- Email: [email protected]
Microinjection of bombesin into the ventrolateral reticular formation inhibits peripherally stimulated gastric acid secretion through spinal pathways in rats
Brain Research 918 (2001) 1–9 www.elsevier.com / locate / bres
Research report
Microinjection of bombesin into the ventrolateral reticular formation inhibits peripherally stimulated gastric acid secretion through spinal pathways in rats Toshio Ishikawa 1 , Hong Yang, Yvette Tache´ * CURE: Digestive Diseases Research Center, Veterans Administration Greater Los Angeles Healthcare System, Department of Medicine, Digestive Diseases Division and Brain Research Institute, University of California at Los Angeles, Los Angeles, CA 90073, USA Accepted 23 July 2001
Abstract Bombesin injected into the cisterna magna potently inhibits gastric acid secretion stimulated by intravenous infusion of pentagastrin. Sites in the medulla oblongata where bombesin acts to suppress gastric acid secretion were investigated in urethane-anesthetized rats with gastric cannula. Bombesin or vehicle was injected into the medullary parenchyma or intracisternally (i.c.) 60 min after the start of an intravenous pentagastrin infusion; gastric acid secretion was monitored every 10 min for 20 min before and 150 min after the start of pentagastrin. Bombesin (0.2, 0.6 or 6.2 pmol) microinjected into the ventrolateral reticular formation (VLRF) inhibited dose-dependently the net acid response to pentagastrin by 40.8611.1, 75.4612.8 and 96.7619.4%, respectively, at the 40–50 min period after microinjection compared with the vehicle group. Bombesin action in the VLRF was long lasting (96% inhibition still observed at 90 min after 6.2 pmol), and completely abolished by cervical spinal cord transection at the C6 level. By contrast, bombesin injected i.c. at 0.2 or 0.6 pmol had no effect while at 6.2 pmol, there was a 79.063.9% peak inhibition of pentagastrin-stimulated acid secretion. Bombesin (6.2 pmol) injected into the dorsal motor nucleus reduced the acid response to pentagastrin by 29%. The parvicellular and gigantocellular reticular nuclei were not responsive to bombesin. These results indicate that bombesin acts in the VLRF to inhibit pentagastrin-stimulated gastric acid secretion through spinal pathways, suggesting a potential role of medullary VLRF area in the sympathetic control of gastric acid secretion. 2001 Elsevier Science B.V. All rights reserved. Theme: Endocrine and autonomic regulation Topic: Gastrointestinal and urogenital regulation Keywords: Bombesin; CRF; Gastric secretion; Spinal cord; Brainstem; Sympathetic
1. Introduction Compelling evidence indicates that bombesin and bombesin-like peptides are potent inhibitors of gastric acid secretion when injected into the cerebrospinal fluid of rats, cats, and dogs [21]. The mapping of hypothalamic sites responsive to bombesin in rats has revealed that vagallystimulated gastric acid secretion was inhibited by the peptide microinjected into the paraventricular nucleus of *Corresponding author. Tel.: 11-310-312-9275; fax: 11-310-2684963. ´ E-mail address: [email protected] (Y. Tache). 1 Current address: Division of Psychosomatic Research, National Institute of Mental Health, 1-7-3 Konodai, Ichikawa, Chiba 272, Japan.
the hypothalamus [13,25], anterior hypothalamus or preoptic area [25] while not modified after microinjection of bombesin into the lateral, dorsomedial or ventromedial hypothalamus and the caudate putamen [13,25]. However, intracisternal (i.c.) bombesin-induced inhibition of vagallyand pentagastrin-stimulated gastric secretion was not altered by electrolytic lesioning of the paraventricular nucleus of the hypothalamus or preoptic-anterior area or by mid-collicular transection [14,25], suggesting the existence of additional hindbrain sites for peptide action. Further studies established that bombesin microinjected into the dorsal motor nucleus of the vagus (DMN) dose-dependently abolished central vagal stimulation of gastric acid secretion induced by co-microinjection of thyrotropin-releasing hormone (TRH) stable analog, RX-77368 [15], a
0006-8993 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 01 )02833-5
T. Ishikawa et al. / Brain Research 918 (2001) 1 – 9
2
peptide established to activate directly preganglionic vagal motor neurons leading to increased gastric vagal efferent discharge [23,37]. By contrast, bombesin suppressed the acid response to an intravenous infusion of pentagastrin less potently when injected into the DMN than intracisternally [15]. These findings indicate that additional sites located in the medulla oblongata are involved in bombesininduced inhibition of gastric acid secretion stimulated by a peripheral secretagogue. Intracerebroventricular (i.c.v.) injection of bombesin elevates plasma levels of catecholamines [6,24] and increases nerve activity in both the sympathetic and adrenal branches of the splanchnic nerve [27]. Intracisternal bombesin-induced inhibition of gastric acid secretion in pylorus-ligated rats is partly prevented by cervical cord transection [36]. Likewise, bombesin injected i.c.-induced inhibition of the acid secretory response to electrical vagal stimulation was reduced by bilateral cutting of the greater splanchnic nerve and abolished by combined chemical sympathectomy and adrenalectomy [26]. Neuroanatomical studies showed that bombesin immunoreactive neurons and receptors are distributed not only in the dorsal vagal complex [8,19,50] but also in the ventrolateral medulla [19,38] where neurons selectively innervate the sympathetic preganglionic neurons in the intermedio-medial and -lateral column of the thoracic spinal cord [10,31,49]. Taken together, these functional and morphological data support a potential action of bombesin in the ventrolateral medulla. In the present study, we investigated the inhibitory effect of bombesin microinjected into the ventrolateral reticular formation (VLRF) on gastric acid secretion stimulated by intravenous infusion of pentagastrin, a well established peripherally acting secretagogue [39] in urethane-anesthetized rats. The site specificity of bombesin action in the VLRF and the mediation through cervical spinal cord pathways were also assessed.
2. Materials and methods
2.1. Animals Male, Sprague Dawley albino rats (Charles Rivers Laboratories) weighing 220–300 g were maintained ad libitum on Purina Laboratory chow and tap water. They were housed under conditions of controlled temperature (20618C) and illumination (06:00 to 18:00 h), and deprived of food, but not water, for 24 h before each experiment. All experiments were conducted under protocols approved by the Veterans Administration Animal Committee and performed in fasted rats anesthetized with urethane (1.5 g / kg, i.p.) throughout the duration of the study.
2.2. Peptides The following peptides were used: bombesin and rat
corticotropin-releasing factor (CRF) (kindly supplied by Dr. J. Rivier, Clayton Foundation Laboratories for Peptide Biology Laboratories, Salk Institute, La Jolla, CA, USA), and pentagastrin solution (Peptavlon, Ayerst Laboratories, New York, NY, USA). Bombesin and CRF in powder form were dissolved in 0.1% bovine serum albumin / saline immediately before use. Pentagastrin was diluted in saline before intravenous infusion.
2.3. Measurement of gastric secretion In urethane anesthetized rats, the femoral vein was catheterized and the esophagus ligated at the cervical level. After performing a laparotomy, the pylorus was ligated and a double lumen cannula was placed through a small incision into the forestomach. Gastric acid secretion was measured by flushing the gastric lumen twice with 5-ml boluses of saline and one 5-ml bolus of air at the end of each 10 min period. Acid output was determined by titration (autotitrator, Radiometer, Copenhagen, Denmark) of the flushed perfusate with 0.01 M NaOH to pH 7.0.
2.4. Medullary injections Urethane anesthetized rats equipped with gastric cannula and intravenous femoral catheter were positioned on a stereotaxic instrument (Kopf Intruments, Model 900). The rat head was fixed at a nose-down position with ear bars and the incisor bar set at 215 mm. For microinjection into the brainstem area, the obex region of the dorsal medulla was exposed. The dorsal cervical musculature was resected and the occipital skull plate was removed. About 23232 mm 3 of ventral–caudal cerebellum tissue covering the caudal half of the forth ventricle was carefully removed by sucking with negative pressure generated from a water pump as in our previous study [15]. General procedure for microinjections were performed as previously reported [15]. A glass micropipette (50–70 mm in diameter) was positioned unilaterally (right side) into the VLRF according to the following coordinates: ventral from the surface of the brainstem (V): 2.2–2.7 mm, anterior from the caudal tip of the area postrema (A): 1.5–2.0 mm, and lateral from the midline (L): 1.5–2.0 mm, or into the right DMN (V: 0.6, A: 0.3, L: 0.5). In one experiment, microinjection was performed into the left side of the VLRF to assess the bilaterality of the observations. Microinjections were carried out using 100 nl volume delivered by pressure ejection over 1 min with a 1-ml Hamilton syringe, the micropipette was left in place for an additional 3 min, then withdrawn. At the end of the experiment, rats were sacrificed by decapitation and brains were removed and fixed in 10% formalin–20% sucrose solution for at least 2 days. Frozen brainstem sections were sliced at 30 mm, and stained with toluidine blue. The locations of microinjection sites were identified by the visualization of the point of termination of the micropipette track on histological sections examined
T. Ishikawa et al. / Brain Research 918 (2001) 1 – 9
microscopically. Sites were marked on plates reproduced from Paxinos and Watson’s atlas [29]. For intracisternal injection, the rat head was fixed with ear bars of the stereotaxic equipment as previously described [36]. The occipital membrane was punctured by the needle of a 50-ml Hamilton syringe for injection in a 10 ml volume. Correctness of the needle placement into the cisterna magna was insured by the presence of cerebrospinal fluid into the Hamilton syringe upon aspiration before the injection.
