1 Nephrology Section, Veterans Affairs Medical Center and New York University School of Medicine, New York, New York 10010; and 2 Division of Digestive Diseases, Veterans Affairs Medical Center and University of Cincinnati College of Medicine, Cincinnati, Ohio 45267
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ABSTRACT |
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We
studied the functional importance of the colonic guanylyl cyclase C
(GCC) receptor in GCC receptor-deficient mice. Mice were anesthetized
with pentobarbital sodium, and colon segments were studied in Ussing
chambers in HCO3 Ringer under short-circuit
conditions. Receptor-deficient mouse proximal colon exhibited similar
net Na+ absorption, lower net Cl
absorption,
and a negative residual ion flux (JR),
indicating net HCO3
absorption compared with that in
normal mice. In normal mouse proximal colon, mucosal addition
of 50 nM Escherichia coli heat-stable enterotoxin (STa)
increased the serosal-to-mucosal flux of Cl
(Js
mCl) and decreased net
Cl
flux (JnetCl) accompanied
by increases in short-circuit current (Isc),
potential difference (PD), and tissue conductance (G).
Serosal STa had no effect. In distal colon neither mucosal nor serosal
STa affected ion transport. In receptor-deficient mice, neither mucosal
nor serosal 500 nM STa affected electrolyte transport in proximal or
distal colon. In these mice, 1 mM 8-bromo-cGMP produced changes in
proximal colon Js
mCl and
JnetCl, Isc, PD,
G, and JR similar to mucosal STa
addition in normal mice. We conclude that the GCC receptor is necessary
in the mouse proximal colon for a secretory response to mucosal STa.
sodium and chloride ion fluxes; guanosine 3',5'-cyclic monophosphate; anion secretion
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INTRODUCTION |
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ACTIVATION OF COLONIC guanylyl cyclase C (GCC) initiates the secretory events caused by the luminal secretion of guanylin and uroguanylin by enterocytes, specifically enterochromaffin cells (2, 6, 7, 25, 29). Escherichia coli heat-stable enterotoxin (STa), which can induce watery diarrhea, also binds to GCC, a transmembrane receptor on intestinal epithelial cells (5, 15). Agonist binding activates guanylyl cyclase, generating cGMP (11, 27). cGMP in turn may interact with cGMP-regulated phosphodiesterases or protein kinases or with cGMP-sensitive apical anion channels (34, 35). Ultimately, the cystic fibrosis transmembrane conductance regulator (CFTR) is activated, and electrogenic anion secretion results. Although the clinical consequences of STa stimulation of intestinal secretion are well known, the physiological importance of intestinal GCC itself (and its endogenous ligands) remains uncertain.
The GCC receptor has been identified along the entire small intestine and colon of the mouse (33), rat (6), opossum (13), and human (4, 22) as well as in a number of other mammals (22). However, STa binding to and activation of GCC does not mean that it is the only means of inducing intestinal secretion. For example, despite apparently similar receptor density, intestinal segments in the rat have markedly different secretory responses to STa (3, 24, 36). Whether prostaglandins or enteric nerves are involved is unclear (14, 20), but there certainly are several steps in the cGMP secretory cascade that are subject to endogenous modulation (3, 14, 20, 35).
We examined the importance of this receptor in two ways. We studied the effects of mucosal STa in GCC receptor-deficient mice. In previous studies (23, 30), GCC receptor-deficient mice exhibited a lack of secretagogue-induced intestinal secretion when examined by the suckling mouse assay. This assay is a valuable screening tool to determine whether STa causes secretion (i.e., by measuring net fluxes and the effects of blood flow, motility, and neuronal and hormonal influences). However, it does not involve a direct measurement of active colonic electrolyte transport. Such measurements in normal and knockout mice would serve to document the importance of the GCC receptor in the secretory cascade and characterize the secretory effect. We also examined whether STa could induce secretion when present on the serosal surface of the epithelium. These experiments were of particular interest in GCC receptor-deficient mice, in which upregulation of a serosal mechanism of action may make an effect easier to detect.
