Role of the vasopressin V1
receptor in regulating the epithelial functions of the guinea pig
distal colon
Yoshihiko
Sato1,
Hiroyuki
Hanai2,
Atsuhiro
Nogaki1,
Koki
Hirasawa1,
Eizo
Kaneko1,
Hisayoshi
Hayashi3, and
Yuichi
Suzuki3
1 First Department of Medicine,
2 Department of Endoscopic and
Photodynamic Medicine, Hamamatsu University School of Medicine,
Hamamatsu 431-3192; and
3 Laboratory of Physiology, School
of Food and Nutritional Sciences, University of Shizuoka, Shizuoka
422-8526, Japan
 |
ABSTRACT |
Vasopressin has a wide spectrum of biological
action. In this study, the role of vasopressin in regulating
electrolyte transport in the colon was elucidated by measuring the
short-circuit current (Isc) as well
as the Na+,
K+, and
Cl
flux in a
chamber-mounted mucosal sheet. The cytosolic
Ca2+ concentration
([Ca2+]i)
was also measured in fura 2-loaded cells by fluorescence imaging. Serosal vasopressin decreased
Isc at
10
9 M and increased
Isc at
10
7-10
6
M. The decrease in
Isc was
accompanied by two effects: one was a decrease in the
amiloride-sensitive Na+
absorption, whereas the other was an increase in the
bumetanide-sensitive K+ secretion.
The increase in
Isc was
accompanied by an increase in the
Cl
secretion that can be
inhibited by serosal bumetanide or mucosal diphenylamine-2-carboxylate.
Vasopressin caused an increase in [Ca2+]i
in crypt cells. These responses of
Isc and the
[Ca2+]i
increase in crypt cells were all more potently inhibited by the
vasopressin V1 receptor antagonist
than by the V2 receptor antagonist. These results suggest that vasopressin inhibits
electrogenic Na+ absorption and
stimulates electrogenic K+ and
Cl
secretion. In all of
these responses, the V1 receptor
is involved, and the
[Ca2+]i
increase may play an important role.
sodium absorption; potassium secretion; chloride secretion; intracellular calcium; intestinal transport
 |
INTRODUCTION |
VASOPRESSIN PLAYS a major role in controlling the whole
body water and electrolyte homeostasis (35). Vasopressin also has many
other actions, such as contraction of the vascular smooth muscle and
glycogenolysis in hepatocytes (42). Two kinds of receptor subtypes
specific to vasopressin, i.e., the
V1 receptor and
V2 receptor, have been
demonstrated to be present on the plasma membrane. Stimulation of the
V1 receptor leads to a rise in
cytosolic Ca2+, whereas
stimulation of the V2 receptor
induces a rise in the cytosolic cAMP level (20, 31, 42).
The colon, the terminal part of the gastrointestinal tract, performs
both the absorption and secretion of a variety of electrolytes by the
epithelial transport systems. These transport systems are regulated by
many kinds of endocrine, neurocrine, and paracrine agents and probably
play an important role in maintaining fluid and electrolyte homeostasis
in the whole body (3). It has been shown that vasopressin can affect
electrolyte and water transport in the colon (37). For example,
vasopressin has been reported to inhibit
Na+ and
Cl
absorption in vivo in
the rat and human colon (7, 26). In contrast, with in vitro
preparations of the mouse, rat, and human colon, the stimulation of
NaCl and water absorption, as well as the inhibition of
Cl
secretion by
vasopressin, have been demonstrated (2, 4, 5, 13, 14, 22, 44).
Therefore, these previous findings are contradictory, even within the
same species. Because the interpretation of the in vivo experimental
results could be complicated by vasopressin action not only on the
mucosal transport function but also on the microcirculation (42) and
motility (36), further experiments with isolated mucosae or epithelial
cells would be required to define the effect of vasopressin on
electrolyte transport in the colon. In addition, the cellular
mechanisms for the action of vasopressin, including the receptor
subtype and the intracellular second messenger, are largely unknown.
The aim of the present study was to further characterize the role of
arginine vasopressin (AVP) in the regulation of electrolyte transport
in the mammalian distal colon. To this end, we measured the
short-circuit current
(Isc) and ion
flux in isolated guinea pig distal colon mounted in Ussing chambers. We
used this method to assess the specific effects of AVP on the following
three electrogenic ion transport systems:
1) electrogenic
Na+ absorption,
2) electrogenic
K+ secretion, and
3) electrogenic
Cl
secretion (3, 33). We
also tested which of the vasopressin receptor subtypes
(V1 vs.
V2) was involved in those
effects by using vasopressin receptor antagonists. Furthermore, we
assessed whether AVP could increase the cytosolic free
Ca2+ concentration
([Ca2+]i)
in crypt cells with fluorescence imaging.
 |
METHODS |
Tissue preparation.
Male Hartley guinea pigs weighing 300-500 g were used in the
experiments. All animals were fed ad libitum on a standard diet (type
GM-1, Funabashi Farm, Chiba, Japan) and had free access to water until
the time of the experiments. The animals were then stunned by a blow to
the head and bled to death. A 10-cm segment of the distal colon was
obtained from 5 cm proximal to the anus. The colon was opened
longitudinally into a flat sheet, and the mucosa was separated from the
underlying connective tissue and musculature with glass microscope
slides (46). Histological studies revealed that the plane of division
was between the muscularis mucosae and the submucosa (data not shown).
In several experiments, animals were injected twice with 375 µg/kg
body wt of aldosterone (1.39 mM in saline) to enhance the electrogenic
Na+ absorption, once by a
subcutaneous injection the evening before the experiment, and then by
an intramuscular injection 4 h before the start of the experiment
(referred to as the aldosterone-treated animals). Unless otherwise
indicated, the experiments were conducted on animals without the
aldosterone treatment.
Measurement of the electrical parameters.
Isc and
transepithelial conductance
(Gt) were
measured as previously described (46). Stripped mucosa was mounted
vertically between Ussing-type chambers with an internal surface area
of 0.5 cm2. The volume of the
bathing solution in each chamber was 10 ml, and its temperature was
kept at 37°C in a water-jacketed reservoir. The bathing solution
contained (in mM) 119 NaCl, 21 NaHCO3, 2.4 K2HPO4,
0.6 KH2PO4,
1.2 CaCl2, 1.2 MgCl2, and 10 glucose. To provide a high-K+ solution, 100 mM KCl was
used in place of 100 mM NaCl (total K+ concentration of 105.4 mM),
whereas a low-Ca2+ solution was
prepared by omitting CaCl2 and
adding 0.2 mM EGTA. A
Cl
-free solution was made
with 119 mM sodium-gluconate, 1.2 mM
magnesium-(gluconate)2, and 8 mM
calcium-(gluconate)2 in place of
119 mM NaCl, 1.2 mM MgCl2, and 1.2 mM CaCl2, respectively. Each
solution was gassed with 95%
O2-5%
CO2, pH 7.4. Isc values were
measured with an automatic voltage-clamping device (CEZ9100; Nihon
Kohden, Tokyo, Japan), the value being referred to as
positive when the current flowed from the mucosa to serosa. The
transepithelial potential was measured through 1 M KCl agar bridges
connected to a pair of calomel half-cells. The transepithelial current
was applied across the tissue via a pair of
Ag-AgCl electrodes kept in contact
with the mucosal and serosal bathing solutions, a pair of 1 M NaCl agar
bridges being used, except when the
Cl
flux was determined (see
Measurement of the
Na+,
K+, and
Cl
flux).