2.5. Experimental protocols Following gastric and brain surgeries, basal gastric secretion was measured for 20 min and pentagastrin (16 mg / kg / h) was infused into the femoral vein for the duration of the experiment. One hour after the start of pentagastrin infusion, injection of peptides or vehicle was performed in one of following routes: bombesin (0.2, 0.6 or 6.2 pmol / rat), CRF (62 pmol / rat) or vehicle (100 nl) into the VLRF; bombesin (6.2 pmol / rat) or vehicle (100 nl) into the DMN; bombesin (0.2, 0.6 or 6.2 pmol / rat) or vehicle (10 ml) intracisternally. The stomach was flushed immediately after the injection and this collection was disregarded. The gastric acid secretion was monitored for 90 min after brain injection of vehicle or peptide under conditions of constant pentagastrin intravenous infusion. In one experiment, cervical cord transection at the C6 level or sham operation (exposure of the cervical cord) was performed as previously described [36]. Then, a similar protocol was followed as specified in the other experiments including the placement of the gastric cannula and femoral catheter followed by brain surgery. After 20 min basal acid secretion, pentagastrin (16 mg / kg / h) was infused throughout the experimental period. One hour after the start of pentagastrin intravenous infusion, bombesin (6.2 pmol / rat) was microinjected into the VLRF in sham-operated and C6-transected rats. Gastric acid secretion was monitored for the 90 min period post-injection.
2.6. Statistics Results are expressed as mean6S.E. Due to the individual variability in the magnitude of the plateau gastric secretory response to pentagastrin, which is reached within 1 h of pentagastrin infusion, in some experiments the acid output in the collection at 60 min after the start of pentagastrin infusion was taken as 100% for each animal; results are expressed as percentage change from this value for each other 10 min collection period. The net pentagastrin response was calculated by subtracting the mean basal values of gastric output for the 20 min before pentagastrin infusion, from each of the 10 min postinfusion value. Comparisons between two groups were performed by Student’s t-test and multiple group comparisons by analysis of variance (ANOVA) followed by
3
Duncan’s contrast. A P value less than 0.05 was considered statistically significant.
3. Results In urethane-anesthetized rats with gastric fistula, basal gastric acid secretion was low (2.260.2 mmol / 10 min, n565). Pentagastrin infused intravenously increased gastric acid secretion to 12.460.9 mmol / 10 min (n565) at the 60 min collection period after the start of peptide infusion. Gastric acid output values (mmol / 30 min) for the 30 min pre-injection period were not significantly different from those collected in the 30 min period starting at 20 min after the unilateral microinjection of vehicle into either the right VLRF (pre-injection: 26.063.7, post-injection: 30.265.2, n58) or DMN (pre-injection: 24.964.0, post-injection: 27.964.9, n510). Bombesin microinjected into the right VLRF at 0.2, 0.6 or 6.2 pmol induced a dose-related and sustained inhibition of the gastric acid secretion stimulated by intravenous pentagastrin (Fig. 1). The significant decrease in pentagastrin-stimulated acid secretion occurred within 20 min after bombesin microinjection into the VLRF. The maximal antisecretory effect induced by bombesin microinjected into the VLRF at 0.2, 0.6 and 6.2 pmol was reached at the 40–50 min post injection period and resulted in 40.8611.1, 75.4612.8 and 96.7619.4% inhibition of the acid response to intravenous pentagastrin, respectively. Thereafter, the gastric acid secretion either returned to levels of the vehicle group at 80 min after bombesin (0.2 pmol), or was maintained significantly inhibited throughout the 40 min remaining observation period (0.6 pmol and 6.2 pmol) (Fig. 1). In a few rats, bombesin microinjected into the left side of the VLRF also resulted in inhibition of acid secretion stimulated by intravenous pentagastrin (data not shown). CRF (62 pmol) microinjected into the right VLRF did not modify the acid secretory response to intravenous pentagastrin (92.867.3% of the pre-injection level, n59) compared with vehicle microinjection (107.565.7%, n510) as monitored during the 20–50 min period after the microinjection. When microinjected into the VLRF, the bombesin inhibitory effect was more potent than that when it was injected into the cisterna magna or DMN. Microinjection of bombesin into the VLRF at 0.2 or 0.6 pmol resulted in a dose-related and significant reduction of gastric acid secretion stimulated by intravenous infusion of pentagastrin while there was no significant decrease when bombesin was injected intracisternally at the same doses (Fig. 2). At 6.2 pmol, bombesin injected into the VLRF or cisterna magna, similarly inhibited the net acid response to intravenous infusion of pentagastrin by 96.7 and 79.1%, respectively, while bombesin microinjected into the DMN resulted in a significant 29% inhibition which was weaker than that induced by i.c. or VLRF injection (Fig. 2). The placement of microinjection sites in medullary coronal sections is illustrated in Fig. 3. Microscopic
4
T. Ishikawa et al. / Brain Research 918 (2001) 1 – 9
Fig. 1. Time course of the dose-related inhibitory effect of bombesin microinjected into the ventrolateral reticular formation (VLRF) on pentagastrinstimulated gastric acid secretion in urethane-anesthetized rats. Gastric acid output at the 60-min collection period after pentagastrin infusion was taken as 100% for each animal and gastric acid output was expressed as a percentage of change from this value for each collection period. Each point represents mean6S.E. of number of rats indicated in the parentheses. *P,0.05 compared with vehicle-microinjected group.
examination showed that responsive sites to bombesin located in the right VLRF encompassed the area surrounding the nucleus ambiguus and expended to the reticular rostroventralateralis and dorsal boundaries of the nucleus paragigantocellularis lateralis / lateral reticular nucleus (Fig.
3). Ineffective sites were located in the parvicellular reticular nucleus (PCRt, n59), gigantocellular reticular nucleus (Gi, n55), parvicellular lateral reticular nucleus (LRtPC, n52), linear nucleus of the medulla (Li, n51) and rubrospinal tract (rs, n51) (Fig. 3).
Fig. 2. Effects of injection of bombesin intracisternally, into the ventrolateral reticular formation (VLRF), or the dorsal motor nucleus of the vagus (DMN) on pentagastrin-stimulated gastric acid secretion in urethane-anesthetized rats. Bombesin at various doses or vehicle was injected at 60 min after the start of pentagastrin infusion. The gastric output is the mean value of the 40–50 min collection period following bombesin or vehicle injection. Each column represents mean6S.E. of number of rats indicated in the bottom of each column. *P,0.05 compared with respective vehicle (0) groups; [P,0.05 compared with same dose of bombesin injected intracisternally.
T. Ishikawa et al. / Brain Research 918 (2001) 1 – 9
5
time-related decrease was observed (Fig. 4). Cervical cord transection did not influence the acid response to pentagastrin infusion as shown by the similar values of gastric acid output at 60 min after the start of pentagastrin infusion in both sham-operated and C6 cervical cordtransected rats (Table 1).
4. Discussion
Fig. 3. Coronal brainstem sections adapted from the atlas of Paxinos and Watson [29] showing the microinjection sites of bombesin in the ventral medulla in urethane-anesthetized rats. d, Effective sites; m, ineffective sites. Abbreviations: Amb, nucleus ambiguus; C1 /A1, C1 adrenaline cells and A1 noradrenaline cells; CVL, caudoventrolateral reticular nucleus; Gi, gigantocellular reticular nucleus; IRt, intermediate reticular nucleus; li, linear nucleus of medulla; LPGi, lateral paragigantocellular nucleus; LRtPC, lateral reticular nucleus, parvicellular; LRt, lateral reticular nucleus; MdV, medullary reticular nucleus, ventral part; PCRt, parvicellular reticular nucleus; rs, rubrospinal tract; RVL, rostroventrolateral reticular nucleus; RVRG, rostral ventral respiratory group.
In cervical cord (C6 level) transected rats, bombesin (6.2 pmol) microinjection into the VLRF no longer inhibited gastric acid secretion induced by intravenous infusion of pentagastrin while in sham-operated rats, a
The present study showed that bombesin microinjected into the VLRF at low doses inhibits gastric acid secretion stimulated by intravenous infusion of pentagastrin in urethane-anesthetized rats. Bombesin action in the VLRF was dose-related resulting in a 41–97% net peak inhibition at 0.2–6.2 pmol and long lasting (96% inhibition at 90 min post-injection at 6.2 pmol). By contrast, CRF microinjected into the VLRF at 62 pmol did not modify the acid response to intravenous pentagastrin. CRF injected i.c. or into the lateral hypothalamus in doses ranging from 0.2 to 2.1 nmol was previously reported to inhibit gastric acid secretion stimulated by intravenous infusion of pentagastrin in urethane-anesthetized rats [12,35]. Whether the absence of biological action of CRF microinjected into the VLRF reflects the use of a subthreshold dose or the peptide injection into a non responsive site will need to be further assessed. These results, however, indicate that the unrelated peptide CRF microinjected into the VLRF did not mimic the antisecretory action of bombesin when delivered at a dose that was 10-fold the maximal effective pmolar dose of bombesin, supporting the peptide specificity of the response. These data are also consistent with the notion that bombesin is so far the most potent peptide to inhibit gastric acid secretion when injected into the rat brain [21]. Histological mapping of microinjection placements shows that the responsive sites to bombesin are located in an area centered around the right nucleus ambiguus, the rostroventrolateral reticular nucleus, and dorsal boundary of the lateral paragigantocellular nucleus / lateral reticular nucleus. The inhibition of intravenous pentagastrin-stimulated gastric secretion was also observed after bombesin microinjection into the left side of the VLRF in the few cases tested. The spreading of responsive sites in the VLRF may reflect internal connections among these nuclei in the reticular formation, which have been established by tracing and functional studies [7,11,48]. However, several sites in specific reticular nuclei namely the parvicellular and gigantocellular nuclei were found to be unresponsive to bombesin. This shows the site specificity of bombesin action. In addition, the net acid response to intravenous pentagastrin infusion was more potently inhibited by bombesin microinjected at 6.2 pmol into the VLRF (97% inhibition) than into the DMN (29% inhibition). Lastly, bombesin injected i.c. at 0.2 or 0.6 pmol did not reduce gastric acid secretion stimulated by intravenous pentagastrin while the same doses into the VLRF resulted in a
T. Ishikawa et al. / Brain Research 918 (2001) 1 – 9
6
Fig. 4. Cervical spinal cord transection completely prevented the inhibitory action of bombesin microinjected into the VLRF on pentagastrin-stimulated gastric acid secretion in urethane-anesthetized rats. Gastric acid output at the 60-min collection period after pentagastrin infusion was taken as 100% for each animal and gastric acid output was expressed as a percentage of change from this value for each collection period. Each point represents mean6S.E. of number of rats indicated in the parentheses. [P,0.05 compared with sham-operated group.
