Prevention of Ethanol-induced Gastric Mucosal Microcirculatory Disturbances by Mild Irritant Through the Actions of Calcitonin Gene-Related Peptide and Prostaglandin I

Prevention of Ethanol-induced Gastric Mucosal Microcirculatory Disturbances by Mild Irritant Through the Actions of Calcitonin Gene-Related Peptide and Prostaglandin I ₂ in Rats

Takeo Saeki

¹

, Takashi Ohno

²

, Kazuhisa Kamata

¹

, Katsuharu Arai

¹

, Sumito Mizuguchi

¹

, Kouichiro Hattori

¹

, Katsunori Saigenji

¹

, and Masataka Majima

³

Key words.

Microcirculation, Gastric mucosal blood flow, Calcitonin gene- related peptide, Prostaglandin I

2

, 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 there- fore 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 medi- ators 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

93

1Department of Internal Medicine, Kitasato University School of Medicine, Sagamihara, Kanagawa 228-8555, Japan

2Department of Internal Medicine, Isehara Kyodo Hospital, Isehara, Kanagawa 259-1132, Japan

3Departments of Pharmacology and Molecular Pharmacology, Kitasato University School of Medicine, Kitasato University Graduate School of Medical Sciences, Sagamihara, Kanagawa 228-8555, Japan

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 E

2

(PGE

2

; 1 nM–10 mM; 20 ml) and a prostaglandin I

2

(PGI

2

) 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 col- lecting 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 ethanol- induced 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 ethanol- induced 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).

These results were similar to those obtained from indomethacin-pretreated rats. Thus, it is suggested that the protective effect of 1 M NaCl against ethanol- induced mucosal injury was mediated by both endogenous PGs and CGRP.

Effects of Administration of PGs (PGE

2

or Beraprost) on Ethanol-Induced Changes in the Microcirculation of the Basal Part of the Gastric Mucosa

Administration of PGE

2

(1 nM–10 mM) or beraprost (1 nM–10 mM) to the

observation window caused a rapid and dose-dependent dilatation of

the arterioles. PGE

2

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

Fig. 1. Changes of the diameters of microvessels in the basal part of the mucosal micro- circulation 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 ethanol- induced rapid constrictions of the collecting venules and venules were completely inhibited (D)

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

action, PGE

2

or beraprost was applied to the window 3 min before the mucosal application of 50% ethanol. The low doses of PGE

2

or beraprost which did not influence the original size of the gastric mucosal vessels including arterioles are given. Both PGE

2

(100 nM) and beraprost (1 nM) inhibited ethanol- induced rapid constrictions of the collecting venules. The dilatation of arte- rioles observed during ethanol exposure was not affected by applications of these doses of PGE

2

and beraprost.

To test whether or not the preventive effects of PGE

2

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 (PGE

2

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 PGE

2

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 uti- lizing CGRP, but PGE

2

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 con- striction of collecting venules and venules, via endogenous PGs and CGRP.

Second, it was seen that the PGI

2

analogue, which did not dilate the arterioles, collecting venules, or venules themselves, inhibited the ethanol-induced con- striction of the collecting venules and venules, suggesting that endogenous PGI

2

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

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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 Pharma- col Ther 13:1243–1250

4. Saeki T, Ohno T, Boku K, et al (2000) Mechanism of prevention by capsaicin of ethanol- induced gastric mucosal injury: a study using intravital microscopy. Aliment Pharma- col Ther 14:135–144