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Intrathecal injection of bombesin inhibits gastric acid secretion in the rat
GASTROENTEROLOGY
1989;96:1403-9
Intrathecal Injection of Bombesin Inhibits Gastric Acid Secretion in the Rat H. YANG, F. CUTTITTA,
H. RAYBOULD,
and Y. TACHfi
Center for Ulcer Research and Education, Veterans Administration Medical Center and Department of Medicine and Brain Research Institute, University of California, Los Angeles, California; National Cancer Institute-Navy Medical Oncology Branch, National Cancer Institute and Naval Hospital, Bethesda, Maryland _
Bombesin (100-500ng) injected intrathecally (T910) inhibited gastric acid secretion stimulated by pentagastrin and the GABA, agonist baclofen in urethane-anesthetized rats and basal gastric acid secretion in conscious, pylorus-ligated rats. Peptide action was dose-related, occurred within 30 min, and lasted for >l h. Bombesin-induced inhibition of pentagastrin-stimulated gastric acid secretion was not altered by cervical cord transection. Intravenous infusion of the monoclonal bombesin antibody 2All abolished intravenous bombesin (10&kg . h)induced 33% inhibition of gastric response to pentagastrin but did not alter intrathecal bombesin (200 ng)-induced 38% inhibition of gastric response to pentagastrin. The inhibitory effect of bombesin (200 ng) on pentagastrin-stimulated gastric secretion was reversed by bilateral adrenalectomy or removal of celiac and mesenteric ganglia. Intrathecal injections of rat calcitonin gene-related peptide, neuromedin B, neuromedin U, and the stable substance P analogue (pGlu’, MePhe’, MeGlyg)-substance P(5-11) did not alter pentagastrin-stimulated gastric acid secretion. These results demonstrate that bombesin injected into the subarachnoid space of the spinal cord inhibits vagally stimulated and pentagastrinstimulated gastric secretion in rats. Bombesin action is peptide specific, exerted at a spinal site, and expressed through the sympathetic nervous system.
S
tudies on the effects of electrical and chemical stimulation of brain nuclei on gastric function have demonstrated that specific hypothalamic and medullary nuclei are involved in the regulation of gastric: acid secretion (1). The spinal cord has recently been the focus of attention as a site independent from the brain that is able to influence gastrointestinal function. In particular, intrathecal injection of bombesin and opioid peptides elicits alteration of gastrointestinal motility and transit in
rats and mice (2-4). Whether the spinal cord is also a chemoresponsive site for peptides to influence gastric acid secretion has not been investigated. Bombesin is a well-established inhibitor of gastric acid secretion when injected into the cerebrospinal fluid in rats, cats, and dogs (5). The central nervous system-mediated effect of bombesin on gastric acid secretion is exerted at specific hypothalamic and medullary nuclei (6-8) and requires the integrity of the spinal cord and the sympathetic nervous system (9,lO). The presence of bombesinlike immunoreactivity and receptors in the spinal cord (11-13) and the spinal sympathetic pathways involved in the gastric inhibitory effect of injection of bombesin into the cerebrospinal fluid (9,lO) are strong reasons to investigate whether bombesin may also act directly at the spinal level to alter gastric secretion. Part of this work has been reported in abstract form (14,15).
Materials
and Methods
Animals Male Sprague-Dawley rats weighing 200-250 g (Simonsen Laboratories, Gilroy, Calif.) were housed in group cages under conditions of controlled temperature (22”-24°C) and illumination (12 h light cycle starting at 6 AM). The animals were maintained ad libitum on Purina Laboratory Chow (Ralston Purina, St. Louis, MO.) and tap water. All experiments were performed in rats deprived of food for 24 h but given free access to water up to the beginning of the study.
Abbreviations used in this paper: CGRP, calcitonin generelated peptide; PCP-GABA, P-(P-chlorophenyl)-yaminobutyric acid. 0 1969 by the American Gastroenterological Association 0016-5065/69/$3.50
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GASTROENTEROLOGYVol. 96, No. 6
YANG ET AL.
Surgery Rats were anesthetized with urethane (1.25 g/kg, i.p.) for the duration of the experiments, and in some experiments, the following surgeries were performed: spinal cord transection at the seventh cervical vertebral segment using scissors, gastric sympathectomy by careful removal of the celiac and mesenteric ganglia under dissecting microscope, or bilateral adrenalectomy through an abdominal incision. Intrathecal injections were made according to the method described by Yaksh and Rudy (16). The cisternal membrane was exposed, and a polyethylene catheter (PE-10) previously filled with 0.9% saline was inserted through a transverse slit into the cisternal membrane and passed distally into the spinal subarachnoid space. The length of the catheter within the spinal cord was 6 cm (volume 7 ~1) and the tip rests at vertebral levels T9-10. In cervical cord-transected animals, the catheter (3.5 cm) was inserted in the subarachnoid space from the seventh cervical vertebra to the T9-10 vertebral levels. At the end of each experiment, 17 ~1 of a blue dye was injected into the catheter, then rats were killed by an intravenous injection of a 5-ml bolus of air. Autopsy was performed to probe the location of the catheter. Data from rats in which the catheters were not positioned into the subarachnoid space were eliminated. In one experiment, the subarachnoid catheter was positioned in rats anesthetized with ether, and the animals regained righting reflex after surgery.
Measurement
of Gastric Secretion
After implantation of the intrathecal catheter in rats anesthetized with urethane, a cannula was inserted into the trachea, the abdomen opened, the pylorus ligated, and a double-lumen cannula placed through a small incision in the forestomach. The lumen was flushed twice with 5-ml boluses of saline and one 5-ml bolus of air at the end of each lo-min period. Acid output was determined by titration (autotitrator: Radiometer Corp., Copenhagen) of the flushed perfusate with 0.1 N NaOH to pH 7.0. Basal gastric secretions were collected for 30 min, then pentagastrin or baclofen was infused intravenously throughout the experiment and peptides or saline were injected intrathecally 1 h after the start of the gastric stimulant. In one experiment, after positioning the catheter in rats anesthetized with ether, the pylorus was ligated and 2 h later, conscious rats were decapitated. Gastric secretions were collected and centrifuged, and the volume and pH were measured. Acid concentration was determined by titration and acid output was calculated by multiplying the volume by the acid concentration.
Drugs and Treatment The following substances were used: pentagastrin solution (Peptavlon; Ayerst Laboratories, New York, N.Y.), P-(p-chlorophenyl)-y-aminobutyric acid (PCP-GABA, baclofen, Lioresal; Geigy Pharmaceuticals, Summit, N.J.), bombesin, and rat calcitonin gene-related peptide (CGRP;
supplied by Dr. J. Rivier, Salk Institute, La Jolla, Calif.), neuromedin B, neuromedin U, and (pGlu’, MePhea, MeGly’)-substance P(5-11) (Peninsula Laboratories, Belmont, Calif.). The bombesin monoclonal antibody 2All was prepared as previously described (17). All drugs were freshly dissolved or diluted in saline before administration. Intravenous infusions were given at a rate of 1.08 ml/h through a catheter (PE-50) inserted into the external ileal veins. Intrathecal injections were given in a volume of 5-17 ~1.