significant 41% and 75% net inhibition, respectively. Taken together, these observations reveal that the VLRF is a potent new site of action for bombesin to inhibit peripherally stimulated gastric acid secretion. The observations that bombesin injected i.c. at 6.2 pmol inhibited the acid response to pentagastrin is in agreement with previous reports showing that i.c. injection of bombesin in doses ranging from 6.2 to 300 pmol inhibited pentagastrin-stimulated acid secretion in urethane-anesthetized rats [3,14,15,36]. The equipotency of i.c. bombesin at 6.2 pmol compared with VLRF microinjection may result from combined sites of action at various sites including the VLRF and DMN when the peptide was i.c. injected into cerebrospinal fluid. Studies related to cerebrospinal fluid flow and penetration into the brain parenchyma following
Table 1 Effect of cervical spinal cord transection on gastric acid response to pentagastrin Treatment a
Control Spinal cord transection
n
10 6
Gastric acid output b (mmol / 10 min) Basal
Pentagastrin-stimulated
2.560.3 2.060.3
13.260.8* 14.863.2*
*P,0.05 compared to respective basal values. a In rats under urethane anesthesia, spinal cord transection at the C6 level or sham operation was performed before implanting the gastric cannula and femoral vein catheter. b Gastric acid output (mean6S.E.) was measured before (basal) and 1 h after the start of pentagastrin infusion (16 mg / kg / h).
i.c. injection of [ 14 C]inulin revealed that the inert tracer within 5 min labeled the subarachnoid space outer brain surface of the basal forebrain, ventral and anterior hypothalamus, midbrain, medulla as well as cervical spinal cord [30]. Bombesin conveys its antisecretory action from the VLRF to the stomach through spinal pathways. This is supported by the demonstration that spinal cord transection at the C6 level to eliminate the descending spinal input, completely prevented the inhibition of pentagastrin-stimulated gastric acid secretion induced by bombesin microinjected into the VLRF at the maximal effective dose. The blockade of bombesin action occurred under conditions in which the gastric acid responses to pentagastrin alone was not influenced by acute cervical cord transection in urethane-anesthetized rats as previously observed [41]. Likewise, surgical sympathectomy by extirpation of the coeliac and superior mesenteric ganglia did not influence the stimulation of acid secretion induced by pentagastrin in rats [2,41]. These results indicate that gastric acid secretion elicited by pentagastrin is not modulated by central spinal input in urethane anesthetized rats. Converging evidence indicates that bombesin action may be primarily mediated through descending spinal pathways controlling sympathetic outflow to the gut. Existing evidence does not support a major role of the parasympathetic nervous system in bombesin inhibitory action. Direct microinjection of bombesin into the DMN, the main medullary nucleus influencing preganglionic vagal motor neurons projecting to the stomach [4,33],
T. Ishikawa et al. / Brain Research 918 (2001) 1 – 9
resulted in a weak inhibition of pentagastrin-stimulated acid secretion compared with that induced by microinjection into the VLRF or the cisterna magna ([15], present study). In addition, i.c. injection of bombesin at 6.2 pmol did not significantly influence gastric vagal efferent discharge in urethane-anesthetized rats [47] while inhibiting the acid response to pentagastrin ([15], present study). By contrast, bombesin injected into the cerebrospinal fluid increased nerve activity of both sympathetic and adrenal branches of the splanchnic nerves in urethane anesthetized rats and elevated circulating levels of catecholamines at low doses in conscious rats [5,6,24,27]. Functional studies showed that electrical stimulation of the splanchnic nerve inhibits pentagastrin- or vagally-stimulated acid secretion in urethane-anesthetized rats [45,46]. In addition, i.c.v. injection of bombesin-induced inhibition of vagally stimulated gastric secretion was completely reversed by combined bilateral cutting of the greater splanchnic nerve and chemical sympathectomy in urethane-anesthetized rats [26]. Lastly, the locations of bombesin effective sites in the present study are consistent with established anatomical basis for sympathetic influence on gastric function. Retrograde labeling following pseudorabies virus injected into the stomach was found in the intermediolateral column of the spinal cord at mid- to low-thoracic levels and preautonomic neurons in the caudal and rostral ventrolateral medulla [32]. Other studies have identified direct spinal projections from the VLRF, including the ventrolateral medulla and lateral paragigantocellular reticular nucleus, to the intermediolateral columns at the T5–T12 levels of the spinal cord [10,31,49] where preganglionic neurons innervating the adrenal gland and stomach are located [18,22,32]. However, the spreading of bombesin responsive sites, particularly those located around and within the nucleus ambiguus indicate that in addition to the bombesin action at VLRF sites with direct descending influence on spinal sympathetic activity, bombesin may most likely act through interareticular pathways providing anatomic connections between nuclei of the ventromedial medulla [7,11,48]. Bombesin actions are mediated by binding to receptors of which three pharmacologically distinct members have been identified by molecular cloning in mammals: the neuromedin B receptor, gastrin releasing peptide receptor (GRP-R), and bombesin receptor subtype-3 [20,34]. In addition, a new bombesin receptor subtype-4 has recently been cloned in amphibian [16]. The pharmacologic characteristics of these bombesin receptor subtypes in transfected cells [16,20] and the rank order of potency for bombesin ligands to inhibit gastric acid secretion when injected into the cerebrospinal fluid [21], suggest that the antisecretory action of centrally mediated bombesin involves GRP-R and / or the mammalian counterpart of amphibian bombesin-4 [21]. The involvement of GRP-R is further supported by morphological studies showing high density of GRP binding and expression of GRP-R gene in the
7
VLRF [17,38]. However, the exact receptor(s) mediating the inhibitory action of bombesin in the VLRF is(are) to be identified. The VLRF is well established to play an important role in the regulation of cardiovascular and respiratory functions [1,9]. The present data unraveled the VLRF as a potential medullary site regulating other visceral functions such as gastric function through spinal cord pathways. The presence of GRP receptors [17,38] and bombesin-like immunoreactive fibers / terminals [19] at VLRF sites where microinjection of bombesin at low doses inhibits gastric acid secretion suggests a possible role of endogenous bombesin-like peptides in the regulation of gastric acid secretion at these sites. In the medulla oblongata, previous studies have localized the dorsal vagal complex, parapyramidal region, raphe pallidus and raphe obscurus as nuclei regulating gastric acid secretion through vagal pathways in rats [40,42–44]. Electrical stimulation of the nucleus ambiguus complex did not influence basal gastric acid and pepsin secretion while stimulating motility through vagal pathways in chloralose anesthetized cats [28]. Bombesin action in the VLRF to inhibit pentagastrinstimulated acid secretion through spinal cord pathways may be indicative of VLRF participation in the central sympathetic regulation of gastric function.
Acknowledgements This work was supported by the National Institute of Health, grants DK-30110, DK 33061 and Center Grant DK-41301 (Animal core). The authors thank Dr. J. Rivier (Peptide Biology Laboratory, Salk Institute, La Jolla, CA, USA) for the generous donation of bombesin and rat CRF.