Gt was calculated
from the change in current in response to periodic (every 1 min)
voltage pulses according to Ohm's law.
Measurement of the
Na+,
K+, and
Cl
flux.
The transepithelial bidirectional flux of both
22Na+
and
42K+
was determined simultaneously under short-circuit conditions. The mucosal-to-serosal and serosal-to-mucosal flux were measured in adjacent tissues that had
Gt values
differing within 30%. Thirty minutes was allowed for the isotopic
steady state to be reached after the bathing solution on one side of
the tissue had been labeled with both
22NaCl and
42KCl. Six samples (0.5-ml each)
were then taken from the unlabeled side at 10-min intervals, each being
replaced with an equal volume of the unlabeled solution. AVP was added
immediately after 20 min of basal flux measurements.
42K+
was assayed immediately with a Packard AutoGamma counter (model 5650),
and
22Na+
was assayed with a Beckman scintillation counter (model LS 8000) after
at least 7 days had passed to allow
42K+
(half-life of 12.5 h) to decay. The transepithelial
Cl
flux was determined only
from the serosa to mucosa. The mucosal side was bathed with a
Cl
-free solution, whereas a
normal solution was used for the serosal side, and the change in
Cl
concentration of the
mucosal solution was measured by ion-exchange chromatography. Six
samples (0.1-ml each) were taken from the mucosal side at 10-min
intervals. AVP was added after 20 min of basal flux measurements. Each
experiment was performed while the transepithelial potential was
clamped at 0 mV.
K2SO4
(0.5 M) agar bridges were used for potential measurements, and 0.5 M
sodium-gluconate agar bridges were used for current application. The
difference in liquid junction potential developed at the interface of
the potential bridge was ~9 mV between serosal control solution and mucosal Cl
-free solution,
this being determined by utilizing a saturated KCl-flowing bridge. This
means that, under the 0 mV-clamped conditions, the transepithelial
potential was actually clamped at 9 mV with mucosal-side negativity.
This potential was not corrected for, because mucosal-side negativity
would presumably have suppressed passive
Cl
secretion more than
active Cl
secretion,
whereby changes in the level of active components would be determined
more accurately because of less contamination of the passive
components. Accordingly, this specific experiment was performed under
the "apparent short-circuit condition." The Cl
concentration of the
collected mucosal fluids was determined with the use of an
anion-exchange column (Shimpack IC-A1; Shimadzu, Tokyo, Japan) and a
conductivity detector (CDD-6A; Shimadzu). A 20-µl amount of a sample
was injected and eluted at a flow rate of 2.5 ml/min with a mobile
phase of 2.5 mM phthalic acid plus 2.4 mM Tris.
Measurement of
[Ca2+]i.
[Ca2+]i
was measured in crypt cells with fura 2 and fluorescence imaging.
Colonic crypts were obtained as previously described (8). Small
fragments of mucosa were incubated for 10 min at 37°C in a
Ca2+-free solution containing
0.1% BSA. Fura 2-AM (5-10 µM) was also added to the solution.
The fura 2-loaded mucosal fragments were then vibrated manually in the
Ca2+-free solution to separate
intact crypts. The Ca2+-free
solution contained (in mM) 107 NaCl, 25 NaHCO3, 4.5 KCl, 0.2 NaH2PO4,
1.8 Na2HPO4,
5 EDTA, 12 glucose, and 2.5 L-glutamine, gassed with 95%
O2-5%
CO2, pH 7.4. The isolated crypt
cells loaded with fura 2 were attached to the bottom of a small chamber
with Cell-Tak. This chamber was then mounted on the stage of an
inverted microscope equipped for epifluorescence (Diaphot; Nikon,
Tokyo, Japan), and the cells were continuously superfused with a
modified Hank's balanced salt solution containing (in mM) 137 NaCl, 5 KCl, 1 CaCl2, 0.8 MgCl2, 10 HEPES, 5 glucose, and
2.5 L-glutamine, gassed with
100% O2, pH 7.4. The
Ca2+-free solution used for
[Ca2+]i
measurements was prepared by omitting
CaCl2 from the previous solution
and adding 1 mM EGTA. The temperature of the solution in the chamber
was kept at 33-34°C by prewarming it.
[Ca2+]i
of each preparation loaded with fura 2 was measured by
ratiometry with the dual-wavelength excitation
technique (17). Digital imaging of the fura 2 fluorescence emitted at
510 nm was carried out during alternate excitation at 340 and 380 nm
with a digital image analysis system (ARGUS-100; Hamamatsu Photonics,
Hamamatsu, Japan), with the use of a silicon-intensified target camera.
Pairs of digital images were successively obtained at specific time intervals (mostly 5-10 s) and stored. The ratio of each image pair
was calculated off line with background correction, first pixel by
pixel and then continued with the mean ratio value for the area of
interest. The relationship between this ratio and [Ca2+]i
was obtained by perfusing a cell-free, fura 2-containing solution with
various Ca2+ concentrations.
Although the relationship determined in this way could be somewhat
different from that in situ (inside the cell), it may not devalue the
main conclusions in this report, which were based on the relative
changes in
[Ca2+]i.
Materials.
AVP, amiloride, and bumetanide were purchased from Sigma (St. Louis,
MO). The vasopressin V1 receptor
antagonist, Des-Gly-[phenylacetic acid1,D-Tyr(Et)2,Lys6,Arg8]vasopressin,
and V2 receptor antagonist,
[1-(
-mercapto-
,
-cyclopentamethylene-propionic acid),D-Ile2,Ile4,Arg8]vasopressin,
were purchased from Peninsula Laboratories (Belmont, MA). Fura 2-AM was
purchased from Molecular Probes (Eugene, OR), Cell-Tak was purchased
from Becton Dickinson Labware (Bedford, MA), and
diphenylamine-2-carboxylate (DPC) and the other chemicals were
purchased from Wako Pure Chemicals (Osaka, Japan). Each drug was added
from a concentrated stock solution dissolved in water, except DPC,
bumetanide, and fura 2-AM, which were dissolved in DMSO. The final
volume of DMSO in an experimental solution was always 0.1%.
22Na+
was purchased from Dupont NEN (Boston, MA), and
42K+
was purchased from Japan Atomic Energy Research Institute (Tokyo, Japan).
Statistical analyses.
Values are expressed as means ± SE, and
n = number of guinea pigs. Comparisons
among multiple groups were tested by applying Scheffé's
F-test to a one-way ANOVA. Comparisons
between two groups were tested with the two-tailed paired or unpaired
Student's t-test. Results were
considered significantly different at
P < 0.05.
 |
RESULTS |
Effects of AVP on Isc
and Gt.
