
effect. Antagonistic interaction of glibenclamide and cromakalim has generally been accepted as evidence for the
involvement of KATP channels (Standen et al., 1989; Quayle
et al., 1995). Cromakalim has been shown previously (Akar et
al., 1999) to prevent indomethacin-induced injury. Our results demonstrate that cromakalim not only reverses the
glibenclamide-induced inhibition of gastroprotection, but can
also directly antagonize ethanol-induced gastric mucosal
damage. In the dosage used, this effect is, however, observed
only after additional treatment with indomethacin. This result is in agreement with data of Armstead (2001) showing
that cyclooxygenase-dependent superoxide generation impairs the vasodilatory effect of cromakalim and that the
potency of cromakalim is increased after inhibition of cyclooxygenase by indomethacin.
In addition, gastroprotection by submaximally effective
doses of sodium salicylate, 20% ethanol, lithium chloride,
KATP Channels and Gastroprotection
diethylmaleate, and dimercaprol seems to rely on functioning
prostaglandin biosynthesis, since pretreatment with indomethacin at a dose that near-maximally inhibits gastric prostaglandin formation (Peskar et al., 1988; Gretzer et al., 1998)
has an inhibitory effect. The results suggest that under such
conditions, endogenous prostaglandins are the mediators of
activation of KATP channels and, thus, of gastroprotection
afforded by the various agents tested. However, the protective effects of much higher doses of sodium salicylate, diethylmaleate, lithium chloride, and dimercaprol are not modified by indomethacin (Robert, 1981; Robert et al., 1984;
Gretzer et al.,1998) or glibenclamide (unpublished observation). Thus, in this type of gastroprotection, additional mechanisms seem to prevail.
Although the effects of glibenclamide and indomethacin on
gastric mucosal integrity are qualitatively parallel, their
mechanisms of action are completely different. Whereas indomethacin is a potent cyclooxygenase inhibitor (Vane, 1971;
Peskar et al., 1988; Gretzer et al., 1998), glibenclamide does
not suppress prostaglandin biosynthesis ex vivo and in vitro.
Whereas glibenclamide antagonized the effect of various gastroprotective compounds, it exhibited protective activity in
higher doses when given alone. Therefore, the present results
were obtained with low doses of glibenclamide after careful
titration experiments to avoid such effects. In any case, the
effects of glibenclamide and KATP channel-opening drugs on
blood glucose levels were found not to correlate with their
effects on gastric mucosal integrity (Akar et al., 1999).
The present results with 16,16-dimethyl-PGE2 correspond
to those in which protective effects of natural and synthetic
prostaglandins have been demonstrated against ischemiareperfusion models of cardiac infarction (Hide et al., 1995;
Hide and Thiemermann, 1996; Zacharowski et al., 1999; Thiemermann and Zacharowski, 2000). These cardioprotective
effects of prostaglandins are independent of great hemodynamic changes but are mediated by activation of prostanoid
EP3 receptors and involve activation of KATP channels and
protein kinase C (Thiemermann and Zacharowski, 2000).
The EP receptor subtype responsible for cardioprotection has
been identified as EP3 using subtype-specific receptor agonists and antagonists. Here we describe protective prosta-
glandin effects in another organ, and against another noxious stimulus, that also involve activation of KATP channels,
whereas a possible contribution of protein kinase C has so far
not been elucidated. The EP receptor subtype responsible for
the protective effects observed under our experimental conditions has not yet been determined. Araki et al. (2000),
using EP1, EP2, EP3, and EP3/4 receptor agonists, concluded
that gastroprotection against 0.15 N HCl in 60% ethanol is
mediated by EP1 receptors. Their results were strongly supported by data obtained in knock-out mice. Although both
EP1 and EP3 knock-out mice reacted with gastric mucosal
damage to HCl/ethanol, only EP1 knock-out mice could not be
protected by exogenous PGE2, whereas EP3 knock-out mice
did not differ from wild-type mice. Neither EP1 nor EP3
receptors are involved in increased blood flow (Araki et al.,
2000; Thiemermann and Zacharowski, 2000). Further experiments are necessary to clarify exactly the prostaglandin
receptor subtypes involved in various types of organ protection.
Whereas in our study gastric mucosal damage induced by
70% ethanol was not affected by oral glibenclamide at the
doses used, glibenclamide administered intravenously aggravated mucosal injury induced by gastric perfusion with 15%
ethanol/0.15 N HCl (Iwata et al., 1997; Doi et al., 1998).
Mucosal hyperemia induced by acidified ethanol or capsaicin
was attenuated by glibenclamide, suggesting that under
these experimental conditions, the KATP channel blocker
most likely acts at the arteriolar level to attenuate the vasodilatory effect of endogenous calcitonin gene-related peptide
(CGRP) released from afferent nerve terminals in the gastric
mucosa (Iwata et al., 1997). This interpretation is supported
by data showing that the increase in gastric mucosal blood
flow induced by exogenous CGRP was also attenuated by
glibenclamide (Doi et al., 1998). Mucosal lesions produced by
intragastric superfusion with 15% ethanol/0.15 N HCl were
exacerbated by glibenclamide but ameliorated by exogenous
CGRP. The authors concluded that CGRP protects the gastric mucosa, at least in part, through the activation of KATP
channels (Doi et al., 1998). It should be pointed out, however,
that gastroprotective effects are not necessarily associated
with mucosal hyperemia but can occur when gastric mucosal
blood flow remains unchanged or is even substantially decreased. Examples are PGF2␣ (Whittle et al., 1985), 16,16dimethyl-PGE2 (Arakawa et al., 1989), and tachykinin neurokinin-2 receptor agonists (Stroff et al., 1996). It is not
known which structures, such as vasculature, epithelium,
etc., of the gastric mucosa contain the KATP channels activated by prostaglandins.
In conclusion, our results indicate an essential contribution of KATP channels to gastroprotection afforded by submaximally effective doses of 16,16-dimethyl-PGE2 as well as
of other agents such as 20% ethanol, sodium salicylate, lithium chloride, diethylmaleate and dimercaprol. The protection conferred by these compounds in the dosage used was
additionally found to be indomethacin-sensitive and thus
depends on an intact prostaglandin system. The results suggest that under these conditions endogenous prostaglandins
act as activators of KATP channels and this mechanism, at
least in part, mediates gastroprotection.
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