References [1] S.A. Aicher, T.A. Milner, V.M. Pickel, D.J. Reis, Anatomical substrates for baroreflex sympathoinhibition in the rat, Brain Res. Bull. 51 (2000) 107–110. [2] P. Alm, G. Liedberg, C. Owman, Gastric and pancreatic sympathetic denervation in the rat. Technique and results, Scand. J. Gastroenterol. 6 (1971) 307–312. [3] B. Beltran, M.D. Barrachina, A. Mendez, E. Quintero, J.V. Esplugues, Synthesis of nitric oxide in the dorsal motor nucleus of the vagus mediates the inhibition of gastric acid secretion by central bombesin, Br. J. Pharmacol. 127 (1999) 1603–1610. [4] H.R. Berthoud, E.A. Fox, T.L. Powley, Abdominal pathways and central origin of rat vagal fibers that stimulate gastric acid, Gastroenterology 100 (1991) 627–637. [5] M.R. Brown, L.A. Fisher, Brain peptide regulation of adrenal epinephrine secretion, Am. J. Physiol. 247 (1984) E41–E46. [6] K. Carver-Moore, T.S. Gray, M.R. Brown, Central nervous system site of action of bombesin to elevate plasma concentrations of catecholamines, Brain Res. 541 (1991) 225–231. [7] S.H. Chan, J.Y. Chan, B.T. Ong, Anatomic connections between
T. Ishikawa et al. / Brain Research 918 (2001) 1 – 9
8
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21] [22]
[23]
[24]
[25]
[26]
nucleus reticularis rostroventrolateralis and some medullary cardiovascular sites in the rat, Neurosci. Lett. 71 (1986) 277–282. J.F. Costello, M.R. Brown, T.S. Gray, Bombesin immunoreactive neurons in the hypothalamic paraventricular nucleus innervate the dorsal vagal complex in the rat, Brain Res. 542 (1991) 77–82. A.K. Curran, G. Chen, R.A. Darnall, J.J. Filiano, A. Li, E.E. Nattie, Lesion or muscimol in the rostral ventral medulla reduces ventilatory output and the CO(2) response in decerebrate piglets, Respir. Physiol. 123 (2000) 23–37. A.K. Goodchild, I.J. Llewellyn-Smith, Q.J. Sun, J. Chalmers, A.M. Cunningham, P.M. Pilowsky, Calbindin-immunoreactive neurons in the reticular formation of the rat brainstem: catecholamine content and spinal projections, J. Comp. Neurol. 424 (2000) 547–562. A.R. Granata, D.A. Ruggiero, Evidence of disynaptic projections from the rostral ventrolateral medulla to the thoracic spinal cord, Brain Res. 781 (1998) 329–334. ´ M. Yoneda, H. Monnikes, ¨ ´ Central M. Gue, J.-L. Junien, Y. Tache, neuropeptide Y and sigma ligand, JO 1784, reverse corticotropinreleasing factor-induced inhibition of gastric acid secretion in rats, Br. J. Pharmacol. 107 (1992) 642–647. ´ Bombesin microinfusion into the paravenM.W. Gunion, Y. Tache, tricular nucleus suppresses gastric acid secretion in the rat, Brain Res. 411 (1987) 156–161. ´ Fore- and hindbrain mediation of gastric M.W. Gunion, Y. Tache, hypoacidity after intracerebral bombesin, Am. J. Physiol. 15 (1987) G675–G684. ´ Bombesin microinjected into the dorsal vagal T. Ishikawa, Y. Tache, complex inhibits vagally stimulated gastric acid secretion in the rat, Regul. Pept. 24 (1989) 187–194. T. Katsuno, T.K. Pradhan, R.R. Ryan, S.A. Mantey, W. Hou, P.J. Donohue, M.A. Akeson, E.R. Spindel, J.F. Battey, D.H. Coy, R.T. Jensen, Pharmacology and cell biology of the bombesin receptor subtype 4 (BB4-R), Biochemistry 38 (1999) 7307–7320. E.E. Ladenheim, R.T. Jensen, S.A. Mantey, T.H. Moran, Distinct distributions of two bombesin receptor subtypes in the rat central nervous system, Brain Res. 593 (1992) 168–178. S. Leman, O. Viltart, H. Sequeira, Expression of Fos protein in adrenal preganglionic neurons following chemical stimulation of the rostral ventrolateral medulla of the rat, Brain Res. 854 (2000) 189–196. R.B. Lynn, T.M. Hyde, R.R. Cooperman, R.R. Miselis, Distribution of bombesin-like immunoreactivity in the nucleus of the solitary tract and dorsal motor nucleus of the rat and human: colocalization with tyrosine hydroxylase, J. Comp. Neurol. 369 (1996) 552–570. S.A. Mantey, H.C. Weber, E. Sainz, M. Akeson, R.R. Ryan, T.K. Pradhan, R.P. Searles, E.R. Spindel, J.F. Battey, D.H. Coy, R.T. Jensen, Discovery of a high affinity radioligand for the human orphan receptor, bombesin receptor subtype 3, which demonstrates that it has a unique pharmacology compared with other mammalian bombesin receptors, J. Biol. Chem. 272 (1997) 26062–26071. ´ Bombesin and the brain–gut axis, Peptides 21 V. Martinez, Y. Tache, (2000) 1617–1625. S.F. Morrison, W.H. Cao, Different adrenal sympathetic preganglionic neurons regulate epinephrine and norepinephrine secretion, Am. J. Physiol. 279 (2000) R1763–R1775. ´ Intracisternal TRH and RX 77368 T.J. O-Lee, J.Y. Wei, Y. Tache, potently activate vagal efferent discharge in rats, Peptides 18 (1997) 213–219. Y. Okuma, K. Yokotani, Y. Murakami, Y. Osumi, Brain histamine mediates the bombesin-induced central activation of sympatho– adrenomedullary outflow, Life Sci. 61 (1997) 2521–2528. Y. Okuma, K. Yokotani, Y. Osumi, Central site of inhibitory action of bombesin on gastric acid secretion, Jpn. J. Pharmacol. 45 (1987) 129–133. Y. Okuma, K. Yokotani, Y. Osumi, Sympatho–adrenomedullary
[27]
[28]
[29] [30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
[39] [40]
[41]
[42]
[43]
[44]
[45]
[46]
[47]
system mediation of the bombesin-induced central inhibition of gastric acid secretion, Eur. J. Pharmacol. 139 (1987) 73–78. Y. Okuma, K. Yokotani, Y. Osumi, Centrally applied bombesin increases nerve activity of both sympathetic and adrenal branch of the splanchnic nerves, Jpn. J. Pharmacol. 68 (1995) 227–230. F.D. Pagani, W.P. Norman, D.K. Kasbekar, R.A. Gillis, Effects of stimulation of nucleus ambiguus complex on gastroduodenal function, Am. J. Physiol. 246 (1984) G253–G262. G. Paxinos, C. Watson, in: The Rat Brain in Stereotaxic Coordinates, Academic Press, Orlando, FL, 1998, pp. 1–116. M.G. Proescholdt, B. Hutto, L.S. Brady, M. Herkenham, Studies of cerebrospinal fluid flow and penetration into brain following lateral ventricle and cisterna magna injections of the tracer [ 14 C]inulin in rat, Neuroscience 95 (2000) 577–592. S. Pyner, J.H. Coote, Rostroventrolateral medulla neurons preferentially project to target-specified sympathetic preganglionic neurons, Neuroscience 83 (1998) 617–631. L. Rinaman, M.R. Roesch, J.P. Card, Retrograde transynaptic pseudorabies virus infection of central autonomic circuits in neonatal rats, Brain Res. Dev. Brain Res. 114 (1999) 207–216. R.E. Shapiro, R.R. Miselis, The central organization of the vagus nerve innervating the stomach of the rat, J. Comp. Neurol. 238 (1985) 473–488. E.R. Spindel, E. Giladi, T.P. Segerson, S. Nagalla, Bombesin-like peptides: of ligands and receptors, Recent Prog. Horm. Res. 48 (1993) 365–391. ´ Y. Goto, M.W. Gunion, W. Vale, J. Rivier, M. Brown, Y. Tache, Inhibition of gastric acid secretion in rats by intracerebral injection of corticotropin-releasing factor, Science 222 (1983) 935–937. ´ D. Lesiege, Y. Goto, Neural pathways involved in Y. Tache, intracisternal bombesin-induced inhibition of gastric secretion in rats, Dig. Dis. Sci. 31 (1986) 412–417. R.A. Travagli, R.A. Gillis, S. Vicini, Effects of thyrotropin-releasing hormone on neurons in the rat dorsal motor nucleus of the vagus, in vitro, Am. J. Physiol. 263 (1992) G508–G517. E. Wada, S. Wray, S. Key, J. Battey, Comparison of gene expression for two distinct bombesin receptor subtypes in postnatal rat central nervous system, Mol. Cell. Neurosci. 3 (1992) 446–460. J.H. Walsh, Physiology and pathology of gastrin, Clinics Gastoenterol. 9 (1980) 567–591. R.L. White Jr., C.D. Rossiter, P.J. Hornby, J.W. Harmon, D.K. Kasbekar, R.A. Gillis, Excitation of neurons in the medullary raphe increases gastric acid and pepsin production in cats, Am. J. Physiol. 260 (1991) G91–96. ´ Intrathecal injection of H. Yang, F. Cuttitta, H. Raybould, Y. Tache, bombesin inhibits gastric acid secretion in the rat, Gastroenterology 96 (1989) 1403–1409. ´ Peptide YY in brain stem nuclei induces vagal H. Yang, Y. Tache, stimulation of gastric acid secretion in rats, Am. J. Physiol. 268 (1995) G943–G948. ´ Prepro-TRH-(160–169) potentiates gastric acid H. Yang, Y. Tache, secretion stimulated by TRH microinjected into the dorsal motor nucleus of the vagus, Neurosci. Lett. 174 (1994) 43–46. ´ Activation of the H. Yang, P.Q. Yuan, L. Wang, Y. Tache, parapyramidal region in the ventral medulla stimulates gastric acid secretion through vagal pathways in rats, Neuroscience 95 (2000) 773–779. K. Yokotani, M. Muramatsu, M. Fujiwara, Y. Osumi, Effects of the sympathoadrenal system on vagally induced gastric acid secretion and mucosal blood flow in rats, J. Pharmacol. Exp. Ther. 224 (1983) 436–442. K. Yokotani, Y. Osumi, Alpha-1 adrenoceptors mediate splanchnic nerve inhibition of pentagastrin-induced gastric acid secretion and mucosal blood flow in rats, J. Pharmacol. Exp. Ther. 236 (1986) 743–747. ´ Bombesin acts in the brain to E. Yoshida-Yoneda, J.Y. Wei, Y. Tache,
T. Ishikawa et al. / Brain Research 918 (2001) 1 – 9 decrease gastric vagal efferent discharge in rats, Peptides 14 (1993) 339–343. [48] A. Zagon, Internal connections in the rostral ventromedial medulla of the rat, J. Auton. Nerv. Syst. 53 (1995) 43–56. [49] A. Zagon, A.D. Smith, Monosynaptic projections from the rostral
9
ventrolateral medulla oblongata to identified sympathetic preganglionic neurons, Neuroscience 54 (1993) 729–743. [50] M.A. Zarbin, M.J. Kuhar, T.L. O’Donohue, S.S. Wolf, T.W. Moody, Autoradiographic localization of ( 125 I-Tyr 4 ) bombesin-binding sites in rat brain, J. Neurosci. 5 (1985) 429–437.
