Introduction

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Opioid peptides have effects on a number of gastrointestinal functions including motility, acid secretion, and intestinal electrolyte and fluid transport. Moreover, opioids and opioid peptides influence the different types of mucosal damage; both protection (e.g., Ferri et al., 1983; Gyires, 1990, 1997; Scoto et al., 1990; Tazi-Saad et al., 1991; Bhounsule et al., 1994) and aggravation (e.g., Gyires et al., 1985; Del Tacca et al., 1987; Esplugues et al., 1989) have been described. Our recent findings showed that -opioid receptor agonists, given peripherally, exerted a dose-dependent inhibition on gastric mucosal damage induced by ethanol in the rat (Gyires et al., 1997). Since gastric acid is not involved in the pathomechanism of mucosal damage induced by ethanol, the protective effect is likely to be due to an increase of mucosal defense but not to inhibition of acid secretion. The mucosal protective effect of -opioid receptor agonists is in good correlation with the results of Nishimura et al. (1984), who found µ- and -binding sites in gastric fundus antrum and corpus, where binding sites were located primarily in the submucosal plexus, deep muscular plexus, and mucosa. The gastroprotective effect of opioid peptides following subcutaneous administration is likely peripheral since these peptides very poorly, if at all, pass the blood-brain barrier. However, numerous gastrointestinal functions are regulated by central mechanism as well, like gastric acid secretion (Fox and Burks, 1988; Improta and Broccardo, 1994) and gastrointestinal motility (Porreca et al., 1984). The present investigation was designed to investigate whether central components are involved also in the gastric mucosal protective effect of opioid peptides. Therefore we examined the effect of centrally administered different opioid peptides on an ethanol ulcer model where the gastric mucosal lesion is due to peripheral mechanisms and central components are not likely to be involved in the lesion formation.

	Materials and Methods

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Animals. Experiments were performed in male Wistar rats weighing 140 to 160 g. The rats were deprived of food for 24 h with free access to tap water. They were housed in wire mesh-bottom cages to prevent coprophagy. The rats were kept on a 12-h light/dark cycle and under condition of controlled temperature.

Gastric Mucosal Damage Induced by Acidified Ethanol. After 24 h of food deprivation, the animals were given orally 0.5 ml of acidified ethanol (98% ethanol in 200 mmol/l HCl). One hour later, the animals were killed by overdose of ether, and the stomachs were removed, opened along the greater curvature, rinsed with saline, and examined for lesions. The total number of mucosal lesions was assessed in a blind manner by calculation of the lesion index based on a 0 to 4 scoring system described previously (Gyires, 1990). The lesion index was calculated as the total number of lesions multiplied by the respective severity factor. The percentage of inhibition of mucosal damage was calculated as follows:
The intracerebroventricular (i.c.v.) injection to the lateral ventricle was performed according to Noble et al. (1967) in conscious rats. The site of i.c.v. injection was 2 mm from either side of the midline on the line drawn through the anterior base of the ears. The substances were injected i.c.v. 10 min before the ethanol challenge. The volume of i.c.v. injection was 10 µl. The opioid antagonists were administered i.c.v. 10 min before the i.c.v. administration of opioid peptides.

Vagotomy. Under ether anesthesia, the cervical section of the vagus nerves was exposed and bilateral cervical truncal vagotomy was performed. Sham-operated control rats had their vagus similarly exposed, but the vagal trunks were not sectioned. The incisions were closed, and all animals were allowed 3 h of recovery from the operation.

Statistical Analysis. All data are presented as the means ± S.E.M. Statistical analysis of the data was evaluated by means of the nonparametric statistical procedure, the Mann-Whitney U test. A probability of p < 0.05 was considered statistically significant.

