Arginine vasopressin inhibits fluid secretion in guinea pig
pancreatic duct cells
S. B. H.
Ko1,
S.
Naruse1,
M.
Kitagawa1,
H.
Ishiguro1,
M.
Murakami2, and
T.
Hayakawa1
1 Internal Medicine II, Nagoya
University School of Medicine, Showa-ku, Nagoya 466-8550; and
2 National Institute of
Physiological Sciences, Okazaki 444-0867, Japan
 |
ABSTRACT |
The effects of arginine vasopressin (AVP) on
pancreatic ductal secretion were studied in guinea pigs. In the
isolated vascularly perfused pancreas, AVP reduced secretin-stimulated
fluid secretion and increased the vascular resistance when the
perfusion rate was held constant. In the isolated interlobular duct
segments, AVP inhibited secretin-stimulated fluid secretion, indicating the direct inhibitory action of AVP on the duct cells. AVP affected neither the basal nor the secretin-induced cAMP productions, suggesting that AVP inhibits the fluid secretion at a point distal to the production of cAMP. AVP increased intracellular
Ca2+ concentration
([Ca2+]i)
in the absence of extracellular
Ca2+. When
[Ca2+]i
was elevated by the application of thapsigargin, AVP caused a rapid
decrease in
[Ca2+]i.
AVP seems to activate both Ca2+
release from intracellular stores and
Ca2+ efflux across the plasma
membrane, but its relation to the inhibition of fluid secretion remains
to be clarified. It is concluded that AVP directly inhibits
secretin-stimulated ductal fluid secretion in the guinea pig pancreas.
isolated vascularly perfused pancreas; isolated interlobular duct
segment; luminal volume; adenosine 3',5'-cyclic
monophosphate; intracellular calcium concentration
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INTRODUCTION |
ARGININE VASOPRESSIN (AVP) stimulates water and
Na+ absorption across several
epithelia and plays an important role in homeostasis of water and
electrolytes in the body. The major target of AVP is the collecting
duct of the kidney, where it regulates expression of aquaporin (15).
AVP also enhances the absorption of
Na+ and water across colonic
mucosa (6), induces Na+ absorption
in fetal lung (9), and inhibits pancreatic exocrine secretion (4, 5).
We have previously reported that an intravenous administration of AVP
inhibited pancreatic juice flow and
HCO
3 secretion, but not protein
secretion, in conscious dogs (20), suggesting that the site of AVP
action is the ductular epithelium of the pancreas. Beijer et al. (5)
observed in anesthetized dogs that a decrease in pancreatic secretory
flow induced by AVP always occurred just after a decrease in blood flow
and concluded that the inhibition of pancreatic secretion was due to a
reduction in oxygen supply caused by AVP-induced vasoconstriction.
Because both studies were carried out in vivo, it is difficult to
distinguish the direct effect of AVP on secretion from indirect ones,
such as vascular and systemic effects.
In this paper, we have attempted to further clarify the inhibitory
mechanisms of AVP on pancreatic exocrine secretion using two
experimental preparations of the guinea pig pancreas. First, to exclude
the systemic effects of AVP, we prepared an isolated vascularly
perfused pancreas from the guinea pig and examined effects of AVP on
fluid secretion and vascular resistance simultaneously. Second, to
examine whether AVP exerts a direct action on the duct cell, isolated
interlobular duct segments were prepared from guinea pig pancreas and
we studied effects of AVP on ductal secretion (i.e., fluid secretion
into the luminal space), cAMP production, and intracellular
Ca2+ concentrations
([Ca2+]i).
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METHODS |
Solutions
Solutions for bath and arterial perfusate.
The standard HCO
3-buffered solution
contained (in mM) 115 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 D-glucose, and 25 NaHCO3 and was equilibrated with
5% CO2 and 95%
O2. The
Ca2+-free
HCO
3-buffered solution was prepared by replacing CaCl2 with 5 mM EGTA.
The standard HEPES-buffered solution contained (in mM) 140 NaCl, 5 KCl,
2 CaCl2, 1 MgCl2, 10 D-glucose, and 10 HEPES and was
equilibrated with 100% O2. All
solutions were adjusted to pH 7.4 at 37°C.
Luminal injection solution.