We were also interested in characterizing electrolyte transport in the normal mouse colon. The study of electrolyte transport has benefited greatly from the use of rat, rabbit, and guinea pig intestine. Models of altered electrolyte physiology have been developed (17, 23, 35) in the mouse, including models of gene deletion. Although electrical parameters are often measured and the in situ suckling mouse assay is often used, to date there are few data describing electrolyte transport in the normal mouse intestine. We (17) recently described the electrolyte transport pattern in the mouse distal colon in a study of the effects of acid-base variables in carbonic anhydrase II-deficient mice. Of interest was the finding that active NaCl absorption in the mouse distal colon was stimulated by changes in ambient pH rather than in CO2, the important acid-base variable in the rat colon (16). Such subtle interspecies differences underscore the importance of directly measuring electrolyte flux. The present study includes the first ion flux measurements reported in mouse proximal colon.
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MATERIALS AND METHODS |
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Animals.
After approval of the Veterans Affairs Subcommittee for Animal Studies,
male and female mice were housed in Plexiglas cages with both food
(standard chow pellets) and water available ad libitum. Mice lacking
the GCC receptor were generated as previously described
(23). In brief, the mouse gene encoding GCC was disrupted at the 5' end and chimeric mice were generated. Chimeras were crossed
with Black Swiss mice, and the resulting heterozygous littermates
(hybrids of 129/Sv and Black Swiss) were bred to produce homozygous GCC
(/
) and GCC (+/+) mice. Wild-type (normal) and homozygous knockout
(GCC receptor-deficient) mice used in these experiments are the progeny
of these F2 mice. The absence of GCC mRNA and protein was
verified by Northern blot, RT-PCR, and Western blot analyses.
Histological examination of major organ systems and arterial blood gas
analyses were normal. These mice did not exhibit STa-induced
secretion in the suckling mouse assay (23).
Ion flux experiments. Flux experiments were conducted as described previously (17). While mice were under pentobarbital sodium anesthesia (5 mg/100 g body wt), the entire colon was removed and rinsed with 0.9% saline. Pairs of resected, unstripped proximal and distal colons were mounted in modified Ussing half-chambers exposing a surface area of 0.385 cm2. Tissues were studied under short-circuit conditions. Periodic bipolar pulses of 0.5 mV yielded electrical current values that were used to calculate tissue conductance (G). Tissues were paired for ion flux studies only when differences in G were not >25%. The short-circuit current (Isc) divided by G yielded the active transport potential difference (PD), which was referenced to the mucosal side.
Unidirectional fluxes of Na+ and ClSolutions.
Reagent grade chemicals were purchased from Sigma Chemical (St. Louis,
MO) unless otherwise indicated. All solutions were maintained at
37°C. The HCO3 Ringer contained (in mmol/l) 10 glucose, 96 NaCl, 4 KCl, 2.4 Na2HPO4, 0.4 NaH2PO4, 1 CaSO4, 1.2 MgSO4, 21 NaHCO3, and 18 Na+
gluconate. This solution was gassed with 5% CO2-95%
O2, yielding a PCO2 of 35 mmHg and
a pH of 7.40.
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Data analysis. All data are expressed as means ± SE and were compared by paired or unpaired Student's t-test. Two-tailed values of P < 0.05 were considered significant.
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RESULTS |
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Colonic transport in GCC (+/+) mice. No differences were found in the ion flux rates across the proximal colon of male and female mice; therefore, all data were combined. The effect of mucosal 0.25-500 nM STa on the Isc of the proximal and distal colon is shown in Fig. 1. The lowest concentration that produced a maximal response in Isc in both segments was 50 nM, and this concentration was used in all flux experiments. The increase in Isc occurred immediately after STa addition and was sustained for at least 72 min.
