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Effect of pituitary adenylate cyclase-activating polypeptide on exocrine and endocrine secretion in the ovine pancreas
Comp. Biochem. Physiol. Vol. 115C, No. 3, pp. 185-193, CopyrIght 0 1996 Elsevier Science Inc.
1996
ISSN 0742-8413/96/$15.00 PI1 SO74208413(96)00078-3
E.L.SEWER
Effect of Pituitary Adenylate Cyclase-Activating Polypeptide on Exocrine and Endocrine Secretion in the Ovine Pancreas Tukenori Onaga, * Megumi Uchidu, ’ Musanori Kimura, ’ Masato Miyazaki,’ Hitoshi Mineo,’ Seiyu Kate,’ and Romuukf Zabielsk? ‘DEPARTMENTOF VETERINARY MEDICINE,RAKUNO GAKUEN UNIVERSITY,EBESTU,HOKKAIDO069, JAPAN; AND ‘DEPARTMENTOF ANIMAL PHYSIOLOGY, SCHOLLOF VETERINARY MEDICINE,WARSAW AGRICULTURAL UNIVERSITI, NOWOLJR~YNOWSKA 166, 02-787 WARSAW, POLAND
ABSTRACT. exocrine
The role of pituitary
and endocrine
PACAP-
adenylate
pancreas was investigated
(1, 3, and 10 pmol/kg/ min)
complex
accelerated
pancreatic
protein
cyclase-activating
polypeptide
in conscious
sheep. Intravenous
at the highest doses were inhibited
Vasoactive
polypeptide
outputs, although VIP increased highest
(VIP)
pancreatic
juice flow and bicarbonate
pmol/kg) did not influence
PACAP-
(l-100
pmol/kg) altered neither
accelerated
pancreatic
protein
of insulin,
endocrine
but not amylase
influenced by atropine. however,
On the other hand, intravenous
basal plasma concentration
myoelectric
The responses in enzyme secre-
output dose-dependently;
by atropine.
migrating
glucagon,
PACAP-
and
the responses
to the
injection
of PACAP-
and glucose.
to accelerate Inc.
COMP
of exocrine
pancreatic
BIOCHEM
of the ovine pancreas but not to endocrine
enzyme secretion
PHYSIOL
KEY WORDS. PACAP,
secretion
mostly via the cholinergic
115c;3:185-193.
contributes
PACAP
appears
1996 Efseoier Science
1996.
VIP, sheep, ovine, pancreas, exocrine,
INTRODUCTION Pituitary adenylate CAP) was originally
secretion.
nerves. Copyright 0
Moreover,
infusion of glucose
response to intravenous
(20 pmol/kg/min) nor that to n-butyric acid (33 pmol/kg/ min). These results suggest that PACAP to the regulation
of and
by atropine infusion (14.4 nmol/kg/min).
significantly
the response to the highest dose was not significantly
dose were not altered significantly
38 (100
significantly
at 3 pmol/kg/min
in the regulation
infusions of PACAP-
for 10 min during phase II of the duodenal
and amylase outputs dose-dependently.
tion to both PACAPs intestinal
(PACAP)
islets, cholinergic
nerve, short-chain
fatty acids
differ from that in electrolyte secretion (3,26,28), cyclase-activating polypeptide (PAisolated from ovine hypothalamic ex-
tracts (24). PACAP possesses two types of molecular form: PACAPand PACAP-38, which are classified in the secretin-glucagon family because of a high homology with vasoactive intestinal polypeptide (VIP) and secretin in amino acid sequence (4). Although
PACAP
was found in the cen-
tral nervous system for the first time, it has been demonstrated that PACAP-immunoreactive fibers are distributed widely in the gastrointestinal tract and pancreas in several species, including sheep (5,19,34,35). In the digestive tract, PACAP has been shown to stimulate pancreatic exocrine secretion in rats (3,16,26,30) and dogs (28). The order of molar potency of PACAP and the related peptides in stimulating pancreatic enzymes seems to
Address reprint requesrs to: Takenori Onaga, Department of Veterinary Medicine, Rakuno Gakuen University, Bunkyo-dai Midori-machi 582-1, Ebetsu, Hokkaido 069, Japan. Tel 81-11-386-1112, ext. 4336; Fax 81-ll387-5890. Received 7 July 1995; accepted 17 April 1996.
suggesting
that a different mechanism is involved in each of the secretory actions. Recently, the stimulatory effect of PACAP on exocrine
pancreas
was demonstrated
VIP in preruminating
calves (40),
in comparison
indicating
with
that PACAP
plays a physiological role in the regulation of exocrine pancreas in neonatal ruminants. However, although PACAPimmunoreactive fibers were found in mature sheep (19), the effect of PACAP on ovine exocrine pancreas has not been examined. In dogs, the stimulatory effect of PACAP on the exocrine pancreas is blocked by atropine (28). Although the regulation of exocrine pancreas was shown to depend greatly on the cholinergic nerve in mature sheep (17) and on the vagus nerve in ruminating calves during the interdigestive period (39), it is still unknown whether PACAP acts on exocrine pancreas directly or indirectly via the cholinergic nerve in ruminants. On the other hand, it was demonstrated for the first time that PACAP stimulates endocrine pancreas in mice in viva (lo), in which PACAPat a comparatively high dose increased basal insulin, glucagon secretion, and carbacholstimulated glucagon secretion while it inhibited carbachol-
T. Onaga et al.
186
stimulated insulin secretion. Subsequently it was shown that PACAPaugmented glucose-induced insulin secretion at a concentration in the nanomolar range in isolated
of Oddi and another into the duct between the pancreas and gallbladder. A third silastic cannula (3 mm I.D., 5 mm O.D.) was introduced into the duodenum at a point more
perfused rat pancreas (25), and that PACAPat concentrations lower than the picomolar range also stimulated in-
than 7 cm caudad to the electrode. The three cannulae and
sulin release from rat islets in a glucose-dependent
tems, Inc., Everett, WA, USA) of the electrode were exteriorized from the right flank. Bile and pancreatic fluid were
manner
(37). These observations suggest that PACAP may play a physiological role in the regulation of endocrine pancreas as well as in that of exocrine pancreas. It should be noted that PACAP potentiates glucose-stimulating insulin secretion at concentrations lower than the picomolar range. In contrast to monogastric animals, in which blood glucose is the main source of energy, ruminants use short-chain fatty acids (SCFAs) as a major energy source, and ovine pancreatic islets respond to not only glucose but also SCFAs
(11).
The effect of n-butyric acid was most potent on both insulin and glucagon secretion among SCFAs in sheep (23). However, there is no information concerning the effect of PACAP on the ovine endocrine pancreas, especially on SCFAinduced insulin and glucagon secretion in ruminants. Therefore, the present study was performed to examine (a) the effect of PACAP-27,
PACAP-38,
and VIP on the
ovine exocrine pancreas and the contribution of the cholinergic nerves to their action; and (b) the effect of PACAP on basal and nutrient-stimulated secretion of insulin and glucagon in conscious sheep. In the latter experiment, PACAP-38 was used because the molar potency of PACAP38 in stimulating insulin release was shown to be greater than that of PACAPin rats (25,38), and glucose and nbutyric acid were used as stimulants
MATERIALS Animals
AND
in blood.
METHODS
the wires (Teflon-coated
stainless steel, 7 strd., A-M Sys-
collected in a glass bottle and were always returned into the duodenum via a peristaltic pump (SJ-1220, Atto, Tokyo, Japan) at the rate of the secretions. In sheep in the endocrine group, the left common carotid artery was carefully divided from the vagus nerve and surrounding tissue under pentobarbital anesthesia (2 5 mg/kg IV) and then wrapped chronically length of about 12 cm. Penicillin
(procaine
penicillin
in a skin loop with a G-Meiji,
Meiji-Seika,
Tokyo, Japan) treatment was continued for 3 days after surgery. The animals were allowed a recovery period of 1 week in the crates and were trained to remain in a standing position for 3 h. All sheep showed normal appetites l-3 days after surgery. An indwelling catheter was inserted into a jugular vein for administration at least 1 h before the start of the experiment. Another catheter was also inserted into the carotid loop for blood collection in the endocrine group. These catheters were filled with a sterilized solution of 3.8% (w/v) trisodium citrate to prevent coagulation.
Effect of PACAP
and VIP on Exocrine
Pancreas
PACAP(human, ovine, rat), PACAP(human, ovine, rat), and VIP (human, porcine) were purchased from Peptide Institute, Inc. (Osaka, Japan). These peptides were
Twelve Suffolk-strain male sheep weighing 34-53 kg were used. All animals were treated according to the Laboratory
dissolved in a sterilized 154 mM NaCl solution containing 0.02% bovine serum albumin (fraction V, RIA grade, Sigma, St. Louis, MO, U.S.A.) at 10 ,uM, divided into small
Animal Control Guidelines
amounts,
of Rakuno Gakuen University,
and kept at -40°C.
