Central CGRP inhibits pancreatic enzyme secretion
by modulation of vagal parasympathetic outflow
YING LI, YI CHENG JIANG, AND CHUNG OWYANG
Gastroenterology Research Unit, Department of Internal Medicine,
University of Michigan Medical Center, Ann Arbor, Michigan 48109
cholecystokinin; vagal pathways; sympathetic nervous system; chronic vagotomy; calcitonin gene-related peptide
CALCITONIN GENE-RELATED
peptide (CGRP), a 37-amino
acid peptide, is a product of alternative splicing of RNA
from the calcitonin gene (2). It is widely distributed in
the peripheral (2) and central nervous system (CNS) (9,
12, 24). Several lines of evidence indicate that CGRP is
a putative neurotransmitter in the CNS. Central administration of CGRP has been shown to inhibit gastric
acid secretion (15, 32, 33), gastrointestinal motility (6,
13), duodenal bicarbonate secretion (14), and food
intake (10). CGRP injected into the cerebrospinal fluid
has been shown to increase noradrenergic outflow (7),
blood pressure, and heart rate (11). Little is known
about CNS control of pancreatic secretion. Intracerebroventricular (ICV) administration of CGRP inhibits
unstimulated (basal) pancreatic secretion in freemoving rats. This inhibition appears to be mediated by
sympathetic noradrenergic efferents through the a-adrenergic receptor (25). Previously, we demonstrated
that peripheral administration of CGRP inhibits pancreatic enzyme secretion stimulated by 2-deoxy-D-glucose
(2-DG) and CCK-8, which act through vagal pathways
(16). Our studies indicate that CGRP does not act on
peripheral vagal afferent or efferent pathways or directly on pancreatic acini; it appears to exert its
inhibitory action at a central vagal site (16). The effects
of central administration of CGRP on stimulated pancreatic secretion are unknown.
In this study, we investigated the CNS effects of
CGRP on pancreatic enzyme secretion induced by
stimulants that act on different sites and we defined
the neural pathways that mediate the action of CGRP.
We used the following four stimulants: 1) 2-DG, a
central vagal stimulant that acts by stimulating the
hypothalamus, which in turn activates the dorsal
nucleus of vagus nerve; 2) CCK- and non-CCKdependent luminal pancreatic stimuli, which act
through vagal afferent pathways to stimulate pancreatic secretion (17, 18, 20–22); 3) electrical vagus nerve
stimulation, which stimulates pancreatic secretion
through vagal release of ACh in the pancreas; and 4)
bethanechol, which directly activates pancreatic muscarinic receptors. To determine if intact vagus nerves are
required to mediate the central effect of CGRP, we used
rats with a chronic vagotomy, in which we have shown
that CCK, at physiological concentrations, activates
enteropancreatic gastrin-releasing peptide neural pathways to mediate pancreatic secretion (19).
METHODS
Materials
Atropine sulfate, bethanechol chloride, 2-DG, phentolamine, guanethedine, and maltose were purchased from
Sigma Chemical (St. Louis, MO). Rat a-CGRP and CCK-8
were purchased from Peninsula Laboratories (Belmont, CA).
Animal Preparations
Male Sprague-Dawley rats were used (body wt, 250–300 g).