Statistics Statistical probabilities for comparisons between groups were calculated by two-way analysis of variance, and log sigmoid dose-response curves were fit and analyzed by nonlinear regression. A probability of ~0.05 was considered statistically significant.
Results Inhibition of Stimulated Secretion by Intrathecal Bombesin
Gastric Acid Injection of
Rats under urethane anesthesia have a low basal gastric acid secretion (0.69 5 0.13 pmol/lO min). Intravenous infusion of pentagastrin induced a steady increase in gastric acid output and plateau values (10.1 k 0.6 pmol/lO min, n = 33) are reached at 1 h after infusion. Intrathecal injection of bombesin (100, 200, or 500 ng) induced a significant dosedependent inhibition of pentagastrin-stimulated gastric acid secretion by 28%, 44%, and 56%, respectively, for the 20-TO-min period after peptide injection. Intrathecal injection of saline did not significantly alter gastric acid secretion as compared with preinjection values (Figure 1). The inhibitory effect lasts for >60 min (Figure 1). Other peptides, including CGRP (500 ng), (pGlu’, MePhe’, MeGlyg)substance P(5-11) (550 ng), neuromedin U (550 ng), and neuromedin B (350 ng) injected intrathecally did not influence the gastric secretory response to pentagastrin in urethane-anesthetized rats (Table 1). Intravenous infusion of PCP-GABA in rats under urethane anesthesia stimulated gastric acid secretion. Gastric acid output before intrathecal injection was 26.0 * 3.2 pmol/lO min, which is statistically higher than that elicited by pentagastrin infusion (p 5 0.05). In rats injected intrathecally with saline, gastric acid secretion continued to increase by 24% over the next hour, whereas intrathecal injection of bombesin (200 ng) elicited within 30 min a peak inhibition of 70% as compared with the salinetreated group (Figure 2). Intrathecal injection of bombesin (25-1000 ng) induced a significant dose-dependent inhibition of
June 198!3
INTRATHECAL BOMBESIN INHIBITS GASTRIC SECRETION
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Time (min) 0- -0Saline A-ABombesin
Figure
(n=5) 200ng
A-A Bombesin (n=9)0-•Bombesin
gastric acid output by 37%77% in conscious rats with pylorus ligation for 2 h [Table 2). The inhibition of gastric secretion resulted mostly from a dosedependent reduction of the concentration of acid and acid output and a rise in gastric pH, whereas the volume of gastric secretion was not dose-dependently altered (Table 2). Calcitonin gene-related peptide (10 pg) did not significantly modify gastric acid output in conscious pylorus-ligated rats [Table 2).
0-
Peptides” Control CGRP Neuromedin Neuromedin
500 B U
(pGlu’, MePhe*, MeGlyg)substance
350 550 550
n
Hourly acid outputb (%I
5 4 4 4 4
109.1 102.1 106.0 115.1 103.6
t + t ? t
4.6 2.1 7.2 8.8 7.7
P( 5-l 1)
CGRP, c,alcitonin gene-related peptide. a Rats fasted for 24 h that were under urethane anesthesia and had a intrathecal catheter and a gastric fistula were infused intravenously with pentagastrin (24 pg/kg h) throughout the experiment. One hour after pentagastrin infusion, peptides were injected intrathecally. Gastric secretions were collected every 10 min. b Mean 2 SE. The average gastric acid output from the 50- and 60-min collection periods after pentagastrin administration was taken as 100% and acid output from 10 to 70 min after peptide injection was expressed as a percentage change from this value for each rat.
-0Soline
80
(min)
?? -•Bombesin
(n=4)
0 120
100
200ng
(n=4)
Site and Pathways Involved in lntrathecal Bombesin-Induced Inhibition of Pentagastrin-Stimulated Gastric Acid Secretion To assess whether the inhibitory effect of intrathecal injection of bombesin reflects a direct action at the spinal level, experiments were performed in urethane-anesthetized rats with cervical cord transection. Cervical cord transection did not alter the effect of bombesin (200 ng) as the peptide injected intrathecally below the site of the lesion induced a 39% suppression of pentagastrin-stimulated acid secretion (Figure 3), which was similar to
1. Effect of Intrathecal Injection of Peptides on Pentagastrin-Stimulated Gastric Acid Secretion in Urethane-Anesthetized Rats Dose (ng/rat)
60
Figure 2. Inhibition of PCP-GABA-induced stimulation of gastric acid secretion by intrathecal injection of bombesin in urethane-anesthetized rats. Each point represents the mean + SE of 4 rats per group.
Table Table
40 Time
1OOng (n=9) 500ng (n=6)
1. Dose-dependent inhibition of pentagastrin-stimulated gastric acid secretion by intrathecal injection of bombesin in urethane-anesthetized rats. The 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 change from this value for each collection period. Each point represents the mean 2 SE. The number of rats (n) is indicated in parentheses.
20
2. Effect of Intrathecal Injection of Bombesin and Calcitonin Gene-Related Peptide on Gastric Secretion in Conscious Pylorus-Ligated Rats Gastric acid secretionb
Treatment”
Dose (us) u 17
Saline Bombesin Bombesin Bombesin Bombesin Bombesin Saline CGRP
25 50 100 300 1000 10*
4 8 7 5 5 4 4
Volume (ml/ 2 h)
Concentration PH
Output (pmoli
(mmol/L)
2 h)
2.0
‘- 0.3
1.44
+ 0.08
81 + 6
173
f
32
1.4 1.2 1.4 0.9 1.7 1.8 1.7
? r+ C ? + 5 f
1.59 1.34 2.37 2.94 3.90 1.78 1.43
? 0.18 + 0.13 C 0.53 t 0.58 YY0.88 2 0.31 If: 0.01
71 57 48 45 31 66 68
109 72 68 40 41 136 120
2 4 ? 2 c ? +
44 24 33 14 10 63 30
0.4 0.4 0.4 0.3 0.5 0.4 0.4
? 2 C 2 2 2 *
12 4 10 10 11 18 3
CGRP, calcitonin gene-related peptide. a Rats fasted for 24 h were acutely implanted under ether anesthesia with an intrathecal PE-10 catheter placed through the cisterna magna. Saline, bom-
besin, or CGRP was injected in a volume of was flushed with 5 ~1 of saline. The pylorus later, conscious rats were decapitated for secretion. b Mean + SE. Dose-response was acid concentration, output, and pH.