The
peptide solution
was
which are basically in conformity with the American Association of Laboratory Animal Control Guidelines from the National Institutes of Health.
thawed at 4°C on the day of the experiment and diluted appropriately with a sterilized isotonic solution of NaCl for administration.
The animals were kept in individual crates and were fed hay (100 g) and lucerne pellets (2.5% of body weight) at 19:00 once per day and allowed free access to water, except The animals were divided into exo-
The animals were kept in a standing position during the experiment. Duodenal myoelectric activity was recorded with an amplifier and a thermal recorder (types AB-601G and RTA-1 lOOM, Nihon-Koden, Tokyo, Japan) and
crine and endocrine groups, which consisted of eight and four sheep, respectively. Before surgery, the animals were fasted for 1 day. Following premeditation with atropine (0.05 mg/kg, IM) and xylazine (0.5 mg/kg, IM), the animals in the exocrine group were anesthetized by halothane-oxygen gas through an intratracheal cannula. The animals were equipped with a bipolar silver electrode by a suture on the duodenum close to
MacLab System (A-D Instruments Pty Ltd., Castle Hill, Australia). Pancreatic juice was collected every 5 min during the experiment. After measuring the volu~~w, 120 ~1 of the juice was taken for analysis and the remainder was returned to the collection bottle. In sheep, pancreatic exocrinc secretion changes cyclically, corresponding with duodenal migrating myoelectric activity (MMC) during the interdigestive period (Onaga, unpublished data); a similar
the opening of the common bile duct. Two silastic cannulae (2 mm I.D., 4 mm O.D.) were inserted into the common bile duct at two points: one into the duct near the sphincter
pattern is seen in humans and other species (l&9,12,21, 22,36,39). It was also observed that intravenous infusion of CCK was most effective during phase 1 of MMC on exocrine
during the experiment.
PACAP
pancreas
187
and Ovine Pancreas
in milk-fed
calves (40).
tides were infused intravenously
Consequently,
the pep-
for 10 min starting from
10 min after the end of duodenal regular spiking activity (phase III). The doses used were 1,3, and 10 pmol/kg/min. In the experiment on cholinergic blockade, atropine was infused intravenously at 14.4 nmol/kg/min for 80 min from 10 min after the end of duodenal phase III, and the peptide was infused at 10 pmol/kg/min for 10 min starting 30 min after the onset of the atropine infusion. Recording of duodenal MMC and collection of pancreatic juice were continued until 50 min after the end of the peptide infusion. The infusion rates were constant
at 0.05 ml/kg/min.
Protein concentration and amylase activity of the pancreatic juice from -30 to 50 min were determined by the
stored at -40°C
until radioimmunoassay.
The plasma con-
centration of insulin was determined by the method of Sasaki and Takahashi (31) using guinea pig anti-bovine insulin serum (ICN, Israel) and “‘I-insulin (NEN Research Products, Boston, MA, USA). Intra- and interassay coefficients of variation for the insulin determination were 5.8% (n = 6) and 8.6% (n = 5), respectively. Plasma glucagon was determined using dextran-charcoal, 1251-glucagon (NEN Research Products, Boston, MA, USA), and antiserum OAL-123, which was specific to the carboxyl-terminal portion of pancreatic glucagon (29), as described previously (32). Intra- and interassay coefficients of variation for the glucagon
determination
were 8.5%
(n =
6) and 10.6%
(n = 5), respectively.
methods of Lowry et nl. (20) and Bernfeld (7) as modified by Kanno (15), respectively. the pancreatic
The bicarbonate
juice from -10
concentration
of
to 50 min was measured by
Statistical
Analysis
PACAPsolution was prepared as described previously. Glucose was dissolved at 0.4 M in a sterilized 154 mM sodium chloride solution. n-Butyric acid (Wako Pure Chemical, Osaka, Japan) was diluted to a concentration of 2.0 M
Pancreatic protein, amylase, and bicarbonate output were calculated from protein concentration, amylase activity, and bicarbonate concentration, respectively. The values represent mean -c- SEM as absolute values or increments to values immediately before administration. Data were compared either between before and after the onset of infusion, or between the control and peptide infusions at the same time, using paired and unpaired t-tests for statistical analy-
and adjusted to pH 7.4 with 1 N NaOH. The solution was
sis, in which p < 0.05 was considered
titration.
Effect of PACAP-
administered
on Endocrine
after appropriate
Pancreas
dilution
significant.
with the sterilized
isotonic NaCl solution. Three milliliters of arterial blood was withdrawn from the carotid artery at -20, -10, 0, 5, 10, 15, 20,30, 50, and 60 min. To examine the effect of PACAPalone, PACAP38 solution was injected at 100 pmol/kg into the jugular vein at 0 min, followed by an intravenous infusion of the
RESULTS Effect on the Exocrine
Pancreas
Pancreatic juice flow was lowest during the period of duode-
isotonic NaCl solution at 7.5 pmol/kg/min (0.05 ml/kg/ min) from 1 min to 11 min. The NaCl solution was infused
nal phase I, which corresponded to the time from -10 to -5 min in Fig. 1. The flow increased gradually, coinciding with the increase of duodenal spiking activity in controls. In comparison with the control, pancreatic juice flow in-
as a control. To examine
creased rapidly during intravenous infusion of PACAP-27, PACAP-38, and VIP and decreased immediately after ces-
the effect of PACAP-
butyric acid-induced
on glucose- or n-
insulin and glucagon
secretion,
PA-
CAP-38 was injected at 1, 3, 10,30, and 100 pmol/kg into the jugular vein at 0 min, followed by an intravenous infusion of glucose at 20 pmol/kg/min or n-butyric
acid at 33
sation of the peptide infusions (Fig. 1). The increments in juice flow were augmented dose-dependently, and those at the highest dose were statistically significant in all the peptide infusions (Fig. 2). The effect of PACAPand PACAP-
was most
pmol/kg/min from 1 min to 11 min. In a preliminary experiment, the dose of glucose used was confirmed to elevate plasma concentration of glucose by 20 mg/dl. The dose of n-butyric acid used was the lowest effective dose among 1, 3.3, 10, 33, and 100 pmol/kg/min to elevate plasma con-
potent,
and VIP were equipotent
at the
centration of insulin in sheep. Blood samples were immediately transferred into a tube containing EDTA at 1.2 mg/ml blood and kept at 4°C. Plasma was separated by centrifugation at 27,200 X g for 10 min at 4°C after the end of the experiment. The glucose concentration was determined by the glucose-oxidase method (14) using 10 ~1 of plasma in a glucose analyzer (GLU-11, TOA, Tokyo, Japan). The remaining plasma was combined with benzamidine (50 pmol/ml plasma) and
the control period (Fig. 1). Although the juice flow at nadir clearly increased in response to the peptides, the increment in PACAPinfusion at 10 pmol/kg/min was inhibited significantly by atropine (Fig. 2). Atropine did not significantly inhibit either the response in juice flow to PACAP27 or that to VIP, although it caused a slight decrease in increment of juice flow to PACAP(Fig. 2). Pancreatic protein and amylase output showed similar responses to PACAPand -38 (Fig. 1). The output of both
highest dose. It is notable that only PACAPat 1 pmol/ kg/min inhibited the juice flow, although the change was not significant. Intravenous infusion of atropine (14.4 nmol/kg/min) effectively decreased the juice flow during
T. Onaga et al.
188
-Ck Control * 1Oprllol -l-J 1Opmol in atropine infusion
(4
1207
w
@I
PACAP
‘21
y
PACAP
+zj
PACAP
i
FIG. 1. Effect of intravenous w :
:
VIP ::
80 40 0 -30 -20 -10 0
10 20 30 Time (min)
G>
3.0
fl
40
50
-30 -20
-10 0
10 20
30
40
50
Time (min)
@I
PACAP
1201
yzz~zy PACAP
120-
/=?+
1 2.0
1.0 0.0 i
P
i :&
;
/
6
PACAP
W
i i
PACAP : i
j?l=zj VIP : : 40 -30 -20 -10
0
10
20
Time (min)
30
40
50
0
-10
0
10
20
30
Time (min)
40
50
infusion of PACAP-27, PACAP-38, and VIP on pancre= atic exocrine secretion in conscious sheep. The results show the response in (A) pro= tein output, (B) amyIase out4 put, (C) pancreatic juice flow, and (D) bicarbonate output to peptide infusion for 10 min at 10 pmol/kg/min with and without atropine. Atropine was infused at 14.4 nmol/kg/ min from 30 min before the peptide infusion. Values represent mean L SEM (n = 7 in PACAPand VIP, R = 6 in PACAP.38). Closed marks indicate significant ( P < 0.05) differ&&s from the control of physiological saline info* sion.