Drugs. The following drugs were used: -endorphin (Sigma, St. Louis, MO), capsaicin (8-methyl-N-vanillyl-nonanamide) (Sigma), [D-Ala²,Phe⁴,Gly⁵-ol]-enkephalin (DAGO), [D-Ala²,D-Leu⁵]-enkephalin (DADLE) (both were synthesized by A. Magyar, Eotvos University of Hungary, Budapest, Hungary), [D-Pen²,Pen⁵]-enkephalin (DPDPE) (Sigma), D-arginine (Sigma), deltorphin II (synthesized by G. Toth, Biological Research Center of Hungarian Academy of Sciences, Szeged, Hungary), indomethacin (Chinoin-Rt., Budapest, Hungary), L-arginine (Sigma), naltrindole HCl (Sigma), L-NNA (Sigma).

	Results

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The Effect of -Endorphin, DAGO, DADLE, Deltorphin II, and DPDPE on Gastric Mucosal Damage Induced by Ethanol following i.c.v. and i.c. Administration. The opioid peptides given either i.c.v. or i.c. inhibited the gastric mucosal lesions induced by acidified ethanol in a dose-dependent manner. Following i.c.v. administration, the ED₅₀ values for -endorphin, DAGO, DADLE, deltorphin II, and DPDPE were 3.5, 6.8, 75, 120.0, and 1100 pmol/rat, respectively. Following i.c. administration, the selective -opioid receptor agonists deltorphin II and DPDPE were much more potent (ED₅₀: 0.25 and 7.0 pmol/rat, respectively), DAGO was less potent (ED₅₀: 9 pmol/rat), while -endorphin and DADLE were slightly, 4.4 and 1.5 times, more potent than after i.c.v. administration (Fig. 1; Fig. 2; Table 1). The figures do not show, however, that when increasing the doses the gastroprotective effect of the compounds was reduced; in the case of -endorphin, the dose-response curve tended to be bell-shaped.

View larger version (14K): [in this window] [in a new window]   Fig. 1.   The effect of -endorphin (), DAGO (), DADLE (), deltorphin II (), and DPDPE () on the acidified ethanol-induced gastric damage following i.c.v. administration in the rat. Each point represents mean ± S.E. of seven rats.

View larger version (14K): [in this window] [in a new window]   Fig. 2.   The effect of deltorphin II (), -endorphin (), DPDPE (), DAGO (), and DADLE () on acidified ethanol-induced gastric mucosal damage following i.c. administration in the rat. Each point represents mean ± S.E. of seven rats.

View this table: [in this window] [in a new window]   TABLE 1 The ED50 values of -endorphin, DAGO, DADLE, deltorphin II, and DPDPE given either i.c.v. or i.c. on the ethanol ulcer model in the rat

The Effect of Naltrindole on the Gastroprotective Effect of DADLE, DPDPE, and DAGO. DADLE (411 pmol/rat), DPDPE (9000 pmol/rat), and DAGO (38 pmol/rat) reduced the gastric mucosal lesions by 75, 70, and 73%, respectively. Naltrindole, the selective -opioid receptor antagonist (500 pmol/rat i.c.v.), slightly aggravated (by 33%) the mucosal damage induced by acidified ethanol and inhibited the gastroprotective effect of DADLE and DPDPE in a significant manner but failed to influence that of DAGO (Fig. 3).

View larger version (21K): [in this window] [in a new window]   Fig. 3.   The effect of naltrindole (500 pmol/rat i.c.v.) on the gastroprotective effect of DADLE, DPDPE, and DAGO against acidified ethanol-induced gastric damage in the rat. SAL., saline. Each bar represents mean ± S.E. of seven rats. *p < 0.05, **p < 0.01; acompared with saline-saline group; bcompared with the same drug in saline (i.c.v.)-treated group; ccompared with saline-naltrindole group.

The effect of Vagotomy on the Gastroprotective Effect of DADLE, Deltorphin II, -Endorphin, and DAGO. Bilateral cervical vagotomy resulted in a slight aggravation of ethanol-induced mucosal damage. DADLE (411 pmol/rat), deltorphin II (560 pmol/rat), -endorphin (10 pmol/rat), and DAGO (38 pmol/rat) inhibited the formation of gastric mucosal lesions by more than 80%. Although bilateral cervical vagotomy reduced the protective effect of the opioid peptides in a significant manner, no complete reversal of the protective action was observed (Fig. 4).