The solution injected into the lumen of isolated ducts contained (in
mM) 139 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 10 D-glucose, and 1 HEPES. The
luminal solution was adjusted to pH 7.2 at 37°C and contained a
dextran conjugate of the pH-sensitive fluoroprobe 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein
(BCECF-dextran, 70 kDa, 20 µM).
Isolated Vascularly Perfused Pancreas
Preparation.
Female Hartley guinea pigs (250-400 g) were purchased from Japan
SLC, (Hamamatsu, Japan) and housed under constant temperature (22°C) and controlled lighting (12:12-h light-dark cycle)
conditions. The animals were deprived of food for 12 h before study and
were anesthetized by an intraperitoneal injection of sodium
pentobarbital (30 mg/kg). The pancreas was isolated and prepared for
vascular perfusion using a modification of the method of Matsumoto and Kanno (21). After division of the rectum, the colon was dissected away
from the pancreas. The bile duct and all vessels supplying the pancreas
except for the celiac and superior mesenteric arteries were ligated,
the remainder of the intestine was removed, and the pancreas was
separated from the spleen, stomach, and duodenum. The main pancreatic
duct was cannulated retrogradely with a polyethylene tube (0.25 mm ID,
0.76 mm OD). The celiac artery was cannulated with a polyethylene tube
(0.51 mm ID, 1.52 mm OD), and the superior mesenteric arteries were
ligated. The pancreas was placed in a thermostatic perfusion chamber
(37°C) and perfused arterially at 8 ml/min with the standard
HCO
3-buffered solution by a
peristaltic pump.
Measurement of fluid secretory rate and vascular resistance.
The fluid secretory rate from the vascularly perfused pancreas was
measured using an electric balance as a weight-differentiating flowmeter (22). The secreted fluid was collected in a cup on an
electric balance, which was connected to a personal computer. The data
were collected at 15-s intervals, and the sensitivity of the balance
was 0.1 mg. The time differentiation of the value gave us the mass flow
rate of the secretion. The secretory rate was normalized to the gland
weight. The perfusion pressure was monitored continuously via a sidearm
in the arterial line by a pressure transducer (Ohmeda, Trenton, New
Zealand). When the perfusion rate is held constant, the measured
perfusion pressure reflects the vascular resistance.
Isolated Interlobular Ducts
Preparation.
Female Hartley guinea pigs (250-400 g) were killed by cervical
dislocation. The body and tail of the pancreas were removed, and
interlobular ducts (diameter 100-150 µm) were isolated and cultured overnight as described previously (18). The layer of connective tissue surrounding the ducts was carefully stripped off; the
morphology was similar to that of interlobular ducts obtained from rats
maintained on a copper-deficient diet (1).
Measurement of the fluid secretory rate.
During overnight culture, both ends of the interlobular duct segments
sealed spontaneously, thus isolating the luminal space from the bathing
medium. The rate of fluid secretion into the closed luminal space of
the isolated duct segment was measured as reported previously (17). The
lumen of the cultured interlobular ducts was micropunctured, and the
luminal fluid was replaced by injecting a solution containing
BCECF-dextran (20 µM) to monitor the luminal pH
(pHL). Images of BCECF-dextran
fluorescence were obtained at 1-min intervals using a charge-coupled
device camera. The volume of the duct lumen was calculated from the
projected area of the lumen of each image. The rate of fluid secretion
was then calculated from the increment in the luminal volume and
expressed as secretory rate per unit luminal area of epithelium
(nl · min
1 · mm
2).
The net HCO
3 flux was calculated from
the fluid secretory rate and the rate of change of
pHL using the
Henderson-Hasselbalch equation (17).
Measurement of cAMP production.
cAMP production was measured in individual ducts using a modification
of the method of Evans et al. (12). The length and diameter of each
duct were measured to calculate the surface area. The ducts were placed
in 20 µl of the standard HEPES-buffered solution containing 1 mM
IBMX. After a preincubation for 10 min at 37°C, 20 µl of the
solution containing agonists (at double the desired final
concentration) were added and the ducts were further incubated for 4 min at 37°C. At the end of the incubation period, 200 µl of
formic acid in ethanol (5% vol/vol) were added to stop the reaction.
The samples were dried and frozen at 20°C before analysis. cAMP
contents were measured using an enzyme-immunoassay kit (Amersham,
Tokyo, Japan). cAMP production was expressed as the amount per unit
area of epithelium (fmol/mm2).