5 ~1 and the catheter was ligated and, 2 h collection of gastric significant for gastric
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GASTROENTEROLOGY
YANG ET AL.
Pentagastrin
24pg/kg/h
26T
pentagostrin
I
I
I
I
Vol. 96, No. 6
24&kg/h
1
Antbody or solins
1 i.t Bombssin 2OOng
24
16 12 8
I
0 0-
Figure
20
40
- 0 Saline
60
80
100
Time
(min)
120
140
?? -•Bombesin
(n=4)
160
200ng
180
01: 0
Pentagasbin
A
Antibody w
24&kg/h Bomb&sin
I
16T
I
saline 1 O&kg/h
60
80 Time
the 38% inhibition observed in intact rats (Figure 1). To rule out the possibility that the peptide may exert its action by leaking into the periphery after intrathecal injection, experiments were performed in rats infused intravenously with monoclonal bombesin antibody 2All. Bombesin infused intravenously (200 ng] de(10 pg/kg . h) or injected intrathecally creased pentagastrin-stimulated gastric secretion by
I
40
(n=7)
3. Effect of intrathecal injection of saline or bombesin on pentagastrin-induced stimulation of gastric secretion in cervical cord-transected rats under urethane anesthesia. Each point represents the mean k SE. The number of rats (n) is given in parentheses.
I
I
20
I
0-
Figure
- 0 Saline (n=5)
100
120
140
160
180
(min)
?? -•Antibody
3.55mg/kg/h
(n=4)
5. Effect of intravenous infusion of the bombesin monoclonal antibody 2All on intrathecal bombesin-induced inhibition of pentagastrin-stimulated gastric acid secretion in urethane-anesthetized rats. Details of the experimental protocol are similar to those described in Figure 4 except that bombesin was administered intrathecally.
33% and 38% respectively (Figures 4 and 5). Intravenous infusion of the bombesin monoclonal anti(3.55 mg/kg . h) completely abolished the body 2All inhibitory effect of intravenous bombesin (Figure 4) but did not alter intrathecal bombesin-induced 38% inhibition of the gastric response to pentagastrin (Figure 5). In rats with celiacisuperior mesenteric ganglionectomy or adrenalectomy, intrathecal bombesin (200 ng) no longer modified the acid response to pentagastrin (Figure 6). Before intrathecal injections, the gastric acid output after 60 min of pentagastrin infusion was 11.9 ? 3.0 PmolllO min in adrenalectomized rats (n = 6) and 11.7 t 1.6 PmolllO min in sympathectomized rats (n = 6), which was not significantly different from that observed in intact rats (10.1 ? 0.6 pmol/lO min; n = 33).
Discussion 0
20
40
60
00 Time
0-
Figure
-0Saline
(n=5)
100
120
140
160
I 180
(min)
@---@Antibody
3.55mg/kg/h
(n=4)
4. Effect of intravenous infusion of the bombesin monoclonal antibody 2A1 I on intravenous bombesin-induced inhibition of pentagastrin-stimulated gastric acid secretion in urethane-anesthetized rats. Basal gastric secretion was monitored for 36 min and then pentagastrin was infused throughout the experiments. The monoclonal bombesin antibody 2All or saline was infused 30 min after the start of pentagastrin infusion for 60 min and bombesin was infused 60 min after pentagastrin for 90 min. Each point represent the mean 2 SE. The number of rats (n) is given in parentheses.
The present results clearly indicate that bombesin delivered locally into the thoracic subarachnoid space inhibits gastric acid secretion stimulated by pentagastrin, PCP-GABA, and pylorus ligation in conscious or urethane-anesthetized rats. In urethane-anesthetized rats, the inhibitory effect was dose related, occurred within 20-30 min after intrathecal injection, and was long lasting. The inhibition of gastric output after intrathecal injection of bombesin in pylorus-ligated rats results primarily from a decrease in gastric concentration. Intrathecal bombesin was more effective against vagal stimulants such as PCP-GABA (18) or pylorus ligation than pentagas-
June 198!3
INTRATHECAL BOMBESIN INHIBITS GASTRIC SECRETION
Wvam operation (n=9) 0
Figure
Before
bomb&n
Adrenalectomy (n-6) E4
After
Sympathactomy (n=6) bomb&n
200ng
6. Effect of adrenalectomy and sympathectomy on intrathecal bombesin-induced inhibition of pentagastrinstimulated gastric secretion. Animals received sham operation, bilateral adrenalectomy, or removal of celiac and mesenteric ganglia, then basal gastric secretions were collected for 30 min and pentagastrin (24 pg/ kg. h) was infused throughout the experiment. Bombesin was injected intrathecally 1 h after pentagastrin infusions. Bar graphs represent the mean 2 SE. Open columns display the gastric acid output of the two last collection periods before intrathecal injection multiplied by 3; hatched columns represent gastric acid output from 29 to 70 min after bombesin injection. The number of rats per group is given in parentheses. *p 5 0.05, analysis of variance with contrasts.