AVP applied to the serosal bathing solution caused a dose-dependent,
biphasic Isc
response in the mucosa from the guinea pig distal colon (Fig.
1): a low concentration of AVP
(10
9 M) decreased
Isc, whereas a
high concentration of AVP
(10
7-10
6
M) increased Isc.
The Isc change
induced by 10
8 M AVP varied
according to individual tissue samples. An increase in
Gt generally
accompanied the
Isc response
(Fig. 1), but, in some tissue samples, a small
Gt decrease was
observed at a low concentration of AVP. In contrast, AVP
(10
10-10
6
M) applied to the mucosal solution changed neither the
Isc nor Gt value (data
not shown).

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Fig. 1.
Effect of arginine vasopressin (AVP) on short-circuit current
(Isc) and
transepithelial conductance
(Gt) of mucosa
from guinea pig distal colon. Arrows show when AVP was added to serosal
bathing solution, with cumulative dose indicated. A representative
result is presented.
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The mammalian distal colon has at least three electrogenic active ion
transport systems that can underlie these
Isc changes induced by AVP, i.e., Na+
absorption and K+ and
Cl
secretions (3, 33).
Electrogenic Na+ absorption
involves the amiloride-sensitive
Na+ channel for the apical
membrane step and
Na+-K+-ATPase
for the basolateral membrane step. On the other hand, electrogenic
K+ and
Cl
secretions involve the
K+ and
Cl
channels, respectively,
for the apical membrane step and the bumetanide-sensitive
Na+-K+-Cl
cotransporter, and
Na+-K+-ATPase
for the basolateral membrane step. We studied the effect of AVP on each
of these electrogenic transport systems.
Effect on amiloride-sensitive
Na+ absorption.
We first assessed whether AVP would affect amiloride-sensitive
electrogenic Na+ absorption. The
data presented in this section were obtained from the
aldosterone-treated animals, in which the activity of electrogenic
Na+ absorption had been enhanced
(see METHODS), but similar results were obtained from the animals without the aldosterone injection, which
exhibited a small amiloride-sensitive
Isc (data not
shown). Isc was
measured in the presence of serosal bumetanide
(10
4 M) to exclude a large
portion of the electrogenic secretions of
K+ and
Cl
(3, 33). Under these
conditions, AVP applied to the serosal solution caused only decreases
in Isc and
Gt (Fig.
2A): the
increases in Isc
and Gt resulting
from a high concentration of AVP (cf. Fig. 1) were largely inhibited.
The decreases in
Isc and
Gt by AVP under
these conditions were virtually removed when the electrogenic Na+ absorption was mostly
inhibited by mucosal amiloride at
10
4 M (34) (Fig.
2A), suggesting that AVP could
inhibit amiloride-sensitive electrogenic
Na+ absorption (3, 33). Serosal
bumetanide alone caused an increase in
Isc and a
decrease in Gt
(Fig. 2A), consistent with the
inhibition of ongoing electrogenic
K+ secretion (21, 34).

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Fig. 2.
Effect of AVP on amiloride-sensitive
Isc and
Gt. Animals used
in these experiments were treated with aldosterone (see
METHODS).
A: representative records obtained
from 2 adjacent tissues. Bumetanide (Bum,
10 4 M) and AVP were applied
at times indicated by arrows to serosal bathing solution, whereas
amiloride (Amil, 10 4 M) was
applied at time indicated to mucosal bathing solution. Large increase
in Isc with
addition of bumetanide may be explained by enhanced level of
bumetanide-sensitive electrogenic
K+ secretion induced by
aldosterone (3). B: concentration
dependency of AVP-induced inhibition of amiloride-sensitive
Isc and
Gt. Percent
inhibition of amiloride-sensitive
Isc was
calculated from
Isc decrease
induced by a cumulative dose of serosal AVP and finally by mucosal
amiloride (10 4 M) in
presence of bumetanide.
Gt represents
magnitude of change in
Gt from pre-AVP
level; n = 5.
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Figure 2B shows that the inhibition of
amiloride-sensitive
Isc by AVP was
concentration dependent with ED50 = 0.8 nM, the maximal inhibition of ~70% being attained at
10
8 M AVP. The decrease in
Gt induced by AVP
largely paralleled the decrease in
Isc, the maximum
decrease being
1.15 ± 0.18 mS/cm2 (Fig.
2B).
Effects on bumetanide-sensitive
K+ and
Cl
secretion.
We next examined the effect of AVP on
Isc and
Gt in the
presence of mucosal amiloride with the use of tissues from animals without the aldosterone treatment. Under this condition, AVP still caused a concentration-dependent biphasic
Isc response,
i.e., a decrease at low concentrations and an increase at high
concentrations of AVP, these being accompanied only by an increase in
Gt (Fig. 3). Both the decrease and increase in
Isc induced by
AVP, as well as the increase in
Gt by AVP, were
all largely inhibited by serosal bumetanide at
10
4 M (Fig. 3), suggesting
that the Isc
decrease was due to the activation of electrogenic
K+ secretion and that the
Isc increase was
due to the activation of electrogenic
Cl
secretion (3, 21, 33,
34, 38, 46). In addition, when
10
9 M AVP was applied, a
small transient
Isc increase was
seen before the
Isc decrease
(Fig. 3A; see also Figs.
4A and
5A). Although this
Isc increase has
not been characterized any further, it could have been due to weak
activation of the electrogenic
Cl
secretion induced by
this concentration of AVP.

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Fig. 3.
Effect of serosal bumetanide (Bum,
10 4 M) on AVP-induced
changes in Isc
and Gt. Tissues
were pretreated with mucosal amiloride
(10 4 M).
A: representative records in absence
(solid line) and presence (dashed line) of bumetanide obtained from two
adjacent tissues. AVP was applied, with cumulative dose indicated, at
time shown by arrows to serosal bathing solution of each tissue.
B: summary of 4 similar experiments in
absence (open bars) and presence (hatched bars) of bumetanide.
Isc and
Gt represent
magnitude of changes in
Isc and
Gt from pre-AVP
levels. * 0.01 < P < 0.05;
** P < 0.01; NS, not
significant.
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To test whether the
Isc decrease in
the presence of amiloride was actually due to the activation of
K+ secretion, we used a
high-K+ solution
([K+] = 105.4 mM) as
the mucosal bathing solution. Under this condition, K+ secretion would be suppressed
as a result of the decrease in electrochemical driving force for
K+ to pass over to the mucosal
side. As shown in Fig. 4, the
Isc response
induced by 10
9 M AVP was
reversed from the decrease to a small increase by the high-K+ mucosal solution,
supporting the idea that the
Isc decrease
reflected the activation of electrogenic
K+ secretion. The increase in
Gt induced by AVP
was significantly greater in the
high-K+ mucosal solution than in
the low-K+ normal mucosal
solution. In contrast to the response to
10
9 M AVP, the magnitude of
the increases in
Isc and
Gt induced by cumulatively added 10
8 M
AVP above the level in the presence of
10
9 M AVP was not
significantly different between the normal and high-K+ mucosal solutions: the
Isc increases in
the normal and high-K+ solutions
were 81 ± 24 and 132 ± 30 µA/cm2
(n = 5), respectively, and
the Gt increases
were 3.1 ± 0.9 and 4.2 ± 1.6 mS/cm2, respectively.