189
PACAP and Ovine Pancreas
I a
Control 1pmol/kg/min
m
3pmobkglm in
1Opmol/kg/min 1OpmoJ/kg/minin atropine infusion at 14.4nmof/kg/min *
J
vIP -“.5PACAP27~PACAP38i FIG. 2. Effect of intravenous infusion of PACAP-27, PACAP-38, and VIP on pancreatic exocrine secretion in conscious sheep. The values represent increments during 10 min of the infusion period in comparison with values during 10 min immediately before the infusion. The peptides were infused intravenously at 1,3, and 10 pmollkglmin for 10 min with or without atropine. Open and closed circles indicate significant (p < 0.05) differences in increment from the control and from 10 pmol/kg/min and VIP, n = 6 in PACAP-38). without atropine, respectively. Values represent mean + SEM (n = 7 in PACAP-
increased dose-dependently in response to infusion of PACAP-27 and -38 (Fig. 2). The increments at the highest dose were evidently blocked by the atropine infusion, whereas the responses did not disappear completely upon atropine infusion (Fig. 1). As well as the juice flow, protein and amylase output also decreased during PACAP-
sponse was clearly delayed compared with that of protein and amylase outputs. The increments in bicarbonate output in response to PACAPand VIP at the highest dose decreased slightly during atropine were not significant.
infusion,
but the changes
infu-
sion at 1 pmol/kg/min. As was the case with PACAP, 3 pmol of VIP significantly accelerated the protein output, and the increment was comparable to the response to 10 pmol of PACAP-27. Although the highest dose of VIP raised the juice flow slightly, it did not further increase protein output. The increment of protein output at the highest dose of VIP was not influenced by the atropine infusion. The pattern of the response of amylase output to VIP resembled that of protein output, though the changes were not significant at all. Bicarbonate secretion was accelerated by PACAPand VIP, but not by PACAP-38. The greatest response was obtained in infusion of PACAPat 10 pmol/kg/min (Figs. 1, 2) as well as in juice flow, although the peak of the re-
Effect 071the Endocrine
Pancreas
Intravenous injection of PACAPat 100 pmol/kg/min did not alter the plasma concentration of insulin, glucagon, and glucose in unstimulated periods (Fig. 3). Intravenous infusion of glucose at 20 pmol/kg/min significantly increased the plasma concentration of glucose and insulin (Fig. 4), whereas the plasma concentration of glucagon did not change. PACAPinjections at doses from 1 to 100 pmol/kg prior to glucose infusion did not alter insulin responses to glucose. The plasma concentrations of insulin and glucagon rose effectively during intravenous infusion of n-butyric acid at 33 pmol/kg/min without detectable change in plasma glu-
190
T. Onaga et al.
-c-o-
control
protein secretion
PACAP38-1 OOpmoVkg
potency in pancreatic shown to be PACAP-
saline infusion
in dogs at all. In rats, the order of molar protein and amylase output was > PACAP2 VIP (26). How-
ever, the order in protein and amylase secretion in sheep was VIP > PACAPz PACAP-38, which was obviously different from the order in dogs and rats. The effect of VIP on acceleration of protein output in sheep was especially noteworthy, where it seems to be more potent than PACAP, because the increment of protein output to 3 pmol of VIP is comparable to that to 10 pmol of PACAP. However, the highest dose of VIP only slightly accelerated the juice flow, whereas it did not further increase the protein output. The protein response to the highest dose of VIP was not inhibited by atropine. These results suggest that the effect of VIP on enzyme secretion
does not depend on the
cholinergic nerves, and the mechanism is likely to be different from that of PACAP. The mechanism of the action of VIP remains to be clarified. In contrast to VIP, PACAPand PACAPincreased protein and amylase output in sheep, and the responses to the highest dose were inhibited
by atropine. These results
in sheep coincided with those in dogs (28), suggesting that PACAPand PACAPstimulate pancreatic enzyme secretion by indirect action via cholinergic nerves by acting on PACAP-27/-38 preferring receptors in these species. Of course, we cannot rule out the direct action of PACAP on the exocrine
Time (min) FIG. 3. Effect of PACAP-
on the plasma concentrations
of insulin, glucagon, and glucose in conscious sheep. PACAP-38 was injected rapidly at 0 min into the jugular vein at a dose of 100 pmollkg, followed by intravenous infusion of physiological saline from 1 min to 11 min. Values represent mean f SEM (n = 4).
case (Fig. 4). However, as was the case with glucose infusion, PACAPinjections altered neither the response of plasma insulin nor glucagon to n-butyric acid.
DISCUSSION
The present study provides clear evidence that both molecular forms of PACAP increase the interdigestive secretion of the exocrine pancreas in mature sheep. PACAP has been shown to stimulate pancreatic exocrine secretion in viva in rats (26), dogs (28), and calves (40). Receptors for VIP and PACAP have been classified into PACAP-I receptors preferring only PACAP, and VIP/PACAP-II receptors preferring both VIP and PACAP, according to in vitro radioreceptor assays using rat pancreas (34). In conscious dogs, the molar potency in pancreatic protein secretion was highest for PACAP-27, and the potency of PACAPwas onetenth of that of PACAP(28). VIP did not increase the
pancreas because the response to PACAP,
al-
though slight, remained during atropine infusion at doses sufficient to inhibit cholinergic transmission in the exocrine pancreas (17). However, the action of PACAP
on pancre-
atic enzyme secretion appears to depend greatly on the cholinergic nerves. Atropine decreased the increment of juice flow during PACAPinfusion, which might be due to the cholinergic dependence of the action of PACAPon enzyme secretion. Although it remains unknown whether vagus cholinergic nerves or pancreatic intrinsic cholinergic nerves mediate in the action of PACAP, both lZ51-PACAP27 and “‘I-PACAPinjected intravenously were shown to enter the brain, passing through the blood-brain
barrier
in the mouse (6), suggesting that PACAP may act on the central nervous system to activate vagus cholinergic nerves. Molar potency in pancreatic fluid and bicarbonate secretion was highest for secretin, intermediate for PACAPand VIP, and lowest for PACAPin conscious dogs (28). Although the effect of secretin was not tested in the present study, the order of the molar potencies of the peptides in juice flow and bicarbonate output in sheep was similar to that in dogs. In sheep, the effects of PACAPand VIP were similar and were not altered significantly by atropine, but PACAPscarcely stimulated bicarbonate secretion. Bicarbonate response to the highest dose of PACAPseems to decrease during atropine infusion, as shown in Fig. 2, although the change was not significant. And the bicarbonate response to the highest dose of PACAPwithout atropine was apparently delayed in comparison with the
PACAP
and Ovine
Pancreas
3
75-
.?
50-
j
25-
-=
0
+
control
--b-
PACAP
10pmoVkg
-
PACAP
lpmol/kg
---Bi-
PACAP
30pmoVkg
-c-
PACAP
3pmoVkg
+
PACAP
1OOpmollkg
1 -
loo-
E
191
&zzQ4
glucose (20 p mol/kg/min)
: n-butyrate
h
(33 p moVkg/min)
E
3
Y
I
E 300aI Js 200s 3 loo% E On
I
.
7
r
I
I
I
I 600 400 200
I
aloo
I :
I *
:
1
I
I
I
I
I
: 2
g;_
% 8
25-
3
0
75
.
