Peripheral Mechanisms Involved in Gastric Mucosal Protection by Intracerebroventricular and Intraperitoneal Nociceptin in Rats
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《内分泌学杂志》
Department of Experimental Medicine and Public Health (C.P., M.M.), University of Camerino, 62032 Camerino
Department of Pharmaceutical Sciences (R.G.), University of Ferrara, 44100 Ferrara
Department of Human Anatomy, Pharmacology, and Forensic Medicine (D.G., D.L., G.M.), University of Parma, 43100 Parma, Italy
Abstract
Nociceptin (N/OFQ) exerts multiple effects in the gastrointestinal tract after central or peripheral administration. In the present study, we examined the possible peripheral mechanisms mediating gastric protection by N/OFQ in rats. Gastric mucosal lesions were induced by 50% ethanol (1 ml/rat intragastrically). N/OFQ, administered either intracerebroventricularly (3 μg/rat) or ip (10 μg/kg), significantly reduced macroscopic and histological damage. The protective effect of intracerebroventricular N/OFQ was blocked by atropine, subdiaphragmatic vagotomy, and bretylium. The effect of both central and peripheral N/OFQ was blocked by functional ablation of afferent nerves produced by capsaicin, by the antagonist of calcitonin gene-related peptide, CGRP8–37, and by the nitric oxide synthase inhibitor, NG-nitro-L-arginine methyl ester. These results indicate that N/OFQ increases gastric mucosal resistance to ethanol by operating both in the central nervous system and in the periphery. Vagal cholinergic and sympathetic pathways mediate the central activity of N/OFQ, whereas vagal nonmuscarinic pathways mediate the peripheral activity of the peptide. The neuronal circuit involving extrinsic sensory neurons, calcitonin gene-related peptide, and nitric oxide is activated by central as well as peripheral N/OFQ. The study provides evidence that N/OFQ contributes to neurally mediated gastric mucosal protection.
Introduction
NOCICEPTIN/ORPHANIN FQ (N/OFQ) is a heptadecapeptide, isolated from brain extract, which shows a structural homology to the opioid peptide dynorphin A and binds to the orphan opioid receptor-like 1 receptor, now termed NOP receptor (1, 2). Since the early studies, it became evident that N/OFQ was involved in modulation of pain, anxiety, feeding, and memory processes (3, 4, 5, 6). Central and peripheral administration of the peptide has also been reported to exert multiple effects in the gastrointestinal tract, affecting gastric and intestinal motility (7, 8, 9) and secretion (10, 11).
We have recently shown that N/OFQ protects against gastric mucosal damage induced by intragastric (ig) ethanol exposure in the rat and that its effect that is reversed by the selective antagonist UFP-101, suggesting the peptide exerts gastric protection through the NOP receptor (12). The evidence that N/OFQ protects the gastric mucosa after both central and peripheral administration suggests that the peptide interacts with distinct central and peripheral pathways. Accordingly, the NOP receptor has been identified in the central and peripheral nervous systems and along the entire gastrointestinal tract of the rat (13, 14). Nerve fibers immunoreactive to N/OFQ were found in the myenteric plexus and circular muscle layer of the rat colon (7).
Resistance of the gastric mucosa against damage by endogenous and exogenous aggressors is ensured by a complex system of mediators. A crucial role has been attributed to the neuropeptide calcitonin gene-related peptide (CGRP), released by capsaicin-sensitive extrinsic nerve fibers and to nitric oxide (NO), synthesized by the neuronal and endothelial enzymes. The sensory neuropeptide and NO appear to act in concert in the modulation of mucosal integrity (15, 16). There is also evidence for an interaction between autonomic nervous system and peripheral CGRP/NO pathways (17). Indeed, neuropeptides like TRH, peptide YY (PYY), and adrenomedullin, acting at the central level, reduce gastric lesions caused by ethanol in rats through vagal cholinergic pathways associated with peripheral release of CGRP and activation of NO synthesis (18, 19, 20).
The present study was aimed to further characterize the protective activity of N/OFQ, injected centrally or peripherally, against ethanol damage in rats, by evaluating the possible peripheral neural mechanisms mediating the effect of the peptide. In particular, we have examined the role of NO and of afferent nerve fibers as well as of parasympathetic and sympathetic systems.
Materials and Methods
Animals
Male Wistar rats, weighing 180–200 g (Harlan, S. Pietro al Natisone, Italy) were individually housed in hanging stainless-steel cages with grid floors, at constant room temperature (25 ± 1 C) and humidity (60 ± 5%), with an artificial 12-h light,12-h dark cycle. Seven to 10 d before the experiments, groups of rats were implanted with lateral ventricular cannulas for the intracerebroventricular (icv) administration of N/OFQ, as previously described (12). All the animals were deprived of food but not of water for 24 h before the experiments and randomly assigned to the different treatment groups. Experimental procedures were approved by the Italian Animal Care and Use Committee.
Experimental protocol
Rats received N/OFQ either by the icv route at 3 μg in a volume of 2 μl/rat or by the ip route at 10 μg/kg in a volume of 2 ml/kg. Control rats received an equal volume of saline. The doses of N/OFQ were chosen from previous dose-response studies, as shown to almost completely prevent ethanol-induced gastric damage in the rat gastric mucosa (12). Thirty minutes later, gastric lesions were induced by ig administration of 1 ml of 50% ethanol. Five minutes after ethanol administration, the rats were killed, and the stomachs were removed, opened along the lesser curvature, and examined. The presently described protocol was common to all the treatment groups.