After an overnight fast, the rats were anesthetized with a
mixture of xylazine and ketamine (13 and 87 mg/kg body wt
im, respectively) and maintained with small doses, as required (one-third of the initial dose every 2 h). Rats were
positioned stereotaxically, and a small hole was drilled through
the skull. A 27-gauge stainless steel cannula attached to
PE-20 polyethylene tubing was placed so that the tip of the
cannula protruded into the middle of the left cerebral ventricle. The cannula was secured by two screws inserted into
the surface of the parietal bone, using cranioplastic powder
(Plastics One). Coordinates from the bregma were as follows:
anteroposterior, 0.6 mm; lateral, 2 mm; and ventral, 4 mm
0193-1857/98 $5.00 Copyright r 1998 the American Physiological Society
G957
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Li, Ying, Yi Cheng Jiang, and Chung Owyang. Central
CGRP inhibits pancreatic enzyme secretion by modulation of
vagal parasympathetic outflow. Am. J. Physiol. 275 (Gastrointest. Liver Physiol. 38): G957–G963, 1998.—Calcitonin
gene-related peptide (CGRP) is a potent inhibitor of pancreatic enzyme secretion in vivo. Recent studies have shown that
CGRP exerts its inhibitory action at a central vagal site. The
present study investigates the mechanism responsible for the
central action of CGRP. Rats were fitted with lateral cerebroventricular cannulas, using stereotaxic instruments, 4 days
before pancreatic secretion studies. In anesthetized rats,
administration of 2-deoxy-D-glucose (2-DG) (75 mg/kg iv) or
CCK-8 (40 pmol · kg21 · h21 ) produced a 100 and 75% increase
in protein secretion, respectively, which was completely
blocked by atropine. Intracerebroventricular (ICV) administration of CGRP (0.03–0.6 nmol/h) resulted in a dose-related
inhibition of pancreatic protein secretion evoked by 2-DG or
CCK-8. CGRP administered by the ICV route was 10–40
times more potent than CGRP given by the intravenous
route. In contrast, ICV administration of CGRP had no
significant effect on pancreatic protein secretion evoked by
electrical vagal stimulation or bethanechol, which directly
activates the pancreatic muscarinic receptor. Chemical sympathectomy induced by pretreatment with guanethedine (20
mg/kg ip, 2 days) or a-adrenergic receptor blockade with
phentolamine did not alter the inhibitory effects of CGRP. We
recently demonstrated that CCK stimulated the enteropancreatic neural pathways to mediate pancreatic secretion in rats
with a chronic vagotomy. ICV-administered CGRP did not
affect CCK-stimulated pancreatic secretion in rats with a
chronic vagotomy. In conclusion, CGRP in the central nervous
system inhibits pancreatic enzyme secretion stimulated by
2-DG and CCK-8, which act through vagal pathways. The
inhibitory action of CGRP is not mediated by the sympathetic
nervous system but appears to depend on intact vagus nerves.
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CENTRAL CGRP INHIBITS PANCREATIC SECRETION
Pancreatic Secretion Study
After a 30-min stabilization period, combined bile-pancreatic secretions were collected for 15-min periods. The volume
was measured, and aliquots were taken and diluted with
distilled water for protein determination. The remainder of
the undiluted bile-pancreatic juice was pumped back into the
rat through the duodenal cannula during the next collection
period at the secretion rate of the preceding collection period.
Assays
Pancreatic biliary juice protein was measured spectrophotometrically, using the assay method of Bradford (4). The
total protein output was calculated as multiplied by volume
and protein concentration and expressed as milligrams per
collection period. We confirmed in a previous study (23) that
the increase in protein output in the bile-pancreatic juice
after CCK infusion largely reflected protein from the pancreatic source; biliary protein did not increase with CCK
stimulation.
Experimental Design
2-DG studies. 2-DG, a central vagal stimulant, was dissolved in saline and administered as a bolus intravenous
injection (75 mg/kg) at the end of a 30-min basal collection
period. Pancreatic secretion was collected for 120 min. To
investigate the CNS effects of CGRP on 2-DG-stimulated
pancreatic secretion, graded doses of CGRP (0.03, 0.3, or 0.6
nmol every 2 h) were injected cerebroventricularly (0.7 µl),
beginning 15 min before basal collection periods. Each rat
received two doses of CGRP or saline given cerebroventricularly with a 1-h resting period between experiments.
CCK studies. CCK-8 was dissolved in 0.9% NaCl containing
1% BSA and stored in 20-µl aliquots at 220°C until the time
of study. CCK-8 or 0.9% NaCl alone was infused at a rate of
1.08 ml/h. After two 15-min basal periods, rats were given an
intravenous infusion of CCK-8 for 60 min at a dose of 40
pmol · kg21 · h21, which produced plasma CCK levels similar to
peak postprandial levels (22). An intracerebral microinjection
of CGRP (0.3 nmol) or saline was administered 15 min before
the basal period.