trin. The peak inhibition elicited by intrathecal bombesin (200-300 ng) as compared with the salinetreated groups was 70%77% when gastric secretion was stimulated by PCP-GABA or pylorus ligation, and 4% when gastric secretion was stimulated by pentagastrin. The difference in the intensity of the gastric secretory response to the various stimulants cannot account for such a difference as PCP-GABA elicited. a significantly higher increase in acid output than pentagastrin. The fact that the inhibitory effect of intrathecal bombesin on gastric secretion can be observed after cervical cord transection at the C-i’ level demonstrates that peptide action is not mediated by neural and/or cerebrospinal fluid pathways to supraspinal centers. By contrast, intrathecal injection of bombesin-induced inhibition of gastrointestinal transit was shown to depend on the integrity of ascending spinalsupraspinal pathways as it was blocked by T-Z cord transection in rats and mice (3,19,20). A study using labeled substance P injected intrathetally (T-9 level), followed by monitoring the radioactivity into the blood collected from the femoral vein, revealed that 0.8°Y&3.5% of the total radioactivity injected passed into the circulation (21). Intravenous administration of bombesin in rats and cats has been reported to inhibit pentagastrin-stimulated or histamine-stimulated gastric secretion (22-24,
1407
present observation). However, bombesin action does not appear to be related to peptide leaking into the peripheral circulation as intravenous infusion of the bombesin antibody 2All abolished the inhibitory effect of intravenous but not intrathecal bombesin. Taken together, these data establish the spinal cord as another target site for bombesin to inhibit gastric acid secretion in addition to previously established supraspinal sites: the paraventricular nucleus, preoptic area, anterior hypothalamus, dorsal motor nucleus, and nucleus ambiguus (68,25). These findings also provide the first evidence that the spinal cord can be a site independent from the brain that is able to influence gastric acid secretion, The precise spinal site and mechanisms of action of intrathecal bombesin are not known. At this time very little information is available on the diffusion and penetration into the grey matter of peptides injected intrathecally. A recent study using radiolabeled substance P injected intrathecally at the T-9 vertebra demonstrated that the labeled peptide penetrates 700-1800 pm from the surface of the spinal cord and this penetration extends about 0.5 cm rostrocaudally within 10 min (21). To what extent bombesin may act on the intermediolateral nucleus, lateral funiculus, nucleus intercalatus, and central autonomic area of segment T-7 to T-12 of spinal cord that contain sympathetic preganglionic neurons projecting to the adrenals and stomach (26) needs to be investigated. Neural pathways through which intrathecal bombesin inhibits pentagastrin-stimulated gastric secretion involved the sympathetic nervous system as demonstrated by the blockade of peptide action after surgical gastric sympathectomy or adrenalectomy. The inhibitory effect of bombesin injected into the lateral cerebroventricle or the cisterna magna is also conveyed through the sympathetic nervous system as peptide action was markedly reduced by spinal cord transection (9) or bilateral cutting of the greater splanchnic nerves and abolished by chemical sympathectomy combined with adrenalectomy (10). However, bilateral adrenalectomy alone prevented the effect of bombesin injected intrathecally (present observation) but not intracisternally or intracerebroventricularly (10,27). These latter findings, added to the fact that in pylorus-ligated rats intrathecal injection of bombesin did not completely inhibit gastric acid concentration and secretory volume as observed after injection of similar or lower doses of the peptide into the cisterna magna (27) or the paraventricular nucleus (6), suggest the involvement of additional inhibitory mechanisms after supraspinal injection of the peptide. Previous studies also demonstrated that bombesin was a less potent inhibitor of gastrointestinal transit when injected intrathe-
1408
YANG
tally
than when
GASTROENTEROLOGY
ET AL.
injected
References
intracerebroventricularly
Gw Other peptides such as CGRP (28,29), neuromedin B (30,31), neuromedin U (32), and substance P (33) have been characterized immunohistochemically or chemically in the spinal cord as well as their specific receptors. Intrathecal injection of CGRP, neuromedin B, neuromedin U, and the stable substance P P(5analogue (pGlu’, MePhe’, MeGlyg)-substance 11) did not alter gastric acid secretion stimulated by pentagastrin or pylorus ligation. The inactivity of intrathecal CGRP indicates that the peptide is not acting on sympathetic pathways influencing gastric secretion. These results are in agreement with a previous demonstration that intracisternal or intracerebroventricular injection of CGRP-induced inhibition of gastric secretion is not mediated by the sympathetic component of the autonomic nervous system (34,35). Neuromedin B was previously reported to be inactive to influence gastric secretion when injected intracisternally or intracerebroventricularly (36,37). As neuromedin B shares structural homology with bombesin (30) with the exception of three amino acid substitutions in the biologically active part of the bombesin fragment, these results show the structure specificity of bombesin action. Pentagastrin response was not altered by intrathecal injection of (pGlu5, MePhe’, MeGlyg)-substance P(511). The stable substance P analogue, which is resistant to enzymatic degradation due to the protection of cleavage sites by methylated amino acids (38), has been shown to bind specifically to substance P receptors in the spinal cord with no binding interaction with bombesin (39). In addition, intrathecal injection of the substance P analogue at a dose of 29 ,ug activates spinal sympathetic outflow as measured by the increase in plasma norepinephrine and epinephrine (39). The low dose used in our study (0.5 pg) may explain the lack of biological activity although a higher dose tested (15 pg) was also inactive (data not shown). Previous studies show that intracisternal injection of substance P in doses up to 10 pg did not alter gastric acid secretion in rats (27). Unlike other peptides, the ability of intrathecal injection of bombesin to inhibit gastric acid secretion provides a new chemical tool to assess spinal mechanisms and autonomic pathways regulating gastric secretion. However, further neuroanatomic, neurochemical, and electrophysiologic studies are required to establish whether bombesinlike peptides and receptors present in the spinal cord (1 l-l 3) may have relevant implications in the regulation of gastric secretion.
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32.
INTRATHECAL BOMBESIN INHIBITS GASTRIC SECRETION
Distribution of label after intrathecal administration of l*‘Isubstance P in the rat. Peptides 1987;8:213-21. Solornon TE, Andriulli A, Grossman MI. Bombesin releases gastrin but inhibits gastric secretion in rat (abstr). Gastroenterology 1981;80:1289. Bado A, Lewin MJM, Dubrasquet M. The effects of intravenously administered bombesin on pentagastrin-stimulated acid secretion in cats. Regul Pept 1988;21:141-9. Schubert ML, Hightower J, Makhlouf GM. Inhibitory role for fundic bombesin in the regulation of gastric acid secretion (abstr). Gastroenterology 1987;92:1627. Ishikawa T. Tache Y. Nucleus ambiguus: a sensitive site of action for bombesin-induced inhibition of gastric acid secretion in rats (abstr) FASEB J 1988;2:A498. Neuhuber W, Niederle B. Spinal ganglion cells innervating the stomach of the rat as demonstrated by somatopetal transport of horseradish peroxidase (HRP). Anat Embryo1 1979;155:355-62. TachlZ Y, Vale W, Rivier J, Brown M. Brain regulation of gastric secretion: influence of neuropeptides. Proc Nat1 Acad Sci USA 1980;77:5515-9. Goltzman D, Mitchell J. Interaction of calcitonin and calcitonin gene-related peptide at receptor sites in target tissues. Science 1985;227:1343-6. Gibson SJ, Polak JM, Bloom SR, et al. Calcitonin gene-related peptide immunoreactivity in the spinal cord of man and of eight other species. J Neurosci 1984;4:3101-11. Minamino N, Kangawa K, Matsuo H. Neuromedin B: a novel bombesin-like peptide identified in porcine spinal cord. Biothem Biophys Res Commun 1983;114:541-8. Namba M, Ghatei MA, Gibson SJ, Polak JM, Bloom SR. Distribution and localization of neuromedin B-like immunoreactivity in pig, cat and rat spinal cord. Neuroscience 1985;15:1217-26. Domin J, Ghatei MA, Chohan P, Bloom SR. Neuromedin Ua study of its distribution in the rat. Peptides 1987;8:779-84.
33.
34.
35.
36.
37.
38.
39.