Effect of hexamethonium, atropine, and bretylium
Groups of rats were pretreated with the following drugs: hexamethonium, at 20 mg/kg ip, reported to antagonize the gastroprotective effect of centrally injected clonidine in the rat (21); atropine at 1 mg/kg sc, shown to prevent the gastroprotective effect of intracisternal PYY in the rat (19); bretylium at 25 mg/kg sc, shown to reverse the inhibitory effect of intracranial hypertension on gastric emptying in rats (22) or with saline. Thirty minutes later, either N/OFQ or vehicle was administered. After an additional 30-min period, all rats received ethanol.
Effect of subdiaphragmatic vagotomy
Rats were anesthetized by im injection of tiletamine hydrochloride, 200 mg/kg, and zolazepam hydrochloride, 200 mg/kg. After induction of anesthesia, the abdomen was opened by a midline incision and the connective tissue removed from the surface of the esophagus. Under a dissecting microscope, the dorsal and ventral trunks of the vagus nerve attached to the subdiaphragmatic portion of the esophagus were transected and the cut nerve stumps retracted. After closure of the incision, the rats were given 7 d to recover before the treatments. Control rats were sham operated.
Effect of functional ablation of afferent nerves
Rats were treated with increasing doses of capsaicin in one or two daily sc injections over 4 consecutive days (12.5; 12.5 + 12.5; 25 + 25; 25 mg/kg). Capsaicin was dissolved in 10% ethanol, 10% Tween 80 and 80% saline (by volume). Control animals received the vehicle. To counteract respiratory impairment associated with capsaicin administration, 30 min before the first daily injection of capsaicin, rats received an ip injection of atropine (0.2 mg/kg), terbutaline (0.2 mg/kg), and aminophylline (20 mg/kg). Studies were performed 2 wk after the last capsaicin injection. To evaluate the effectiveness of capsaicin pretreatment, wiping movements in response to intraocular instillation of a 0.1% solution of ammonia were evaluated. Capsaicin-pretreated animals showing any wiping movements were excluded from the study.
Effect of CGRP8–37
The antagonist of CGRP, CGRP8–37, at the dose of 25 μg/kg, was administered sc 30 min before either N/OFQ or vehicle. Ethanol was administered 30 min after N/OFQ injection. The dose and route of administration of CGRP8–37 have been shown to effectively reduce amylin gastroprotective activity in the rat (23).
Effect of inhibition of NO biosynthesis
Groups of rats were pretreated with the nonselective NO synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME), 10 mg/kg sc, 60 min before either N/OFQ or vehicle. To evaluate the specificity of the effects of L-NAME, L-arginine (300 mg/kg sc), as a substrate for NO synthase, was coadministered with L-NAME. The doses of the two compounds were selected from previous studies in this model (24). Ethanol was administered 30 min after N/OFQ injection.
Assessment of gastric mucosal damage
For the macroscopic assessment of mucosal lesions, the stomachs were placed on a flat surface under a stereo microscope. The glandular mucosa was examined, and each individual hemorrhagic lesion was measured along its greatest length (<1 mm, rating of 1; 1–2 mm, rating of 2; >2 mm, rating according to their greatest length). The lengths of the lesions were summed to give an overall total, designated as the lesion index, for each stomach. For the histological assessment of mucosal lesions, a strip was excised from the glandular mucosa, 3–4 mm below and parallel to the limiting ridge, so that the greater curvature was approximately located in the middle of the strip. Three different tissue samples were taken from each strip, fixed in 10% formaldehyde, and embedded. Serial sections, 4 μm thick, in which the gastric pits and glands were oriented perpendicular to the mucosal surface, were cut from each block. The sections were stained either with hematoxylin-eosin or periodic acid-Schiff. For the morphometric analysis of gastric lesions, the image of the section was displayed on a color monitor by means of a video camera attached to the microscope (Nikon Optiphot), and quantitations were performed using a color image analysis software system (LUCIA G, Nikon). The damage was scored in different grades on the basis of its depth: grade 0, all gastric mucosal cells appeared intact; grade I, damage to luminal surface mucous cells only; grade II, damage to luminal surface and gastric pit mucous cells, with presence of cell exfoliation; and grade III, damage extending from luminal surface to gastric gland cells. For each stomach, the total length of mucosa examined and the length of mucosa with each grade of damage were measured. The length values of each grade of damage were then expressed as a percentage of the total length of mucosa examined.
Chemicals
The N/OFQ peptide was synthesized as a salt derivative at the Department of Pharmaceutical Sciences of the University of Ferrara, Italy. The purity grade was more than 95% determined by analytical HPLC and electrospray mass spectrometry analyses. The peptide was dissolved in saline solution (0.9% NaCl). Capsaicin was purchased from Serva (Heidelberg, Germany). All the other reagents were purchased from Sigma Chemical Co. (St. Louis, MO).
Statistical analysis
Data shown are means ± SEM. Statistical analysis was performed using ANOVA followed by Newman-Keuls test, and P values < 0.05 were considered significant.
Results
Protective effect of N/OFQ
Exposure to ethanol for 5 min caused the formation of macroscopically visible hemorrhagic lesions in the glandular portion of the stomach; lesion index was 103.2 ± 10.1 (Table 1). Histological examination of gastric tissues 5 min after ethanol revealed that 64.2% of the glandular mucosa was damaged. The superficial portions of the mucosa were largely affected, with surface mucous cells and pit cells being destroyed and exfoliated. Lesions extending into the gland area occupied 24.2% of the total length measured. The depth of these lesions corresponded to the middle of the gland, that is to the parietal cell area, whereas the chief cell area remained unaltered (Fig. 1 and Table 1).