Non-CCK-dependent luminal stimuli studies. Recently, we
have shown (18, 20) that similar to CCK, non-CCK-dependent
luminal stimuli evoked pancreatic enzyme secretion through
stimulation of vagal afferent nerve pathways originating in
the duodenal mucosa. In the present study, we evaluated the
CNS effect of CGRP on pancreatic enzyme secretion stimu-
lated by intraduodenal administration of maltose and hypertonic saline. A 20-cm segment of the proximal small intestine
(the entire duodenum and proximal jejunum) was isolated
between two cannulas positioned at 4 and 24 cm from the
pylorus. After a 30-min basal period, NaCl (500 mosM, pH
6.0) or maltose (300 mM, pH 6.0) was perfused at a rate of
3 ml/h for 60 min, using a peristaltic pump. Free intestinal
drainage was established to avoid an increase in intraluminal
pressure. Each test solution was administered separately
with a 1-h resting period in between experiments to allow
pancreatic secretion to return to basal levels. In a separate
study, an acute vagotomy was performed as described previously (22), and pancreatic secretion studies in response to
maltose and hyperosmolar NaCl solution were performed 30
min after vagotomy. Similar pancreatic secretion studies
were performed in a separate groups of rats that received an
ICV injection of CGRP (0.3 nmol/7 µl) or saline 15 min before
basal collection.
Nerve stimulation studies. Through a midline incision
made on the anterior surface of the neck, the left vagus nerve
was dissected free and severed. The distal end was placed on
a nerve electrode and stimulated with a Grass stimulator for
a period of 1 h (5 V, 4 Hz, 2-ms pulses), beginning at the end of
the basal period. Infusion of atropine (50 µg · kg21 · h21 ) or ICV
injection of CGRP (0.3 or 0.6 nmol) or saline was begun 30
min after stimulation of the vagus nerve.
Bethanechol studies. Bethanechol (0.3 mg/kg) was dissolved in saline and injected subcutaneously as a bolus dose,
after the 30-min basal collection period. ICV injection of
saline or CGRP (0.6 nmol) was performed 15 min before the
basal collection.
Chemical sympathectomy and a-adrenergic receptor blockade studies. To determine if the inhibitory action of central
administration of CGRP on pancreatic secretion stimulated
by 2-DG, CCK, or non-CCK luminal stimuli is mediated by
the sympathetic nervous system, we examined the effect of
the administration of guanethidine and phentolamine. 2-DG,
CCK, and non-CCK luminal stimuli were given after a 30-min
basal period. Phentolamine (1 µmol · kg21 · h21 ), an a-adrenergic antagonist, was dissolved in 0.9% NaCl and given intravenously 30 min before basal collection. The effectiveness of this
dose of phentolamine has been demonstrated previously (5).
To evaluate the effect of chemical sympathectomy on the
inhibitory action of central CGRP, rats were injected with
guanethidine (20 mg/kg ip), once daily for 2 consecutive days.
This dose of guanethidine has been shown to deplete cardiac
norepinephrine by .90% (35). ICV administration of CGRP
(0.3 nmol) was begun 15 min before basal collection. Pancreatic secretion studies in response to 2-DG, CCK, and non-CCKdependent luminal stimuli were performed.
Chronic vagotomy. To determine the role of vagus nerves in
mediating the inhibitory effects of CGRP, we used the chronic
vagotomized rat model. Previously, we have shown that the
pancreatic response to CCK returns to normal within 2 wk
after chronic vagotomy. The response was not blocked by
atropine but was abolished by TTX or surgical interruption of
the enteropancreatic neural pathways (19). This suggests
that adaptation occurs after chronic vagotomy and that CCK
acts through enteropancreatic neural pathways to mediate
pancreatic enzyme secretion in these animals. In the present
study, vagotomy was performed as described earlier (22).