Research report
Microinjection of bombesin into the ventrolateral reticular formation inhibits peripherally stimulated gastric acid secretion through spinal pathways in rats Toshio Ishikawa 1 , Hong Yang, Yvette Tache´ * CURE: Digestive Diseases Research Center, Veterans Administration Greater Los Angeles Healthcare System, Department of Medicine, Digestive Diseases Division and Brain Research Institute, University of California at Los Angeles, Los Angeles, CA 90073, USA Accepted 23 July 2001
Abstract Bombesin injected into the cisterna magna potently inhibits gastric acid secretion stimulated by intravenous infusion of pentagastrin. Sites in the medulla oblongata where bombesin acts to suppress gastric acid secretion were investigated in urethane-anesthetized rats with gastric cannula. Bombesin or vehicle was injected into the medullary parenchyma or intracisternally (i.c.) 60 min after the start of an intravenous pentagastrin infusion; gastric acid secretion was monitored every 10 min for 20 min before and 150 min after the start of pentagastrin. Bombesin (0.2, 0.6 or 6.2 pmol) microinjected into the ventrolateral reticular formation (VLRF) inhibited dose-dependently the net acid response to pentagastrin by 40.8611.1, 75.4612.8 and 96.7619.4%, respectively, at the 40–50 min period after microinjection compared with the vehicle group. Bombesin action in the VLRF was long lasting (96% inhibition still observed at 90 min after 6.2 pmol), and completely abolished by cervical spinal cord transection at the C6 level. By contrast, bombesin injected i.c. at 0.2 or 0.6 pmol had no effect while at 6.2 pmol, there was a 79.063.9% peak inhibition of pentagastrin-stimulated acid secretion. Bombesin (6.2 pmol) injected into the dorsal motor nucleus reduced the acid response to pentagastrin by 29%. The parvicellular and gigantocellular reticular nuclei were not responsive to bombesin. These results indicate that bombesin acts in the VLRF to inhibit pentagastrin-stimulated gastric acid secretion through spinal pathways, suggesting a potential role of medullary VLRF area in the sympathetic control of gastric acid secretion. 2001 Elsevier Science B.V. All rights reserved. Theme: Endocrine and autonomic regulation Topic: Gastrointestinal and urogenital regulation Keywords: Bombesin; CRF; Gastric secretion; Spinal cord; Brainstem; Sympathetic
1. Introduction Compelling evidence indicates that bombesin and bombesin-like peptides are potent inhibitors of gastric acid secretion when injected into the cerebrospinal fluid of rats, cats, and dogs [21]. The mapping of hypothalamic sites responsive to bombesin in rats has revealed that vagallystimulated gastric acid secretion was inhibited by the peptide microinjected into the paraventricular nucleus of *Corresponding author. Tel.: 11-310-312-9275; fax: 11-310-2684963. ´ E-mail address: [email protected] (Y. Tache). 1 Current address: Division of Psychosomatic Research, National Institute of Mental Health, 1-7-3 Konodai, Ichikawa, Chiba 272, Japan.
the hypothalamus [13,25], anterior hypothalamus or preoptic area [25] while not modified after microinjection of bombesin into the lateral, dorsomedial or ventromedial hypothalamus and the caudate putamen [13,25]. However, intracisternal (i.c.) bombesin-induced inhibition of vagallyand pentagastrin-stimulated gastric secretion was not altered by electrolytic lesioning of the paraventricular nucleus of the hypothalamus or preoptic-anterior area or by mid-collicular transection [14,25], suggesting the existence of additional hindbrain sites for peptide action. Further studies established that bombesin microinjected into the dorsal motor nucleus of the vagus (DMN) dose-dependently abolished central vagal stimulation of gastric acid secretion induced by co-microinjection of thyrotropin-releasing hormone (TRH) stable analog, RX-77368 [15], a
0006-8993 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 01 )02833-5
T. Ishikawa et al. / Brain Research 918 (2001) 1 – 9
2
peptide established to activate directly preganglionic vagal motor neurons leading to increased gastric vagal efferent discharge [23,37]. By contrast, bombesin suppressed the acid response to an intravenous infusion of pentagastrin less potently when injected into the DMN than intracisternally [15]. These findings indicate that additional sites located in the medulla oblongata are involved in bombesininduced inhibition of gastric acid secretion stimulated by a peripheral secretagogue. Intracerebroventricular (i.c.v.) injection of bombesin elevates plasma levels of catecholamines [6,24] and increases nerve activity in both the sympathetic and adrenal branches of the splanchnic nerve [27]. Intracisternal bombesin-induced inhibition of gastric acid secretion in pylorus-ligated rats is partly prevented by cervical cord transection [36]. Likewise, bombesin injected i.c.-induced inhibition of the acid secretory response to electrical vagal stimulation was reduced by bilateral cutting of the greater splanchnic nerve and abolished by combined chemical sympathectomy and adrenalectomy [26]. Neuroanatomical studies showed that bombesin immunoreactive neurons and receptors are distributed not only in the dorsal vagal complex [8,19,50] but also in the ventrolateral medulla [19,38] where neurons selectively innervate the sympathetic preganglionic neurons in the intermedio-medial and -lateral column of the thoracic spinal cord [10,31,49]. Taken together, these functional and morphological data support a potential action of bombesin in the ventrolateral medulla. In the present study, we investigated the inhibitory effect of bombesin microinjected into the ventrolateral reticular formation (VLRF) on gastric acid secretion stimulated by intravenous infusion of pentagastrin, a well established peripherally acting secretagogue [39] in urethane-anesthetized rats. The site specificity of bombesin action in the VLRF and the mediation through cervical spinal cord pathways were also assessed.
2. Materials and methods
2.1. Animals Male, Sprague Dawley albino rats (Charles Rivers Laboratories) weighing 220–300 g were maintained ad libitum on Purina Laboratory chow and tap water. They were housed under conditions of controlled temperature (20618C) and illumination (06:00 to 18:00 h), and deprived of food, but not water, for 24 h before each experiment. All experiments were conducted under protocols approved by the Veterans Administration Animal Committee and performed in fasted rats anesthetized with urethane (1.5 g / kg, i.p.) throughout the duration of the study.
2.2. Peptides The following peptides were used: bombesin and rat
corticotropin-releasing factor (CRF) (kindly supplied by Dr. J. Rivier, Clayton Foundation Laboratories for Peptide Biology Laboratories, Salk Institute, La Jolla, CA, USA), and pentagastrin solution (Peptavlon, Ayerst Laboratories, New York, NY, USA). Bombesin and CRF in powder form were dissolved in 0.1% bovine serum albumin / saline immediately before use. Pentagastrin was diluted in saline before intravenous infusion.
2.3. Measurement of gastric secretion In urethane anesthetized rats, the femoral vein was catheterized and the esophagus ligated at the cervical level. After performing a laparotomy, the pylorus was ligated and a double lumen cannula was placed through a small incision into the forestomach. Gastric acid secretion was measured by flushing the gastric lumen twice with 5-ml boluses of saline and one 5-ml bolus of air at the end of each 10 min period. Acid output was determined by titration (autotitrator, Radiometer, Copenhagen, Denmark) of the flushed perfusate with 0.01 M NaOH to pH 7.0.
2.4. Medullary injections Urethane anesthetized rats equipped with gastric cannula and intravenous femoral catheter were positioned on a stereotaxic instrument (Kopf Intruments, Model 900). The rat head was fixed at a nose-down position with ear bars and the incisor bar set at 215 mm. For microinjection into the brainstem area, the obex region of the dorsal medulla was exposed. The dorsal cervical musculature was resected and the occipital skull plate was removed. About 23232 mm 3 of ventral–caudal cerebellum tissue covering the caudal half of the forth ventricle was carefully removed by sucking with negative pressure generated from a water pump as in our previous study [15]. General procedure for microinjections were performed as previously reported [15]. A glass micropipette (50–70 mm in diameter) was positioned unilaterally (right side) into the VLRF according to the following coordinates: ventral from the surface of the brainstem (V): 2.2–2.7 mm, anterior from the caudal tip of the area postrema (A): 1.5–2.0 mm, and lateral from the midline (L): 1.5–2.0 mm, or into the right DMN (V: 0.6, A: 0.3, L: 0.5). In one experiment, microinjection was performed into the left side of the VLRF to assess the bilaterality of the observations. Microinjections were carried out using 100 nl volume delivered by pressure ejection over 1 min with a 1-ml Hamilton syringe, the micropipette was left in place for an additional 3 min, then withdrawn. At the end of the experiment, rats were sacrificed by decapitation and brains were removed and fixed in 10% formalin–20% sucrose solution for at least 2 days. Frozen brainstem sections were sliced at 30 mm, and stained with toluidine blue. The locations of microinjection sites were identified by the visualization of the point of termination of the micropipette track on histological sections examined
T. Ishikawa et al. / Brain Research 918 (2001) 1 – 9
microscopically. Sites were marked on plates reproduced from Paxinos and Watson’s atlas [29]. For intracisternal injection, the rat head was fixed with ear bars of the stereotaxic equipment as previously described [36]. The occipital membrane was punctured by the needle of a 50-ml Hamilton syringe for injection in a 10 ml volume. Correctness of the needle placement into the cisterna magna was insured by the presence of cerebrospinal fluid into the Hamilton syringe upon aspiration before the injection.