N/OFQ significantly reduced the mucosal lesions after both central and peripheral administration. The dose of 3 μg icv appears to be equally effective with the dose of 10 μg/kg ip. Macroscopic lesions were reduced by 71.1% (P < 0.01) and by 72.8% (P < 0.01) after icv and ip administration, respectively (Table 1). Similar results were obtained when damage to the mucosa was evaluated histologically. The length of the mucosa damaged in gastric tissues from rats pretreated with icv or ip N/OFQ before ethanol exposure was markedly reduced. Furthermore, when present, lesions were confined to the superficial portions of the mucosa, whereas the integrity of the gland cell area appeared to be nearly completely preserved (Fig. 1 and Table 1). Control groups receiving either saline or N/OFQ in the absence of ethanol exposure had normal appearing gastric mucosa.
Effect of hexamethonium, atropine, and bretylium
Hexamethonium (20 mg/kg ip) alone significantly reduced by 68.9% gastric mucosal damage consequent to the exposure to 50% ethanol. Pretreatment with hexamethonium did not apparently influence the effectiveness of icv or ip N/OFQ in preventing damage by ethanol (Fig. 2). Atropine (1 mg/kg sc) alone was ineffective in modifying ethanol-induced damage, and it did not influence the protective activity of ip N/OFQ (Fig. 2). Conversely, atropine significantly reduced the effect of icv N/OFQ (Fig. 2). Bretylium (25 mg/kg sc) had no significant effect on damage caused by 50% ethanol and on protection exerted by ip N/OFQ. In contrast, it reversed gastroprotective activity of icv N/OFQ (Fig. 2). In the absence of ethanol exposure, hexamethonium, atropine, and bretylium did not modify the macroscopic appearance of the gastric mucosa.
Effect of subdiaphragmatic vagotomy
In sham-operated rats, formation of gastric lesions induced by 50% ethanol and protective activity exerted by icv or ip N/OFQ were not different from that observed in nonoperated rats. Subdiaphragmatic vagotomy itself did not influence 50% ethanol-induced gastric lesions in rats injected either icv or ip with saline, with the lesion index being 88.3 ± 13.2. In contrast, vagotomy completely prevented the protective effect of N/OFQ when given by icv or ip injection (Fig. 3).
Effect of functional ablation of afferent nerves and of CGRP8–37
Pretreatment with capsaicin (112.5 mg/kg sc), 2 wk before study, caused a slight, although not significant, increase in the extent of gastric mucosal damage induced by ethanol, with the lesion index being 114.3 ± 14.2. In capsaicin-pretreated rats, the lesion index after both icv and ip administration of N/OFQ was comparable to that observed in the control group (Fig. 4). Pretreatment with CGRP8–37, which itself did not influence the damaging effect of 50% ethanol, reversed protection resulting from both icv and ip N/OFQ (Fig. 5).
Effect of inhibition of NO biosynthesis
Previous treatment with L-NAME (10 mg/kg sc) did not significantly modify gastric mucosal lesions induced by 50% ethanol. The lesion index remained unaltered when L-NAME was administered concurrently with L-arginine (300 mg/kg sc). In rats pretreated with L-NAME, the protective effect exerted by either icv or ip administered N/OFQ was significantly reduced, reduction of ip N/OFQ activity being more pronounced than that of icv N/OFQ (Fig. 6). In both conditions, the protective activity of N/OFQ was restored by the concurrent pretreatment with L-NAME and L-arginine (Fig. 6). When L-NAME and L-arginine were administered alone, no effect on the macroscopic appearance of the gastric mucosa was observed.
Discussion
The present findings confirm a role of N/OFQ in the maintenance of the integrity of the gastric mucosa in conscious rats and validate the evidence of macroscopic protection of the mucosa with quantitative histology of gastric epithelium in rats pretreated with N/OFQ before ethanol exposure. Histologically, pretreatment with N/OFQ was associated with reduction of the total extension of ethanol-induced damage. Furthermore, after N/OFQ, damage, where present, was confined to the most superficial layer of the mucosa, whereas damage by ethanol alone was distributed throughout the different regions of fundic mucosa. The reduction of the depth of damage is considered of special relevance, because it facilitates a rapid repair and the consequent reestablishment of the epithelial continuity. It is reported to occur under conditions in which mucosal blood flow is maintained or enhanced (25, 26), suggesting that mucosal microcirculation could be primarily affected by N/OFQ. Macroscopic and histological protection afforded by the icv N/OFQ was also observed after ip N/OFQ, indicating that quantitatively similar degrees of protection could be achieved by the two routes of administration.
Pharmacological and surgical approaches were used to determine pathways mediating gastroprotective activity of N/OFQ. Subdiaphragmatic vagotomy abolished the response to central and peripheral N/OFQ, indicating the involvement of vagal pathways. Cholinergic receptor blockade by atropine effectively reduced protection exerted by central but not by peripheral N/OFQ. Vagal postganglionic activation through muscarinic receptors appears to have a role only in central protective activity of N/OFQ. The involvement of autonomic ganglia remains uncertain. The ganglionic blocking agent hexamethonium caused a significant decrease in ethanol-induced lesions and hence made the interpretation of its effect on protection by N/OFQ difficult. Sympathetic nerves appear to mediate central, but not peripheral, activity of N/OFQ, as supported by the fact that the adrenergic blocking agent, bretylium, completely reversed protection only by centrally injected N/OFQ. Vagal cholinergic and sympathetic nerves are likely to mediate the central activity of N/OFQ, whereas vagal nonmuscarinic pathways mediate the peripheral activity of the peptide.
