1 Institut Curie, UMR 144, 75248 Paris cedex 05; and U.410 and E.0112, Faculté Xavier Bichat, 75870 Paris cedex 18, France
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ABSTRACT |
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This study was done to establish and
validate a single-pass perfusion method for measuring the absorption of
water and electrolytes by the mouse small intestine. The method was
then used to study intestinal absorption in mice whose villin gene had
been invalidated (v/
). The single-pass perfusion of the
jejunum measures the absorption of water, Cl
,
Na+, K+, HCO
/
mice in vivo.
We measured absorption under basal and stimulated conditions
(carbachol, vasoactive intestinal polypeptide, intralumen
PGE2). Basal absorption and stimulated secretions were
similar to those previously obtained in rats. There was no difference
between wild-type and v
/
mice in animals with mixed
genetic background or in pure C57BL6 mice. We conclude that this in
vivo perfusion method is suitable for studying the absorption/secretion
of electrolytes in the mouse intestine and that a lack of villin does
not significantly alter basal and secretagogue-stimulated electrolyte
movements across the epithelium of the mouse jejunum in vivo.
intestinal hormones; transport; brush-border epithelium; jejunum
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INTRODUCTION |
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TRANSGENIC MICE ARE POWERFUL tools for studying the physiological functions of many genes and their products. A foreign gene may alter the function of a native gene and so modify its function. Functions directly or indirectly affected must be screened to detect changes in physiological parameters.
Because mice are small, most published studies have used histological or biochemical tests on fragments of tissues or suspensions of isolated cells to test the effects of the gene of interest. Few data have been obtained in mice by measuring organ function in physiological conditions in vivo, except for general parameters such as blood pressure, body weight, or oxygen consumption.
Tissue and animal models are required to study the functions of epithelial cells, such as absorption and secretion of electrolytes in the small intestine. These functions are well documented in vivo in the rat (5, 21, 22, 24), whereas most studies on mice have used Ussing chamber experiments (6, 10, 13). However, these are more suitable for exploring mechanisms than for establishing physiological secretory conditions.
The present study was done to establish and validate a single-pass
perfusion method for measuring the intestinal absorption of water and
electrolytes by the mouse small intestine. We then used this method to
study intestinal absorption in mice in which the villin gene had been
invalidated (v/
mice) (9).
Villin is an actin-binding protein located in the intestinal and
renal brush borders. In vitro, villin presents F-actin
bundling and severing activities depending on the Ca2+
concentration (3). In vivo pharmacological (carbachol
stimulation) or pathophysiological situations that increase the
intracellular Ca2+ concentration result in impaired changes
in the actin cytoskeleton in v/
mice (9).
The F-actin fragmentation is also necessary for carbachol to inhibit
NaCl absorption in the ileum (14). Villin could therefore
be implicated in modulating transporter activities by altering the
dynamic stability of the actin cytoskeleton. We have measured the basal
absorption of water and electrolytes and the effect of hormones that
modulate their transport (4, 7), such as carbachol,
vasoactive intestinal polypeptide (VIP), and PGE2, in
wild-type and v
/
mice.
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METHODS |
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The method used was adapted from the intestinal perfusion method, used in rats, developed by Schanker et al. (20) and described in Berlioz et al. (1).
Surgery
Each mouse was fasted for 12 h and allowed drinking water ad libitum before it was anesthetized with 1.5 g/kg ip ethylurethane. Each mouse was then placed under a heating lamp to keep its core temperature at 38 ± 0.2°C. It breathed spontaneously, and the normal color of the ears and feet indicated good oxygenation throughout the experiment. The abdominal wall was opened, and the jejunum was ligated 1 cm below the ligament of Treitz. A Silastic perfusion catheter (Silastic 602.205, ID 1.02 mm, OD 2.16 mm) bearing two silicone swellings ~3 mm apart was inserted into the gut just below the ligature and secured in place by a silk ligature between the two swellings. A collecting cannula (Silastic 602.305, ID 1.98 mm, OD 3.18 mm) was inserted into the lumen of the jejunum 10 cm further down to collect the perfusion fluid (Fig. 1). A third catheter (Silastic 602.155, ID 0.64 mm, OD 1.19 mm) was placed in the peritoneum for intraperitoneal injections. The abdominal wall was closed with three sewing points of 4-0 silk. The isolated segment of jejunum was quickly rinsed with perfusion fluid, the input catheter was attached to a perfusion pump (Minipuls II, Gilson Instruments, speed 80, Technicon Tygon tubes black/black R3607, producing a flow of ~2 ml/15 min), and the output catheter was placed over a fraction collector set to collect 15-min samples. The total perfusion time was ~135 min. Each mouse was killed at the end of the perfusion, and the segment of jejunum was removed and its length measured. Macroscopic examination of the abdomen contents indicated no local necrosis or hypoperfusion.