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Fig. 4.
Effect of high-K+ mucosal solution
on AVP-stimulated changes in
Isc and
Gt in presence of
mucosal amiloride (10 4 M).
A: 2 representative records obtained
from 2 adjacent tissues, 1 with a 5.4 mM
K+ solution (solid line) and other
with a 105.4 mM K+ solution
(dashed line) on mucosal side. AVP was applied, with cumulative dose
indicated, at time shown by arrows to serosal bathing solution of each
tissue. B: summary of 6 similar
experiments with normal (open bars) and
high-K+ (hatched bars) solution.
Isc and
Gt represent
magnitude of changes in
Isc and
Gt from pre-AVP
levels. * 0.01 < P < 0.05; NS, not significant.
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To test whether the increase in
Isc induced by a
high concentration of AVP was the result of the activation of
electrogenic Cl
secretion,
we examined the effect of DPC, a chloride channel blocker (1, 6), on
the AVP-induced
Isc increase in
the presence of mucosal amiloride (Fig. 5).
We found that mucosal DPC (30 µM) hardly affected the
Isc decrease from
a low concentration of AVP but inhibited most of the
Isc increase
induced by a high concentration of AVP. The increase in
Gt induced by AVP
was slightly, but significantly, reduced by DPC. These results support
the proposition that an Isc increase due
to a high concentration of AVP reflected the activation of electrogenic
Cl
secretion. DPC alone
slightly increased
Isc and decreased
Gt (
Isc = 9 ± 1 µA/cm2,
Gt =
1.8 ± 1.2 mS/cm2,
n = 4).

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Fig. 5.
Effect of mucosal diphenylamine-2-carboxylate (DPC, 30 µM) on
AVP-stimulated changes in
Isc and
Gt in presence of
mucosal amiloride (10 4 M).
A: representative records in absence
(solid line) and presence (dashed line) of DPC obtained from 2 adjacent
tissues. AVP was applied, with cumulative dose indicated, at time shown
by arrows to serosal bathing solution of each tissue.
B: summary of 4 similar experiments in
absence (open bar) and presence (hatched bar) of DPC.
Isc and
Gt represent
magnitude of changes in
Isc and
Gt from pre-AVP
levels. * 0.01 < P < 0.05;
NS, not significant.
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Na+,
K+, and
Cl
flux measurements.
The foregoing results suggest that the
Isc responses to
AVP (Fig. 1) can be explained by the inhibition of
Na+ absorption and activation of
K+ and
Cl
secretion. To confirm
the foregoing hypothesis for an ionic basis to the changes in
electrical parameters induced by AVP, we determined the effect of AVP
on bidirectional
22Na+
and
42K+
flux under the short-circuit condition (Table
1; aldosterone-treated animals were used).
AVP (10
7 M) decreased net
22Na+
absorption mainly due to a decrease in mucosal-to-serosal
22Na+
flux, with little change in serosal-to-mucosal
22Na+
flux. In addition, AVP stimulated net
42K+
secretion mainly due to the increase in serosal-to-mucosal
42K+
flux, with a small decrease in mucosal-to-serosal
42K+
flux. These changes in flux were accompanied by a decrease in Isc. The
magnitude of the
Isc decrease (3.9 µmol · cm
2 · h
1)
was, however, not large enough to account for the sum of the changes in
net
22Na+
absorption (4.0 µmol · cm
2 · h
1)
and net
42K+
secretion (1.5 µmol · cm
2 · h
1).
This discrepancy may be explained by a partial offset of
Isc decrease due
to the concomitant activation of electrogenic
Cl
secretion.
We also determined the effect of AVP on
Cl
flux from serosa to
mucosa by ion-exchange chromatography under the apparent short-circuit condition (see methods) (Table
2). AVP
(10
6 M) significantly
increased Cl
flux from the
serosa to mucosa in association with the increase in apparent
Isc. The increase
in apparent Isc
induced by AVP was ~60% larger than the change in
Cl
flux. One possible
explanation for this discrepancy is that electrogenic HCO
3 secretion was also activated by
AVP.
AVP receptor subtypes involved in regulating ion transport.
We next examined which vasopressin receptor subtype, the
V1 or
V2 receptor, would mediate the
effects of AVP on the electrogenic transport systems in the colon with
the V1 antagonist and the V2 antagonist (Table
3) (29, 30). In this and the next sections, an AVP concentration of 10
9
M was used for evaluating the K+
secretion, of
10
8-10
7
M for Na+ absorption, and of
10
7 M for
Cl
secretion, so that,
together with pharmacological agents, the Isc component for
each transport pathway could be separately determined (still responses
are at or close to the maximum level). The reduction of electrogenic
Na+ absorption and the enhancement
of electrogenic K+ and
Cl
secretion induced by AVP
were all inhibited more potently by the
V1 than by the
V2 antagonist. These antagonists
had no effect on the baseline
Isc and
Gt levels.
Influence of the serosal
low-Ca2+
solution on the Isc
response to AVP.
Ca2+ has been suggested to be an
important intracellular second messenger for vasopressin
V1 receptor activation (20, 42). To study the role of Ca2+ in the
regulation of colonic ion transport by AVP, we first examined the
effect of the serosal low-Ca2+
solution (nominally Ca2+ free,
containing 0.2 mM EGTA) on AVP-induced changes to the
electrogenic ion transport systems (Table
4). The stimulation of
K+ and
Cl
secretion by AVP were
both almost completely inhibited when
Ca2+ was removed from the serosal
solution. On the other hand, serosal Ca2+ removal had no effect on the
inhibition of electrogenic Na+
absorption (in terms of the percent inhibition) induced by AVP. It was
noticed, however, that the magnitude of reduction of
amiloride-sensitive Isc was somewhat
smaller under the serosal low-Ca2+
condition than under the control condition. This was because of a
reduced basal level of amiloride-sensitive
Isc under the serosal low-Ca2+ condition (data
not shown), the reason for which is unknown at present. We could not
assess the effect of removing Ca2+
from both the mucosal and serosal solutions, because
Gt was greatly increased under those conditions.
Effect of AVP on
[Ca2+]i
in crypt cells.
We next measured microfluorometrically by fluorescence imaging the
changes in
[Ca2+]i
induced by AVP in colonic crypt cells. In the crypt cells, the baseline
[Ca2+]i
value was 90 ± 4 nM (n = 4). AVP
(10
7 M) increased
[Ca2+]i
in most portions of the crypt (Fig. 6). The
increase in
[Ca2+]i
reached a maximum within 30 s after the application of AVP, the value
being substantially sustained thereafter (Fig.