It is well established that intact vagal innervation is necessary to afford protection to the gastric mucosa. Although controversial, there are studies in which vagotomy is reported to cause a tendency to worsen ethanol-induced lesions (27, 28). Protection by peripherally administered prostaglandin E2, prostacyclin, and mild irritants (28, 29) and by centrally administered TRH (18) is reduced in vagotomized rats. Fibers contained in the vagus nerve are both efferent and afferent, with 90% of vagal fibers in the subdiaphragm being afferent fibers (30). In the rat gastric mucosa, extrinsic afferent nerve fibers are both vagal and spinal, and they appear to subserve different roles. Spinal afferents should enhance the resistance of gastric mucosa to injury, whereas vagal afferents, many of them being resistant to capsaicin (31), should convey information from the gastric mucosa to the brain stem (15, 32). Capsaicin-sensitive extrinsic afferent fibers release from their peripheral endings several peptides, including substance P and CGRP. Afferent nerve fibers and CGRP are of great relevance in promoting gastric mucosal defense (15). They appear to act primarily on the regulation of gastric mucosal blood flow, by influencing the arterial and arteriolar system and promoting vasodilation (33). Vasodilation by CGRP involves the formation of NO. NO, produced both by endothelial cells and in the intrinsic neurons, provokes vasodilation and mucus secretion and exerts a protective activity against experimental gastric damage (16, 17).
The present results show that gastric protection by N/OFQ, acting both at central and at peripheral sites, is prevented by capsaicin pretreatment, by administration of the CGRP antagonist, and by the peripheral NO synthase inhibitor L-NAME. It is likely that N/OFQ activates the neuronal circuit involving extrinsic sensory nerves, CGRP, and NO. The central activity of N/OFQ, like that of the neuropeptides TRH, PYY, and adrenomedullin (18, 19, 20), could be mediated in part by the influence on vagal efferent activity, which in turn activates CGRP-containing nerve fibers and NO formation.
In our experiments, response to central N/OFQ was also dependent on sympathetic pathways, because it was completely blocked by bretylium. There is little information regarding the role of the sympathetic nervous system in gastric mucosal defense. Sympathectomy has been reported to worsen damage by ethanol and to abolish adaptive protection in rats (27). N/OFQ is reported to decrease peripheral resistance and to increase blood flow to peripheral organs, including stomach, in the rat (34, 35). Peripheral vasodilation by N/OFQ, contributing to its protective activity, could result from inhibition of sympathetic pathways. In support of this hypothesis is the finding that hypotension caused in the rat by iv nociceptin, in the dose range 20–200 μg/kg, was reduced by the adrenergic neuron blocker guanethidine and completely abolished by guanethidine plus vagotomy, indicating a concomitant inhibitory influence on sympathetic outflow and activation of vagal outflow (36). However, in the present study, we did not observe any influence of the adrenergic neuron blocker bretylium on peripheral activity of N/OFQ, possibly because of the lower dose range used in the present study as compared with that used by Giuliani et al. (36) or alternatively of a minor role of sympathetic nerve fibers in mediating the peripheral activity of the peptide. It is worth noting that the neuropeptide corticotropin-releasing factor (CRF) when centrally administered protects the gastric mucosa, stimulates gastric mucosal blood flow, and influences gastrointestinal motor and secretory functions in rats, and its effects have been ascribed to the modulation of both vagal efferent and sympathetic pathways (37, 38, 39, 40). Central CRF is hypothesized to alter the balance of the autonomic nervous system, particularly shifting the system toward the sympathetic component. Such studies demonstrate that gastrointestinal functions could be modulated at the central level by the simultaneous influence on parasympathetic and sympathetic outflow. Both pathways could similarly be implicated in the protective activity of central N/OFQ. Conversely, peripheral N/OFQ appears to affect almost exclusively vagal noncholinergic and afferent capsaicin-sensitive fibers. The difference in the neuronal pathways mediating central and peripheral effects of the peptide strengthens the hypothesis of the existence of distinct sites of action.
There is a general consensus that the effects of N/OFQ on gastrointestinal functions are mediated neurally. The possible mechanisms at the basis of the effects of N/OFQ in vivo have been partly explored. The effect of centrally administered N/OFQ on gastric acid secretion in rats was completely inhibited by atropine and vagotomy, suggesting the involvement of the vagus cholinergic nerve (10). Mechanisms of motor effects have been evaluated only after peripheral administration of N/OFQ. An increase in antral and colonic contractility in rats was unaffected by L-NAME, vagotomy, and splanchnicotomy, leading to suppose that the peptide acts at the level of the intramural myenteric plexus (8, 9). N/OFQ could be considered a neuropeptide operating both in the central nervous system and at the gastrointestinal level.
In addition to N/OFQ, a number of peptides, such as ghrelin, cholecystokinin, PYY, and CRF, are implicated both in the modulation of feeding (41, 42, 43) and in the regulation of gastrointestinal functions (43, 44, 45). Similarly to N/OFQ, these peptides are present centrally, in brain areas involved in feeding behavior pathways, and peripherally, in neurons and/or endocrine cells in the gut. Importantly, CRF, after acute central injection, is well known to produce a decrease of feeding behavior in rats (43), and its effect can be reversed by central N/OFQ injection, through a functional antagonism (46). So, CRF and N/OFQ display a comparable protective effect on gastric mucosa, although they differ in relation to the effects on feeding behavior, suggesting that feeding controlling pathway systems differ from those involved in the maintenance of mucosal epithelial integrity.
The present results strongly suggest that N/OFQ could be a component of a multifactorial system regulating energy/homeostasis, gastrointestinal motility and secretion, and neurally mediated gastric mucosal protection.
Footnotes
This work was supported by a grant (COFIN 2002) from the Italian Ministry of Education, University and Research, Rome, Italy.
Abbreviations: CGRP, Calcitonin gene-related peptide; CRF, corticotropin-releasing factor; ig, intragastric; icv, intracerebroventricular;L-NAME, NG-nitro-L-arginine methyl ester; NO, nitric oxide; N/OFQ, nociceptin/orphanin FQ; PYY, peptide YY.