Measurement of
[Ca2+]i.
Free
[Ca2+]i
was estimated by microfluorometry in isolated duct segments loaded with
fura 2 using a modification of the method of Ashton et al. (3). The
cultured pancreatic ducts were incubated for 60-90 min at room
temperature with the acetoxymethyl ester fura 2-AM (3-5 µM) in
the standard HCO
3-buffered solution.
Fura 2-AM was first dissolved in DMSO at a concentration of 10 mM and
mixed with an equal volume of 20% pluronic acid solution in DMSO
immediately before use. The ducts were washed and kept for 45-60
min at room temperature in the fresh
HCO
3-buffered solution to allow
completion of hydrolysis. The fura 2-loaded ducts were attached to
glass coverslips coated with Cell-Tak. The coverslips were then mounted
on the base of a 400-µl chamber that was superfused at 1.2 ml/min
with the standard HCO
3-buffered solution and maintained at 37°C on the stage of an inverted
microscope (Olympus IX). Microfluorometry was performed on a small area
of the ductal epithelium (10-20 cells), which was illuminated
alternately at 340 nm and 380 nm. The fluorescence intensities
(F340 and
F380) were measured at 510 nm.
Calibration of the
F340-to-F380
fluorescence ratio was performed in situ as described by Grynkiewicz et
al. (14), using values that were determined for the ratios in zero Ca2+ concentration and saturating
(20 mM) Ca2+ concentration and a
dissociation constant for fura 2 of 224 nM at 37°C.
Materials
Fura 2-AM, BCECF-dextran, and pluronic acid were obtained from
Molecular Probes (Eugene, OR); Cell-Tak was from Becton Dickinson Labware (Bedford, MA); AVP and secretin were from the Peptide Institute
(Osaka, Japan); and ACh and thapsigargin were from Sigma (St. Louis, MO).
Statistical Analysis
Data are presented as means ± SE unless otherwise indicated.
Statistical analysis was carried out by ANOVA followed by Student's t-test.
P < 0.05 was considered significant.
 |
RESULTS |
Fluid Secretion and Vascular Responses in Perfused Pancreas
Figure 1 shows the effect of 1 nM secretin
on fluid secretion and perfusion pressure in an isolated pancreas
perfused with the standard
HCO
3-buffered solution at 8 ml/min. After a 30-min equilibration period, spontaneous pancreatic secretion was 9.6 ± 2.7 µg · min
1 · g
1
(means ± SE, n = 4) and the
perfusion pressure was 40.5 ± 3.9 mmHg (Fig. 1). When 1 nM secretin
was applied, the secretory rate reached an initial peak (76.3 ± 2.4 µg · min
1 · g
1)
within 2 min and then declined to a plateau (51.8 ± 4.7 µg · min
1 · g
1)
within 20 min. The sustained increase in fluid secretion lasted for at
least 90 min during secretin stimulation. The perfusion pressure was
not affected by 1 nM secretin (42.2 ± 4.3 mmHg after 20 min of
secretin stimulation). Figure 2 shows the
effect of 1 nM AVP on fluid secretion and the perfusion pressure during stimulation with 1 nM secretin. After an application of AVP, the secretory rate decreased by 15.1 ± 2.7%
(n = 3) in 10 min and then gradually
recovered to control level. There remained no inhibitory effect at 20 min. The application of 1 nM AVP increased the perfusion pressure from
40.5 ± 3.9 to the maximum of 95.4 ± 12.6 mmHg within 3 min. The
perfusion pressure gradually decreased during the application of AVP
but remained above control levels (70.3 ± 7.5 mmHg) at 20 min. When
the AVP infusion was halted, the pressure quickly returned to baseline.

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Fig. 1.
Effect of 1 nM secretin on fluid secretion and perfusion pressure in an
isolated guinea pig pancreas perfused at 8 ml/min with the standard
HCO 3-buffered solution.
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Fig. 2.
Effect of arginine vasopressin (AVP) on fluid secretion and vascular
resistance in an isolated pancreas perfused 8 ml/min with the standard
HCO 3buffered solution containing 1 nM secretin.