Secretion studies were performed 2 wk after vagotomy in a
similar manner as described for intact rats. Complete vagal
section was confirmed by an absence of the pancreatic secretion response after administration of 2-DG (16). To show that
CCK acts through enteropancreatic neural pathways after
chronic vagotomy, the duodenal part of the pancreas was
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(34). Immediately before rats were killed, we administered
2 µl of methylene blue by ICV injection to verify the injection
site. Data from animals that did not display the dye throughout the ventricular system were excluded from the analyses.
After stereotaxic surgery, one or more polyethylene catheters
were placed in the external jugular vein for intravenous
infusion with a syringe-driven pump. A polyethylene cannula
(Clay-Adams PE-100) was inserted through a midline incision into the common bile-pancreatic duct at the sphincter of
Oddi. To permit intraduodenal infusion of pancreaticobiliary
juice, a second cannula was placed in the duodenum, slightly
above the sphincter of Oddi. Because CGRP is a potent
central inhibitor of gastric secretion (15, 32), it is necessary to
avoid any potential contribution of gastric acid on pancreatic
secretion. A cannula was placed in the stomach to drain
gastric juice, thus preventing gastric secretion from reaching
the duodenum. The abdominal wound was covered with
saline-moistened gauze. The rats were maintained at 37°C
with a heating pad.
CENTRAL CGRP INHIBITS PANCREATIC SECRETION
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meticulously dissected away from its vascular and neural
connections to the duodenal wall and pyloric region. CCK-8
infusion studies were performed after a 30-min basal period.
In a separate group of rats with chronic vagotomies, ICV
injections of CGRP or saline were administered 15 min before
basal period.
Statistical analysis. Results were expressed as means 6
SE. The multivariate ANOVA method was used to evaluate
the effects of repeated measurements over time and treatment effect and the interactions between them. Significance
was determined using Student’s t-test and was accepted at
the 5% level.
RESULTS
Effect of ICV Administration of CGRP on Pancreatic
Protein Secretion Stimulated by 2-DG and CCK-8
stimulated protein secretion in a dose-related manner
(Fig. 1). The lowest dose of CGRP to produce significant
inhibition was 0.03 nmol (7 µl); this dose caused a
reduction of pancreatic secretion from the control volume of 108 6 6 to 85 6 9 mg/30 min. Complete
inhibition was produced with 0.6 nmol of CGRP (Fig. 1).
Intravenous infusion of CCK-8 (40 pmol · kg21 · h21 )
produced a significant increase in pancreatic protein
output, averaging 246 6 18 mg/h, an 80% increase over
basal. This increase was completely blocked by ICV
administration of CGRP (0.3 nmol) (Fig. 2).
Effect of ICV Administration of CGRP on Pancreatic
Secretion Stimulated by Non-CCK Luminal Stimuli
Infusion of hyperosmolar NaCl (500 mosM) and
maltose (300 mM) at 3 ml/h increased protein secretion
to 227 6 12 and 230 6 16 mg/h, respectively (Fig. 3).
This represents a 70% increase in protein secretion
over basal. Previously, we have shown that administration of hyperosmolar NaCl or maltose did not elevate
plasma CCK levels (18, 20). Acute vagotomy did not
affect basal secretion but abolished the response to
hyperosmolar NaCl or maltose solution (Fig. 3A). This
indicates that non-CCK-dependent luminal stimuli
evoked pancreatic secretion by way of vagal pathways.
ICV administration of CGRP (0.3 nmol) completely
abolished the pancreatic protein response to hyperosmolar NaCl or maltose solution (Fig. 3B).