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Department of Pharmaceutical Sciences (R.G.), University of Ferrara, 44100 Ferrara
Department of Human Anatomy, Pharmacology, and Forensic Medicine (D.G., D.L., G.M.), University of Parma, 43100 Parma, Italy
Abstract
Nociceptin (N/OFQ) exerts multiple effects in the gastrointestinal tract after central or peripheral administration. In the present study, we examined the possible peripheral mechanisms mediating gastric protection by N/OFQ in rats. Gastric mucosal lesions were induced by 50% ethanol (1 ml/rat intragastrically). N/OFQ, administered either intracerebroventricularly (3 μg/rat) or ip (10 μg/kg), significantly reduced macroscopic and histological damage. The protective effect of intracerebroventricular N/OFQ was blocked by atropine, subdiaphragmatic vagotomy, and bretylium. The effect of both central and peripheral N/OFQ was blocked by functional ablation of afferent nerves produced by capsaicin, by the antagonist of calcitonin gene-related peptide, CGRP8–37, and by the nitric oxide synthase inhibitor, NG-nitro-L-arginine methyl ester. These results indicate that N/OFQ increases gastric mucosal resistance to ethanol by operating both in the central nervous system and in the periphery. Vagal cholinergic and sympathetic pathways mediate the central activity of N/OFQ, whereas vagal nonmuscarinic pathways mediate the peripheral activity of the peptide. The neuronal circuit involving extrinsic sensory neurons, calcitonin gene-related peptide, and nitric oxide is activated by central as well as peripheral N/OFQ. The study provides evidence that N/OFQ contributes to neurally mediated gastric mucosal protection.
Introduction
NOCICEPTIN/ORPHANIN FQ (N/OFQ) is a heptadecapeptide, isolated from brain extract, which shows a structural homology to the opioid peptide dynorphin A and binds to the orphan opioid receptor-like 1 receptor, now termed NOP receptor (1, 2). Since the early studies, it became evident that N/OFQ was involved in modulation of pain, anxiety, feeding, and memory processes (3, 4, 5, 6). Central and peripheral administration of the peptide has also been reported to exert multiple effects in the gastrointestinal tract, affecting gastric and intestinal motility (7, 8, 9) and secretion (10, 11).
We have recently shown that N/OFQ protects against gastric mucosal damage induced by intragastric (ig) ethanol exposure in the rat and that its effect that is reversed by the selective antagonist UFP-101, suggesting the peptide exerts gastric protection through the NOP receptor (12). The evidence that N/OFQ protects the gastric mucosa after both central and peripheral administration suggests that the peptide interacts with distinct central and peripheral pathways. Accordingly, the NOP receptor has been identified in the central and peripheral nervous systems and along the entire gastrointestinal tract of the rat (13, 14). Nerve fibers immunoreactive to N/OFQ were found in the myenteric plexus and circular muscle layer of the rat colon (7).
Resistance of the gastric mucosa against damage by endogenous and exogenous aggressors is ensured by a complex system of mediators. A crucial role has been attributed to the neuropeptide calcitonin gene-related peptide (CGRP), released by capsaicin-sensitive extrinsic nerve fibers and to nitric oxide (NO), synthesized by the neuronal and endothelial enzymes. The sensory neuropeptide and NO appear to act in concert in the modulation of mucosal integrity (15, 16). There is also evidence for an interaction between autonomic nervous system and peripheral CGRP/NO pathways (17). Indeed, neuropeptides like TRH, peptide YY (PYY), and adrenomedullin, acting at the central level, reduce gastric lesions caused by ethanol in rats through vagal cholinergic pathways associated with peripheral release of CGRP and activation of NO synthesis (18, 19, 20).
The present study was aimed to further characterize the protective activity of N/OFQ, injected centrally or peripherally, against ethanol damage in rats, by evaluating the possible peripheral neural mechanisms mediating the effect of the peptide. In particular, we have examined the role of NO and of afferent nerve fibers as well as of parasympathetic and sympathetic systems.
Materials and Methods
Animals
Male Wistar rats, weighing 180–200 g (Harlan, S. Pietro al Natisone, Italy) were individually housed in hanging stainless-steel cages with grid floors, at constant room temperature (25 ± 1 C) and humidity (60 ± 5%), with an artificial 12-h light,12-h dark cycle. Seven to 10 d before the experiments, groups of rats were implanted with lateral ventricular cannulas for the intracerebroventricular (icv) administration of N/OFQ, as previously described (12). All the animals were deprived of food but not of water for 24 h before the experiments and randomly assigned to the different treatment groups. Experimental procedures were approved by the Italian Animal Care and Use Committee.
Experimental protocol
Rats received N/OFQ either by the icv route at 3 μg in a volume of 2 μl/rat or by the ip route at 10 μg/kg in a volume of 2 ml/kg. Control rats received an equal volume of saline. The doses of N/OFQ were chosen from previous dose-response studies, as shown to almost completely prevent ethanol-induced gastric damage in the rat gastric mucosa (12). Thirty minutes later, gastric lesions were induced by ig administration of 1 ml of 50% ethanol. Five minutes after ethanol administration, the rats were killed, and the stomachs were removed, opened along the lesser curvature, and examined. The presently described protocol was common to all the treatment groups.
Effect of hexamethonium, atropine, and bretylium
Groups of rats were pretreated with the following drugs: hexamethonium, at 20 mg/kg ip, reported to antagonize the gastroprotective effect of centrally injected clonidine in the rat (21); atropine at 1 mg/kg sc, shown to prevent the gastroprotective effect of intracisternal PYY in the rat (19); bretylium at 25 mg/kg sc, shown to reverse the inhibitory effect of intracranial hypertension on gastric emptying in rats (22) or with saline. Thirty minutes later, either N/OFQ or vehicle was administered. After an additional 30-min period, all rats received ethanol.