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Fluid Secretion in Isolated Pancreatic Ducts
When the bath solution was switched from standard
HCO
3-free HEPES-buffered solution to
standard HCO
3-buffered solution,
spontaneous fluid secretion (1.26 ± 0.38 nl · min
1 · mm
2,
n = 4) was observed (Fig.
3). When 10 nM secretin was then added to
the bath, the fluid secretory rate increased to 3.15 ± 0.16 nl · min
1 · mm
2.
During stimulation with secretin, the rate of fluid secretion was
constant. In this situation, 100 nM AVP added to the bath for a period
of 5 min significantly (P < 0.05)
decreased the fluid secretory rate by 30 ± 6% to 2.14 ± 0.14 nl · min
1 · mm
2.
pHL remained above 8.0 during
stimulation with secretin and was not affected by the application of
AVP. Thus the net HCO
3 flux was
significantly (P < 0.05) reduced
from 0.99 ± 0.09 to 0.65 ± 0.12 nmol · min
1 · mm
2
when AVP was added.

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Fig. 3.
Effects of AVP fluid secretion in interlobular duct segments isolated
from guinea pig pancreas. An isolated duct segment filled with the
injection solution containing
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein
(BCECF)-dextran was initially superfused with a standard
HCO 3free HEPES-buffered solution.
After a 3-min period, bath solution was switched to standard
HCO 3-buffered solution. After a
further 4 min, 10 nM secretin was applied, and then 100 nM AVP was
added to the bath for a period of 5 min during the stimulation with
secretin. Changes in fluid secretory rate (means ± SE of 4 experiments) are shown.
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cAMP Productions in Isolated Pancreatic Ducts
The cAMP production of the resting ducts was 14 ± 2 fmol/mm2 duct epithelium
(n = 13, Fig.
4). Secretin (from 1 pM to 100 nM) increased cAMP production in a concentration-dependent manner, and the
concentration of secretin required for half-maximal increase was ~4
nM. Secretin (10 nM) caused a 10-fold increase (151 ± 43 fmol/mm2,
n = 12) in cAMP production, which was
comparable to the response to 1 µM of forskolin (142 ± 39 fmol/mm2,
n =12). AVP (100 nM) affected neither
the basal (16 ± 3 fmol/mm2,
n = 12) nor the secretin-stimulated
cAMP production (114 ± 20 fmol/mm2,
n = 12).

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Fig. 4.
Effects of 100 nM AVP, 10 nM secretin, and 10 nM secretin + 100 nM AVP
on cAMP production in isolated interlobular duct segments. Means ± SE of at least 12 experiments are shown. * Significant difference
(P < 0.05) from control. NS, not
significant.
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[Ca2+]i
in Isolated Pancreatic Ducts
AVP, like ACh, increased
[Ca2+]i
in a concentration-dependent manner (Fig.
5). The
[Ca2+]i
in unstimulated cells was 103 ± 12 nM
(n = 10), and the peak values of
[Ca2+]i
after an AVP application were 139 ± 80 nM
(n = 3) with 1 nM AVP, 157 ± 65 nM
(n = 3) with 10 nM AVP, and 397 ± 84 nM with 100 nM AVP (n = 5). During
a sustained AVP application, the
[Ca2+]i
slowly declined toward baseline and a sustained plateau phase was not
observed. In the absence of extracellular
Ca2+, the
[Ca2+]i
increase caused by 100 nM AVP was transient and smaller (Fig. 6A) than
that in the presence of extracellular
Ca2+ (Fig.
5C). In some ducts (4 of 15 ducts),
a transient small increase in
[Ca2+]i
(Fig. 5B) was observed when AVP was
removed from the perfusate. To investigate
Ca2+ mobilization across the
plasma membrane in response to AVP, thapsigargin, a selective inhibitor
of Ca2+-ATPase of the endoplasmic
reticulum, was applied. Thapsigargin (1 µM) evoked a sustained
increase of
[Ca2+]i
(Fig. 6, B and
C). The addition of ACh (10 µM) in
the presence of thapsigargin caused an additional small increase in
[Ca2+]i
(Fig. 6B). In contrast to ACh, the
addition of AVP (100 nM) in the presence of thapsigargin caused a
transient
[Ca2+]i
increase (due to an additional
Ca2+ release) followed by a rapid
decline (Fig. 6C).

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Fig. 5.