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Perfusion Fluid and Protocol
The perfusion fluid was a Krebs-Ringer-bicarbonate solution (KRB) containing (in mM): 120 NaCl, 4.5 KCl, 0.7 Na2HPO4, 1.5 NaH2PO4, 1.2 CaCl2, 0.5 MgCl2, and 10 glucose. Perfusion began with an equilibration period of 45 min; these samples were discarded. Three 15-min samples were then collected (t =Determinations
Volume. The volume of each sample was determined by weight (assuming that 1 ml = 1 g). The actual input volume was determined before each perfusion by averaging the weight of three 15-min samples directly from the pump. The absorbed or secreted volume was calculated as collected volume minus perfused volume and expressed per centimeter of perfused bowel. Negative values indicate absorption, and positive values indicate secretion.
Ion transport.
Na+, K+ (ion electrodes), Cl
(interaction with mercury sulfocyanide and subsequent photometry of
iron sulfocyanide), total CO2 (enzymatic method with
phosphoenol pyruvate carboxylase), and glucose (hexokinase) were
measured by standard established clinical methods using a laboratory
autoanalyzer (Kone Specific Supra 4.4, Kone Instruments, Evry, France).
The net fluxes of ions and glucose were calculated from the
concentrations in the influx and efflux fluids and the flow volume.
Animals.
v/
Mice were obtained as previously described
(9). The following experiments were performed on sibling
v
/
mice and wild-type animals with either a mixed
genetic background (M2) or on mice obtained by multiple crosses with
the C57BL6 genetic background, which gave pure C57BL6 wild-type and
v
/
mice (B5). The Curie Institute has approved the
protocol used for animal testing.
Statistical Analysis
Groups were compared by two-way ANOVA, which separated the effects of treatment vs. control and v ![]() |
RESULTS |
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Basal Absorption in M2 and B5 Wild-Type Mice
The average values of all of the variables (fluid, Na+, K+, Cl
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Hormone Stimulation of Wild-Type Animals
Carbachol decreased fluid absorption by ~
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VIP (50 nmol/kg ip) produced a decrease in water absorption in M2
wild-type mice (n = 6) (Table 2, Fig.
3) that was larger than that produced by
carbachol. The increase in secretion peaked in the first (0-15
min) fraction collected after VIP injection and returned to the
preinjection level 45 min after VIP injection (Fig. 3). The other
variables measured were also shifted toward secretion after VIP
injection (P < 0.001), except for glucose absorption.
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PGE2 (10 µg/ml) was dissolved in KRB containing 1%
ethanol and perfused into the jejunum lumen (n = 6).
The ethanol in the perfusing fluid produced a slight, transient
increase in the volume absorbed during the first 15-min period (30 to
15 min; Fig. 4). The volume absorbed
returned to the preethanol values in the
15- to 0-min period (Fig.
4). PGE2 produced a progressive and significant (Table 2,
Fig. 4) decrease in absorption that affected all of the variables
measured (even glucose absorption was slightly decreased after
PGE2). Total CO2 net flux turned to clear
secretion during PGE2 infusion. The peak secretion for all
variables occurred during the 15- to 30-min period, and this secretion
remained constant during the 30- to 45-min period of PGE2
infusion (Fig. 4).
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Basal Absorption in M2 and B5 v/
Mice
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Hormone Stimulation in v/
Mice
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DISCUSSION |
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Our studies on the absorption of water and electrolytes by the
mouse jejunum gave basal and hormone-stimulated values of the same
order of magnitude as those found for the rat. We found no major
differences between the values for v/
mice and their
wild-type littermates for both mixed and pure (C57BL6) genetic backgrounds.
This is the first report, to our knowledge, of intestinal transport in
vivo in mice measured by using a perfusion method. Our unpublished
studies on Wistar rats under similar conditions provided reference
basal values of intestinal absorption. The basal absorption of water in
Wistar rats was greater per centimeter of intestine than the absorption
in mice (45.9 ± 2.9 to
64.8 ± 5.6 µl · cm
1 · 15 min
1 from 7 series of 6 rats each). This may be due to differences in the
absorptive capacity of the jejunal epithelium or to differences in the
absorptive surfaces and/or in the rate of perfusion (2 ml/15 min in
mice vs. 4 ml/15 min in rats). This perfusion flow rate was chosen to
take into account the smaller diameter of the mouse jejunum. Rats
absorbed the various electrolytes [basal values from 7 different
series of experiments including 5-7 Wistar rats each (in
µmol · cm
1 · 15 min
1);
Na+:
3.31 ± 0.65 to
6.84 ± 0.65;
K+:
0.08 ± 0.08 to
0.31 ± 0.06;
Cl
:
5.65 ± 0.26 to
7.07 ± 0.34; and
glucose
1.45 ± 0.15 to
1.72 ± 0.13] at about the same
rates as mice, except that HCO
1.43 ± 0.15 to
1.74 ± 0.19 µmol · cm
1 · 15 min
1) seems to be less readily taken up by the
mouse jejunum, and this mainly in M2 mice.
