Prevention of Ethanol-induced Gastric
Mucosal Microcirculatory Disturbances
by Mild Irritant Through the Actions of
Calcitonin Gene-Related Peptide and
Prostaglandin I2 in Rats
Takeo Saeki1, Takashi Ohno2, Kazuhisa Kamata1, Katsuharu Arai1,
Sumito Mizuguchi1, Kouichiro Hattori1, Katsunori Saigenji1,
and Masataka Majima3
Key words. Microcirculation, Gastric mucosal blood flow, Calcitonin generelated peptide, Prostaglandin I2, Sodium chloride, Ethanol
Introduction
Disturbances in the gastric mucosal microcirculation are thought to be an
important cause of injury, and observation of the microcirculation is therefore important for revealing the pathophysiology of that injury. In the present
study, we investigated the mechanism of adaptive cytoprotection of 1 M NaCl
against ethanol [1] by observing the microcirculation in the basal part of the
gastric mucosa of rats using intravital microscopy [2–4] to clarify the mediators involved in the maintenance of the integrity of the mucosal blood flow.
Materials and Methods
The mucosal microcirculation of male Sprague-Dawley rats anesthetized with
urethane was observed using the methods that we previously developed [2–4].
The microcirculation of the basal part of the mucosa was examined with a
light microscope with a long working distance objective lens. One arteriole,
one venule, and two collecting venules were selected in one observation
Department of Internal Medicine, Kitasato University School of Medicine, Sagamihara,
Kanagawa 228-8555, Japan
2
Department of Internal Medicine, Isehara Kyodo Hospital, Isehara, Kanagawa 259-1132,
Japan
3
Departments of Pharmacology and Molecular Pharmacology, Kitasato University School
of Medicine, Kitasato University Graduate School of Medical Sciences, Sagamihara,
Kanagawa 228-8555, Japan
1
93
94
T. Saeki et al.
window, and their internal diameters were measured using an adjustable
electronic microscaler. The diameters of the arterioles were expressed as
percentages of the original diameter. Ethanol (50%; 1 ml) and NaCl (1 M;
1 ml) were placed between the chamber and the gastric mucosa. Calcitonin
gene-related peptide (CGRP)-(8–37) (10 mM; 20 ml), prostaglandin E2
(PGE2; 1 nM–10 mM; 20 ml) and a prostaglandin I2 (PGI2) analogue, beraprost
sodium (1 nM–10 mM; 20 ml) were applied in the window. Indomethacin
(1 mg/kg; Banyu Pharmaceutical, Tokyo, Japan) was administered
intravenously.
Results and Discussion
Microcirculatory Changes in the Basal Part of the Gastric
Mucosa After Exposure of the Mucosa to 50% Ethanol
The gastric mucosa was exposed for 3 min to 50% ethanol, which was then
washed out with Tyrode’s solution.Application of 50% ethanol on the mucosal
side caused marked dilatation of the arterioles, but the collecting venules and
the venules were strongly constricted, the internal cross-section of the collecting venules in particular becoming very small (Fig. 1B).
When 1 M NaCl was applied to the mucosa 5 min before mucosal treatment
with 50% ethanol, the ethanol-induced rapid constrictions of the collecting
venules and venules were completely inhibited (Fig. 1D), although 1 M NaCl
alone caused some constriction of both the collecting venules and the venules
(Fig. 2). The diameter of the arterioles during ethanol exposure was not
affected by prior administration of 1 M NaCl, although they were markedly
dilated during 1 M NaCl treatment (Fig. 2).
Furthermore, to investigate whether or not this preventive effect exerted by
1 M NaCl was attributable to the release of endogenous PGs, indomethacin
(10 mg/kg, i.v.) was infused before the application of 1 M NaCl. The ethanolinduced constriction of the collecting venules, which had been blocked by
1 M NaCl pretreatment, appeared again in rats treated with indomethacin. The
dilatation of arterioles observed during ethanol exposure was not affected by
indomethacin preapplication.
Next, we applied CGRP-(8–37) (10 mM), a CGRP receptor antagonist, to the
microvasculature 3 min before treatment of 1 M NaCl to investigate the
involvement of endogenous CGRP. The treatment with CGRP-(8–37) did not
affect the size of microvessels under 1 M NaCl application, but the ethanolinduced constriction of the collecting venules, which had been blocked by
1 M NaCl pretreatment, appeared again on treatment with a CGRP antagonist.
The dilatation of arterioles observed during ethanol exposure was not
affected by the applications of CGRP-(8–37).