Effect of ICV Administration of CGRP on Electrical
Vagal Stimulation of Pancreatic Secretion
Fig. 1. A: dose-dependent inhibition of 2-deoxy-D-glucose (2-DG)stimulated pancreatic protein secretion by intracerebroventricular
(ICV) administration of calcitonin gene-related peptide (CGRP). A
bolus dose of 2-DG (75 mg/kg) was given intravenously at the end of a
30-min basal collection period. ICV injections of saline or graded
doses of CGRP were given 15 min before basal collection. B: pancreatic protein output during the last 30 min of the study. Values are
means 6 SE for 5 rats in each group. * P , 0.01, significant difference
vs. control (without CGRP).
Direct electrical stimulation of the left cervical vagus
nerve with pulses of 5 V, 4 Hz, and 2-ms duration
resulted in a twofold increase in pancreatic protein
secretion over basal, similar to the increase produced
by CCK-8 and 2-DG in this study. Atropine markedly
inhibited this response (Fig. 4). ICV administration of
CGRP at a dose as high as 0.6 nmol did not affect
nerve-stimulated pancreatic protein secretion (Fig. 4).
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In the ketamine-xylazine anesthetized rat model,
basal pancreatic protein secretion remained stable and
averaged 64.2 6 5 mg/30 min (Fig. 1). ICV administration of CGRP did not significantly alter basal pancreatic secretion in our anesthetized rat. Intravenous
administration of 2-DG (75 mg/kg iv) increased protein
secretion to 112 6 4 mg/30 min, similar to the increase
produced by a physiological concentration of CCK-8.
Central administration of CGRP inhibited 2-DG-
Fig. 2. Effect of ICV administration of CGRP on CCK-8-stimulated
pancreatic protein secretion. CCK-8 (40 pmol · kg21 · h21 ) was administered at the end of a 30-min basal collection period by continuous
infusion for 60 min. ICV administration of saline or CGRP (0.3 nmol)
was performed 15 min before basal collection. Protein content of
pancreatic secretion was measured in samples collected at 15-min
intervals. Values are means 6 SE for 5 rats in each group. * P , 0.01.
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CENTRAL CGRP INHIBITS PANCREATIC SECRETION
basal when injected subcutaneously at a dose of 0.3
mg/kg (Fig. 5). ICV microinjection of CGRP at doses up
to 0.6 nmol did not alter the protein output in response
to bethanechol (Fig. 5).
Effect of Guanethidine and Phentolamine on Inhibitory
Effect of Central CGRP
Effect of ICV Administration of CGRP
on CCK-Stimulated Pancreatic Secretion
in Rats With Chronic Vagotomy
Fig. 3. Intraduodenal infusion of hyperosmolar NaCl and maltose. A:
after a 30-min basal period, NaCl (500 mosM) or maltose (300 mM)
was given for 60 min at a constant perfusion rate of 3 ml/h. In a
separate group of rats, acute vagotomy was performed 15 min before
basal collection. B: effect of ICV administration of CGRP on maltoseor hyperosmolar NaCl-stimulated pancreatic secretion. ICV injection
of saline or CGRP (0.3 nmol) was performed 15 min before basal
collection. Values are means 6 SE for 5 rats in each group. * P , 0.01.
Effect of ICV Administration of CGRP on Pancreatic
Secretion Stimulated by Bethanechol
Bethanechol, a muscarinic receptor antagonist without significant nicotinic receptor effect, stimulated pancreatic protein secretion approximately twofold above
Fig. 4. Effect of ICV administration of CGRP on electrical nerve
stimulation of pancreatic protein secretion. The left cervical vagus
nerve was stimulated beginning at 30 min and ending at 105 min (5
V, 4 Hz, 2-ms pulses). An ICV injection of CGRP (0.3 or 0.6 nmol) was
given 30 min after the beginning of nerve stimulation. In a separate
group of rats, atropine (50 µg · kg21 · h21 ) was administered intravenously, 30 min after the beginning of electrical vagal stimulation.
Protein content of pancreatic secretion was measured in samples
collected at 15-min intervals. Values are means 6 SE for 5 rats in
each group. * P , 0.01.