Effect of subdiaphragmatic vagotomy
Rats were anesthetized by im injection of tiletamine hydrochloride, 200 mg/kg, and zolazepam hydrochloride, 200 mg/kg. After induction of anesthesia, the abdomen was opened by a midline incision and the connective tissue removed from the surface of the esophagus. Under a dissecting microscope, the dorsal and ventral trunks of the vagus nerve attached to the subdiaphragmatic portion of the esophagus were transected and the cut nerve stumps retracted. After closure of the incision, the rats were given 7 d to recover before the treatments. Control rats were sham operated.
Effect of functional ablation of afferent nerves
Rats were treated with increasing doses of capsaicin in one or two daily sc injections over 4 consecutive days (12.5; 12.5 + 12.5; 25 + 25; 25 mg/kg). Capsaicin was dissolved in 10% ethanol, 10% Tween 80 and 80% saline (by volume). Control animals received the vehicle. To counteract respiratory impairment associated with capsaicin administration, 30 min before the first daily injection of capsaicin, rats received an ip injection of atropine (0.2 mg/kg), terbutaline (0.2 mg/kg), and aminophylline (20 mg/kg). Studies were performed 2 wk after the last capsaicin injection. To evaluate the effectiveness of capsaicin pretreatment, wiping movements in response to intraocular instillation of a 0.1% solution of ammonia were evaluated. Capsaicin-pretreated animals showing any wiping movements were excluded from the study.
Effect of CGRP8–37
The antagonist of CGRP, CGRP8–37, at the dose of 25 μg/kg, was administered sc 30 min before either N/OFQ or vehicle. Ethanol was administered 30 min after N/OFQ injection. The dose and route of administration of CGRP8–37 have been shown to effectively reduce amylin gastroprotective activity in the rat (23).
Effect of inhibition of NO biosynthesis
Groups of rats were pretreated with the nonselective NO synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME), 10 mg/kg sc, 60 min before either N/OFQ or vehicle. To evaluate the specificity of the effects of L-NAME, L-arginine (300 mg/kg sc), as a substrate for NO synthase, was coadministered with L-NAME. The doses of the two compounds were selected from previous studies in this model (24). Ethanol was administered 30 min after N/OFQ injection.
Assessment of gastric mucosal damage
For the macroscopic assessment of mucosal lesions, the stomachs were placed on a flat surface under a stereo microscope. The glandular mucosa was examined, and each individual hemorrhagic lesion was measured along its greatest length (<1 mm, rating of 1; 1–2 mm, rating of 2; >2 mm, rating according to their greatest length). The lengths of the lesions were summed to give an overall total, designated as the lesion index, for each stomach. For the histological assessment of mucosal lesions, a strip was excised from the glandular mucosa, 3–4 mm below and parallel to the limiting ridge, so that the greater curvature was approximately located in the middle of the strip. Three different tissue samples were taken from each strip, fixed in 10% formaldehyde, and embedded. Serial sections, 4 μm thick, in which the gastric pits and glands were oriented perpendicular to the mucosal surface, were cut from each block. The sections were stained either with hematoxylin-eosin or periodic acid-Schiff. For the morphometric analysis of gastric lesions, the image of the section was displayed on a color monitor by means of a video camera attached to the microscope (Nikon Optiphot), and quantitations were performed using a color image analysis software system (LUCIA G, Nikon). The damage was scored in different grades on the basis of its depth: grade 0, all gastric mucosal cells appeared intact; grade I, damage to luminal surface mucous cells only; grade II, damage to luminal surface and gastric pit mucous cells, with presence of cell exfoliation; and grade III, damage extending from luminal surface to gastric gland cells. For each stomach, the total length of mucosa examined and the length of mucosa with each grade of damage were measured. The length values of each grade of damage were then expressed as a percentage of the total length of mucosa examined.
Chemicals
The N/OFQ peptide was synthesized as a salt derivative at the Department of Pharmaceutical Sciences of the University of Ferrara, Italy. The purity grade was more than 95% determined by analytical HPLC and electrospray mass spectrometry analyses. The peptide was dissolved in saline solution (0.9% NaCl). Capsaicin was purchased from Serva (Heidelberg, Germany). All the other reagents were purchased from Sigma Chemical Co. (St. Louis, MO).
Statistical analysis
Data shown are means ± SEM. Statistical analysis was performed using ANOVA followed by Newman-Keuls test, and P values < 0.05 were considered significant.
Results
Protective effect of N/OFQ
Exposure to ethanol for 5 min caused the formation of macroscopically visible hemorrhagic lesions in the glandular portion of the stomach; lesion index was 103.2 ± 10.1 (Table 1). Histological examination of gastric tissues 5 min after ethanol revealed that 64.2% of the glandular mucosa was damaged. The superficial portions of the mucosa were largely affected, with surface mucous cells and pit cells being destroyed and exfoliated. Lesions extending into the gland area occupied 24.2% of the total length measured. The depth of these lesions corresponded to the middle of the gland, that is to the parietal cell area, whereas the chief cell area remained unaltered (Fig. 1 and Table 1).