Effects of AVP or ACh on intracellular
Ca2+ concentration
([Ca2+]i)
in isolated interlobular duct segments superfused with a standard
HCO 3buffered solution; 1 nM
(A), 10 nM
(B), or 100 nM
(C) AVP or 10 µM ACh
(D) were added to the bath for 3 min. Each trace is representative of at least 3 experiments.
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Fig. 6.
A: effect of 100 nM AVP on
[Ca2+]i
in an isolated interlobular duct superfused with the
Ca2+-free
HCO 3-buffered solution. A
representative of 4 experiments is shown.
B and
C: ducts were superfused with a
standard HCO 3-buffered solution. In
the presence of 1 µM thapsigargin (Tg), 10 µM ACh
(B) or 100 nM AVP
(C) was applied for 3 min. Each
trace is representative of 3 experiments.
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DISCUSSION |
In the present investigation in guinea pigs, we have confirmed the
observation in conscious (4, 20) and anesthetized dogs (5) that AVP
inhibits pancreatic secretion. Because AVP is a potent vasoconstrictor,
AVP can act on both secretory cells and the blood vessels. In the
isolated vascularly perfused pancreas, we can strictly control the
composition and the flow rate of the arterial perfusate and analyze
secretion and vascular resistance with good time resolution and
accuracy. Because the secretory rate is a linear function of the rate
of arterial perfusion (i.e., the rate of oxygen delivery) in the
isolated vascularly perfused pancreas from cat (24), in our preparation
the rate of vascular perfusion was held at a constant value.
Secretin at 1 nM stimulated pancreatic secretion but did not affect the
perfusion pressure. AVP at 1 nM reversibly inhibited secretin-stimulated fluid secretion by 15% and raised the pressure by
99%. Although the rate of vascular perfusion was held at a constant
value, there remains a possibility that a part of the perfusate was
functionally shunted and the distribution of flow within the pancreas
was modified by AVP. A reduction of the effective flow through the duct
area may cause a decrease in fluid secretion. Thus, although the
systemic secondary effects of AVP were excluded in the isolated
preparation of the pancreas, we cannot completely exclude the
possibility that AVP affected the pancreatic secretion solely through
vasoconstriction or redistribution of flow in this preparation. Another
vasomotor substance, noradrenaline, also inhibited pancreatic
electrolyte secretion in the isolated cat pancreas, and the inhibitory
effect was attributed to both a direct action on the secretory cells
and an indirect action via vasoconstriction (11). Takeuchi et al. (25)
examined secretory and vascular responses of the isolated
blood-perfused canine pancreas to various gastrointestinal hormones and
neuropeptides and found that fluid secretion and local blood flow were
independently regulated by those agents.
Therefore, the direct action of AVP on fluid secretion in pancreatic
duct cells was tested using a newly developed technique in which fluid
secretion was continuously monitored by measurement of luminal volume
of isolated interlobular duct segments (17). The isolated interlobular
duct segments from the guinea pig pancreas were found to produce
HCO
3-rich (>130 mM) fluid secretion
into the closed luminal space in response to secretin (3 nl · min
1 · mm
2
with 10 nM secretin). AVP (100 nM) significantly inhibited
secretin-stimulated fluid and HCO
3
secretion by 30%. Because the connective tissue layer surrounding the
duct was carefully removed in our preparation, this result suggests
that AVP has a direct action on pancreatic duct cells.
Ductal fluid transport in the pancreas is mainly regulated by secretin
or vasoactive intestinal peptide via a cAMP-mediated transduction
pathway (8). In the present study, secretin increased cAMP production
in isolated guinea pig pancreatic duct segments. The Michaelis-Menten
constant (Km)
was ~4 nM, which is slightly higher than the
Km (0.9 nM) for
fluid secretion in the same preparation (17). The
Km values for
cAMP production in two other reports are 2 nM in the same type of
preparation (13) and 0.15 nM in acutely isolated guinea pig main and
interlobular ducts (10). AVP affected neither the basal nor the
secretin-stimulated cAMP production (Fig. 4), indicating that AVP did
not alter cAMP metabolism in guinea pig pancreatic ducts. Thus it is
likely that AVP inhibited the ductal fluid secretion from guinea pig
pancreas at a point distal to the production of cAMP.