2.5. Experimental protocols Following gastric and brain surgeries, basal gastric secretion was measured for 20 min and pentagastrin (16 mg / kg / h) was infused into the femoral vein for the duration of the experiment. One hour after the start of pentagastrin infusion, injection of peptides or vehicle was performed in one of following routes: bombesin (0.2, 0.6 or 6.2 pmol / rat), CRF (62 pmol / rat) or vehicle (100 nl) into the VLRF; bombesin (6.2 pmol / rat) or vehicle (100 nl) into the DMN; bombesin (0.2, 0.6 or 6.2 pmol / rat) or vehicle (10 ml) intracisternally. The stomach was flushed immediately after the injection and this collection was disregarded. The gastric acid secretion was monitored for 90 min after brain injection of vehicle or peptide under conditions of constant pentagastrin intravenous infusion. In one experiment, cervical cord transection at the C6 level or sham operation (exposure of the cervical cord) was performed as previously described [36]. Then, a similar protocol was followed as specified in the other experiments including the placement of the gastric cannula and femoral catheter followed by brain surgery. After 20 min basal acid secretion, pentagastrin (16 mg / kg / h) was infused throughout the experimental period. One hour after the start of pentagastrin intravenous infusion, bombesin (6.2 pmol / rat) was microinjected into the VLRF in sham-operated and C6-transected rats. Gastric acid secretion was monitored for the 90 min period post-injection.
2.6. Statistics Results are expressed as mean6S.E. Due to the individual variability in the magnitude of the plateau gastric secretory response to pentagastrin, which is reached within 1 h of pentagastrin infusion, in some experiments the acid output in the collection at 60 min after the start of pentagastrin infusion was taken as 100% for each animal; results are expressed as percentage change from this value for each other 10 min collection period. The net pentagastrin response was calculated by subtracting the mean basal values of gastric output for the 20 min before pentagastrin infusion, from each of the 10 min postinfusion value. Comparisons between two groups were performed by Student’s t-test and multiple group comparisons by analysis of variance (ANOVA) followed by
3
Duncan’s contrast. A P value less than 0.05 was considered statistically significant.
3. Results In urethane-anesthetized rats with gastric fistula, basal gastric acid secretion was low (2.260.2 mmol / 10 min, n565). Pentagastrin infused intravenously increased gastric acid secretion to 12.460.9 mmol / 10 min (n565) at the 60 min collection period after the start of peptide infusion. Gastric acid output values (mmol / 30 min) for the 30 min pre-injection period were not significantly different from those collected in the 30 min period starting at 20 min after the unilateral microinjection of vehicle into either the right VLRF (pre-injection: 26.063.7, post-injection: 30.265.2, n58) or DMN (pre-injection: 24.964.0, post-injection: 27.964.9, n510). Bombesin microinjected into the right VLRF at 0.2, 0.6 or 6.2 pmol induced a dose-related and sustained inhibition of the gastric acid secretion stimulated by intravenous pentagastrin (Fig. 1). The significant decrease in pentagastrin-stimulated acid secretion occurred within 20 min after bombesin microinjection into the VLRF. The maximal antisecretory effect induced by bombesin microinjected into the VLRF at 0.2, 0.6 and 6.2 pmol was reached at the 40–50 min post injection period and resulted in 40.8611.1, 75.4612.8 and 96.7619.4% inhibition of the acid response to intravenous pentagastrin, respectively. Thereafter, the gastric acid secretion either returned to levels of the vehicle group at 80 min after bombesin (0.2 pmol), or was maintained significantly inhibited throughout the 40 min remaining observation period (0.6 pmol and 6.2 pmol) (Fig. 1). In a few rats, bombesin microinjected into the left side of the VLRF also resulted in inhibition of acid secretion stimulated by intravenous pentagastrin (data not shown). CRF (62 pmol) microinjected into the right VLRF did not modify the acid secretory response to intravenous pentagastrin (92.867.3% of the pre-injection level, n59) compared with vehicle microinjection (107.565.7%, n510) as monitored during the 20–50 min period after the microinjection. When microinjected into the VLRF, the bombesin inhibitory effect was more potent than that when it was injected into the cisterna magna or DMN. Microinjection of bombesin into the VLRF at 0.2 or 0.6 pmol resulted in a dose-related and significant reduction of gastric acid secretion stimulated by intravenous infusion of pentagastrin while there was no significant decrease when bombesin was injected intracisternally at the same doses (Fig. 2). At 6.2 pmol, bombesin injected into the VLRF or cisterna magna, similarly inhibited the net acid response to intravenous infusion of pentagastrin by 96.7 and 79.1%, respectively, while bombesin microinjected into the DMN resulted in a significant 29% inhibition which was weaker than that induced by i.c. or VLRF injection (Fig. 2). The placement of microinjection sites in medullary coronal sections is illustrated in Fig. 3. Microscopic
4
T. Ishikawa et al. / Brain Research 918 (2001) 1 – 9
Fig. 1. Time course of the dose-related inhibitory effect of bombesin microinjected into the ventrolateral reticular formation (VLRF) on pentagastrinstimulated gastric acid secretion in urethane-anesthetized rats. Gastric acid output at the 60-min collection period after pentagastrin infusion was taken as 100% for each animal and gastric acid output was expressed as a percentage of change from this value for each collection period. Each point represents mean6S.E. of number of rats indicated in the parentheses. *P,0.05 compared with vehicle-microinjected group.
examination showed that responsive sites to bombesin located in the right VLRF encompassed the area surrounding the nucleus ambiguus and expended to the reticular rostroventralateralis and dorsal boundaries of the nucleus paragigantocellularis lateralis / lateral reticular nucleus (Fig.
3). Ineffective sites were located in the parvicellular reticular nucleus (PCRt, n59), gigantocellular reticular nucleus (Gi, n55), parvicellular lateral reticular nucleus (LRtPC, n52), linear nucleus of the medulla (Li, n51) and rubrospinal tract (rs, n51) (Fig. 3).
Fig. 2. Effects of injection of bombesin intracisternally, into the ventrolateral reticular formation (VLRF), or the dorsal motor nucleus of the vagus (DMN) on pentagastrin-stimulated gastric acid secretion in urethane-anesthetized rats. Bombesin at various doses or vehicle was injected at 60 min after the start of pentagastrin infusion. The gastric output is the mean value of the 40–50 min collection period following bombesin or vehicle injection. Each column represents mean6S.E. of number of rats indicated in the bottom of each column. *P,0.05 compared with respective vehicle (0) groups; [P,0.05 compared with same dose of bombesin injected intracisternally.
T. Ishikawa et al. / Brain Research 918 (2001) 1 – 9
5
time-related decrease was observed (Fig. 4). Cervical cord transection did not influence the acid response to pentagastrin infusion as shown by the similar values of gastric acid output at 60 min after the start of pentagastrin infusion in both sham-operated and C6 cervical cordtransected rats (Table 1).
4. Discussion
Fig. 3. Coronal brainstem sections adapted from the atlas of Paxinos and Watson [29] showing the microinjection sites of bombesin in the ventral medulla in urethane-anesthetized rats. d, Effective sites; m, ineffective sites. Abbreviations: Amb, nucleus ambiguus; C1 /A1, C1 adrenaline cells and A1 noradrenaline cells; CVL, caudoventrolateral reticular nucleus; Gi, gigantocellular reticular nucleus; IRt, intermediate reticular nucleus; li, linear nucleus of medulla; LPGi, lateral paragigantocellular nucleus; LRtPC, lateral reticular nucleus, parvicellular; LRt, lateral reticular nucleus; MdV, medullary reticular nucleus, ventral part; PCRt, parvicellular reticular nucleus; rs, rubrospinal tract; RVL, rostroventrolateral reticular nucleus; RVRG, rostral ventral respiratory group.
In cervical cord (C6 level) transected rats, bombesin (6.2 pmol) microinjection into the VLRF no longer inhibited gastric acid secretion induced by intravenous infusion of pentagastrin while in sham-operated rats, a
The present study showed that bombesin microinjected into the VLRF at low doses inhibits gastric acid secretion stimulated by intravenous infusion of pentagastrin in urethane-anesthetized rats. Bombesin action in the VLRF was dose-related resulting in a 41–97% net peak inhibition at 0.2–6.2 pmol and long lasting (96% inhibition at 90 min post-injection at 6.2 pmol). By contrast, CRF microinjected into the VLRF at 62 pmol did not modify the acid response to intravenous pentagastrin. CRF injected i.c. or into the lateral hypothalamus in doses ranging from 0.2 to 2.1 nmol was previously reported to inhibit gastric acid secretion stimulated by intravenous infusion of pentagastrin in urethane-anesthetized rats [12,35]. Whether the absence of biological action of CRF microinjected into the VLRF reflects the use of a subthreshold dose or the peptide injection into a non responsive site will need to be further assessed. These results, however, indicate that the unrelated peptide CRF microinjected into the VLRF did not mimic the antisecretory action of bombesin when delivered at a dose that was 10-fold the maximal effective pmolar dose of bombesin, supporting the peptide specificity of the response. These data are also consistent with the notion that bombesin is so far the most potent peptide to inhibit gastric acid secretion when injected into the rat brain [21]. Histological mapping of microinjection placements shows that the responsive sites to bombesin are located in an area centered around the right nucleus ambiguus, the rostroventrolateral reticular nucleus, and dorsal boundary of the lateral paragigantocellular nucleus / lateral reticular nucleus. The inhibition of intravenous pentagastrin-stimulated gastric secretion was also observed after bombesin microinjection into the left side of the VLRF in the few cases tested. The spreading of responsive sites in the VLRF may reflect internal connections among these nuclei in the reticular formation, which have been established by tracing and functional studies [7,11,48]. However, several sites in specific reticular nuclei namely the parvicellular and gigantocellular nuclei were found to be unresponsive to bombesin. This shows the site specificity of bombesin action. In addition, the net acid response to intravenous pentagastrin infusion was more potently inhibited by bombesin microinjected at 6.2 pmol into the VLRF (97% inhibition) than into the DMN (29% inhibition). Lastly, bombesin injected i.c. at 0.2 or 0.6 pmol did not reduce gastric acid secretion stimulated by intravenous pentagastrin while the same doses into the VLRF resulted in a
T. Ishikawa et al. / Brain Research 918 (2001) 1 – 9
6
Fig. 4. Cervical spinal cord transection completely prevented the inhibitory action of bombesin microinjected into the VLRF on pentagastrin-stimulated gastric acid secretion in urethane-anesthetized rats. Gastric acid output at the 60-min collection period after pentagastrin infusion was taken as 100% for each animal and gastric acid output was expressed as a percentage of change from this value for each collection period. Each point represents mean6S.E. of number of rats indicated in the parentheses. [P,0.05 compared with sham-operated group.