N/OFQ significantly reduced the mucosal lesions after both central and peripheral administration. The dose of 3 μg icv appears to be equally effective with the dose of 10 μg/kg ip. Macroscopic lesions were reduced by 71.1% (P < 0.01) and by 72.8% (P < 0.01) after icv and ip administration, respectively (Table 1). Similar results were obtained when damage to the mucosa was evaluated histologically. The length of the mucosa damaged in gastric tissues from rats pretreated with icv or ip N/OFQ before ethanol exposure was markedly reduced. Furthermore, when present, lesions were confined to the superficial portions of the mucosa, whereas the integrity of the gland cell area appeared to be nearly completely preserved (Fig. 1 and Table 1). Control groups receiving either saline or N/OFQ in the absence of ethanol exposure had normal appearing gastric mucosa.
Effect of hexamethonium, atropine, and bretylium
Hexamethonium (20 mg/kg ip) alone significantly reduced by 68.9% gastric mucosal damage consequent to the exposure to 50% ethanol. Pretreatment with hexamethonium did not apparently influence the effectiveness of icv or ip N/OFQ in preventing damage by ethanol (Fig. 2). Atropine (1 mg/kg sc) alone was ineffective in modifying ethanol-induced damage, and it did not influence the protective activity of ip N/OFQ (Fig. 2). Conversely, atropine significantly reduced the effect of icv N/OFQ (Fig. 2). Bretylium (25 mg/kg sc) had no significant effect on damage caused by 50% ethanol and on protection exerted by ip N/OFQ. In contrast, it reversed gastroprotective activity of icv N/OFQ (Fig. 2). In the absence of ethanol exposure, hexamethonium, atropine, and bretylium did not modify the macroscopic appearance of the gastric mucosa.
Effect of subdiaphragmatic vagotomy
In sham-operated rats, formation of gastric lesions induced by 50% ethanol and protective activity exerted by icv or ip N/OFQ were not different from that observed in nonoperated rats. Subdiaphragmatic vagotomy itself did not influence 50% ethanol-induced gastric lesions in rats injected either icv or ip with saline, with the lesion index being 88.3 ± 13.2. In contrast, vagotomy completely prevented the protective effect of N/OFQ when given by icv or ip injection (Fig. 3).
Effect of functional ablation of afferent nerves and of CGRP8–37
Pretreatment with capsaicin (112.5 mg/kg sc), 2 wk before study, caused a slight, although not significant, increase in the extent of gastric mucosal damage induced by ethanol, with the lesion index being 114.3 ± 14.2. In capsaicin-pretreated rats, the lesion index after both icv and ip administration of N/OFQ was comparable to that observed in the control group (Fig. 4). Pretreatment with CGRP8–37, which itself did not influence the damaging effect of 50% ethanol, reversed protection resulting from both icv and ip N/OFQ (Fig. 5).
Effect of inhibition of NO biosynthesis
Previous treatment with L-NAME (10 mg/kg sc) did not significantly modify gastric mucosal lesions induced by 50% ethanol. The lesion index remained unaltered when L-NAME was administered concurrently with L-arginine (300 mg/kg sc). In rats pretreated with L-NAME, the protective effect exerted by either icv or ip administered N/OFQ was significantly reduced, reduction of ip N/OFQ activity being more pronounced than that of icv N/OFQ (Fig. 6). In both conditions, the protective activity of N/OFQ was restored by the concurrent pretreatment with L-NAME and L-arginine (Fig. 6). When L-NAME and L-arginine were administered alone, no effect on the macroscopic appearance of the gastric mucosa was observed.
Discussion
The present findings confirm a role of N/OFQ in the maintenance of the integrity of the gastric mucosa in conscious rats and validate the evidence of macroscopic protection of the mucosa with quantitative histology of gastric epithelium in rats pretreated with N/OFQ before ethanol exposure. Histologically, pretreatment with N/OFQ was associated with reduction of the total extension of ethanol-induced damage. Furthermore, after N/OFQ, damage, where present, was confined to the most superficial layer of the mucosa, whereas damage by ethanol alone was distributed throughout the different regions of fundic mucosa. The reduction of the depth of damage is considered of special relevance, because it facilitates a rapid repair and the consequent reestablishment of the epithelial continuity. It is reported to occur under conditions in which mucosal blood flow is maintained or enhanced (25, 26), suggesting that mucosal microcirculation could be primarily affected by N/OFQ. Macroscopic and histological protection afforded by the icv N/OFQ was also observed after ip N/OFQ, indicating that quantitatively similar degrees of protection could be achieved by the two routes of administration.
Pharmacological and surgical approaches were used to determine pathways mediating gastroprotective activity of N/OFQ. Subdiaphragmatic vagotomy abolished the response to central and peripheral N/OFQ, indicating the involvement of vagal pathways. Cholinergic receptor blockade by atropine effectively reduced protection exerted by central but not by peripheral N/OFQ. Vagal postganglionic activation through muscarinic receptors appears to have a role only in central protective activity of N/OFQ. The involvement of autonomic ganglia remains uncertain. The ganglionic blocking agent hexamethonium caused a significant decrease in ethanol-induced lesions and hence made the interpretation of its effect on protection by N/OFQ difficult. Sympathetic nerves appear to mediate central, but not peripheral, activity of N/OFQ, as supported by the fact that the adrenergic blocking agent, bretylium, completely reversed protection only by centrally injected N/OFQ. Vagal cholinergic and sympathetic nerves are likely to mediate the central activity of N/OFQ, whereas vagal nonmuscarinic pathways mediate the peripheral activity of the peptide.