View larger version (24K): [in this window] [in a new window]   Fig. 4.   The effect of acute bilateral cervical vagotomy on the gastroprotective effect of DADLE, deltorphin II (DELTOR.), -endorphin (-END.), and DAGO against acidified ethanol-induced gastric damage in the rat. SAL., saline. Each bar represents mean ± S.E. of seven rats. *p < 0.05, **p < 0.01; acompared with saline-group (sham operation); bcompared with same drug in sham-operated group; ccompared with saline-group (vagotomy).

The Effect of Vagotomy on the Gastroprotective Effect of Capsaicin Given Orally. Capsaicin in the dose of 0.5 mg/kg p.o. decreased the ethanol-induced gastric mucosal damage in a significant manner. After bilateral cervical vagotomy, the gastroprotective effect of capsaicin remained unchanged (Fig. 5).

View larger version (12K): [in this window] [in a new window]   Fig. 5.   The effect of acute bilateral cervical vagotomy on the gastroprotective effect of orally administered capsaicin (0.5 mg/kg) in the rat. Each bar represents mean ± S.E. of seven rats. **p < 0.01

The Effect of N^G-Nitro-L-arginine on the Gastroprotective Effect of DADLE, -Endorphin, and DAGO. Intravenous administration of L-NNA (3 mg/kg i.v.) resulted in a slight increase of ethanol-induced gastric mucosal damage. DADLE (411 pmol/rat), -endorphin (10 pmol/rat), and DAGO (38 pmol/rat) inhibited the lesion formation highly effectively. When L-NNA was injected prior to the compounds, the gastroprotective effects were reduced in a significant manner (Fig. 6). The inhibitory effect of L-NNA was reversed by L-arginine but not by D-arginine (not shown).

View larger version (22K): [in this window] [in a new window]   Fig. 6.   The effect of L-NNA on the gastroprotective effect of DADLE, -endorphin (-END.), and DAGO against acidified ethanol-induced gastric damage in the rat. SAL., saline. Each bar represents mean ± S.E. of seven rats. *p < 0.05, **p < 0.01; acompared with saline-saline group; bcompared with same drug in saline (i.v.)-treated group; ccompared with saline-L-NNA group.

The Effect of Indomethacin on the Protective Effect DADLE, Deltorphin II, DPDPE, and DAGO. Indomethacin (20 mg/kg p.o.) resulted in a slight increase of mucosal damage induced by ethanol and antagonized the gastroprotective effect of opioid peptides (Fig. 7).

View larger version (24K): [in this window] [in a new window]   Fig. 7.   The effect of indomethacin on the gastroprotective effect of DADLE, deltorphin II (DELTOR.), DPDPE, and DAGO against acidified ethanol-induced gastric damage in the rat. SAL., saline. Each bar represents mean ± S.E. of seven rats. *p < 0.05, **p < 0.01; acompared with saline-methylcellulose group; bcompared with the same drug in methylcellulose (p.o.)-treated group; ccompared with saline-indomethacin group.