significant 41% and 75% net inhibition, respectively. Taken together, these observations reveal that the VLRF is a potent new site of action for bombesin to inhibit peripherally stimulated gastric acid secretion. The observations that bombesin injected i.c. at 6.2 pmol inhibited the acid response to pentagastrin is in agreement with previous reports showing that i.c. injection of bombesin in doses ranging from 6.2 to 300 pmol inhibited pentagastrin-stimulated acid secretion in urethane-anesthetized rats [3,14,15,36]. The equipotency of i.c. bombesin at 6.2 pmol compared with VLRF microinjection may result from combined sites of action at various sites including the VLRF and DMN when the peptide was i.c. injected into cerebrospinal fluid. Studies related to cerebrospinal fluid flow and penetration into the brain parenchyma following
Table 1 Effect of cervical spinal cord transection on gastric acid response to pentagastrin Treatment a
Control Spinal cord transection
n
10 6
Gastric acid output b (mmol / 10 min) Basal
Pentagastrin-stimulated
2.560.3 2.060.3
13.260.8* 14.863.2*
*P,0.05 compared to respective basal values. a In rats under urethane anesthesia, spinal cord transection at the C6 level or sham operation was performed before implanting the gastric cannula and femoral vein catheter. b Gastric acid output (mean6S.E.) was measured before (basal) and 1 h after the start of pentagastrin infusion (16 mg / kg / h).
i.c. injection of [ 14 C]inulin revealed that the inert tracer within 5 min labeled the subarachnoid space outer brain surface of the basal forebrain, ventral and anterior hypothalamus, midbrain, medulla as well as cervical spinal cord [30]. Bombesin conveys its antisecretory action from the VLRF to the stomach through spinal pathways. This is supported by the demonstration that spinal cord transection at the C6 level to eliminate the descending spinal input, completely prevented the inhibition of pentagastrin-stimulated gastric acid secretion induced by bombesin microinjected into the VLRF at the maximal effective dose. The blockade of bombesin action occurred under conditions in which the gastric acid responses to pentagastrin alone was not influenced by acute cervical cord transection in urethane-anesthetized rats as previously observed [41]. Likewise, surgical sympathectomy by extirpation of the coeliac and superior mesenteric ganglia did not influence the stimulation of acid secretion induced by pentagastrin in rats [2,41]. These results indicate that gastric acid secretion elicited by pentagastrin is not modulated by central spinal input in urethane anesthetized rats. Converging evidence indicates that bombesin action may be primarily mediated through descending spinal pathways controlling sympathetic outflow to the gut. Existing evidence does not support a major role of the parasympathetic nervous system in bombesin inhibitory action. Direct microinjection of bombesin into the DMN, the main medullary nucleus influencing preganglionic vagal motor neurons projecting to the stomach [4,33],
T. Ishikawa et al. / Brain Research 918 (2001) 1 – 9
resulted in a weak inhibition of pentagastrin-stimulated acid secretion compared with that induced by microinjection into the VLRF or the cisterna magna ([15], present study). In addition, i.c. injection of bombesin at 6.2 pmol did not significantly influence gastric vagal efferent discharge in urethane-anesthetized rats [47] while inhibiting the acid response to pentagastrin ([15], present study). By contrast, bombesin injected into the cerebrospinal fluid increased nerve activity of both sympathetic and adrenal branches of the splanchnic nerves in urethane anesthetized rats and elevated circulating levels of catecholamines at low doses in conscious rats [5,6,24,27]. Functional studies showed that electrical stimulation of the splanchnic nerve inhibits pentagastrin- or vagally-stimulated acid secretion in urethane-anesthetized rats [45,46]. In addition, i.c.v. injection of bombesin-induced inhibition of vagally stimulated gastric secretion was completely reversed by combined bilateral cutting of the greater splanchnic nerve and chemical sympathectomy in urethane-anesthetized rats [26]. Lastly, the locations of bombesin effective sites in the present study are consistent with established anatomical basis for sympathetic influence on gastric function. Retrograde labeling following pseudorabies virus injected into the stomach was found in the intermediolateral column of the spinal cord at mid- to low-thoracic levels and preautonomic neurons in the caudal and rostral ventrolateral medulla [32]. Other studies have identified direct spinal projections from the VLRF, including the ventrolateral medulla and lateral paragigantocellular reticular nucleus, to the intermediolateral columns at the T5–T12 levels of the spinal cord [10,31,49] where preganglionic neurons innervating the adrenal gland and stomach are located [18,22,32]. However, the spreading of bombesin responsive sites, particularly those located around and within the nucleus ambiguus indicate that in addition to the bombesin action at VLRF sites with direct descending influence on spinal sympathetic activity, bombesin may most likely act through interareticular pathways providing anatomic connections between nuclei of the ventromedial medulla [7,11,48]. Bombesin actions are mediated by binding to receptors of which three pharmacologically distinct members have been identified by molecular cloning in mammals: the neuromedin B receptor, gastrin releasing peptide receptor (GRP-R), and bombesin receptor subtype-3 [20,34]. In addition, a new bombesin receptor subtype-4 has recently been cloned in amphibian [16]. The pharmacologic characteristics of these bombesin receptor subtypes in transfected cells [16,20] and the rank order of potency for bombesin ligands to inhibit gastric acid secretion when injected into the cerebrospinal fluid [21], suggest that the antisecretory action of centrally mediated bombesin involves GRP-R and / or the mammalian counterpart of amphibian bombesin-4 [21]. The involvement of GRP-R is further supported by morphological studies showing high density of GRP binding and expression of GRP-R gene in the
7
VLRF [17,38]. However, the exact receptor(s) mediating the inhibitory action of bombesin in the VLRF is(are) to be identified. The VLRF is well established to play an important role in the regulation of cardiovascular and respiratory functions [1,9]. The present data unraveled the VLRF as a potential medullary site regulating other visceral functions such as gastric function through spinal cord pathways. The presence of GRP receptors [17,38] and bombesin-like immunoreactive fibers / terminals [19] at VLRF sites where microinjection of bombesin at low doses inhibits gastric acid secretion suggests a possible role of endogenous bombesin-like peptides in the regulation of gastric acid secretion at these sites. In the medulla oblongata, previous studies have localized the dorsal vagal complex, parapyramidal region, raphe pallidus and raphe obscurus as nuclei regulating gastric acid secretion through vagal pathways in rats [40,42–44]. Electrical stimulation of the nucleus ambiguus complex did not influence basal gastric acid and pepsin secretion while stimulating motility through vagal pathways in chloralose anesthetized cats [28]. Bombesin action in the VLRF to inhibit pentagastrinstimulated acid secretion through spinal cord pathways may be indicative of VLRF participation in the central sympathetic regulation of gastric function.
Acknowledgements This work was supported by the National Institute of Health, grants DK-30110, DK 33061 and Center Grant DK-41301 (Animal core). The authors thank Dr. J. Rivier (Peptide Biology Laboratory, Salk Institute, La Jolla, CA, USA) for the generous donation of bombesin and rat CRF.