As shown in Table 1, under control conditions GCC (+/+) mice exhibited a serosal positive PD, net Na+ absorption, and minimal net Cl
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Colonic transport in GCC (/
) mice.
Table 3 shows the basal flux rates and
effects of mucosal STa in GCC receptor-deficient mice. When perfused
with HCO3
-Ringer under control conditions, GCC
(
/
) mice exhibited a serosal positive PD and net Na+
absorption in proximal colon similar to GCC (+/+) mice. However, net
Cl
absorption was 0 compared with minimal absorption in
normal mice [
0.9 ± 0.8 (n = 11) vs. 1.0 ± 0.5 µeq · cm
2 · h
1
(n = 20 derived from Tables 1 and 2); P < 0.03], and a negative JR was observed,
indicating net HCO3
absorption [
3.4 ± 0.9 (n = 11) vs.
0.4 ± 0.8 µeq · cm
2 · h
1;
n = 20; P < 0.02].
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DISCUSSION |
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The development of genetically altered mice has increased the use
of mouse intestine in physiological research. The control data in this
study are the first measures of active electrolyte transport in murine
proximal colon. Together with our recent publication describing
(17) the mouse distal colon, a more complete picture of
electrolyte transport has emerged. Both proximal and distal colon
exhibit a lumen-positive PD and net Na+ absorption when
perfused with HCO3-Ringer. Like human, rat, and
rabbit colon, the mouse colon has a proximal-to-distal gradient of
conductance, although it appears to be somewhat more permeable than
these tissues (3, 31). Of interest is that mouse distal
colon absorbs Cl
at a greater rate than the proximal
colon. It is unclear whether this reflects the experimental conditions
or whether there are fewer or different Cl
absorptive
processes along the apical membranes of proximal colonic epithelial cells.
The values for Na+ transport and various electrical
parameters were similar in normal and GCC receptor-deficient mice.
However, net Cl absorption was lower in GCC (
/
) mice
than in normal mice, and a negative JR was
observed (indicating net HCO3
absorption). These
differences most likely reflect a lower level of apical membrane
Cl
/HCO3
exchange in receptor-deficient
mice. Another possibility is that a basal HCO3
secretory process, normally active, is absent or reduced in GCC (
/
)
mice. Guanylin is secreted by mouse colonic mucosa, and active
Cl
and HCO3
secretion could be present
in normal mice under basal conditions. However, if a basal anion
secretory process is absent, JnetCl should
be greater, not smaller, in GCC (
/
) mice than in GCC (+/+) mice
(because of a lower Js
mCl).
Indeed, when basal secretion was tested for in normal mice with
bumetanide and butyrate, no evidence for active, basal Cl
secretion was found in either proximal or distal colon. Bumetanide inhibits Na+-K+-2Cl
cotransport
and thereby inhibits Cl
entry across the basolateral
membrane (1). Butyrate appears to compete for apical anion
channels in intestinal epithelia (10). By the use of these
same inhibitors, rat distal colon (but not proximal colon) exhibits
active, basal Cl
secretion (3, 10). An
increase in intracellular HCO3
concentration also
inhibits active Cl
secretion in rat distal colon
(9) but does not affect basal Js
mCl in mouse distal colon
(17), confirming the present findings.
Another finding in this study is the confirmation that GCC
receptor-deficient mice do not respond to mucosal exposure to STa. Although the suckling mouse assay was initially used to confirm the
gene disruption that generated these mice (23, 30), this assay does not involve measurement of unidirectional or active electrolyte transport. In addition, there is a minimal but reproducible degree of specific binding of STa to colonic mucosal cell membranes in
GCC (/
) mice (23). This suggests the presence of an
apical membrane receptor other than GCC and/or a serosal membrane
receptor (see below). However, our data indicate that GCC (
/
) mice
do not exhibit any of the STa-induced changes in colonic
Isc, PD, G,
Js
mCl, or
JnetCl that are typical of normal mice. This
finding appears to exclude a second apical membrane receptor and the
possibility that prostaglandin release mediates a portion of the
secretory response in the mouse, as has been found in pig colon
(20). Because mucosal nerves were present in our
unstripped mouse preparation, a role for the enteric nervous system,
which may be significant in rat colon (36), also appears
unlikely. Our data clearly indicate that mucosal STa cannot access any
secretory step between guanylyl cyclase activation and anion flux
across the apical membrane other than the GCC receptor itself and, at
least in vitro, no other secretory pathway.