1409
Shults CW. Quirion R, Chronwall B, Chase TN, O’Donohue TL. A comparison of the anatomical distribution of substance P and substance P receptors in the rat central nervous system. Peptides 1984;5:1097-128, Tache Y, Gunion M, Lauffenberger M, Goto Y. Inhibition of gastric acid secretion by intracerebral injection of calcitonin gene related peptide in rats. Life Sci 1984:35:871-8. Lenz HJ, Mortrud MT, Rivier JE, Brown MR. Central nervous system actions of calcitonin gene-related peptide on gastric acid secretion in the rat. Gastroenterology 1985;88:539-44. Tache Y. Intracisternal bombesin induced inhibition of gastric secretion is not mediated through prostaglandin or opioid pathways. Peptides 1985;6(Suppl 3):69-73. Guglietta A, Strunk CL, Irons BJ, Lazarus LH. Central neuromodulation of gastric acid secretion by bombesin-like peptides. Peptides 1985;6(Suppl 3):75-81. Eison AS, Iversen SD, Sandberg BE, Watson SP, Hanley MR, Iversen LL. Substance P analog, DiMe-C7: evidence for stability in rat brain and prolonged central actions. Science 1982;215:188-90. Keeler JR, Charlton CG, Helke CJ. Cardiovascular effects of spinal cord substance P: studies with a stable receptor agonist. J Pharmacol Exp Ther 1985;233:755-60.
Received June 27, 1988. Accepted December 6, 1988. Address requests for reprints to: Yvette Tache, Ph.D., Veterans Administration Wadsworth Medical Center, CURE, Building 115, Room 203, Wilshire and Sawtelle Boulevards, Los Angeles, California 90073. This study was supported by grant DK-30110 from the National Institute of Diabetes and Digestive and Kidney Diseases, and grant MH-00663 from the National Institute of Mental Health. La Jolla, The authors thank Dr. Jean Rivier (Salk Institute, California] for donating bombesin and calcitonin gene-related peptide, Dr. T. Reedy for helping in the statistical analysis of the data, and D. Hamel for technical assistance.
1989;96:1403-9
Intrathecal Injection of Bombesin Inhibits Gastric Acid Secretion in the Rat H. YANG, F. CUTTITTA,
H. RAYBOULD,
and Y. TACHfi
Center for Ulcer Research and Education, Veterans Administration Medical Center and Department of Medicine and Brain Research Institute, University of California, Los Angeles, California; National Cancer Institute-Navy Medical Oncology Branch, National Cancer Institute and Naval Hospital, Bethesda, Maryland _
Bombesin (100-500ng) injected intrathecally (T910) inhibited gastric acid secretion stimulated by pentagastrin and the GABA, agonist baclofen in urethane-anesthetized rats and basal gastric acid secretion in conscious, pylorus-ligated rats. Peptide action was dose-related, occurred within 30 min, and lasted for >l h. Bombesin-induced inhibition of pentagastrin-stimulated gastric acid secretion was not altered by cervical cord transection. Intravenous infusion of the monoclonal bombesin antibody 2All abolished intravenous bombesin (10&kg . h)induced 33% inhibition of gastric response to pentagastrin but did not alter intrathecal bombesin (200 ng)-induced 38% inhibition of gastric response to pentagastrin. The inhibitory effect of bombesin (200 ng) on pentagastrin-stimulated gastric secretion was reversed by bilateral adrenalectomy or removal of celiac and mesenteric ganglia. Intrathecal injections of rat calcitonin gene-related peptide, neuromedin B, neuromedin U, and the stable substance P analogue (pGlu’, MePhe’, MeGlyg)-substance P(5-11) did not alter pentagastrin-stimulated gastric acid secretion. These results demonstrate that bombesin injected into the subarachnoid space of the spinal cord inhibits vagally stimulated and pentagastrinstimulated gastric secretion in rats. Bombesin action is peptide specific, exerted at a spinal site, and expressed through the sympathetic nervous system.
S
tudies on the effects of electrical and chemical stimulation of brain nuclei on gastric function have demonstrated that specific hypothalamic and medullary nuclei are involved in the regulation of gastric: acid secretion (1). The spinal cord has recently been the focus of attention as a site independent from the brain that is able to influence gastrointestinal function. In particular, intrathecal injection of bombesin and opioid peptides elicits alteration of gastrointestinal motility and transit in
rats and mice (2-4). Whether the spinal cord is also a chemoresponsive site for peptides to influence gastric acid secretion has not been investigated. Bombesin is a well-established inhibitor of gastric acid secretion when injected into the cerebrospinal fluid in rats, cats, and dogs (5). The central nervous system-mediated effect of bombesin on gastric acid secretion is exerted at specific hypothalamic and medullary nuclei (6-8) and requires the integrity of the spinal cord and the sympathetic nervous system (9,lO). The presence of bombesinlike immunoreactivity and receptors in the spinal cord (11-13) and the spinal sympathetic pathways involved in the gastric inhibitory effect of injection of bombesin into the cerebrospinal fluid (9,lO) are strong reasons to investigate whether bombesin may also act directly at the spinal level to alter gastric secretion. Part of this work has been reported in abstract form (14,15).
Materials
and Methods
Animals Male Sprague-Dawley rats weighing 200-250 g (Simonsen Laboratories, Gilroy, Calif.) were housed in group cages under conditions of controlled temperature (22”-24°C) and illumination (12 h light cycle starting at 6 AM). The animals were maintained ad libitum on Purina Laboratory Chow (Ralston Purina, St. Louis, MO.) and tap water. All experiments were performed in rats deprived of food for 24 h but given free access to water up to the beginning of the study.
Abbreviations used in this paper: CGRP, calcitonin generelated peptide; PCP-GABA, P-(P-chlorophenyl)-yaminobutyric acid. 0 1969 by the American Gastroenterological Association 0016-5065/69/$3.50
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GASTROENTEROLOGYVol. 96, No. 6
YANG ET AL.
Surgery Rats were anesthetized with urethane (1.25 g/kg, i.p.) for the duration of the experiments, and in some experiments, the following surgeries were performed: spinal cord transection at the seventh cervical vertebral segment using scissors, gastric sympathectomy by careful removal of the celiac and mesenteric ganglia under dissecting microscope, or bilateral adrenalectomy through an abdominal incision. Intrathecal injections were made according to the method described by Yaksh and Rudy (16). The cisternal membrane was exposed, and a polyethylene catheter (PE-10) previously filled with 0.9% saline was inserted through a transverse slit into the cisternal membrane and passed distally into the spinal subarachnoid space. The length of the catheter within the spinal cord was 6 cm (volume 7 ~1) and the tip rests at vertebral levels T9-10. In cervical cord-transected animals, the catheter (3.5 cm) was inserted in the subarachnoid space from the seventh cervical vertebra to the T9-10 vertebral levels. At the end of each experiment, 17 ~1 of a blue dye was injected into the catheter, then rats were killed by an intravenous injection of a 5-ml bolus of air. Autopsy was performed to probe the location of the catheter. Data from rats in which the catheters were not positioned into the subarachnoid space were eliminated. In one experiment, the subarachnoid catheter was positioned in rats anesthetized with ether, and the animals regained righting reflex after surgery.