References [1] S.A. Aicher, T.A. Milner, V.M. Pickel, D.J. Reis, Anatomical substrates for baroreflex sympathoinhibition in the rat, Brain Res. Bull. 51 (2000) 107–110. [2] P. Alm, G. Liedberg, C. Owman, Gastric and pancreatic sympathetic denervation in the rat. Technique and results, Scand. J. Gastroenterol. 6 (1971) 307–312. [3] B. Beltran, M.D. Barrachina, A. Mendez, E. Quintero, J.V. Esplugues, Synthesis of nitric oxide in the dorsal motor nucleus of the vagus mediates the inhibition of gastric acid secretion by central bombesin, Br. J. Pharmacol. 127 (1999) 1603–1610. [4] H.R. Berthoud, E.A. Fox, T.L. Powley, Abdominal pathways and central origin of rat vagal fibers that stimulate gastric acid, Gastroenterology 100 (1991) 627–637. [5] M.R. Brown, L.A. Fisher, Brain peptide regulation of adrenal epinephrine secretion, Am. J. Physiol. 247 (1984) E41–E46. [6] K. Carver-Moore, T.S. Gray, M.R. Brown, Central nervous system site of action of bombesin to elevate plasma concentrations of catecholamines, Brain Res. 541 (1991) 225–231. [7] S.H. Chan, J.Y. Chan, B.T. Ong, Anatomic connections between
T. Ishikawa et al. / Brain Research 918 (2001) 1 – 9
8
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21] [22]
[23]
[24]
[25]
[26]
nucleus reticularis rostroventrolateralis and some medullary cardiovascular sites in the rat, Neurosci. Lett. 71 (1986) 277–282. J.F. Costello, M.R. Brown, T.S. Gray, Bombesin immunoreactive neurons in the hypothalamic paraventricular nucleus innervate the dorsal vagal complex in the rat, Brain Res. 542 (1991) 77–82. A.K. Curran, G. Chen, R.A. Darnall, J.J. Filiano, A. Li, E.E. Nattie, Lesion or muscimol in the rostral ventral medulla reduces ventilatory output and the CO(2) response in decerebrate piglets, Respir. Physiol. 123 (2000) 23–37. A.K. Goodchild, I.J. Llewellyn-Smith, Q.J. Sun, J. Chalmers, A.M. Cunningham, P.M. Pilowsky, Calbindin-immunoreactive neurons in the reticular formation of the rat brainstem: catecholamine content and spinal projections, J. Comp. Neurol. 424 (2000) 547–562. A.R. Granata, D.A. Ruggiero, Evidence of disynaptic projections from the rostral ventrolateral medulla to the thoracic spinal cord, Brain Res. 781 (1998) 329–334. ´ M. Yoneda, H. Monnikes, ¨ ´ Central M. Gue, J.-L. Junien, Y. Tache, neuropeptide Y and sigma ligand, JO 1784, reverse corticotropinreleasing factor-induced inhibition of gastric acid secretion in rats, Br. J. Pharmacol. 107 (1992) 642–647. ´ Bombesin microinfusion into the paravenM.W. Gunion, Y. Tache, tricular nucleus suppresses gastric acid secretion in the rat, Brain Res. 411 (1987) 156–161. ´ Fore- and hindbrain mediation of gastric M.W. Gunion, Y. Tache, hypoacidity after intracerebral bombesin, Am. J. Physiol. 15 (1987) G675–G684. ´ Bombesin microinjected into the dorsal vagal T. Ishikawa, Y. Tache, complex inhibits vagally stimulated gastric acid secretion in the rat, Regul. Pept. 24 (1989) 187–194. T. Katsuno, T.K. Pradhan, R.R. Ryan, S.A. Mantey, W. Hou, P.J. Donohue, M.A. Akeson, E.R. Spindel, J.F. Battey, D.H. Coy, R.T. Jensen, Pharmacology and cell biology of the bombesin receptor subtype 4 (BB4-R), Biochemistry 38 (1999) 7307–7320. E.E. Ladenheim, R.T. Jensen, S.A. Mantey, T.H. Moran, Distinct distributions of two bombesin receptor subtypes in the rat central nervous system, Brain Res. 593 (1992) 168–178. S. Leman, O. Viltart, H. Sequeira, Expression of Fos protein in adrenal preganglionic neurons following chemical stimulation of the rostral ventrolateral medulla of the rat, Brain Res. 854 (2000) 189–196. R.B. Lynn, T.M. Hyde, R.R. Cooperman, R.R. Miselis, Distribution of bombesin-like immunoreactivity in the nucleus of the solitary tract and dorsal motor nucleus of the rat and human: colocalization with tyrosine hydroxylase, J. Comp. Neurol. 369 (1996) 552–570. S.A. Mantey, H.C. Weber, E. Sainz, M. Akeson, R.R. Ryan, T.K. Pradhan, R.P. Searles, E.R. Spindel, J.F. Battey, D.H. Coy, R.T. Jensen, Discovery of a high affinity radioligand for the human orphan receptor, bombesin receptor subtype 3, which demonstrates that it has a unique pharmacology compared with other mammalian bombesin receptors, J. Biol. Chem. 272 (1997) 26062–26071. ´ Bombesin and the brain–gut axis, Peptides 21 V. Martinez, Y. Tache, (2000) 1617–1625. S.F. Morrison, W.H. Cao, Different adrenal sympathetic preganglionic neurons regulate epinephrine and norepinephrine secretion, Am. J. Physiol. 279 (2000) R1763–R1775. ´ Intracisternal TRH and RX 77368 T.J. O-Lee, J.Y. Wei, Y. Tache, potently activate vagal efferent discharge in rats, Peptides 18 (1997) 213–219. Y. Okuma, K. Yokotani, Y. Murakami, Y. Osumi, Brain histamine mediates the bombesin-induced central activation of sympatho– adrenomedullary outflow, Life Sci. 61 (1997) 2521–2528. Y. Okuma, K. Yokotani, Y. Osumi, Central site of inhibitory action of bombesin on gastric acid secretion, Jpn. J. Pharmacol. 45 (1987) 129–133. Y. Okuma, K. Yokotani, Y. Osumi, Sympatho–adrenomedullary
[27]
[28]
[29] [30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
[39] [40]
[41]
[42]
[43]
[44]
[45]
[46]
[47]
system mediation of the bombesin-induced central inhibition of gastric acid secretion, Eur. J. Pharmacol. 139 (1987) 73–78. Y. Okuma, K. Yokotani, Y. Osumi, Centrally applied bombesin increases nerve activity of both sympathetic and adrenal branch of the splanchnic nerves, Jpn. J. Pharmacol. 68 (1995) 227–230. F.D. Pagani, W.P. Norman, D.K. Kasbekar, R.A. Gillis, Effects of stimulation of nucleus ambiguus complex on gastroduodenal function, Am. J. Physiol. 246 (1984) G253–G262. G. Paxinos, C. Watson, in: The Rat Brain in Stereotaxic Coordinates, Academic Press, Orlando, FL, 1998, pp. 1–116. M.G. Proescholdt, B. Hutto, L.S. Brady, M. Herkenham, Studies of cerebrospinal fluid flow and penetration into brain following lateral ventricle and cisterna magna injections of the tracer [ 14 C]inulin in rat, Neuroscience 95 (2000) 577–592. S. Pyner, J.H. Coote, Rostroventrolateral medulla neurons preferentially project to target-specified sympathetic preganglionic neurons, Neuroscience 83 (1998) 617–631. L. Rinaman, M.R. Roesch, J.P. Card, Retrograde transynaptic pseudorabies virus infection of central autonomic circuits in neonatal rats, Brain Res. Dev. Brain Res. 114 (1999) 207–216. R.E. Shapiro, R.R. Miselis, The central organization of the vagus nerve innervating the stomach of the rat, J. Comp. Neurol. 238 (1985) 473–488. E.R. Spindel, E. Giladi, T.P. Segerson, S. Nagalla, Bombesin-like peptides: of ligands and receptors, Recent Prog. Horm. Res. 48 (1993) 365–391. ´ Y. Goto, M.W. Gunion, W. Vale, J. Rivier, M. Brown, Y. Tache, Inhibition of gastric acid secretion in rats by intracerebral injection of corticotropin-releasing factor, Science 222 (1983) 935–937. ´ D. Lesiege, Y. Goto, Neural pathways involved in Y. Tache, intracisternal bombesin-induced inhibition of gastric secretion in rats, Dig. Dis. Sci. 31 (1986) 412–417. R.A. Travagli, R.A. Gillis, S. Vicini, Effects of thyrotropin-releasing hormone on neurons in the rat dorsal motor nucleus of the vagus, in vitro, Am. J. Physiol. 263 (1992) G508–G517. E. Wada, S. Wray, S. Key, J. Battey, Comparison of gene expression for two distinct bombesin receptor subtypes in postnatal rat central nervous system, Mol. Cell. Neurosci. 3 (1992) 446–460. J.H. Walsh, Physiology and pathology of gastrin, Clinics Gastoenterol. 9 (1980) 567–591. R.L. White Jr., C.D. Rossiter, P.J. Hornby, J.W. Harmon, D.K. Kasbekar, R.A. Gillis, Excitation of neurons in the medullary raphe increases gastric acid and pepsin production in cats, Am. J. Physiol. 260 (1991) G91–96. ´ Intrathecal injection of H. Yang, F. Cuttitta, H. Raybould, Y. Tache, bombesin inhibits gastric acid secretion in the rat, Gastroenterology 96 (1989) 1403–1409. ´ Peptide YY in brain stem nuclei induces vagal H. Yang, Y. Tache, stimulation of gastric acid secretion in rats, Am. J. Physiol. 268 (1995) G943–G948. ´ Prepro-TRH-(160–169) potentiates gastric acid H. Yang, Y. Tache, secretion stimulated by TRH microinjected into the dorsal motor nucleus of the vagus, Neurosci. Lett. 174 (1994) 43–46. ´ Activation of the H. Yang, P.Q. Yuan, L. Wang, Y. Tache, parapyramidal region in the ventral medulla stimulates gastric acid secretion through vagal pathways in rats, Neuroscience 95 (2000) 773–779. K. Yokotani, M. Muramatsu, M. Fujiwara, Y. Osumi, Effects of the sympathoadrenal system on vagally induced gastric acid secretion and mucosal blood flow in rats, J. Pharmacol. Exp. Ther. 224 (1983) 436–442. K. Yokotani, Y. Osumi, Alpha-1 adrenoceptors mediate splanchnic nerve inhibition of pentagastrin-induced gastric acid secretion and mucosal blood flow in rats, J. Pharmacol. Exp. Ther. 236 (1986) 743–747. ´ Bombesin acts in the brain to E. Yoshida-Yoneda, J.Y. Wei, Y. Tache,
T. Ishikawa et al. / Brain Research 918 (2001) 1 – 9 decrease gastric vagal efferent discharge in rats, Peptides 14 (1993) 339–343. [48] A. Zagon, Internal connections in the rostral ventromedial medulla of the rat, J. Auton. Nerv. Syst. 53 (1995) 43–56. [49] A. Zagon, A.D. Smith, Monosynaptic projections from the rostral
9
ventrolateral medulla oblongata to identified sympathetic preganglionic neurons, Neuroscience 54 (1993) 729–743. [50] M.A. Zarbin, M.J. Kuhar, T.L. O’Donohue, S.S. Wolf, T.W. Moody, Autoradiographic localization of ( 125 I-Tyr 4 ) bombesin-binding sites in rat brain, J. Neurosci. 5 (1985) 429–437.