Acute vagotomy completely abolished the pancreatic
response to the infusion of CCK-8 (40 pmol · kg21 · h21 ),
which produced a plasma CCK level similar to the peak
postprandial plasma level (22). In rats with chronic
vagal denervation, the pancreatic protein secretion in
response to CCK was fully restored at 20 days postvagotomy (Fig. 7). This normalization of pancreatic function was not due to regeneration of the vagus nerve,
because administration of 2-DG (75 mg/kg) failed to
increase protein secretion in rats with a chronic vagotomy (19). In contrast to rats with intact vagus
nerves, ICV microinjections of CGRP (0.3 and 0.6 nmol)
failed to inhibit CCK-stimulated pancreatic protein
secretion in rats with a chronic vagotomy (Fig. 7B).
Conversely, local denervation by dissecting part of the
pancreas away from its connection with the duodenal
wall (interruption of enteropancreatic neural pathways) completely abolished pancreatic responses to
CCK-8 after chronic vagotomy (Fig. 7B). We have
Fig. 5. Effect of ICV administration of CGRP on pancreatic protein
output stimulated by bethanechol (Bet). Bethanechol (0.3 mg/kg) was
given subcutaneously at the end of a 30-min basal collection period.
ICV injection of saline or CGRP (0.6 nmol) was administered 15 min
before basal collection. Values are means 6 SE for 5 rats in each
group.
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Intravenous administration of phentolamine, an a-adrenergic receptor antagonist, or chemical sympathectomy by guanethidine significantly increased basal
pancreatic protein secretion by 15 6 2 and 15 6 3%
(P , 0.01), respectively, but did not alter the inhibitory
effect of ICV injection of CGRP (0.3 nmol) on 2-DG-,
CCK-, and maltose-induced pancreatic protein secretion (Fig. 6). This indicates that the inhibitory effect of
CGRP on stimulated pancreatic secretion is not mediated by the sympathetic nervous system.
CENTRAL CGRP INHIBITS PANCREATIC SECRETION
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Fig. 6. Guanethidine (Gua) or phentolamine (Phe) failed to reverse
central CGRP-induced inhibition of pancreatic secretion stimulated
by 2-DG, CCK, or maltose. 2-DG and CCK were given intravenously,
and maltose was given intraduodenally at the end of a 30-min basal
collection period. Guanethidine (20 mg/kg ip) was administered once
daily for 2 consecutive days. Intravenous infusion of phentolamine (1
µmol · kg21 · h21 ) was given 30 min before basal collection. ICV
administration of CGRP (0.3 nmol) was given 15 min before basal
collection. Values are means 6 SE for 5 rats in each group. * P , 0.01.
previously shown that the local surgical denervation of
the pancreas did not alter CCK-stimulated pancreatic
protein secretion in rats with intact vagus nerves (19).
DISCUSSION
We have shown that vagal afferent pathways represent the primary targets on which postprandial mediators (e.g., CCK and luminal stimuli) act to stimulate
pancreatic secretion (17, 18, 20–22). In addition, we
have shown that central vagal pathways are the key
sites on which peptides, such as somatostatin (21) and
CGRP (16), act to inhibit pancreatic secretion. Messmer
et al. (25) have shown that ICV administration of CGRP
inhibits basal pancreatic secretion in the conscious rat
and the effects are mediated by the sympathetic nervous system (25). The objective of the present study was
to provide direct evidence that CGRP in the CNS
inhibits stimulated pancreatic enzyme secretion and to
investigate the mechanism.
In the present study, we found that ICV administration of CGRP was highly effective in inhibiting pancreatic protein secretion evoked by 2-DG and CCK-8.
Fig. 7. A: pancreatic secretion in response to CCK after acute
vagotomy and chronic vagotomy. B: effects of ICV administration of
CGRP (0.6 nmol) and local denervation by dissecting part of the
pancreas away from its connection with the duodenal wall on
pancreatic response to CCK-8 in rats with a chronic vagotomy. Values
are means 6 SE for 5 rats in each group. * P , 0.01.