Measurement
of Gastric Secretion
After implantation of the intrathecal catheter in rats anesthetized with urethane, a cannula was inserted into the trachea, the abdomen opened, the pylorus ligated, and a double-lumen cannula placed through a small incision in the forestomach. The lumen was flushed twice with 5-ml boluses of saline and one 5-ml bolus of air at the end of each lo-min period. Acid output was determined by titration (autotitrator: Radiometer Corp., Copenhagen) of the flushed perfusate with 0.1 N NaOH to pH 7.0. Basal gastric secretions were collected for 30 min, then pentagastrin or baclofen was infused intravenously throughout the experiment and peptides or saline were injected intrathecally 1 h after the start of the gastric stimulant. In one experiment, after positioning the catheter in rats anesthetized with ether, the pylorus was ligated and 2 h later, conscious rats were decapitated. Gastric secretions were collected and centrifuged, and the volume and pH were measured. Acid concentration was determined by titration and acid output was calculated by multiplying the volume by the acid concentration.
Drugs and Treatment The following substances were used: pentagastrin solution (Peptavlon; Ayerst Laboratories, New York, N.Y.), P-(p-chlorophenyl)-y-aminobutyric acid (PCP-GABA, baclofen, Lioresal; Geigy Pharmaceuticals, Summit, N.J.), bombesin, and rat calcitonin gene-related peptide (CGRP;
supplied by Dr. J. Rivier, Salk Institute, La Jolla, Calif.), neuromedin B, neuromedin U, and (pGlu’, MePhea, MeGly’)-substance P(5-11) (Peninsula Laboratories, Belmont, Calif.). The bombesin monoclonal antibody 2All was prepared as previously described (17). All drugs were freshly dissolved or diluted in saline before administration. Intravenous infusions were given at a rate of 1.08 ml/h through a catheter (PE-50) inserted into the external ileal veins. Intrathecal injections were given in a volume of 5-17 ~1.
Statistics Statistical probabilities for comparisons between groups were calculated by two-way analysis of variance, and log sigmoid dose-response curves were fit and analyzed by nonlinear regression. A probability of ~0.05 was considered statistically significant.
Results Inhibition of Stimulated Secretion by Intrathecal Bombesin
Gastric Acid Injection of
Rats under urethane anesthesia have a low basal gastric acid secretion (0.69 5 0.13 pmol/lO min). Intravenous infusion of pentagastrin induced a steady increase in gastric acid output and plateau values (10.1 k 0.6 pmol/lO min, n = 33) are reached at 1 h after infusion. Intrathecal injection of bombesin (100, 200, or 500 ng) induced a significant dosedependent inhibition of pentagastrin-stimulated gastric acid secretion by 28%, 44%, and 56%, respectively, for the 20-TO-min period after peptide injection. Intrathecal injection of saline did not significantly alter gastric acid secretion as compared with preinjection values (Figure 1). The inhibitory effect lasts for >60 min (Figure 1). Other peptides, including CGRP (500 ng), (pGlu’, MePhe’, MeGlyg)substance P(5-11) (550 ng), neuromedin U (550 ng), and neuromedin B (350 ng) injected intrathecally did not influence the gastric secretory response to pentagastrin in urethane-anesthetized rats (Table 1). Intravenous infusion of PCP-GABA in rats under urethane anesthesia stimulated gastric acid secretion. Gastric acid output before intrathecal injection was 26.0 * 3.2 pmol/lO min, which is statistically higher than that elicited by pentagastrin infusion (p 5 0.05). In rats injected intrathecally with saline, gastric acid secretion continued to increase by 24% over the next hour, whereas intrathecal injection of bombesin (200 ng) elicited within 30 min a peak inhibition of 70% as compared with the salinetreated group (Figure 2). Intrathecal injection of bombesin (25-1000 ng) induced a significant dose-dependent inhibition of
June 198!3
INTRATHECAL BOMBESIN INHIBITS GASTRIC SECRETION
1601
24 &kg/h
Pentogastrin
I
f
PCP GABA 6mg/kg/h
I
507
I
i.t.
140 T
1405
i.t. J
40--
120 --
I I
loo--
I
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1 -O--o-_6__J
, ‘.
0’
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I
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607-
0
b/“,
40 .20 -0
4
0
20
40
60
80
100
120
140
160
180
0
Time (min) 0- -0Saline A-ABombesin
Figure
(n=5) 200ng
A-A Bombesin (n=9)0-•Bombesin
gastric acid output by 37%77% in conscious rats with pylorus ligation for 2 h [Table 2). The inhibition of gastric secretion resulted mostly from a dosedependent reduction of the concentration of acid and acid output and a rise in gastric pH, whereas the volume of gastric secretion was not dose-dependently altered (Table 2). Calcitonin gene-related peptide (10 pg) did not significantly modify gastric acid output in conscious pylorus-ligated rats [Table 2).
0-
Peptides” Control CGRP Neuromedin Neuromedin
500 B U
(pGlu’, MePhe*, MeGlyg)substance
350 550 550
n
Hourly acid outputb (%I
5 4 4 4 4
109.1 102.1 106.0 115.1 103.6
t + t ? t
4.6 2.1 7.2 8.8 7.7
P( 5-l 1)
CGRP, c,alcitonin gene-related peptide. a Rats fasted for 24 h that were under urethane anesthesia and had a intrathecal catheter and a gastric fistula were infused intravenously with pentagastrin (24 pg/kg h) throughout the experiment. One hour after pentagastrin infusion, peptides were injected intrathecally. Gastric secretions were collected every 10 min. b Mean 2 SE. The average gastric acid output from the 50- and 60-min collection periods after pentagastrin administration was taken as 100% and acid output from 10 to 70 min after peptide injection was expressed as a percentage change from this value for each rat.
-0Soline
80
(min)
?? -•Bombesin
(n=4)
0 120
100
200ng
(n=4)
Site and Pathways Involved in lntrathecal Bombesin-Induced Inhibition of Pentagastrin-Stimulated Gastric Acid Secretion To assess whether the inhibitory effect of intrathecal injection of bombesin reflects a direct action at the spinal level, experiments were performed in urethane-anesthetized rats with cervical cord transection. Cervical cord transection did not alter the effect of bombesin (200 ng) as the peptide injected intrathecally below the site of the lesion induced a 39% suppression of pentagastrin-stimulated acid secretion (Figure 3), which was similar to
1. Effect of Intrathecal Injection of Peptides on Pentagastrin-Stimulated Gastric Acid Secretion in Urethane-Anesthetized Rats Dose (ng/rat)
60
Figure 2. Inhibition of PCP-GABA-induced stimulation of gastric acid secretion by intrathecal injection of bombesin in urethane-anesthetized rats. Each point represents the mean + SE of 4 rats per group.