It is widely recognized that AVP induces vasoconstriction by increasing
[Ca2+]i
in vascular smooth muscle cells (26) via
V1 vasopressin receptors, whereas
AVP increases cAMP production in the collecting duct cells of the
kidney via V2 vasopressin
receptors, thereby regulating expression of aquaporin-2, hence
antidiuretic effects (15). At present, the cellular mechanisms for the
action of AVP on epithelial fluid transport are not well understood
except for its action on kidney collecting duct cells. In the rat
colonic mucosa, AVP decreased the short-circuit current
(serosal-to-mucosal flux of Cl
) and enhanced the
absorption of water (6). It was suggested that the inhibitory effect of
AVP was mediated by a decrease in [Ca2+]i
via V1 vasopressin receptors. In
the sheep fetal lung, AVP activated the
Na+ channel possibly via
V2 vasopressin receptors and
diminished net lung liquid secretion (9).
In this study, AVP mobilized
[Ca2+]i
in the duct cells in a way different from ACh, an agonist of
HCO
3 secretion in the same preparation
(19). AVP increased
[Ca2+]i,
but a sustained plateau phase was not observed. Instead, a transient
small increase in
[Ca2+]i
was observed in some ducts when AVP was removed from the perfusate (Fig. 5B), suggesting that
Ca2+ extrusion or reuptake is
activated during AVP application. Therefore, we investigated
Ca2+ mobilization across the
plasma membrane after depletion of intracellular Ca2+ stores. The addition of AVP
in the presence of thapsigargin caused a rapid decline of
[Ca2+]i,
which suggests that AVP activates
Ca2+ efflux across the plasma
membrane. Although ACh activated both Ca2+ release from intracellular
stores and Ca2+ influx across the
plasma membrane, AVP activated both
Ca2+ release from intracellular
stores and Ca2+ efflux across the
plasma membrane. This activation of
Ca2+ efflux by AVP was also
reported in rat smooth muscle cells (7). In rat pancreatic ducts, ACh
and ionomycin stimulated fluid secretion to an extent similar to
secretin (3), and ACh increased
[Ca2+]i
and depolarized the basolateral membrane voltage (16). However, two
other agonists utilizing the
Ca2+-mediated transduction
pathway, substance P and ATP, induced different effects on ion
transport. Substance P inhibited fluid secretion stimulated by
secretin, bombesin, dibutyryl cAMP, or forskolin (2). ATP increased
[Ca2+]i
but, contrary to ACh, hyperpolarized the basolateral membrane voltage
(16), suggesting that effects of ATP on ion channels and on fluid
secretion are different from ACh. Taken together, although a sustained
increase in
[Ca2+]i
induces fluid secretion in pancreatic duct cells, each agonist that
activates a Ca2+ pathway may exert
an opposite (inhibitory) action by activating another transduction
pathway such as protein kinase C or by directly modifying the activity
of ion transporters. Similar findings have been reported in biliary
epithelial cells (23). ACh and ATP increased
[Ca2+]i
but did not induce any fluid secretion in isolated bile duct units.
In summary, we have shown that AVP inhibits secretin-stimulated ductal
fluid secretion in the isolated vascularly perfused pancreas of the
guinea pig. The direct inhibitory action of AVP on duct cells was
confirmed utilizing isolated interlobular duct segments, although the
precise cellular mechanisms involved remain to be clarified.
Vasopressin controls water conservation, and its release is coordinated
with the thirst center activity that regulates fluid intake. The
pancreas secretes a large amount of fluid into the intestine. When
fluid intake is restricted (thirst) or fluid loss is increased
(diarrhea), a relatively small reduction in fluid secretion (~30%)
by AVP observed in this study may be physiologically important for body
fluid conservation.
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ACKNOWLEDGEMENTS |
We thank Prof. R. M. Case and Dr. M. C. Steward for helpful discussions.
 |
FOOTNOTES |
This study was supported by the Ministry of Health and Welfare (Japan),
a Monbusho international scientific research program grant from the
Ministry of Education, Science, and Culture (Japan), and the British
Council (Tokyo).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: S. Naruse,
Internal Medicine II, Nagoya Univ. School of Medicine, 65 Tsurumai-cho,
Showa-ku, Nagoya 466-8550, Japan (E-mail:
snaruse{at}tsuru.med.nagoya-u.ac.jp).
Received 23 November 1998; accepted in final form 17 March 1999.
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