6D). When tissues were superfused
with the Ca2+-free solution
(nominally Ca2+ free, containing 1 mM EGTA), the baseline
[Ca2+]i
value stabilized within 1 min to 67 ± 12 nM
(n = 4). Under such a
Ca2+-free condition, AVP still
evoked an increase in
[Ca2+]i,
the initial peak being little, although significantly, reduced (
[Ca2+]i:
control = 153 ± 9 nM,
Ca2+-free = 96 ± 7 nM;
P < 0.01, n = 4), whereas the decline during the
late phase was accelerated
([
Ca2+]i
3 min after the
peak/
[Ca2+]i
at the peak: control = 0.72 ± 0.11, Ca2+-free = 0.20 ± 0.03;
P < 0.05, n = 4). This result suggests that the
[Ca2+]i
increase induced by AVP in the crypt cells included both a Ca2+ release from the
intracellular store and a Ca2+
influx from the extracellular fluid.

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|
Fig. 6.
Measurement of
[Ca2+]i
in colonic crypt cells. Optical microscopic image
(A) and fluorescence
(B; excitation at 340 nm and emission
at 510 nm) of isolated colonic crypts loaded with fura 2. C: pseudocolor image of crypt
[Ca2+]i.
Dark is low
[Ca2+]i
and red to white is high
[Ca2+]i.
Application of 10 7 M AVP
(at time 0) caused an increase in
[Ca2+]i
in a large portion of crypt cells. AVP was removed at 3 min, and
[Ca2+]i
decreased toward prestimulation level.
D: time-course plot for
[Ca2+]i
increase above baseline level evoked by
10 7 M AVP under control
(solid line) and Ca2+-free (dashed
line) conditions. Ca2+-free
solution was prepared by including 1 mM EGTA and omitting
CaCl2 (see
methods). Response was analyzed and
averaged in central area of one crypt (area = 20 µm × 20 µm),
and mean values from 4 independent measurements on 4 animals are shown
for both conditions.
|
|
The relationship between the AVP concentration and the peak increase in
[Ca2+]i
(
[Ca2+]i)
was determined in the crypt cells (Fig. 7).
The maximal
[Ca2+]i
value was 160 ± 5 nM, ED50
being ~2 nM.
[Ca2+]i
by 10
7 M AVP was inhibited
more potently by the V1 antagonist
than by the V2 antagonist (Fig.
7B), suggesting that AVP increased
[Ca2+]i
in the crypt cells by activating the
V1 receptor.

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|
Fig. 7.
Concentration dependency and receptor subtype of AVP-evoked
[Ca2+]i
increase in crypt cells. A:
concentration dependency of peak increase in
[Ca2+]i
evoked by AVP
( [Ca2+]i)
in crypt cells; n = 4-6 for each
point. B: effects of
V1 receptor antagonist,
Des-Gly-[phenylacetic
acid1,d-Tyr(Et)2,Lys6,Arg8]vasopressin,
and V2 receptor antagonist,
[1-( -mercapto- , ,cyclopentamethylene-propionic
acid),D-Ile2,Ile4,Arg8]vasopressin,
on
[Ca2+]i
in crypt cells; n = 4 for each point.
Each antagonist was added 5-10 min before applying
10 7 M AVP. An antagonist
alone had no noticeable effect on
[Ca2+]i.
* 0.01 < P < 0.05;
** P < 0.01.
|
|
 |
DISCUSSION |
This study has shown that serosal AVP could regulate the three kinds of
electrogenic ion transport system in the guinea pig distal colon.
First, AVP inhibits electrogenic
Na+ absorption. The evidence for
this is that, in the presence of bumetanide to inhibit
K+ and
Cl
secretions, AVP evoked a
decrease in Isc
in association with a
Gt decrease, both
of these effects being absent in the presence of mucosal
10
4 M amiloride (Fig. 2)
(3, 33). In addition, AVP reduced
22Na+
absorption (Table 1). It is unlikely that the reduction of
22Na+
absorption induced by AVP (4 µmol · cm
2 · h
1)
was due mainly to the inhibition of electroneutral
Na+ absorption rather than the
inhibition of the electrogenic Na+
absorption because, in the aldosterone-treated guinea pig distal colon,
the rate of electroneutral Na+
absorption (estimated from the rate of
22Na+
absorption) in the presence of
10
4 M mucosal amiloride was
<2
µmol · cm
2 · h
1
(T. Yamamoto and Y. Suzuki, unpublished observations). The maximum inhibition of electrogenic Na+
absorption by AVP was ~70%. Second, AVP stimulates electrogenic K+ secretion. When the
electrogenic Na+ absorption was
inhibited by amiloride, AVP at a low concentration (10
9 M) evoked a decrease
in Isc associated
with an increase in
Gt (Fig. 3). This
Isc decrease was
inhibited by serosal bumetanide, an inhibitor of the
Na+-K+-Cl
cotransporter, or when the high-K+
solution was used for the mucosal solution (Figs. 3 and 4). These results are consistent with the notion of AVP stimulating electrogenic K+ secretion (3, 21, 33, 34, 38).
The result of the
42K+
flux study confirms the activation of
K+ secretion by AVP (Table 1).
Third, AVP stimulates electrogenic Cl
secretion. The evidence
for this is that AVP at a relatively high concentration
(>10
7 M) increased
Isc, which was
inhibited by bumetanide (Fig. 3). This
Isc increase,
when corrected for the
Isc decrease due
to K+ secretion, was not
noticeably influenced by the
high-K+ mucosal solution, but was
attenuated by mucosal DPC, a
Cl
channel-blocking
acrylaminobenzoate (1, 6) (Figs. 4 and 5). These findings are
consistent with the activation of electrogenic Cl
secretion (3, 33, 34,
46). The result of the Cl
flux study confirmed the stimulation of
Cl
secretion by AVP (Table
2). Collectively, the present results strongly suggest that AVP would
inhibit electrogenic Na+
absorption and stimulate electrogenic
K+ and
Cl
secretions. It cannot be
excluded, however, that AVP could also regulate such other
ion-transport systems demonstrated in the colon as
Na+ absorption mediated by
Na+/H+
exchange, Cl
absorption/HCO
3 secretion mediated by
Cl
/HCO
3
exchange, and K+
absorption/H+ secretion mediated
by
H+-K+-ATPase
(3, 40). These transport systems are believed to be nonelectrogenic and
thus cannot be assessed from
Isc measurements.