Most published studies on mice have used in vitro methods in an Ussing
chamber, measuring the short-circuit current
(Isc), which reflects global electrogenic ion
transport across the mucosa. Isc is usually
believed to mainly reflect Cl secretion by intestinal
crypts (13). The basal values of
Isc in the mouse duodenum and jejunum are on the
order of 30-50 µA/cm2 of mucosa (6, 13,
22), with large individual variations, when measured in KRB
solution without glucose in the mucosal compartment. There are few
reports of individual ion transport: a net Na+ influx of
5-6 µeq · cm
2 · h
1
was reported in wild-type mouse jejunum with KRB, corresponding to
neutral NaCl transport (6). Our in vivo data of ~12
µeq · cm
2 · h
1 are in the
same range, assuming an equivalent Ussing chamber area of 1 cm/cm2 of jejunum length and a doubling of
Na+ absorption in the presence of 10 mM glucose.
Carbachol, VIP, and PGE2 all stimulated secretion in vivo,
as indicated by studies on the mouse jejunal mucosa in Ussing chambers (7) and in the rat jejunum in vivo (21, 23).
The amplitude and duration of the responses, however, depend greatly on
the conditions of drug administration. Carbachol and VIP were given as
single intraperitoneal injections, and their effects lasted only
15-30 min. PGE2 was added to the lumen perfusion fluid
and had a progressive effect that peaked 30 min after the onset of perfusion and lasted as long as PGE2 remained in the
perfusion fluid. A clear secretory effect was observed with 50 nmol/kg
VIP (166 µg/kg ip), whereas effects of this order of magnitude were obtained with intravenous doses of 30-100
µg · kg1 · h
1 of VIP in
the rat (23). PGE2 was given as a steady-state
perfusion, whereas carbachol and VIP were given as bolus injections.
Thus their secretory and possible motor effects changed the steady state conditions, so that the absolute absorption values obtained should be considered with caution. However, the results for the wild
type and v
/
mice were always parallel. Hence, it is
unlikely that there was any obvious difference between the two groups
of mice.
Villin is mainly expressed in absorptive epithelial cells, such as
those of the small and large intestine and the kidney proximal tubule
(2, 16, 18). Villin belongs to a large family of Ca2+-regulated actin-binding proteins that are structurally
and functionally similar. In vitro, villin presents F-actin
bundling and severing activities, depending on the Ca2+
concentration (3). Villin-severing capacity might be
involved under certain physiological conditions, in bacterial
infection, or in response to fasting and feeding, which are believed to
modulate intracellular Ca2+ (11, 15, 17).
Carbachol activates basolateral muscarinic receptors, leading to an
increase in intracellular Ca2+ (8). We have
shown that carbachol placed directly on jejunum loops isolated in situ
causes the brush-border actin to fragment, which was not observed in
v/
mice (9).
Studies on rabbit designed to analyze the inhibition of NaCl absorption
by carbachol suggest that villin is involved in intestinal absorption
(14). The authors proposed that carbachol inhibits NaCl
absorption by fragmenting actin due to the severing activity of villin
when the intracellular Ca2+ concentration is
increased. Nevertheless, the stabilization of F-actin filaments
by jasplakinolide only slightly increased the carbachol-abolished NaCl
transport: NaCl absorption remained very low, ~15% of the basal
absorption. The present study shows no differences in water and
electrolyte transport by the wild-type and v/
mice under
both basal and stimulated conditions. This suggests that the transport
of these ions is not mainly correlated with villin-induced actin
fragmentation. As our previous data showed that F-actin is stable in
the absence of villin (9), the present results suggest
that the fragmentation of F-actin is not a major parameter in NaCl
absorption. This conclusion is supported by the small inhibitory effect
of jasplakinolide (14). However, studies on the intact
animal level point out the complexity of the interactions that maintain
a normal intestinal function. The present studies on mice were not
intended to specifically study Na+/H+ exchange,
which might be slightly different in wild-type and v
/
mice, but to simulate physiological in vivo conditions and so
determine whether the small differences found by in vitro methods were
important in vivo. Invalidation of the villin gene is apparently not
sufficient to cause significant absorptive changes under our conditions, probably because of the complexity of the many parameters that interact to maintain normal intestinal function.
In conclusion, we have developed an in vivo method for studying
intestinal transport in mice. The absence of any difference between
wild-type and v/
mice confirms the complexity of the interactions between proteins at the cell, tissue, organ, and animal
levels. The exact role of villin under pathological conditions remains
to be further investigated.
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FOOTNOTES |
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Address for reprint requests and other correspondence: S. Robine, Laboratoire de Morphogénèse et Signalisation Cellulaires, Institut Curie, UMR 144, 26, rue d'Ulm, 75248 Paris cedex 05, France (E-mail: sylvie.robine{at}curie.fr).
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. Section 1734 solely to indicate this fact.
First published January 2, 2002;10.1152/ajpgi.00327.2001
Received 31 July 2001; accepted in final form 12 December 2001.
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