50 25 1 -20
I
,: 0
I;
I 20
1
I 40
I
1
60
Time (min)
* I
-20
I
I
0
I
20
I
I
40
1
I
60
Time (min)
FIG. 4. Effect of PACAPon plasma concentrations of insulin, glucagon, and glucose during intravenous infusion of glucose or n-butyric acid in conscious sheep. PACAPwas injected rapidly at 0 min into the jugular vein at 1, 3, 10, 30, and 100 pmollkg, followed by intravenous infusion of glucose or n-butyric acid from 1 min to 11 min. The values represent means from four animals. PACAPinjection did not significantly alter either the endocrine response to glucose or the response to nebutyric acid.
course of the cholinergic-dependent
responses of prodepended on
bicarbonate secretion differed among these species are unknown. Also, the data obtained in the present study are not
cholinergic nerves, bicarbonate would have responded as quickly as did protein and amylase. Consequently, the ac-
sufficient to explain the difference between the cholinergic dependence of the stimulatory effects of PACAP and of VIP
tion of PACAPon bicarbonate secretion does not seem to depend on the cholinergic nerves in sheep as well as in dogs. Thus, it was suggested that PACAPand VIP stim-
on enzyme secretion because of species differences and complexity of in viva experiments involving pharmacokinetics
ulate water and bicarbonate secretion via PACAP-27/VIP preferring receptors that probably exist on pancreatic duct
peptides (2,27,33), and other new types of receptors, such as the apamin-sensitive receptor found in the muscle cells of
cells in sheep and dogs. On the other hand, in neonatal
guinea pig tenia coli (13).
calves, the order of the molar potency in bicarbonate output was also PACAP> PACAP> VIP (40), but the bicarbonate responses to PACAPs were significantly reduced by atropine (R. Zabielski, unpublished data), suggesting that the effect of PACAP on bicarbonate secretion is also mediated by cholinergic nerves in calves. However, the reasons why the cholinergic dependence of PACAP on
receptors and PACAP-27/VIP receptors appear to be involved in the regulation of pancreatic exocrine secretion in sheep, although the full details of the mechanism of peptide actions remains to be clarified. In contrast to the obvious effects of the peptides on exocrine secretion, PACAPinfluenced neither unstimulated nor nutrient-stimulated secretion of insulin and gluca-
time
tein and amylase. If the action of PACAP-
of the peptides in blood, the cardiovascular
Both PACAP-27/-38
effect of the
preferring
T. Onaga et al.
192
gon in conscious
sheep. These
results are in contrast
to
observations in the rat and mouse, in which PACAPand PACAPincreased the basal secretion of islet hormones and augmented glucose-infused
secretion
of insulin
(10,25,37). In rats, PACAPwas demonstrated to increase intracellular concentration of Ca” in islet cells, and PACAP-27-induced insulin release was abolished by the Ltype Ca’+ channel blocker nitrendipine (37). PACAP-38, which also increased the intracellular concentration of Ca” in islet cells, stimulated PACAP-27.
insulin release equipotently
In the present study, however, PACAP-
with did
not solely alter the plasma concentrations of insulin, glucagon, and glucose. In contrast to monogastric animals, ruminants
utilize short-chain
fatty acids as a primary energy
source, and ovine pancreatic islets respond not only to glucose but also to SCFAs (11). Among SCFAs, n-butyric acid was most potent in stimulating insulin secretion in sheep (23). Hence, the effect of PACAPon the glucose- and n-butyric acid-induced hormone secretion by islets was examined. The doses of glucose and n-butyric acid used were submaximal, as per preliminary experiments. However, PACAP-38 did not significantly alter the response of insulin or that of glucagon to these nutrients in sheep. Therefore, it seems unlikely that PACAP is involved in the regulation of insulin and glucagon secretion
in sheep, which is sup-
ported by the observation that the intravenous administration of VIP did not influence the plasma concentration of insulin
and glucagon
in sheep
(H.
Mineo,
unpublished
data). Nevertheless, it should be noted that PACAPat the lowest dose decreased increments in all secretory parameters of the exocrine pancreas. Because endogenous insulin has been demonstrated to inhibit exocrine secretion in the ovine pancreas
(18)
in contrast
to rats, we cannot
com-
pletely rule out the possibility that an undetectable
change
in the local concentration
circula-
of insulin in islets-acinar
tion might occur. Although PACAP-immunoreactive innervate the ovine exocrine acini (19),
the role of PACAP
fibers were shown to and endocrine islets
in the regulation
pancreas has not yet been established.
of the ovine
The present study
demonstrated that PACAPand PACAPstimulate pancreatic enzyme secretion via the cholinergic nerves, whereas PACAPand VIP stimulate bicarbonate secretion, probably by direct action on the duct cells in the exocrine pancreas of sheep. However, PACAPinfluenced neither basal secretion nor nutrient-stimulated secretion of insulin and glucagon in sheep. Therefore, it is concluded that PACAP contributes to the regulation of exocrine secretion of ovine pancreas, but not to endocrine secretion.
of
We are grateful to Emeritis Professor N. Yanaiharu, University Shizuoka School of Pharmaceutical Science, Yad~,Shizuoka 422, Japan, for his supplyof glucagon antiserum OAL-123. The present study was partly funded by the Sasagawa Scientific Research Grant from The ]apan Scientific Society.
References 1. Abello, J.; Laplace, J.P.; Corring, T. Biliary and pancreatic
secretory component of the migrating myoelectric complex in the pig. Effect of intraduodenal pH. Reprod. Nutr. Dev.
28(4A):953-967;1988. 2. Absood, A.; Chen, D.; Wang, Z.-Y.; Hakanson, R. Vascular effects of pituitary adenylate cyclase activating peptide: A comparison with vasoactive intestinal peptide. Regul. Pept. 40:323-329;1992. 3. Alonso, R.M.; Rodriguez, A.M.; Garcia, L.J.; Lopez, M.A.; Calvo, J.J. Comparison between the effect of VIP and the novel peptide PACAP on the exocrine pancreatic secretion of the rat. Pancreas 9:123-128;1994. polypep4. Arimura, A. Pituitary adenylate cyclase-activating tide (PACAP): Discovery and current status of research. Regul. Pept. 37:287-303;1992. 5. Arimura, A.; Somogyvari-Vigh, A.; Miyata, A.; Mizuno, K.; Coy, D.H.; Kitada, C. Tissue distribution of PACAP as determined by RIA: Highly abundant in the rat brain and testes. Endocrinology 129:2787-2789;1991. 6. Banks, W.A.; Kastin, A.J.; Komaki, G.; Arimura, A. Passage of pituitary adenylate cyclase activating polypeptidc,_li and pituitary adenylate cyclase activating polypeptide,_i, across the blood-brain barrier. J. Pharmacol. Exp. Ther. 267:690696;1993. 7. Bernfeld, P. Amylase aand p. In: Colowik, S.P.; Kaplan, N.O. (eds). Methods in enzymology, volume 1. New York: Academic Press; 1955:149-158. 8. Boldyreff, W. Einige neue Seiten der Tatigkeit des Pancreas. Ergeb. Physiol. 11:121-217;1911. 9. DiMagno, E.P.; Hendricks, J.C.; Go, V.L.W.; Dozois, R.R. Relationships among canine fasting pancreatic and biliary secretions, pancreatic duct pressure, and duodenal phase III motor activity-Boldyreff revisited. Dig. Dis. Sci. 24:689-693;1979. 10. Fridolf, T.; Sundler, F.; Ahren, B. Pituitary adenylate cyclaseactivating polypepnde (PACAP): Occurrence in rodent pancreas and effect on insulin and glucagon secretion in the mouse. Cell Tissue Res. 269:275-279;1992. 11. Harmon, D.L. Impact of nutrition on pancreatic exocrine and endocrine secretion in ruminants: A review. J. Anim. Sci. 70: 1290-1301;1992. 12. Itoh, 2.; Takahashl, I.; Nakata, M.; Suzuki, T. Variation in canine exocrine pancreatic secretory activity during the interdigestive state. Am. J. Physiol. 241:098-G103;1981. 13. Jin, J.-G.; Katsoulis, S.; Grider, J.R.; Makhlouf, G.M. Apaminsensitive receptors on dispersed muscle cells of guinea pig tenia coli. Gastroenterology 106:A519;1994. 14. Kabasakalian, P.; Kalliney, S.; Westcott, A. Enzymatic blood glucose determination by calorimetry of N,N diethylaniline4-aminoantipyrine. Clin. Chem. 20:606&607;1974. 15. Kanno, T. The electrogenic pump in the hyperpolarizing and secretory effects of pancreozymin in the pancreatic acinar cell. 1. Physiol. 245:599-616;1975. T.; Kikuchi, Y.; Kolzumi, M.; 16. Kashimura, J.; Shimosegawa, Toyota, T. The stimulatory effect of PACAP 38 on amylase release in dispersed rat pancreatic acini. Tohoku J. Exp. Med. 164:309-318;1991. 17. Kato, S.; Adachi, N.; Ando, T.; Mineo, H.; Ushijima, J. The effect of intravenous infusions of atropine on pancreatic exocrine secretion in sheep. Jpn. J. Zootech. Sci. 56(11):905910;1985. 18. Kato, S.; Matsumoto, S.; Kojima, H.; Mineo, H.; Onaga, T.; Kikuchi, K. Effect of intravenous injections of insulin, glucose and tolbutamide on pancreatic exocrine secretion in sheep. Jpn. J. Zootech. SCI. 61(4):360-365;1990.