	Discussion

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Investigations on gastric mucosal protective mechanisms are focused mainly on the local mucosal processes. Much less is known about how the central nervous system may influence the gastric mucosal defense. However, gastric mucosal protection induced by a central mechanism was described recently (Tache et al., 1994; Gyires, 1997; Guidobono et al., 1998; Kaneko et al., 1998; Yang et al., 1999). In our present study, the role of central opioid receptors was analyzed by means of selective - and µ-opioid receptor agonists. It was found that both the selective - and µ-opioid receptor agonists injected either i.c.v. or i.c. exerted protective effect against acidified ethanol-induced lesions; the rank order of potency was -endorphin > DAGO > DADLE > deltorphin II > DPDPE following i.c.v. injection and deltorphin II >  endorphin > DPDPE > DAGO > DADLE following i.c. administration. The results suggest that activation of supraspinal - and µ-opioid receptors may induce gastric mucosal protection. It is well documented that activation of these receptors induces antinociception as well (e.g., Porreca et al., 1984; Ossipov et al., 1995). However, while the analgesic dose of -endorphin, which binds to both - and µ-opioid receptors, was found to be between 0.5 (Geisow et al., 1977) and 7.7 (Székely et al., 1977) nmol/rat i.c.v., the gastroprotective doses, as our present data suggested, were 0.001 to 0.030 nmol/rat given i.c.v. Moreover, while DPDPE induced antinociceptive effect in the doses of 1 to 100 µg given into the ventromedial medulla (Hurley et al., 1999), it exerted gastroprotective action in the doses of 0.005 to 0.5 µg injected intracisternally. These results suggest that gastroprotection can be induced by much lower doses of opioids than those for antinociception. Consequently, the ethanol-induced ulcer model in the rat is a useful, quick, simple, reliable, and sensitive in vivo method for examination and analysis of the -opioid receptor-selective drugs.

Two subtypes of  receptors, ₁ and ₂, were described; DPDPE activates ₁, while deltorphin II stimulates the ₂ receptors (Mattia et al., 1991). Our present results showed that both ₁- and ₂-opioid receptor subtypes may be involved in the centrally induced gastroprotection, however, since deltorphin II was more effective injected both i.c.v. (9 times) and i.c. (27 times) than DPDPE, the dominant role of central ₂-opioid receptor subtype might be raised.

Since the ED₅₀ values of DADLE, DPDPE, and deltorphin II injected peripherally (s.c.) are much higher35, 1800, and 3500 nmol/kg, respectively (Gyires et al., 1997)than that following central administration, it can be concluded that gastric protection induced by opioid peptides injected into the lateral ventricles or the cisterna magna is mediated through the central nervous system and does not represent a leakage of the peptides into the periphery.

The -opioid receptor agonist peptides were more potent given i.c. than i.c.v., whereas the µ-agonist DAGO proved to be approximately equiactive given either i.c.v. or. i.c. This result may suggest that 1) DAGO may induce a gastroprotective effect acting either in periventricular structures or in brainstem (or in both) since direct projection from paraventricular nucleus to dorsal vagal complex was described (Swanson and Kuypers, 1980), and 2) the primary site of action of the protective effect of -opioid agonists is in the brain stem. This assumption is in good correlation with the findings that µ- but not -receptors were found in periventricular structures, while both - and µ-opioid receptors were described in the brainstem, in nucleus tractus solitarii and vagal fibers (Snyder, 1982; Mansour et al., 1995). The prominent role of the brainstem in opioid-induced central gastroprotection is supported by our previous findings (Gyires et al., 2000). We analyzed the gastroprotective mechanism of clonidine, and on the basis of literature (Giersbergen et al., 1989) we supposed that the release of -endorphin is the key link in the opioid-mediated central gastroprotective effect of clonidine and that the site of action might be tentatively localized to the dorsal vagal complex. Therefore the effect of intracisternally injected yohimbine, naltrindole, as well as -endorphin antiserum was examined on the gastroprotective effect of intracerebroventricularly administered clonidine. It was found that all these agents antagonized the clonidine-induced gastroprotection, indicating that activation of ₂-adrenoceptors--endorphin release-opioid receptor stimulation chain is likely to be localized to the lower brainstem.