We also tested for the integrity of the secretory pathway beyond the
GCC receptor. The addition of cGMP in GCC (/
) mice produced a
secretory effect identical to STa in normal mice and confirmed that the
gene product deletion was limited to the receptor alone. Two other
deletions elsewhere in the secretory pathway have been described.
Humans with cystic fibrosis (CF) and transgenic CF mice both lack the
CFTR anion channel, and their colons are nearly or completely
unresponsive to mucosal STa and guanylin, respectively (8,
18). However, the duodena of these CF patients and CFTR knockout
mice do respond to STa or uroguanylin. The patients have normal
increases in net HCO3
secretion and
Isc in response to 100 nM mucosal STa, and the mice exhibit small but reproducible increases in
Isc at uroguanylin concentrations between 1 and
10 µM (21, 26).
The jejuna of cGMP-dependent protein kinase II-deficient mice also have
a small but significant Isc response to (100 nM)
mucosal STa, although not to cGMP (35). In contrast with
the models of anion channel deletion described above, the proximal
colons of these animals have a nearly normal response to STa both in vivo and in vitro. These findings apparently indicate the existence of
a colonic (and possibly small intestinal) secretory pathway independent
of this kinase, perhaps involving a cGMP-inhibitable phosphodiesterase
or activation of a cAMP-dependent kinase (35). Although
our findings in GCC (/
) mice do not have a bearing on this
possibility, they do suggest that some steps in this secretory pathway
can be bypassed and others cannot. These three models (with specific
deficiencies in the receptor, protein kinase, or anion channel) offer
unique opportunities to examine this secretory mechanism and its
relation to other transport pathways, absorptive and secretory
(28).
Finally, our data suggest that STa has no effect on proximal or distal
colonic electrolyte transport when present on the serosal surface. The
finding has meaning because, as noted above, there is a minimal degree
of specific binding of STa to colonic mucosal cell membranes in GCC
(/
) mice (23). Moreover, uroguanylin, which activates
GCC when present on the luminal surface of intestinal epithelial cells,
also is secreted into the bloodstream. On reaching the kidneys,
uroguanylin can cause natriuresis and kaliuresis (12, 19).
This suggests that uroguanylin may have two functions: aiding the
digestive process by causing luminal fluid secretion as chyme passes
along the bowel and signaling the kidney as intestinal absorption
proceeds to expedite Na+ and K+ excretion.
Although it is doubtful that a serosal GCC receptor and second
signaling pathway exist in the mouse colon, serosal activation of
guanylyl cyclase (or another secretory pathway) may be present in other
tissues or species.
In conclusion, we have described the transport pattern in normal mouse
proximal colon, which is similar but not identical to the rat, rabbit,
and human colon. We also found that the proximal colon of GCC (/
)
mice does not respond to mucosal STa, although the remainder of this
cGMP-stimulated secretory pathway is intact. Neither normal nor
receptor-deficient mice respond to serosal STa. The GCC (
/
) mouse
and other models of genetically engineered breaks in the cGMP secretory
pathway are valuable tools to study the pathogenesis of intestinal secretion.
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ACKNOWLEDGEMENTS |
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This material is based on work supported by the Office of Research and Development, Medical Research Service, Department of Veterans Affairs.
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FOOTNOTES |
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Address for reprint requests and other correspondence: A. N. Charney, Nephrology Section, VA Medical Center, 423 E. 23rd St., New York, NY 10010.
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.
Received 8 June 2000; accepted in final form 1 September 2000.
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