PACAP
and Ovine
Pancreas
19. Koves, K.; Arimura, A.; Vigh, S.; Somogyvari-Vigh, A.; Miller, J. lmmunohistochemical localization of PACAP in the ovine digestive system. Peptide 14:449-455;1993. 20. Lowry, O.H.; Rosebrough, N.J.; Farr, A.L.; Randall, R.J. Protein measurement with Folin phenol reagent. J. Biol. Chem. 193:265-275;1953. 21. Magee, D.F.; Naruse, S. Effect of pancreatic juice on basal pancreatic and gastric secretion in dogs. J. Physiol. 330:489496;1982. 22. Magee, D.F.; Naruse, S. Neural control of periodic secretion of the pancreas and the stomach in fasting dogs. J. Physiol. 344:153-160;1983. 23. Mineo, H.; Hashizume, Y.; Hanaki, Y.; Murata, K.; Maeda, H.; Onaga, T.; Kato, S.; Yanaihara, N. Chemical specificity of short-chain fatty acids in stimulating insulin and glucagon secretion in sheep. Am. J. Physiol. 267:E234-E241;1994. 24. Miyata, A.; Arimura, A.; Darl, R.R.; Minamino, N.; Uehara, A.; Jiang, L.; Culler, M.C.; Coy, D.H. Isolation of a novel 38 residue-hypothalamic polypeptide which stimulates adenylate cyclase in pituitary cells. Biochem. Biophys. Res. Commun. 164:567-574;1989. 25. Mochizuki, T.; Sato, K.; Ohshima, K.; Takahata, H.; Kakuyama, H.; Yanaihara, N. Insulinotropic activity of synthetic pituitary adenylate cyclase activating polypeptide (PACAP)related peptides. Biomed. Res. lJ(Supp1. 2):389-392;1992. 26. Mungan, 2.; Ertan, A.; Hammer, R.A.; Arimura, A. Effect of pituitary adenylate cyclase activating polypeptide on rat pancreatic exocrine secretion. Peptides 12:559-562;1991. 27. Naruse, S.; Sazi, T.; Ozaki, T.; Nokihara, K.; Wray, V. The effect of PACAP and VIP in isolated guinea pig pulmonary artery. Biomed. Res. 15:217-219;1994. 28. Naruse, S.; Suzuki, T.; Ozaki, T. The effect of pituitary adenylate cyclase activating polypeptide (PACAP) on exocrine pancreatic secretion in dogs. Pancreas 5:543-547;1992. 29. Nishino, T.; Kodaira, T.; Shin, S.; Imagawa, K.; Shima, K.; Kumahara, Y.; Yanaihara, C.; Yanaihara, N. Glucagon radioimmunoassay with use of antiserum to glucagon C-terminal fragment. Clin. Chem. 27:1690-1697;1981. a novel 30. Raufman, J.P.; Malhotra, R.; Singh, L. PACAP-38, peptide from ovine hypothalamus, is a potent modulator of
193
31. 32.
33.
34.
35.
36.
37.
38.
39.
40.
amylase release from dispersed acini from rat pancreas. Regul. Pept. 36:121-129;1991. Sasaki, Y.; Takahashi, H. Insulin secretion in sheep exposed to cold. J. Physiol. 306:323-335;1980. Sasaki, Y.; Takahashi, H.; Aso, H.; Ohneda, A.; Weekes, T.E.C. Effect of cold exposure on insulin and glucagon secretion in sheep. Endocrinology 111:2070-2076;1982. Sawangjaroen, K.; Dallemagne, C.R.; Cross, R.B.; Curlewis, J.D. Effects of pituitary adenylate cyclase activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) on the cardiovascular system in sheep. Peptides 13:10291032;1992. Shivers, B.D.; Gores, T.J.; Gottschall, P.E.; Arimura, A. Two high affinity binding sites for pituitary adenylate cyclase-activating polypeptide have differential tissue distributions. Endocrinology 128:3055-3065;1991. Sundler, F.; Ekblad, E.; Absood, A.; Hakanson, R.; Koves, K.; Arimura, A. Pituitary adenylate cyclase activating polypeptide: A novel vasoactive intestinal peptide-like neuropeptide in the gut. Neuroscience 46:439-454;1992. Vantrappen, G.R.; Peeters, T.L.; Jeanssens, J. The secretory component of the interdigestive migrating motor complex in man. Stand. J. Gastroenterol. 14:663-667;1979. Yada, T.; Sakurada, M.; Ihida, K.; Nakata, M.; Murata, F.; Arimura, A.; Kikuchi, M. Pituitary adenylate cyclase activating polypeptide is an extraordinarily potent intra-pancreatic regulator of insulin secretion from islets-cells. J. Biol. Chem. 269: 1290-1293;1994. Yokota, C.; Kawai, K.; Ohashi, S.; Watanabe, Y.; Suzuki, S.; Yamashita, K. Stimulatory effects of pituitary adenylate cyclase-activating polypeptide (PACAP) on insulin and glucagon release from the isolated perfused rat pancreas. Acta Endocrinologica 129:473-479;1993. Zabielski, R.; Onaga, T.; Mineo, H.; Kato, S. Periodic fluctuations in pancreatic secretion and duodenal motility investigated in neonatal calves. Exp. Physiol. 78:675-684;1993. Zabielski, R.; Onaga, T.; Mineo, H.; Okine, E.; Kato, S. Pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates exocrine pancreas in conscious preruminating calves. Comp. Biochem. Physiol. 109( 1):93-99;1994.
1996
ISSN 0742-8413/96/$15.00 PI1 SO74208413(96)00078-3
E.L.SEWER
Effect of Pituitary Adenylate Cyclase-Activating Polypeptide on Exocrine and Endocrine Secretion in the Ovine Pancreas Tukenori Onaga, * Megumi Uchidu, ’ Musanori Kimura, ’ Masato Miyazaki,’ Hitoshi Mineo,’ Seiyu Kate,’ and Romuukf Zabielsk? ‘DEPARTMENTOF VETERINARY MEDICINE,RAKUNO GAKUEN UNIVERSITY,EBESTU,HOKKAIDO069, JAPAN; AND ‘DEPARTMENTOF ANIMAL PHYSIOLOGY, SCHOLLOF VETERINARY MEDICINE,WARSAW AGRICULTURAL UNIVERSITI, NOWOLJR~YNOWSKA 166, 02-787 WARSAW, POLAND
ABSTRACT. exocrine
The role of pituitary
and endocrine
PACAP-
adenylate
pancreas was investigated
(1, 3, and 10 pmol/kg/ min)
complex
accelerated
pancreatic
protein
cyclase-activating
polypeptide
in conscious
sheep. Intravenous
at the highest doses were inhibited
Vasoactive
polypeptide
outputs, although VIP increased highest
(VIP)
pancreatic
juice flow and bicarbonate
pmol/kg) did not influence
PACAP-
(l-100
pmol/kg) altered neither
accelerated
pancreatic
protein
of insulin,
endocrine
but not amylase
influenced by atropine. however,
On the other hand, intravenous
basal plasma concentration
myoelectric
The responses in enzyme secre-
output dose-dependently;
by atropine.
migrating
glucagon,
PACAP-
and
the responses
to the
injection
of PACAP-
and glucose.
to accelerate Inc.
COMP
of exocrine
pancreatic
BIOCHEM
of the ovine pancreas but not to endocrine
enzyme secretion
PHYSIOL
KEY WORDS. PACAP,
secretion
mostly via the cholinergic
115c;3:185-193.
contributes
PACAP
appears
1996 Efseoier Science
1996.
VIP, sheep, ovine, pancreas, exocrine,
INTRODUCTION Pituitary adenylate CAP) was originally
secretion.
nerves. Copyright 0
Moreover,
infusion of glucose
response to intravenous
(20 pmol/kg/min) nor that to n-butyric acid (33 pmol/kg/ min). These results suggest that PACAP to the regulation
of and
by atropine infusion (14.4 nmol/kg/min).
significantly
the response to the highest dose was not significantly
dose were not altered significantly
38 (100
significantly
at 3 pmol/kg/min
in the regulation
infusions of PACAP-
for 10 min during phase II of the duodenal
and amylase outputs dose-dependently.
tion to both PACAPs intestinal
(PACAP)
islets, cholinergic
nerve, short-chain
fatty acids
differ from that in electrolyte secretion (3,26,28), cyclase-activating polypeptide (PAisolated from ovine hypothalamic ex-
tracts (24). PACAP possesses two types of molecular form: PACAPand PACAP-38, which are classified in the secretin-glucagon family because of a high homology with vasoactive intestinal polypeptide (VIP) and secretin in amino acid sequence (4). Although
PACAP
was found in the cen-
tral nervous system for the first time, it has been demonstrated that PACAP-immunoreactive fibers are distributed widely in the gastrointestinal tract and pancreas in several species, including sheep (5,19,34,35). In the digestive tract, PACAP has been shown to stimulate pancreatic exocrine secretion in rats (3,16,26,30) and dogs (28). The order of molar potency of PACAP and the related peptides in stimulating pancreatic enzymes seems to
Address reprint requesrs to: Takenori Onaga, Department of Veterinary Medicine, Rakuno Gakuen University, Bunkyo-dai Midori-machi 582-1, Ebetsu, Hokkaido 069, Japan. Tel 81-11-386-1112, ext. 4336; Fax 81-ll387-5890. Received 7 July 1995; accepted 17 April 1996.