Mild Irritant and Microcirculation
95
Fig. 1. Changes of the diameters of microvessels in the basal part of the mucosal microcirculation before (A) and 4 min after (B) application of 50% ethanol on the mucosal side
of the stomach wall for 3 min. Marked dilatation of arterioles (a), and severe constriction
of the collecting venules (cv) and the venules (v) in the gastric mucosa were observed under
intravital microscopy. The diameters of microvessels in the basal part of the mucosal
microcirculation before application of 1 M NaCl and ethanol (C). When 1 M NaCl was
applied to the window 5 min before mucosal treatment with 50% ethanol, the ethanolinduced rapid constrictions of the collecting venules and venules were completely
inhibited (D)
These results were similar to those obtained from indomethacin-pretreated
rats. Thus, it is suggested that the protective effect of 1 M NaCl against ethanolinduced mucosal injury was mediated by both endogenous PGs and CGRP.
Effects of Administration of PGs (PGE2 or Beraprost) on
Ethanol-Induced Changes in the Microcirculation of the
Basal Part of the Gastric Mucosa
Administration of PGE2 (1 nM–10 mM) or beraprost (1 nM–10 mM) to the
observation window caused a rapid and dose-dependent dilatation of
the arterioles. PGE2 caused a dose-dependent constriction of the collecting
venules, but beraprost induced no changes in the diameter of the collecting
venules. To identify the PGs contributing to 1 M NaCl-induced protective
96
T. Saeki et al.
Fig. 2. Effects of prior application
of 1 M NaCl on the ethanol-induced
changes in the diameter of mucosal
microvessels. The constrictions of
the collecting venules (middle
panel) and venules (lower panel)
induced by 50% ethanol were
markedly inhibited by 1 M NaCl
pretreatment. The upper panel
depicts results in arterioles. Each
value indicates mean + SEM. *p <
0.05, **p < 0.01, ***p < 0.001
Mild Irritant and Microcirculation
97
action, PGE2 or beraprost was applied to the window 3 min before the mucosal
application of 50% ethanol. The low doses of PGE2 or beraprost which did not
influence the original size of the gastric mucosal vessels including arterioles
are given. Both PGE2 (100 nM) and beraprost (1 nM) inhibited ethanolinduced rapid constrictions of the collecting venules. The dilatation of arterioles observed during ethanol exposure was not affected by applications of
these doses of PGE2 and beraprost.
To test whether or not the preventive effects of PGE2 and beraprost were
attributable to the increased release of endogenous CGRP, a sufficient dose of
CGRP-(8–37) (10 mM) was applied to the window 3 min before the application
of PGs (PGE2 100 nM or beraprost 1 nM). The blockade of ethanol-induced
constriction of the collecting venules by beraprost appeared again with the
use of CGRP-(8–37). By contrast, that by PGE2 did not appear even with CGRP(8–37). These suggested that beraprost protected the gastric mucosa from
ethanol through the cancellation of the constriction of collecting venules utilizing CGRP, but PGE2 did offer protection independently, without CGRP.
In the present study, we found first that the mechanism of prevention by 1
M NaCl of ethanol-induced gastric mucosal injury is the inhibition of the constriction of collecting venules and venules, via endogenous PGs and CGRP.
Second, it was seen that the PGI2 analogue, which did not dilate the arterioles,
collecting venules, or venules themselves, inhibited the ethanol-induced constriction of the collecting venules and venules, suggesting that endogenous
PGI2 was responsible for the preventive effect seen when 1 M NaCl was applied
before the ethanol-induced injury through the increased release of CGRP.
Thus, the dilator of the collecting venules, the venules or both may in future
become a useful agent for preventing the induction of gastric mucosal injury
by various necrotizing agents such as ethanol.
References
1. Boku K, Ohno T, Saeki T, et al (2001) Adaptive cytoprotection mediated by prostaglandin
I2 is attributable to sensitization of CGRP-containing sensory nerves. Gastroenterology
120:134–143
2. Ohno T, Katori M, Nishiyama K, et al (1995) Direct observation of microcirculation of
the basal region of rat gastric mucosa. J Gastroenterol 30:557–564
3. Ohno T, Katori M, Majima M, et al (1999) Dilatation and constriction of rat gastric
mucosal microvessels through prostaglandin EP2 and EP3 receptors. Aliment Pharmacol Ther 13:1243–1250
4. Saeki T, Ohno T, Boku K, et al (2000) Mechanism of prevention by capsaicin of ethanolinduced gastric mucosal injury: a study using intravital microscopy. Aliment Pharmacol Ther 14:135–144