The vasopressin receptor is of two different subtypes, the
V1 and
V2 receptors (20, 31, 42). The
present study has demonstrated that the regulation of three kinds of
electrogenic ion transport in the colon by AVP was probably all
mediated by the V1 receptor, because they were each inhibited more potently by the
V1 than by the
V2 receptor antagonist (Table 3)
(29, 30). The dissociation constant of AVP for the
V1 receptor has been reported to
be 0.6-3 nM (42). The ED50
value for inhibiting electrogenic
Na+ absorption (0.8 nM) was within
this range (Fig. 2). The ED50 value for activating K+ secretion
may also be within this range (0.1-1 nM; Figs. 3 and 5), whereas
that for activating Cl
secretion seems to be at a slightly higher value (>10 nM; Fig. 3). It
cannot be excluded, however, that there is also the
V2 receptor in the colonic mucosa,
which is involved in regulating nonelectrogenic transport systems, such
as those just described, or in modulating other physiological
regulators acting on the electrogenic transport systems.
The present study has demonstrated by fluorescence imaging that the AVP
receptor connected with the
[Ca2+]i
increase was present in crypt cells of the distal colon. The AVP
receptor in crypt cells is probably of the
V1 subtype, because the
[Ca2+]i
increase was inhibited more potently by the
V1 receptor antagonist than by the
V2 receptor antagonist (Fig. 7).
This agrees with the general notion that the
V1 receptor is coupled to the
Ca2+ signaling pathway (20, 42).
The relationship between the concentration of AVP and the magnitude of
the
[Ca2+]i
increase in crypt cells reveals an
ED50 value of ~2 nM (Fig. 7),
well within the range for the dissociation constant of AVP for the
V1 receptor (see Fig. 3). It is
possible, although it remains to be demonstrated, that the
V1 receptor coupled to the Ca2+ signaling pathways is also
present in surface cells, because electrogenic
Na+ absorption, which is presumed
to mainly reside in surface cells (10, 24, 28), was regulated by a
V1 receptor-mediated mechanism. We
have attempted to prepare isolated surface cells to measure [Ca2+]i
and found that AVP caused an increase in
[Ca2+]i
(Y. Sato, unpublished observations). However, we cannot exclude the
possibility that such prepartions were contaminated by cell types other
than surface cells.
It is possible that both the AVP-induced
[Ca2+]i
increase in the crypt cells (Fig. 6) and that in the surface cells (as
just proposed) are important parts of the signal transduction pathway for the V1 receptor-mediated
regulation of electrogenic transport systems in the distal colon. We
have shown here that the activation of
Cl
and
K+ secretions induced by AVP
depended on the presence of Ca2+
in the serosal medium (Table 4). It has been shown that
Ca2+ is one of the most important
mediators for activating Cl
(3, 9, 46) and K+ secretions in
the intestine (34, 38). Cl
secretion in the distal colon has been suggested to occur mainly in
crypt cells (18, 39), although Köckerling and Fromm (23) have
demonstrated that Cl
secretion is also considerably performed by surface cells. Thus the
AVP-induced
[Ca2+]i
increase in crypt cells could be intimately related to the AVP-induced
activation of Cl
secretion.
The
[Ca2+]i
increase in crypt cells has been shown to be induced by agents that can
stimulate Cl
secretion,
such as muscarinic agonists and ATP (25, 27). In addition,
Ca2+-dependent activation of the
ion channels that may underlie the activation of transepithelial
Cl
secretion has recently
been reported to exist in colonic crypt cells (15). On the other hand,
the cellular type responsible for electrogenic
K+ secretion is equivocal: both
surface and crypt cells have been suggested to be involved (16, 18, 28,
38). In contrast to the activation of
Cl
and
K+ secretions, the AVP-induced
inhibition of electrogenic Na+
absorption was not influenced by the serosal
low-Ca2+ solution (Table 4).
However, we think that the increase in
[Ca2+]i
could also play a role here, because
Ca2+-mobilizing agents such as a
muscarinic agonist (41) or calcium ionophore A23187 (Y. Suzuki,
unpublished observations) inhibited electrogenic
Na+ absorption in the colon. The
Ca2+-dependent inhibition of
apical amiloride-sensitive Na+
channels has been demonstrated in other
Na+-transporting epithelia (11,
12, 32). Because electrogenic Na+ absorption has been
suggested to occur mainly in the surface cells (10, 24, 28), the
possible
[Ca2+]i
increase in the surface cells could be related to the inhibition of
electrogenic Na+ absorption by
AVP. The failure to influence the effect of AVP on
Na+ absorption by serosal
Ca2+ removal (Table 4) might be
explained by the idea that Ca2+
released from the intracellular
Ca2+ store is more important than
that from an extracellular source, or that
Ca2+ entering from the mucosal
side plays the major role. Accordingly, the increased
[Ca2+]i
in the crypt and surface cells might be involved in regulation of the
Na+ absorption,
Cl
secretion, and
K+ secretion by AVP. Several
findings from the present study are, however, apparently inconsistent
with this conclusion; for example, the
ED50 value for increased
[Ca2+]i
in the crypt cells by AVP was 2 nM, whereas that for
Cl
secretion seems to have
been >10 nM (Fig. 3). Clearly, further studies will be needed to
elucidate the precise role played by [Ca2+]i
in regulating electrolyte transport by AVP in the colon.
Vasopressin has previously been reported to influence fluid and
electrolyte transport in the mammalian small and large intestines. Both
stimulation and inhibition of Na+
and water absorption have been shown (37). For example, in vivo studies
on the human and rat colon have demonstrated that intravenous
vasopressin administration inhibited NaCl and water absorption (7, 26),
whereas K+ secretion was unchanged
(26); this generally agrees with the present findings of the inhibition
of Na+ absorption and the
stimulation of Cl
secretion
by AVP in the guinea pig distal colon. On the other hand, in vitro
studies on isolated human, mouse, and rat colons have reported that AVP
stimulated Na+ absorption (2, 4,
5, 13, 14, 22, 44). It might be difficult, however, with in vivo
studies to entirely exclude the possibility that those AVP-induced
changes in electrolyte and fluid transport were the result of changes
in the mucosal blood supply or in intestinal motility, rather than
changes in epithelial transport per se (7, 26). Extensive studies on the rat colon in vitro (4, 5, 14) have shown that AVP enhanced
amiloride-insensitive
Cl
-dependent
Na+ absorption and inhibited
electrogenic Cl
secretion
in the distal but not in the proximal colon. In addition, AVP had no
effect on electrogenic, amiloride-sensitive
Na+ absorption in the rat colon
(5). We have no explanation other than species difference to explain
the different effects of AVP on the rat colon, in spite of the use of a
similar experimental technique. The effect of AVP on the rat colon has
been suggested to be mediated by a decrease in
[Ca2+]i
(4, 22).
The circulating AVP concentration is ~1 pM in euhydrated mammals,
which is elevated to ~100 pM under severe hypovolemia (35). Thus the
action of plasma AVP on colonic electrolyte transport via the
V1 receptor can only be exerted
under relatively severe pathophysiological conditions. Alternatively,
AVP released by a neurocrine or paracrine mechanism within the mucosa
could be the main factor responsible for regulating colonic ion
transport (19). A recent study has demonstrated the presence of
vasopressin-like immunoreactivity in epithelial, submucosal, and muscle
layers ranging from the stomach to rectum (43, 45).