suggesting
that a different mechanism is involved in each of the secretory actions. Recently, the stimulatory effect of PACAP on exocrine
pancreas
was demonstrated
VIP in preruminating
calves (40),
in comparison
indicating
with
that PACAP
plays a physiological role in the regulation of exocrine pancreas in neonatal ruminants. However, although PACAPimmunoreactive fibers were found in mature sheep (19), the effect of PACAP on ovine exocrine pancreas has not been examined. In dogs, the stimulatory effect of PACAP on the exocrine pancreas is blocked by atropine (28). Although the regulation of exocrine pancreas was shown to depend greatly on the cholinergic nerve in mature sheep (17) and on the vagus nerve in ruminating calves during the interdigestive period (39), it is still unknown whether PACAP acts on exocrine pancreas directly or indirectly via the cholinergic nerve in ruminants. On the other hand, it was demonstrated for the first time that PACAP stimulates endocrine pancreas in mice in viva (lo), in which PACAPat a comparatively high dose increased basal insulin, glucagon secretion, and carbacholstimulated glucagon secretion while it inhibited carbachol-
T. Onaga et al.
186
stimulated insulin secretion. Subsequently it was shown that PACAPaugmented glucose-induced insulin secretion at a concentration in the nanomolar range in isolated
of Oddi and another into the duct between the pancreas and gallbladder. A third silastic cannula (3 mm I.D., 5 mm O.D.) was introduced into the duodenum at a point more
perfused rat pancreas (25), and that PACAPat concentrations lower than the picomolar range also stimulated in-
than 7 cm caudad to the electrode. The three cannulae and
sulin release from rat islets in a glucose-dependent
tems, Inc., Everett, WA, USA) of the electrode were exteriorized from the right flank. Bile and pancreatic fluid were
manner
(37). These observations suggest that PACAP may play a physiological role in the regulation of endocrine pancreas as well as in that of exocrine pancreas. It should be noted that PACAP potentiates glucose-stimulating insulin secretion at concentrations lower than the picomolar range. In contrast to monogastric animals, in which blood glucose is the main source of energy, ruminants use short-chain fatty acids (SCFAs) as a major energy source, and ovine pancreatic islets respond to not only glucose but also SCFAs
(11).
The effect of n-butyric acid was most potent on both insulin and glucagon secretion among SCFAs in sheep (23). However, there is no information concerning the effect of PACAP on the ovine endocrine pancreas, especially on SCFAinduced insulin and glucagon secretion in ruminants. Therefore, the present study was performed to examine (a) the effect of PACAP-27,
PACAP-38,
and VIP on the
ovine exocrine pancreas and the contribution of the cholinergic nerves to their action; and (b) the effect of PACAP on basal and nutrient-stimulated secretion of insulin and glucagon in conscious sheep. In the latter experiment, PACAP-38 was used because the molar potency of PACAP38 in stimulating insulin release was shown to be greater than that of PACAPin rats (25,38), and glucose and nbutyric acid were used as stimulants
MATERIALS Animals
AND
in blood.
METHODS
the wires (Teflon-coated
stainless steel, 7 strd., A-M Sys-
collected in a glass bottle and were always returned into the duodenum via a peristaltic pump (SJ-1220, Atto, Tokyo, Japan) at the rate of the secretions. In sheep in the endocrine group, the left common carotid artery was carefully divided from the vagus nerve and surrounding tissue under pentobarbital anesthesia (2 5 mg/kg IV) and then wrapped chronically length of about 12 cm. Penicillin
(procaine
penicillin
in a skin loop with a G-Meiji,
Meiji-Seika,
Tokyo, Japan) treatment was continued for 3 days after surgery. The animals were allowed a recovery period of 1 week in the crates and were trained to remain in a standing position for 3 h. All sheep showed normal appetites l-3 days after surgery. An indwelling catheter was inserted into a jugular vein for administration at least 1 h before the start of the experiment. Another catheter was also inserted into the carotid loop for blood collection in the endocrine group. These catheters were filled with a sterilized solution of 3.8% (w/v) trisodium citrate to prevent coagulation.
Effect of PACAP
and VIP on Exocrine
Pancreas
PACAP(human, ovine, rat), PACAP(human, ovine, rat), and VIP (human, porcine) were purchased from Peptide Institute, Inc. (Osaka, Japan). These peptides were
Twelve Suffolk-strain male sheep weighing 34-53 kg were used. All animals were treated according to the Laboratory
dissolved in a sterilized 154 mM NaCl solution containing 0.02% bovine serum albumin (fraction V, RIA grade, Sigma, St. Louis, MO, U.S.A.) at 10 ,uM, divided into small
Animal Control Guidelines
amounts,
of Rakuno Gakuen University,
and kept at -40°C.
The
peptide solution
was
which are basically in conformity with the American Association of Laboratory Animal Control Guidelines from the National Institutes of Health.
thawed at 4°C on the day of the experiment and diluted appropriately with a sterilized isotonic solution of NaCl for administration.
The animals were kept in individual crates and were fed hay (100 g) and lucerne pellets (2.5% of body weight) at 19:00 once per day and allowed free access to water, except The animals were divided into exo-
The animals were kept in a standing position during the experiment. Duodenal myoelectric activity was recorded with an amplifier and a thermal recorder (types AB-601G and RTA-1 lOOM, Nihon-Koden, Tokyo, Japan) and
crine and endocrine groups, which consisted of eight and four sheep, respectively. Before surgery, the animals were fasted for 1 day. Following premeditation with atropine (0.05 mg/kg, IM) and xylazine (0.5 mg/kg, IM), the animals in the exocrine group were anesthetized by halothane-oxygen gas through an intratracheal cannula. The animals were equipped with a bipolar silver electrode by a suture on the duodenum close to
MacLab System (A-D Instruments Pty Ltd., Castle Hill, Australia). Pancreatic juice was collected every 5 min during the experiment. After measuring the volu~~w, 120 ~1 of the juice was taken for analysis and the remainder was returned to the collection bottle. In sheep, pancreatic exocrinc secretion changes cyclically, corresponding with duodenal migrating myoelectric activity (MMC) during the interdigestive period (Onaga, unpublished data); a similar
the opening of the common bile duct. Two silastic cannulae (2 mm I.D., 4 mm O.D.) were inserted into the common bile duct at two points: one into the duct near the sphincter
pattern is seen in humans and other species (l&9,12,21, 22,36,39). It was also observed that intravenous infusion of CCK was most effective during phase 1 of MMC on exocrine
during the experiment.
PACAP
pancreas
187
and Ovine Pancreas
in milk-fed
calves (40).
tides were infused intravenously
Consequently,
the pep-
for 10 min starting from
10 min after the end of duodenal regular spiking activity (phase III). The doses used were 1,3, and 10 pmol/kg/min. In the experiment on cholinergic blockade, atropine was infused intravenously at 14.4 nmol/kg/min for 80 min from 10 min after the end of duodenal phase III, and the peptide was infused at 10 pmol/kg/min for 10 min starting 30 min after the onset of the atropine infusion. Recording of duodenal MMC and collection of pancreatic juice were continued until 50 min after the end of the peptide infusion. The infusion rates were constant
at 0.05 ml/kg/min.
Protein concentration and amylase activity of the pancreatic juice from -30 to 50 min were determined by the
stored at -40°C
until radioimmunoassay.
The plasma con-
centration of insulin was determined by the method of Sasaki and Takahashi (31) using guinea pig anti-bovine insulin serum (ICN, Israel) and “‘I-insulin (NEN Research Products, Boston, MA, USA). Intra- and interassay coefficients of variation for the insulin determination were 5.8% (n = 6) and 8.6% (n = 5), respectively. Plasma glucagon was determined using dextran-charcoal, 1251-glucagon (NEN Research Products, Boston, MA, USA), and antiserum OAL-123, which was specific to the carboxyl-terminal portion of pancreatic glucagon (29), as described previously (32). Intra- and interassay coefficients of variation for the glucagon
determination
were 8.5%
(n =
6) and 10.6%
(n = 5), respectively.
methods of Lowry et nl. (20) and Bernfeld (7) as modified by Kanno (15), respectively. the pancreatic
The bicarbonate
juice from -10
concentration
of
to 50 min was measured by
Statistical
Analysis
PACAPsolution was prepared as described previously. Glucose was dissolved at 0.4 M in a sterilized 154 mM sodium chloride solution. n-Butyric acid (Wako Pure Chemical, Osaka, Japan) was diluted to a concentration of 2.0 M
Pancreatic protein, amylase, and bicarbonate output were calculated from protein concentration, amylase activity, and bicarbonate concentration, respectively. The values represent mean -c- SEM as absolute values or increments to values immediately before administration. Data were compared either between before and after the onset of infusion, or between the control and peptide infusions at the same time, using paired and unpaired t-tests for statistical analy-
and adjusted to pH 7.4 with 1 N NaOH. The solution was
sis, in which p < 0.05 was considered
titration.