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2-DG, a well-known central vagal stimulant, acts by
competing with glucose for cell membrane transfer,
causing intracellular glucopenia, especially at the hypothalamus. The resultant hypothalamic glucopenia excites the dorsal nucleus of vagus nerve and stimulates
vagus nerve transmission (28). We confirmed that 2-DG
acts through the vagal cholinergic pathway by demonstrating that pancreatic secretion induced by 2-DG
could be completely abolished by administration of
atropine or by truncal vagotomy (21). In the present
study, ICV administration of CGRP at doses of 0.3 and
0.6 nmol produced 88 and 100% inhibition of pancreatic
protein secretion stimulated by 2-DG, respectively. Our
recent studies (22) have shown that CCK at physiological levels acts through stimulation of vagal afferent
pathways, whereas CCK at supraphysiological levels
acts directly on pancreatic acini to induce pancreatic
enzyme secretion. In this study, CCK-8, at a dose of 40
pmol · kg21 · h21, produced an 80% increase in protein
secretion over basal and a plasma level similar to the
peak postprandial level observed in rats (22). Central
administration of CGRP at 0.3 nmol produced a 94%
inhibition of pancreatic protein secretion induced by
CCK-8 at physiological doses. This inhibition appears
to be directed at a pre-acinar site, because direct
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CENTRAL CGRP INHIBITS PANCREATIC SECRETION
duodenal bicarbonate secretion (14) and basal pancreatic secretion in conscious rats (25) by activation of
sympathetic efferents with subsequent release of norepinephrine, which acts on a-adrenergic receptors. To
determine if the inhibitory actions of central CGRP on
stimulated pancreatic secretion are mediated by the
sympathetic nervous system, we evaluated the effects
of guanethidine and phentolamine on the action of
CGRP. Intravenous administration of phentolamine,
an a-adrenergic receptor antagonist, or chemical sympathectomy by guanethidine, increased basal pancreatic protein output by 15%, but did not alter the
inhibitory effects of CGRP on pancreatic secretion
stimulated by 2-DG, CCK-8, intraluminal maltose, or
hyperosmolar NaCl. This observation indicates that
the inhibitory action of CGRP is not mediated by the
sympathetic nervous system. Therefore, the mechanisms that mediate central CGRP to inhibit pancreatic
secretion under basal conditions in conscious rats and
in response to meal-related stimuli under our experimental conditions are different. It is conceivable that
the mechanisms of action of CGRP in conscious and
anesthetized rats are different. Alternatively, neural
release of norepinephrine may be adequate to inhibit
basal secretion but insufficient to reduce stimulated
pancreatic secretion. In our present study, central
CGRP inhibited pancreatic secretion in response to
2-DG and CCK-8 in rats with intact vagus nerves but
failed to inhibit the response in rats with a chronic
vagotomy, in which CCK acts through enteropancreatic
neuropathways to mediate pancreatic secretion (19).
This suggests that central CGRP inhibits the stimulation arising from central or afferent vagal sites through
a pathway primarily dependent on the vagus nerves.
Central CGRP probably inhibits vagal tone and reduces
vagal stimulation to the pancreas. Note that the central
inhibitory action of CGRP on gastric acid secretion is
also not mediated by the sympathoadrenal axis. A
vagally mediated action is suggested by the abolition of
inhibitory effects of central CGRP in rats with a
vagotomy (15, 33).
Electrophysiological studies in the rat indicate that
the central action of CGRP on the stomach is mediated
through vagal pathways. Intracisternal injection of
a-CGRP inhibits unit efferent discharges recorded from
the gastric branch of the vagus nerve in a dose- and
time-dependent manner (36). In situ hybridization
studies (1, 27, 29) have revealed the distribution and
expression of a-CGRP mRNA in the hypothalamus and
various brain stem nuclei, including the nucleus ambiguus. The demonstration of CGRP-like immunoreactivity and receptors in the lateral hypothalamus, the
nucleus of solitary tract, and the nucleus ambiguus (8,
29, 30, 31) also provides anatomic support for a possible
role of CGRP in the modulation of parasympathetic
outflow.