The physiological consequences of the changes in ion transport induced
by AVP that have been demonstrated here could be lubrication of the
mucosal surface layer of the colon by secreted fluid resulting from the
stimulation of K+ and
Cl
secretion and the
inhibition of Na+ absorption.
These effects of AVP, in concert with the
V1 receptor-mediated contraction
of smooth muscle and the stimulation of peristalsis (36), would help
the smooth passage of intestinal contents. Alternatively, if the
changes in ion transport by AVP are not associated with the net
transepithelial water movement, the inhibition of
Na+ absorption and stimulation of
Cl
and
K+ secretion may be able to
contribute to lowering the plasma osmolarity, a well-known systemic
effect of AVP (35). The source of AVP responsible for regulating ion
transport in the colon as well as the physiological significance of
this regulation remain to be elucidated.
 |
ACKNOWLEDGEMENTS |
We thank Drs. Atsuo Miyakawa and Masahiko Hirano (Hamamatsu
University School of Medicine) for useful advice in the experiments to
measure
[Ca2+]i.
We also thank Drs. Yoshinori Marunaka (University of Toronto) and
Atukazu Kuwahara (University of Shizuoka) for critical reading of the
manuscript and Tony Innes for helping us to edit the English text.
 |
FOOTNOTES |
This work was supported in part by Grant-in-Aid (06670536) for
Scientific Research from the Ministry of Education, Science, Sports and
Culture of Japan.
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: Yoshihiko Sato,
First Dept. of Medicine, Hamamatsu Univ. School of Medicine, 3600 Handa-cho, Hamamatsu 431-3192, Japan (E-mail:
ysato826{at}hama-med.ac.jp).
Received 3 March 1999; accepted in final form 12 July 1999.
 |
REFERENCES |
1.
Anderson, M. P.,
D. N. Sheppard,
H. A. Berger,
and
M. J. Welsh.
Chloride channels in the apical membrane of normal and cystic fibrosis airway and intestinal epithelia.
Am. J. Physiol.
263 (Lung Cell. Mol. Physiol. 7):
L1-L14,
1992[Abstract/Free Full Text].
2.
Aulsebrook, K. A.
Effect of vasopressin on sodium transfer by rat colon in vitro.
Endocrinology
68:
1063-1067,
1961.
3.
Binder, H. J.,
and
G. E. Sandle.
Electrolyte transport in the mammalian colon.
In: Physiology of the Gastrointestinal Tract, edited by L. R. Johnson. New York: Raven, 1994, p. 2133-2171.
4.
Bridges, R. J.,
G. Nell,
and
W. Rummel.
Influence of vasopressin and calcium on electrolyte transport across isolated colonic mucosa of the rat.
J. Physiol. (Lond.)
338:
463-475,
1983[Abstract].
5.
Bridges, R. J.,
W. Rummel,
and
P. Wollenberg.
Effects of vasopressin on electrolyte transport across isolated colon from normal and dexamethasone-treated rats.
J. Physiol. (Lond.)
355:
11-23,
1984[Abstract].
6.
Cabantchik, Z. I.,
and
R. Greger.
Chemical probes for anion transporters of mammalian cell membranes.
Am. J. Physiol.
262 (Cell Physiol. 31):
C803-C827,
1992[Abstract/Free Full Text].
7.
Dennhardt, R.,
B. Lingelbach,
and
F. J. Haberich.
Intestinal absorption under the influence of vasopressin: studies in unanaesthetised rats.
Gut
20:
107-113,
1979[Abstract].
8.
Diener, M.,
W. Rummel,
P. Mestres,
and
B. Lindemann.
Single chloride channels in colon mucosa and isolated colonic enterocytes of the rat.
J. Membr. Biol.
108:
21-30,
1989[Medline].
9.
Donowitz, M.,
and
M. J. Welsh.
Ca2+ and cyclic AMP in regulation of intestinal Na, K, and Cl transport.
Annu. Rev. Physiol.
48:
135-150,
1986[Medline].
10.
Duc, C.,
N. Farman,
C. M. Canessa,
J.-P. Bonvalet,
and
B. C. Rossier.
Cell-specific expression of epithelial sodium channel
,
, and
subunits in aldosterone-responsive epithelia from the rat: localization by in situ hybridization and immunocytochemistry.
J. Cell Biol.
127:
1907-1921,
1994[Abstract].
11.
Frindt, G.,
L. G. Palmer,
and
E. E. Windhager.
Feedback regulation of Na channels in rat CCT IV. Mediation by activation of protein kinase C.
Am. J. Physiol.
270 (Renal Fluid Electrolyte Physiol. 39):
F371-F376,
1996[Abstract/Free Full Text].
12.
Garty, H.,
C. Asher,
and
O. Yeger.
Direct inhibition of epithelial Na+ channels by a pH-dependent interaction with calcium, and by other divalent ions.
J. Membr. Biol.
95:
151-162,
1987[Medline].
13.
Grady, G. F.,
R. C. Duhamel,
and
E. W. Moore.
Active transport of sodium by human colon in vitro.
Gastroenterology
59:
583-588,
1970[Medline].
14.
Green, K.,
and
A. J. Matty.
Effects of vasopressin on ion transport across intestinal epithelia.
Life Sci.
5:
205-209,
1966.
15.
Greger, R.,
J. Bleich,
J. Leipziger,
D. Ecke,
M. Mall,
and
K. Kunzelmann.
Regulation of ion transport in colonic crypts.
News Physiol. Sci.
12:
62-66,
1997.[Abstract/Free Full Text]
16.
Grotjohann, I.,
A. H. Gitter,
A. Köckerling,
M. Bertog,
J. D. Schulzke,
and
M. Fromm.
Localization of cAMP- and aldosterone-induced K+ secretion in rat distal colon by conductance scanning.
J. Physiol. (Lond.)
507:
561-570,
1998[Abstract/Free Full Text].
17.
Grynkiewicz, G.,
M. Poenie,
and
R. Y. Tsien.
A new generation of Ca2+ indicators with greatly improved fluorescence properties.
J. Biol. Chem.
260:
3440-3450,
1985[Abstract].
18.
Halm, D. R.,
and
R. Rick.
Secretion of K and Cl across colonic epithelium: cellular localization using electron microprobe analysis.
Am. J. Physiol.
262 (Cell Physiol. 31):
C1392-C1402,
1992[Abstract/Free Full Text].
19.
Hanley, M. R.,
H. P. Benton,
S. L. Lightman,
K. Todd,
F. A. Bone,
P. Fretten,
S. Palmer,
C. J. Kirk,
and
R. H. Michell.
A vasopressin-like peptide in mammalian sympathetic nervous system.
Nature
309:
258-261,
1984[Medline].
20.
Hays, R. M.
Cell biology of vasopressin.
In: The Kidney (4th ed.), edited by B. M. Brenner,
and F. C. Rector. Philadelphia: W. B. Saunders, 1991, vol. 1, p. 424-444.
21.
Ishida, H.,
and
Y. Suzuki.
Potassium secretion in the guinea pig distal colon.