Table Table
40 Time
1OOng (n=9) 500ng (n=6)
1. Dose-dependent inhibition of pentagastrin-stimulated gastric acid secretion by intrathecal injection of bombesin in urethane-anesthetized rats. The 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 change from this value for each collection period. Each point represents the mean 2 SE. The number of rats (n) is indicated in parentheses.
20
2. Effect of Intrathecal Injection of Bombesin and Calcitonin Gene-Related Peptide on Gastric Secretion in Conscious Pylorus-Ligated Rats Gastric acid secretionb
Treatment”
Dose (us) u 17
Saline Bombesin Bombesin Bombesin Bombesin Bombesin Saline CGRP
25 50 100 300 1000 10*
4 8 7 5 5 4 4
Volume (ml/ 2 h)
Concentration PH
Output (pmoli
(mmol/L)
2 h)
2.0
‘- 0.3
1.44
+ 0.08
81 + 6
173
f
32
1.4 1.2 1.4 0.9 1.7 1.8 1.7
? r+ C ? + 5 f
1.59 1.34 2.37 2.94 3.90 1.78 1.43
? 0.18 + 0.13 C 0.53 t 0.58 YY0.88 2 0.31 If: 0.01
71 57 48 45 31 66 68
109 72 68 40 41 136 120
2 4 ? 2 c ? +
44 24 33 14 10 63 30
0.4 0.4 0.4 0.3 0.5 0.4 0.4
? 2 C 2 2 2 *
12 4 10 10 11 18 3
CGRP, calcitonin gene-related peptide. a Rats fasted for 24 h were acutely implanted under ether anesthesia with an intrathecal PE-10 catheter placed through the cisterna magna. Saline, bom-
besin, or CGRP was injected in a volume of was flushed with 5 ~1 of saline. The pylorus later, conscious rats were decapitated for secretion. b Mean + SE. Dose-response was acid concentration, output, and pH.
5 ~1 and the catheter was ligated and, 2 h collection of gastric significant for gastric
1406
GASTROENTEROLOGY
YANG ET AL.
Pentagastrin
24pg/kg/h
26T
pentagostrin
I
I
I
I
Vol. 96, No. 6
24&kg/h
1
Antbody or solins
1 i.t Bombssin 2OOng
24
16 12 8
I
0 0-
Figure
20
40
- 0 Saline
60
80
100
Time
(min)
120
140
?? -•Bombesin
(n=4)
160
200ng
180
01: 0
Pentagasbin
A
Antibody w
24&kg/h Bomb&sin
I
16T
I
saline 1 O&kg/h
60
80 Time
the 38% inhibition observed in intact rats (Figure 1). To rule out the possibility that the peptide may exert its action by leaking into the periphery after intrathecal injection, experiments were performed in rats infused intravenously with monoclonal bombesin antibody 2All. Bombesin infused intravenously (200 ng] de(10 pg/kg . h) or injected intrathecally creased pentagastrin-stimulated gastric secretion by
I
40
(n=7)
3. Effect of intrathecal injection of saline or bombesin on pentagastrin-induced stimulation of gastric secretion in cervical cord-transected rats under urethane anesthesia. Each point represents the mean k SE. The number of rats (n) is given in parentheses.
I
I
20
I
0-
Figure
- 0 Saline (n=5)
100
120
140
160
180
(min)
?? -•Antibody
3.55mg/kg/h
(n=4)
5. Effect of intravenous infusion of the bombesin monoclonal antibody 2All on intrathecal bombesin-induced inhibition of pentagastrin-stimulated gastric acid secretion in urethane-anesthetized rats. Details of the experimental protocol are similar to those described in Figure 4 except that bombesin was administered intrathecally.
33% and 38% respectively (Figures 4 and 5). Intravenous infusion of the bombesin monoclonal anti(3.55 mg/kg . h) completely abolished the body 2All inhibitory effect of intravenous bombesin (Figure 4) but did not alter intrathecal bombesin-induced 38% inhibition of the gastric response to pentagastrin (Figure 5). In rats with celiacisuperior mesenteric ganglionectomy or adrenalectomy, intrathecal bombesin (200 ng) no longer modified the acid response to pentagastrin (Figure 6). Before intrathecal injections, the gastric acid output after 60 min of pentagastrin infusion was 11.9 ? 3.0 PmolllO min in adrenalectomized rats (n = 6) and 11.7 t 1.6 PmolllO min in sympathectomized rats (n = 6), which was not significantly different from that observed in intact rats (10.1 ? 0.6 pmol/lO min; n = 33).
Discussion 0
20
40
60
00 Time
0-
Figure
-0Saline
(n=5)
100
120
140
160
I 180
(min)
@---@Antibody
3.55mg/kg/h
(n=4)
4. Effect of intravenous infusion of the bombesin monoclonal antibody 2A1 I on intravenous bombesin-induced inhibition of pentagastrin-stimulated gastric acid secretion in urethane-anesthetized rats. Basal gastric secretion was monitored for 36 min and then pentagastrin was infused throughout the experiments. The monoclonal bombesin antibody 2All or saline was infused 30 min after the start of pentagastrin infusion for 60 min and bombesin was infused 60 min after pentagastrin for 90 min. Each point represent the mean 2 SE. The number of rats (n) is given in parentheses.
The present results clearly indicate that bombesin delivered locally into the thoracic subarachnoid space inhibits gastric acid secretion stimulated by pentagastrin, PCP-GABA, and pylorus ligation in conscious or urethane-anesthetized rats. In urethane-anesthetized rats, the inhibitory effect was dose related, occurred within 20-30 min after intrathecal injection, and was long lasting. The inhibition of gastric output after intrathecal injection of bombesin in pylorus-ligated rats results primarily from a decrease in gastric concentration. Intrathecal bombesin was more effective against vagal stimulants such as PCP-GABA (18) or pylorus ligation than pentagas-
June 198!3
INTRATHECAL BOMBESIN INHIBITS GASTRIC SECRETION
Wvam operation (n=9) 0
Figure
Before
bomb&n
Adrenalectomy (n-6) E4
After
Sympathactomy (n=6) bomb&n
200ng
6. Effect of adrenalectomy and sympathectomy on intrathecal bombesin-induced inhibition of pentagastrinstimulated gastric secretion. Animals received sham operation, bilateral adrenalectomy, or removal of celiac and mesenteric ganglia, then basal gastric secretions were collected for 30 min and pentagastrin (24 pg/ kg. h) was infused throughout the experiment. Bombesin was injected intrathecally 1 h after pentagastrin infusions. Bar graphs represent the mean 2 SE. Open columns display the gastric acid output of the two last collection periods before intrathecal injection multiplied by 3; hatched columns represent gastric acid output from 29 to 70 min after bombesin injection. The number of rats per group is given in parentheses. *p 5 0.05, analysis of variance with contrasts.