In summary, we have demonstrated that CGRP in
the CNS inhibits pancreatic secretion stimulated by
2-DG, CCK-8, and non-CCK luminal stimuli, which act
through vagal pathways. The inhibitory action of CGRP
is not mediated by the sympathetic nervous system but
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stimulation of the muscarinic receptor by bethanechol
was not affected by ICV administration of CGRP at a
dose as high as 0.6 nmol.
Previously, we reported (16) that peripheral administration of CGRP at a dose of 25 µg (6.5 nmol) or 50 µg ·
kg21 · h21 completely inhibited 2-DG- and CCK-8stimulated pancreatic enzyme secretion. In our current
study, pancreatic protein secretion was inhibited with
ICV-administered CGRP, at smaller doses compared
with the doses administered peripherally. Complete
inhibition of 2-DG-stimulated pancreatic enzyme secretion was obtained with an ICV injection of CGRP at 0.6
nmol, a dose 10 times less than the dose administered
peripherally. An ICV-administered dose of CGRP at 0.3
nmol completely blocked pancreatic protein secretion
induced by CCK-8 (40 pmol · kg21 · h21 ), a dose 40 times
less than the peripherally administered dose. Therefore, although there is evidence that ICV-administered
neuropeptides can rapidly diffuse into the circulation
(26), the inhibitory effect of ICV injection of CGRP
represents a CNS-mediated action. This effect is not
due to the leakage of the peptide from the central to
peripheral circulation, as only ,8% of the centrally
administered CGRP leaked from the lateral ventricle
into the circulatory system of the rat (3).
Apart from CCK, we used non-CCK-dependent luminal factors to activate the vagal afferent pathway.
Luminal factors such as osmolarity, volume distension,
and nutrients also play a major role in the intestinal
phase of pancreatic secretion. Recent studies in rats
(18, 20) have shown that stimulation of duodenal osmoreceptors, mechanoreceptors, or chemoreceptors induced
pancreatic enzyme secretion through a CCK-independent cholinergic pathway. Perivagal and duodenum
mucosal pretreatment with capsaicin impaired pancreatic responses to duodenal stimuli such as hyperosmolar NaCl solution or maltose. This indicates that nonCCK-dependent luminal stimuli act through duodenal
mucosal vagal afferent fibers to stimulate pancreatic
secretion. We showed that ICV administration of CGRP
completely abolished the pancreatic secretion induced
by intraduodenal infusion of maltose or hyperosmolar
NaCl solution. This observation suggests that cerebral
CGRP can inhibit pancreatic stimulation, which is mediated by the afferent vagal pathways that can be activated
by CCK- and non-CCK-dependent luminal stimuli.
Pancreatic protein secretion induced by electrical
stimulation of the peripheral cut end of the vagus nerve
was markedly inhibited by atropine, indicating that it
is mainly mediated by a cholinergic pathway. This
mode of stimulation directly activates the vagal efferent pathway, bypassing the vagal afferent and central
sites. Cerebral CGRP at a dose of 0.3 and 0.9 nmol was
ineffective in inhibiting pancreatic protein secretion
induced by vagal electrical stimulation, suggesting that
central CGRP is acting at a site proximal to the vagal
efferent pathway and therefore does not inhibit the
release of neurotransmitter from the vagus nerve ending in the pancreas.
CGRP is a potent CNS stimulator of noradrenergic
sympathetic outflow (7) in the rat. CGRP inhibits
CENTRAL CGRP INHIBITS PANCREATIC SECRETION
appears to be mediated by modulation of parasympathetic outflow.
The investigation was supported in part by National Institute of
Diabetes and Digestive and Kidney Diseases Grants R01 DK-32838
and P30 DK-349333.
Address for reprint requests: C. Owyang, 3912 Taubman Center,
Box 0362, Univ. of Michigan Medical Center, Ann Arbor, MI 48109.
Received 3 September 1997; accepted in final form 29 June 1998.
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