Jpn. J. Physiol.
37:
33-48,
1987[Medline].
22.
Knobloch, S. F.,
M. Diener,
and
W. Rummel.
Antisecretory effects of somatostatin and vasopressin in the rat colon descendens in vitro.
Regul. Pept.
25:
75-85,
1989[Medline].
23.
Köckerling, A.,
and
M. Fromm.
Origin of cAMP-dependent Cl
secretion from both crypts and surface epithelia of rat intestine.
Am. J. Physiol.
264 (Cell Physiol. 33):
C1294-C1301,
1993[Abstract/Free Full Text].
24.
Köckerling, A.,
D. Sorgenfrei,
and
M. Fromm.
Electrogenic Na+ absorption of rat distal colon is confined to surface epithelium: a voltage-scanning study.
Am. J. Physiol.
264 (Cell Physiol. 33):
C1285-C1293,
1993[Abstract/Free Full Text].
25.
Leipziger, J.,
D. Kerstan,
R. Nitschke,
and
R. Greger.
ATP increases [Ca2+]i and ion secretion via a basolateral P2Y receptor in rat distal colonic mucosa.
Pflügers Arch.
434:
77-83,
1997[Medline].
26.
Levitan, R.,
and
I. Mauer.
Effect of intravenous antidiuretic hormone administration on salt and water absorption from the human colon.
J. Lab. Clin. Med.
72:
739-746,
1968[Medline].
27.
Lindqvist, S. M.,
P. Sharp,
I. T. Johnson,
Y. Satoh,
and
M. R. Williams.
Acetylcholine-induced calcium signaling along the rat colonic crypt axis.
Gastroenterology
115:
1131-1143,
1998[Medline].
28.
Lomax, R. B.,
C. M. McNicholas,
M. Lobès,
and
G. I. Sandle.
Aldosterone-induced apical Na+ and K+ conductances are located predominantly in surface cells in rat distal colon.
Am. J. Physiol.
266 (Gastrointest. Liver Physiol. 29):
G71-G82,
1994[Abstract/Free Full Text].
29.
Manning, M.,
E. Nawrocka,
A. Misicka,
A. Olma,
W. A. Klis,
J. Seto,
and
W. H. Sawyer.
Potent and selective antagonists of the antidiuretic responses to arginine-vasopressin based on modifications of [1-(
-mercapto-
,
-pentamethylene-propionic acid),2-D-isoleucine,4-valine] arginine-vasopressin at position 4.
J. Med. Chem.
27:
423-429,
1984[Medline].
30.
Manning, M.,
S. Stoev,
A. Kolodziejczyk,
W. A. Klis,
M. Kruszynski,
A. Misicka,
and
A. Olma.
Design of potent and selective linear antagonists of vasopressor (V1 receptor) responses to vasopressin.
J. Med. Chem.
33:
3079-3086,
1990[Medline].
31.
Morel, A.,
S. J. Lolait,
and
M. J. Brownstein.
Molecular cloning and expression of rat V1a and V2 arginine vasopressin receptors.
Regul. Pept.
45:
53-59,
1993[Medline].
32.
Oh, Y.,
P. R. Smith,
A. L. Bradford,
D. Keeton,
and
D. J. Benos.
Regulation by phosphorylation of purified epithelial Na+ channels in planar lipid bilayers.
Am. J. Physiol.
265 (Cell Physiol. 34):
C85-C91,
1993[Abstract/Free Full Text].
33.
Rechkemmer, G.,
and
W. von Engelhardt.
Absorption and secretion of electrolytes and short-chain fatty acids in the guinea pig large intestine.
In: Advances in Comparative and Environmental Physiology 16: Ion Transport in Vertebrate Colon, edited by W. Clauss. Berlin: Springer-Verlag, 1993, p. 139-167.
34.
Rechkemmer, G.,
R. A. Frizzell,
and
D. R. Halm.
Active potassium transport across guinea-pig distal colon: action of secretagogues.
J. Physiol. (Lond.)
493:
485-502,
1996[Abstract].
35.
Robertson, G. L.,
and
T. Berl.
Pathophysiology of water metabolism.
In: The Kidney (4th ed.), edited by B. M. Brenner,
and F. C. Rector. Philadelphia: W. B. Saunders, 1991, p. 677-736.
36.
Schang, J.-C.,
M. Dapoigny,
and
G. Devroede.
Stimulation of colonic peristalsis by vasopressin: electromyographic study in normal subjects and patients with chronic idiopathic constipation.
Can. J. Physiol. Pharmacol.
65:
2137-2141,
1986.
37.
Schapiro, H.,
and
L. G. Britt.
The action of vasopressin on the gastrointestinal tract. A review of the literature.
Am. J. Dig. Dis.
17:
649-667,
1972[Medline].
38.
Smith, P. L.,
S. K. Sullivan,
and
R. D. McCabe.
Potassium absorption and secretion by the intestinal epithelium.
In: Textbook of Secretory Diarrhea, edited by E. Lebenthal,
and E. Duffey. New York: Raven, 1990, p. 109-118.
39.
Strong, T. V.,
K. Boehm,
and
F. S. Collins.
Localization of cystic fibrosis transmembrane conductance regulator mRNA in the human gastrointestinal tract by in situ hybridization.
J. Clin. Invest.
93:
347-354,
1994[Medline].
40.
Suzuki, Y.,
T. Sano,
and
H. Hayashi.
Muscarinic receptor-mediated inhibition of electrogenic Na+ absorption in guinea pig distal colon (Abstract).
Jpn. J. Physiol.
45:
S98,
1995.
41.
Suzuki, Y.,
T. Watanabe,
and
K. Kaneko.
A novel H+,K+-ATPase in the colonic apical membrane.
Jpn. J. Physiol.
43:
291-298,
1993[Medline].
42.
Thibonnier, M.
Signal transduction of V1-vascular vasopressin receptors.
Regul. Pept.
38:
1-11,
1992[Medline].
43.
Vasallo, J. L.,
R. Arreaza,
and
L. Munoz-Barragán.
Immunocytochemical study of vasopressin-like immunoreactive material in the gastrointestinal tract of the hedgehog Erinanceus europeus.
Bol. Asoc. Med. PR
84:
67-69,
1992.
44.
Vincentini-Paulino, M. L. M.
In vitro action of vasopressin on water absorption by rat colon.
Braz. J. Med. Biol. Res.
25:
1041-1043,
1992[Medline].
45.
Ward, S. M.,
O. P. Bayguinov,
H. K. Lee,
and
K. M. Sanders.
Excitatory and inhibitory action of vasopressin on colonic excitation-contraction coupling in dogs.
Gastroenterology
113:
1233-1245,
1997[Medline].
46.
Yajima, T.,
T. Suzuki,
and
Y. Suzuki.
Synergism between calcium-mediated and cyclic AMP-mediated activation of chloride secretion in isolated guinea pig distal colon.
Jpn. J. Physiol.
38:
427-443,
1988[Medline].
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