Effect of PACAP-
administered
on Endocrine
after appropriate
Pancreas
dilution
significant.
with the sterilized
isotonic NaCl solution. Three milliliters of arterial blood was withdrawn from the carotid artery at -20, -10, 0, 5, 10, 15, 20,30, 50, and 60 min. To examine the effect of PACAPalone, PACAP38 solution was injected at 100 pmol/kg into the jugular vein at 0 min, followed by an intravenous infusion of the
RESULTS Effect on the Exocrine
Pancreas
Pancreatic juice flow was lowest during the period of duode-
isotonic NaCl solution at 7.5 pmol/kg/min (0.05 ml/kg/ min) from 1 min to 11 min. The NaCl solution was infused
nal phase I, which corresponded to the time from -10 to -5 min in Fig. 1. The flow increased gradually, coinciding with the increase of duodenal spiking activity in controls. In comparison with the control, pancreatic juice flow in-
as a control. To examine
creased rapidly during intravenous infusion of PACAP-27, PACAP-38, and VIP and decreased immediately after ces-
the effect of PACAP-
butyric acid-induced
on glucose- or n-
insulin and glucagon
secretion,
PA-
CAP-38 was injected at 1, 3, 10,30, and 100 pmol/kg into the jugular vein at 0 min, followed by an intravenous infusion of glucose at 20 pmol/kg/min or n-butyric
acid at 33
sation of the peptide infusions (Fig. 1). The increments in juice flow were augmented dose-dependently, and those at the highest dose were statistically significant in all the peptide infusions (Fig. 2). The effect of PACAPand PACAP-
was most
pmol/kg/min from 1 min to 11 min. In a preliminary experiment, the dose of glucose used was confirmed to elevate plasma concentration of glucose by 20 mg/dl. The dose of n-butyric acid used was the lowest effective dose among 1, 3.3, 10, 33, and 100 pmol/kg/min to elevate plasma con-
potent,
and VIP were equipotent
at the
centration of insulin in sheep. Blood samples were immediately transferred into a tube containing EDTA at 1.2 mg/ml blood and kept at 4°C. Plasma was separated by centrifugation at 27,200 X g for 10 min at 4°C after the end of the experiment. The glucose concentration was determined by the glucose-oxidase method (14) using 10 ~1 of plasma in a glucose analyzer (GLU-11, TOA, Tokyo, Japan). The remaining plasma was combined with benzamidine (50 pmol/ml plasma) and
the control period (Fig. 1). Although the juice flow at nadir clearly increased in response to the peptides, the increment in PACAPinfusion at 10 pmol/kg/min was inhibited significantly by atropine (Fig. 2). Atropine did not significantly inhibit either the response in juice flow to PACAP27 or that to VIP, although it caused a slight decrease in increment of juice flow to PACAP(Fig. 2). Pancreatic protein and amylase output showed similar responses to PACAPand -38 (Fig. 1). The output of both
highest dose. It is notable that only PACAPat 1 pmol/ kg/min inhibited the juice flow, although the change was not significant. Intravenous infusion of atropine (14.4 nmol/kg/min) effectively decreased the juice flow during
T. Onaga et al.
188
-Ck Control * 1Oprllol -l-J 1Opmol in atropine infusion
(4
1207
w
@I
PACAP
‘21
y
PACAP
+zj
PACAP
i
FIG. 1. Effect of intravenous w :
:
VIP ::
80 40 0 -30 -20 -10 0
10 20 30 Time (min)
G>
3.0
fl
40
50
-30 -20
-10 0
10 20
30
40
50
Time (min)
@I
PACAP
1201
yzz~zy PACAP
120-
/=?+
1 2.0
1.0 0.0 i
P
i :&
;
/
6
PACAP
W
i i
PACAP : i
j?l=zj VIP : : 40 -30 -20 -10
0
10
20
Time (min)
30
40
50
0
-10
0
10
20
30
Time (min)
40
50
infusion of PACAP-27, PACAP-38, and VIP on pancre= atic exocrine secretion in conscious sheep. The results show the response in (A) pro= tein output, (B) amyIase out4 put, (C) pancreatic juice flow, and (D) bicarbonate output to peptide infusion for 10 min at 10 pmol/kg/min with and without atropine. Atropine was infused at 14.4 nmol/kg/ min from 30 min before the peptide infusion. Values represent mean L SEM (n = 7 in PACAPand VIP, R = 6 in PACAP.38). Closed marks indicate significant ( P < 0.05) differ&&s from the control of physiological saline info* sion.
189
PACAP and Ovine Pancreas
I a
Control 1pmol/kg/min
m
3pmobkglm in
1Opmol/kg/min 1OpmoJ/kg/minin atropine infusion at 14.4nmof/kg/min *
J
vIP -“.5PACAP27~PACAP38i FIG. 2. Effect of intravenous infusion of PACAP-27, PACAP-38, and VIP on pancreatic exocrine secretion in conscious sheep. The values represent increments during 10 min of the infusion period in comparison with values during 10 min immediately before the infusion. The peptides were infused intravenously at 1,3, and 10 pmollkglmin for 10 min with or without atropine. Open and closed circles indicate significant (p < 0.05) differences in increment from the control and from 10 pmol/kg/min and VIP, n = 6 in PACAP-38). without atropine, respectively. Values represent mean + SEM (n = 7 in PACAP-
increased dose-dependently in response to infusion of PACAP-27 and -38 (Fig. 2). The increments at the highest dose were evidently blocked by the atropine infusion, whereas the responses did not disappear completely upon atropine infusion (Fig. 1). As well as the juice flow, protein and amylase output also decreased during PACAP-
sponse was clearly delayed compared with that of protein and amylase outputs. The increments in bicarbonate output in response to PACAPand VIP at the highest dose decreased slightly during atropine were not significant.
infusion,
but the changes
infu-
sion at 1 pmol/kg/min. As was the case with PACAP, 3 pmol of VIP significantly accelerated the protein output, and the increment was comparable to the response to 10 pmol of PACAP-27. Although the highest dose of VIP raised the juice flow slightly, it did not further increase protein output. The increment of protein output at the highest dose of VIP was not influenced by the atropine infusion. The pattern of the response of amylase output to VIP resembled that of protein output, though the changes were not significant at all. Bicarbonate secretion was accelerated by PACAPand VIP, but not by PACAP-38. The greatest response was obtained in infusion of PACAPat 10 pmol/kg/min (Figs. 1, 2) as well as in juice flow, although the peak of the re-
Effect 071the Endocrine
Pancreas
Intravenous injection of PACAPat 100 pmol/kg/min did not alter the plasma concentration of insulin, glucagon, and glucose in unstimulated periods (Fig. 3). Intravenous infusion of glucose at 20 pmol/kg/min significantly increased the plasma concentration of glucose and insulin (Fig. 4), whereas the plasma concentration of glucagon did not change. PACAPinjections at doses from 1 to 100 pmol/kg prior to glucose infusion did not alter insulin responses to glucose. The plasma concentrations of insulin and glucagon rose effectively during intravenous infusion of n-butyric acid at 33 pmol/kg/min without detectable change in plasma glu-
190
T. Onaga et al.
-c-o-
control
protein secretion
PACAP38-1 OOpmoVkg
potency in pancreatic shown to be PACAP-
saline infusion
in dogs at all. In rats, the order of molar protein and amylase output was > PACAP2 VIP (26). How-
ever, the order in protein and amylase secretion in sheep was VIP > PACAPz PACAP-38, which was obviously different from the order in dogs and rats. The effect of VIP on acceleration of protein output in sheep was especially noteworthy, where it seems to be more potent than PACAP, because the increment of protein output to 3 pmol of VIP is comparable to that to 10 pmol of PACAP. However, the highest dose of VIP only slightly accelerated the juice flow, whereas it did not further increase the protein output. The protein response to the highest dose of VIP was not inhibited by atropine. These results suggest that the effect of VIP on enzyme secretion
does not depend on the
cholinergic nerves, and the mechanism is likely to be different from that of PACAP. The mechanism of the action of VIP remains to be clarified. In contrast to VIP, PACAPand PACAPincreased protein and amylase output in sheep, and the responses to the highest dose were inhibited
by atropine. These results
in sheep coincided with those in dogs (28), suggesting that PACAPand PACAPstimulate pancreatic enzyme secretion by indirect action via cholinergic nerves by acting on PACAP-27/-38 preferring receptors in these species. Of course, we cannot rule out the direct action of PACAP on the exocrine
Time (min) FIG. 3. Effect of PACAP-
on the plasma concentrations
of insulin, glucagon, and glucose in conscious sheep. PACAP-38 was injected rapidly at 0 min into the jugular vein at a dose of 100 pmollkg, followed by intravenous infusion of physiological saline from 1 min to 11 min. Values represent mean f SEM (n = 4).
case (Fig. 4). However, as was the case with glucose infusion, PACAPinjections altered neither the response of plasma insulin nor glucagon to n-butyric acid.