Moreover, the role of the dorsal vagal complex in gastric mucosal protection is well documented. For example, intracisternal injection of the thyrotropin-releasing hormone analog RX-773668 (Tache et al., 1994), neuropeptide Y (Yang et al., 1999), or adrenomedullin (Kaneko et al., 1998) induced vagally mediated gastric protection; moreover, the vagal stimulant 2-deoxy-D-glucose produced gastroprotection against ethanol through a vagally mediated pathway (Henagan et al., 1984). In our experiments, a significant reduction of the protective effect of opioid peptides was observed following acute vagotomy, indicating that the dorsal vagal complex is likely to be involved in conveying the central effect of opioids to the periphery. The reduced peripheral action of opioid peptides after acute vagotomy may be either due to mechanical arrest of centrifugal impulses in the efferent fibers and/or to a functional impairment of the secretory function of sensory nerve endings, which are thought to be involved in mucosal defense (Lambrecht et al., 1993). To examine whether the sensory nerve endings of vagus is functionally intact after acute vagotomy, we compared the effect of orally administered capsaicin on ethanol-induced lesions in sham-operated and vagotomized rats. Namely, a single oral dose of capsaicin induces the release of sensory neuropeptides from primary afferent nerve terminals and exerts a gastroprotective effect (Holzer and Lippe, 1988). Our recent data showed that gastroprotection induced by a single oral dose of capsaicin produced the same gastroprotective effect in acutely vagotomized rats as in sham-operated animals, indicating that the nerve terminal was intact functionally. Consequently, the highly reduced gastroprotective effect of centrally injected opioid peptides in vagotomized rats may be due to a mechanical failure of the transmission of central impulses to the periphery by vagal efferents and not to a functional impairment of sensory nerve terminals. However, the reduction of the gastroprotective effect was not complete, indicating that a vagal-independent mechanism may also be involved in the centrally initiated gastroprotective effect of opioid peptides.

Vagal nerve is likely involved in the effect of some peripherally acting gastroprotective agents, like prostacyclin, atropine, -carotene, cimetidine (Mózsik et al., 1991), and prostaglandin E₂ (PGE₂), as well as in adaptive cytoprotection (Henagan et al., 1984). On the other hand, a subpopulation of GABA_A receptors in the stomach may mediate gastroprotection, and this effect was not altered by vagotomy (Erdö et al., 1989), indicating that gastroprotection can be induced by a vagal-independent pathway as well.

In the periphery, numerous factors are involved in the maintenance of the integrity of gastric mucosa. Whittle et al. (1990) suggested that nitric oxide (NO), PGs, and sensory neuropeptides are responsible for the integrity of mucosa. The present data showed N^G-nitro L-arginine, an inhibitor of NO synthase, reduced the protective effect of DAGO, -endorphin, and, in a lesser extent, DADLE. The effect was reversed in a specific manner by an excess of L-arginine, a substrate of NO synthase, but not by D-arginine, which is not a substrate. The results indicate that mucosal NO is likely to be involved in the opioid-induced gastroprotection. Moreover, inhibition of PG synthesis by indomethacin also resulted in a significant reduction of the mucosal protective effect of opioids, suggesting that in addition to NO, prostaglandins may also be involved in the mucosal protective mechanism of opioid peptides.

Our previous results showed that the -receptor-selective opioid peptides were also effective following peripheral (subcutaneous) application against ethanol-induced mucosal damage. However, since the substances were given peripherally, the site of action is likely to be peripheral because the peptides do not pass the blood-brain barrier. It may be raised that development of a systemically bioavailable  (₂)-receptor-selective opioid, which passes the blood-brain barrier, may result in a highly effective gastroprotective compound with a dual mechanism, both central and peripheral. Recently it was found that nonpeptidic, naltrindole-related -opioid receptor agonists exerted a gastric mucosal-protective effect against ethanol-induced gastric lesions in the rat (Rónai et al., 1997); however, it has yet to be analyzed to which extent the central and peripheral component is involved in the gastroprotective action. Other structurally dissimilar, systemically bioavailable nonpeptidic -receptor agonists, e.g., BW373U86 (Chang et al., 1993), SNC 80 (Bilsky et al., 1995), and TAN67 (Suzuki et al., 1995), have been developed recently; they exhibited limited efficacy and potency in different pain models in rats and mice following systemic administration. Further studies are required to elucidate and analyze the probable gastroprotective effect of these nonpeptidic opioids.

In summary, the present data demonstrated that activation of supraspinal µ- and -opioid receptors induces a series of events that result in a mucosal protective action. The primary site of action of -agonists is likely to be the brain stem. In the periphery, both the mucosal nitric oxide and prostaglandins are thought to be involved in the gastric protective processes.