trin. The peak inhibition elicited by intrathecal bombesin (200-300 ng) as compared with the salinetreated groups was 70%77% when gastric secretion was stimulated by PCP-GABA or pylorus ligation, and 4% when gastric secretion was stimulated by pentagastrin. The difference in the intensity of the gastric secretory response to the various stimulants cannot account for such a difference as PCP-GABA elicited. a significantly higher increase in acid output than pentagastrin. The fact that the inhibitory effect of intrathecal bombesin on gastric secretion can be observed after cervical cord transection at the C-i’ level demonstrates that peptide action is not mediated by neural and/or cerebrospinal fluid pathways to supraspinal centers. By contrast, intrathecal injection of bombesin-induced inhibition of gastrointestinal transit was shown to depend on the integrity of ascending spinalsupraspinal pathways as it was blocked by T-Z cord transection in rats and mice (3,19,20). A study using labeled substance P injected intrathetally (T-9 level), followed by monitoring the radioactivity into the blood collected from the femoral vein, revealed that 0.8°Y&3.5% of the total radioactivity injected passed into the circulation (21). Intravenous administration of bombesin in rats and cats has been reported to inhibit pentagastrin-stimulated or histamine-stimulated gastric secretion (22-24,
1407
present observation). However, bombesin action does not appear to be related to peptide leaking into the peripheral circulation as intravenous infusion of the bombesin antibody 2All abolished the inhibitory effect of intravenous but not intrathecal bombesin. Taken together, these data establish the spinal cord as another target site for bombesin to inhibit gastric acid secretion in addition to previously established supraspinal sites: the paraventricular nucleus, preoptic area, anterior hypothalamus, dorsal motor nucleus, and nucleus ambiguus (68,25). These findings also provide the first evidence that the spinal cord can be a site independent from the brain that is able to influence gastric acid secretion, The precise spinal site and mechanisms of action of intrathecal bombesin are not known. At this time very little information is available on the diffusion and penetration into the grey matter of peptides injected intrathecally. A recent study using radiolabeled substance P injected intrathecally at the T-9 vertebra demonstrated that the labeled peptide penetrates 700-1800 pm from the surface of the spinal cord and this penetration extends about 0.5 cm rostrocaudally within 10 min (21). To what extent bombesin may act on the intermediolateral nucleus, lateral funiculus, nucleus intercalatus, and central autonomic area of segment T-7 to T-12 of spinal cord that contain sympathetic preganglionic neurons projecting to the adrenals and stomach (26) needs to be investigated. Neural pathways through which intrathecal bombesin inhibits pentagastrin-stimulated gastric secretion involved the sympathetic nervous system as demonstrated by the blockade of peptide action after surgical gastric sympathectomy or adrenalectomy. The inhibitory effect of bombesin injected into the lateral cerebroventricle or the cisterna magna is also conveyed through the sympathetic nervous system as peptide action was markedly reduced by spinal cord transection (9) or bilateral cutting of the greater splanchnic nerves and abolished by chemical sympathectomy combined with adrenalectomy (10). However, bilateral adrenalectomy alone prevented the effect of bombesin injected intrathecally (present observation) but not intracisternally or intracerebroventricularly (10,27). These latter findings, added to the fact that in pylorus-ligated rats intrathecal injection of bombesin did not completely inhibit gastric acid concentration and secretory volume as observed after injection of similar or lower doses of the peptide into the cisterna magna (27) or the paraventricular nucleus (6), suggest the involvement of additional inhibitory mechanisms after supraspinal injection of the peptide. Previous studies also demonstrated that bombesin was a less potent inhibitor of gastrointestinal transit when injected intrathe-
1408
YANG
tally
than when
GASTROENTEROLOGY
ET AL.
injected
References
intracerebroventricularly
Gw Other peptides such as CGRP (28,29), neuromedin B (30,31), neuromedin U (32), and substance P (33) have been characterized immunohistochemically or chemically in the spinal cord as well as their specific receptors. Intrathecal injection of CGRP, neuromedin B, neuromedin U, and the stable substance P P(5analogue (pGlu’, MePhe’, MeGlyg)-substance 11) did not alter gastric acid secretion stimulated by pentagastrin or pylorus ligation. The inactivity of intrathecal CGRP indicates that the peptide is not acting on sympathetic pathways influencing gastric secretion. These results are in agreement with a previous demonstration that intracisternal or intracerebroventricular injection of CGRP-induced inhibition of gastric secretion is not mediated by the sympathetic component of the autonomic nervous system (34,35). Neuromedin B was previously reported to be inactive to influence gastric secretion when injected intracisternally or intracerebroventricularly (36,37). As neuromedin B shares structural homology with bombesin (30) with the exception of three amino acid substitutions in the biologically active part of the bombesin fragment, these results show the structure specificity of bombesin action. Pentagastrin response was not altered by intrathecal injection of (pGlu5, MePhe’, MeGlyg)-substance P(511). The stable substance P analogue, which is resistant to enzymatic degradation due to the protection of cleavage sites by methylated amino acids (38), has been shown to bind specifically to substance P receptors in the spinal cord with no binding interaction with bombesin (39). In addition, intrathecal injection of the substance P analogue at a dose of 29 ,ug activates spinal sympathetic outflow as measured by the increase in plasma norepinephrine and epinephrine (39). The low dose used in our study (0.5 pg) may explain the lack of biological activity although a higher dose tested (15 pg) was also inactive (data not shown). Previous studies show that intracisternal injection of substance P in doses up to 10 pg did not alter gastric acid secretion in rats (27). Unlike other peptides, the ability of intrathecal injection of bombesin to inhibit gastric acid secretion provides a new chemical tool to assess spinal mechanisms and autonomic pathways regulating gastric secretion. However, further neuroanatomic, neurochemical, and electrophysiologic studies are required to establish whether bombesinlike peptides and receptors present in the spinal cord (1 l-l 3) may have relevant implications in the regulation of gastric secretion.
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Tache
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of gastric
acid
secretion.
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Received June 27, 1988. Accepted December 6, 1988. Address requests for reprints to: Yvette Tache, Ph.D., Veterans Administration Wadsworth Medical Center, CURE, Building 115, Room 203, Wilshire and Sawtelle Boulevards, Los Angeles, California 90073. This study was supported by grant DK-30110 from the National Institute of Diabetes and Digestive and Kidney Diseases, and grant MH-00663 from the National Institute of Mental Health. La Jolla, The authors thank Dr. Jean Rivier (Salk Institute, California] for donating bombesin and calcitonin gene-related peptide, Dr. T. Reedy for helping in the statistical analysis of the data, and D. Hamel for technical assistance.