DISCUSSION
The present study provides clear evidence that both molecular forms of PACAP increase the interdigestive secretion of the exocrine pancreas in mature sheep. PACAP has been shown to stimulate pancreatic exocrine secretion in viva in rats (26), dogs (28), and calves (40). Receptors for VIP and PACAP have been classified into PACAP-I receptors preferring only PACAP, and VIP/PACAP-II receptors preferring both VIP and PACAP, according to in vitro radioreceptor assays using rat pancreas (34). In conscious dogs, the molar potency in pancreatic protein secretion was highest for PACAP-27, and the potency of PACAPwas onetenth of that of PACAP(28). VIP did not increase the
pancreas because the response to PACAP,
al-
though slight, remained during atropine infusion at doses sufficient to inhibit cholinergic transmission in the exocrine pancreas (17). However, the action of PACAP
on pancre-
atic enzyme secretion appears to depend greatly on the cholinergic nerves. Atropine decreased the increment of juice flow during PACAPinfusion, which might be due to the cholinergic dependence of the action of PACAPon enzyme secretion. Although it remains unknown whether vagus cholinergic nerves or pancreatic intrinsic cholinergic nerves mediate in the action of PACAP, both lZ51-PACAP27 and “‘I-PACAPinjected intravenously were shown to enter the brain, passing through the blood-brain
barrier
in the mouse (6), suggesting that PACAP may act on the central nervous system to activate vagus cholinergic nerves. Molar potency in pancreatic fluid and bicarbonate secretion was highest for secretin, intermediate for PACAPand VIP, and lowest for PACAPin conscious dogs (28). Although the effect of secretin was not tested in the present study, the order of the molar potencies of the peptides in juice flow and bicarbonate output in sheep was similar to that in dogs. In sheep, the effects of PACAPand VIP were similar and were not altered significantly by atropine, but PACAPscarcely stimulated bicarbonate secretion. Bicarbonate response to the highest dose of PACAPseems to decrease during atropine infusion, as shown in Fig. 2, although the change was not significant. And the bicarbonate response to the highest dose of PACAPwithout atropine was apparently delayed in comparison with the
PACAP
and Ovine
Pancreas
3
75-
.?
50-
j
25-
-=
0
+
control
--b-
PACAP
10pmoVkg
-
PACAP
lpmol/kg
---Bi-
PACAP
30pmoVkg
-c-
PACAP
3pmoVkg
+
PACAP
1OOpmollkg
1 -
loo-
E
191
&zzQ4
glucose (20 p mol/kg/min)
: n-butyrate
h
(33 p moVkg/min)
E
3
Y
I
E 300aI Js 200s 3 loo% E On
I
.
7
r
I
I
I
I 600 400 200
I
aloo
I :
I *
:
1
I
I
I
I
I
: 2
g;_
% 8
25-
3
0
75
.
50 25 1 -20
I
,: 0
I;
I 20
1
I 40
I
1
60
Time (min)
* I
-20
I
I
0
I
20
I
I
40
1
I
60
Time (min)
FIG. 4. Effect of PACAPon plasma concentrations of insulin, glucagon, and glucose during intravenous infusion of glucose or n-butyric acid in conscious sheep. PACAPwas injected rapidly at 0 min into the jugular vein at 1, 3, 10, 30, and 100 pmollkg, followed by intravenous infusion of glucose or n-butyric acid from 1 min to 11 min. The values represent means from four animals. PACAPinjection did not significantly alter either the endocrine response to glucose or the response to nebutyric acid.
course of the cholinergic-dependent
responses of prodepended on
bicarbonate secretion differed among these species are unknown. Also, the data obtained in the present study are not
cholinergic nerves, bicarbonate would have responded as quickly as did protein and amylase. Consequently, the ac-
sufficient to explain the difference between the cholinergic dependence of the stimulatory effects of PACAP and of VIP
tion of PACAPon bicarbonate secretion does not seem to depend on the cholinergic nerves in sheep as well as in dogs. Thus, it was suggested that PACAPand VIP stim-
on enzyme secretion because of species differences and complexity of in viva experiments involving pharmacokinetics
ulate water and bicarbonate secretion via PACAP-27/VIP preferring receptors that probably exist on pancreatic duct
peptides (2,27,33), and other new types of receptors, such as the apamin-sensitive receptor found in the muscle cells of
cells in sheep and dogs. On the other hand, in neonatal
guinea pig tenia coli (13).
calves, the order of the molar potency in bicarbonate output was also PACAP> PACAP> VIP (40), but the bicarbonate responses to PACAPs were significantly reduced by atropine (R. Zabielski, unpublished data), suggesting that the effect of PACAP on bicarbonate secretion is also mediated by cholinergic nerves in calves. However, the reasons why the cholinergic dependence of PACAP on
receptors and PACAP-27/VIP receptors appear to be involved in the regulation of pancreatic exocrine secretion in sheep, although the full details of the mechanism of peptide actions remains to be clarified. In contrast to the obvious effects of the peptides on exocrine secretion, PACAPinfluenced neither unstimulated nor nutrient-stimulated secretion of insulin and gluca-
time
tein and amylase. If the action of PACAP-
of the peptides in blood, the cardiovascular
Both PACAP-27/-38
effect of the
preferring
T. Onaga et al.
192
gon in conscious
sheep. These
results are in contrast
to
observations in the rat and mouse, in which PACAPand PACAPincreased the basal secretion of islet hormones and augmented glucose-infused
secretion
of insulin
(10,25,37). In rats, PACAPwas demonstrated to increase intracellular concentration of Ca” in islet cells, and PACAP-27-induced insulin release was abolished by the Ltype Ca’+ channel blocker nitrendipine (37). PACAP-38, which also increased the intracellular concentration of Ca” in islet cells, stimulated PACAP-27.
insulin release equipotently
In the present study, however, PACAP-
with did
not solely alter the plasma concentrations of insulin, glucagon, and glucose. In contrast to monogastric animals, ruminants
utilize short-chain
fatty acids as a primary energy
source, and ovine pancreatic islets respond not only to glucose but also to SCFAs (11). Among SCFAs, n-butyric acid was most potent in stimulating insulin secretion in sheep (23). Hence, the effect of PACAPon the glucose- and n-butyric acid-induced hormone secretion by islets was examined. The doses of glucose and n-butyric acid used were submaximal, as per preliminary experiments. However, PACAP-38 did not significantly alter the response of insulin or that of glucagon to these nutrients in sheep. Therefore, it seems unlikely that PACAP is involved in the regulation of insulin and glucagon secretion
in sheep, which is sup-
ported by the observation that the intravenous administration of VIP did not influence the plasma concentration of insulin
and glucagon
in sheep
(H.
Mineo,
unpublished
data). Nevertheless, it should be noted that PACAPat the lowest dose decreased increments in all secretory parameters of the exocrine pancreas. Because endogenous insulin has been demonstrated to inhibit exocrine secretion in the ovine pancreas
(18)
in contrast
to rats, we cannot
com-
pletely rule out the possibility that an undetectable
change
in the local concentration
circula-
of insulin in islets-acinar
tion might occur. Although PACAP-immunoreactive innervate the ovine exocrine acini (19),
the role of PACAP
fibers were shown to and endocrine islets
in the regulation
pancreas has not yet been established.
of the ovine
The present study
demonstrated that PACAPand PACAPstimulate pancreatic enzyme secretion via the cholinergic nerves, whereas PACAPand VIP stimulate bicarbonate secretion, probably by direct action on the duct cells in the exocrine pancreas of sheep. However, PACAPinfluenced neither basal secretion nor nutrient-stimulated secretion of insulin and glucagon in sheep. Therefore, it is concluded that PACAP contributes to the regulation of exocrine secretion of ovine pancreas, but not to endocrine secretion.
of
We are grateful to Emeritis Professor N. Yanaiharu, University Shizuoka School of Pharmaceutical Science, Yad~,Shizuoka 422, Japan, for his supplyof glucagon antiserum OAL-123. The present study was partly funded by the Sasagawa Scientific Research Grant from The ]apan Scientific Society.
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