It is well established that intact vagal innervation is necessary to afford protection to the gastric mucosa. Although controversial, there are studies in which vagotomy is reported to cause a tendency to worsen ethanol-induced lesions (27, 28). Protection by peripherally administered prostaglandin E2, prostacyclin, and mild irritants (28, 29) and by centrally administered TRH (18) is reduced in vagotomized rats. Fibers contained in the vagus nerve are both efferent and afferent, with 90% of vagal fibers in the subdiaphragm being afferent fibers (30). In the rat gastric mucosa, extrinsic afferent nerve fibers are both vagal and spinal, and they appear to subserve different roles. Spinal afferents should enhance the resistance of gastric mucosa to injury, whereas vagal afferents, many of them being resistant to capsaicin (31), should convey information from the gastric mucosa to the brain stem (15, 32). Capsaicin-sensitive extrinsic afferent fibers release from their peripheral endings several peptides, including substance P and CGRP. Afferent nerve fibers and CGRP are of great relevance in promoting gastric mucosal defense (15). They appear to act primarily on the regulation of gastric mucosal blood flow, by influencing the arterial and arteriolar system and promoting vasodilation (33). Vasodilation by CGRP involves the formation of NO. NO, produced both by endothelial cells and in the intrinsic neurons, provokes vasodilation and mucus secretion and exerts a protective activity against experimental gastric damage (16, 17).
The present results show that gastric protection by N/OFQ, acting both at central and at peripheral sites, is prevented by capsaicin pretreatment, by administration of the CGRP antagonist, and by the peripheral NO synthase inhibitor L-NAME. It is likely that N/OFQ activates the neuronal circuit involving extrinsic sensory nerves, CGRP, and NO. The central activity of N/OFQ, like that of the neuropeptides TRH, PYY, and adrenomedullin (18, 19, 20), could be mediated in part by the influence on vagal efferent activity, which in turn activates CGRP-containing nerve fibers and NO formation.
In our experiments, response to central N/OFQ was also dependent on sympathetic pathways, because it was completely blocked by bretylium. There is little information regarding the role of the sympathetic nervous system in gastric mucosal defense. Sympathectomy has been reported to worsen damage by ethanol and to abolish adaptive protection in rats (27). N/OFQ is reported to decrease peripheral resistance and to increase blood flow to peripheral organs, including stomach, in the rat (34, 35). Peripheral vasodilation by N/OFQ, contributing to its protective activity, could result from inhibition of sympathetic pathways. In support of this hypothesis is the finding that hypotension caused in the rat by iv nociceptin, in the dose range 20–200 μg/kg, was reduced by the adrenergic neuron blocker guanethidine and completely abolished by guanethidine plus vagotomy, indicating a concomitant inhibitory influence on sympathetic outflow and activation of vagal outflow (36). However, in the present study, we did not observe any influence of the adrenergic neuron blocker bretylium on peripheral activity of N/OFQ, possibly because of the lower dose range used in the present study as compared with that used by Giuliani et al. (36) or alternatively of a minor role of sympathetic nerve fibers in mediating the peripheral activity of the peptide. It is worth noting that the neuropeptide corticotropin-releasing factor (CRF) when centrally administered protects the gastric mucosa, stimulates gastric mucosal blood flow, and influences gastrointestinal motor and secretory functions in rats, and its effects have been ascribed to the modulation of both vagal efferent and sympathetic pathways (37, 38, 39, 40). Central CRF is hypothesized to alter the balance of the autonomic nervous system, particularly shifting the system toward the sympathetic component. Such studies demonstrate that gastrointestinal functions could be modulated at the central level by the simultaneous influence on parasympathetic and sympathetic outflow. Both pathways could similarly be implicated in the protective activity of central N/OFQ. Conversely, peripheral N/OFQ appears to affect almost exclusively vagal noncholinergic and afferent capsaicin-sensitive fibers. The difference in the neuronal pathways mediating central and peripheral effects of the peptide strengthens the hypothesis of the existence of distinct sites of action.
There is a general consensus that the effects of N/OFQ on gastrointestinal functions are mediated neurally. The possible mechanisms at the basis of the effects of N/OFQ in vivo have been partly explored. The effect of centrally administered N/OFQ on gastric acid secretion in rats was completely inhibited by atropine and vagotomy, suggesting the involvement of the vagus cholinergic nerve (10). Mechanisms of motor effects have been evaluated only after peripheral administration of N/OFQ. An increase in antral and colonic contractility in rats was unaffected by L-NAME, vagotomy, and splanchnicotomy, leading to suppose that the peptide acts at the level of the intramural myenteric plexus (8, 9). N/OFQ could be considered a neuropeptide operating both in the central nervous system and at the gastrointestinal level.
In addition to N/OFQ, a number of peptides, such as ghrelin, cholecystokinin, PYY, and CRF, are implicated both in the modulation of feeding (41, 42, 43) and in the regulation of gastrointestinal functions (43, 44, 45). Similarly to N/OFQ, these peptides are present centrally, in brain areas involved in feeding behavior pathways, and peripherally, in neurons and/or endocrine cells in the gut. Importantly, CRF, after acute central injection, is well known to produce a decrease of feeding behavior in rats (43), and its effect can be reversed by central N/OFQ injection, through a functional antagonism (46). So, CRF and N/OFQ display a comparable protective effect on gastric mucosa, although they differ in relation to the effects on feeding behavior, suggesting that feeding controlling pathway systems differ from those involved in the maintenance of mucosal epithelial integrity.
The present results strongly suggest that N/OFQ could be a component of a multifactorial system regulating energy/homeostasis, gastrointestinal motility and secretion, and neurally mediated gastric mucosal protection.
Footnotes
This work was supported by a grant (COFIN 2002) from the Italian Ministry of Education, University and Research, Rome, Italy.
Abbreviations: CGRP, Calcitonin gene-related peptide; CRF, corticotropin-releasing factor; ig, intragastric; icv, intracerebroventricular;L-NAME, NG-nitro-L-arginine methyl ester; NO, nitric oxide; N/OFQ, nociceptin/orphanin FQ; PYY, peptide YY.
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