1 Department of Pharmacology, Uroguanylin is an intestinal peptide hormone
that may regulate epithelial ion transport by activating a receptor
guanylyl cyclase on the luminal surface of the intestine. In this
study, we examined the action of uroguanylin on anion transport in
different segments of freshly excised mouse intestine, using
voltage-clamped Ussing chambers. Uroguanylin induced larger increases
in short-circuit current
(Isc) in
proximal duodenum and cecum compared with jejunum, ileum, and distal
colon. The acidification of the lumen of the proximal duodenum (pH
5.0-5.5) enhanced the stimulatory action of uroguanylin. In
physiological Ringer solution, a significant fraction of the
Isc stimulated by
uroguanylin was insensitive to bumetanide and dependent on
cyclic GMP; bicarbonate transport; chloride transport; cystic
fibrosis; cystic fibrosis transmembrane conductance regulator; guanylyl
cyclase; mouse intestine; proximal duodenum
UROGUANYLIN is an intestinal peptide that is closely
related to guanylin, another intestinal peptide that is secreted onto the intestinal epithelial surface and regulates transepithelial salt
and water transport through a receptor-mediated action (19, 26, 27, 34,
50). Guanylin was first discovered in attempts to identify an
endogenous ligand for the apical membrane-bound guanylyl cyclase C,
which serves as the receptor for Escherichia coli heat-stable enterotoxin (STa) (6, 13, 20, 35, 36, 48, 54, 55), the causative agent of traveler's diarrhea (12). Guanylin
binding to the receptor increases intracellular cGMP, resulting in
activation of protein kinase G II in the intestinal epithelial cell
(18, 45). The subsequent intracellular events include stimulation of
anion secretion via the cystic fibrosis transmembrane conductance
regulator (CFTR) (3, 7, 16, 21) and possibly the inhibition of
electroneutral NaCl absorption (37, 52). Much less is known about the
intestinal actions of uroguanylin. However, it has been shown that the
peptide stimulates intracellular cGMP production and transepithelial
Cl Although uroguanylin and guanylin share nearly 50% identity in their
primary amino acid sequences, uroguanylin differs from guanylin with
regard to intestinal expression and intrinsic biochemical properties
(26, 27, 34). First, both uroguanylin and guanylin are found throughout
the length of the rat intestinal mucosa, but uroguanylin mRNA is most
abundant in the proximal small intestine, whereas guanylin mRNA is
greatest in the distal small intestine and large bowel (39, 40, 42). In
opossums, uroguanylin mRNA is abundant in the duodenum but also has
high levels of expression in the large intestine compared with guanylin
(11). Second, the amino acid sequence of uroguanylin has a conserved
asparagine residue, instead of the phenylalanine (in opossum) or
tyrosine (in other species) found in guanylin, which makes uroguanylin resistant to proteolysis by the pancreatic enzyme chymotrypsin (25).
Third, uroguanylin has two additional acidic amino acids that render it
a more strongly acidic molecule. Interestingly, uroguanylin is more
potent at acidic pH, whereas guanylin is more potent at alkaline pH, in
stimulating cGMP production and
Cl The effects of uroguanylin on anion secretion have not been previously
examined in the intact mammalian intestinal mucosa. On the basis of its
higher expression in duodenum and its pH dependence of action, it was
reasoned that uroguanylin may be more effective in regions of the
intestinal tract where the mucosa is exposed to acidic luminal
conditions. Therefore, different segments of the murine intestine were
investigated for responsiveness to uroguanylin by measuring the
short-circuit current
(Isc), an index
of anion secretion. In the most responsive intestinal segments
(proximal duodenum and cecum), we examined the pH dependence of
uroguanylin in stimulating transepithelial secretion of
Cl Animals
ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
in the bathing medium.
Experiments using pH-stat titration revealed that uroguanylin stimulates serosal-to-luminal
secretion (
)
together with a larger increase in
Isc. Both
and Isc were
significantly augmented when luminal pH was reduced to pH 5.15. Uroguanylin also stimulated the
and Isc across
the cecum, but luminal acidity caused a generalized decrease in the
bioelectric responsiveness to agonist stimulation. In cystic fibrosis
transmembrane conductance regulator (CFTR) knockout mice, the duodenal
Isc response to
uroguanylin was markedly reduced, but not eliminated, despite having a
similar density of functional receptors. It was concluded that
uroguanylin is most effective in acidic regions of the small intestine,
where it stimulates both
and
Cl
secretion primarily via
a CFTR-dependent mechanism.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
secretion in T84 cells,
a cell line derived from a colonic adenocarcinoma (17, 24, 27). Both
uroguanylin and guanylin are abundantly expressed in the intestinal
epithelium as inactive precursors, or propeptides, that undergo
enzymatic cleavage to yield the bioactive peptides (11, 29, 43).
Uroguanylin and, to a lesser extent, guanylin are expressed in other
tissues and have been isolated from plasma and urine (11, 25, 27, 28,
40, 44, 46). Recent studies demonstrate that uroguanylin induces
natriuresis in the perfused rat kidney (M. Fonteles, personal
communication), suggesting that (intestinal) uroguanylin may also be
elaborated into the blood as an endocrine mediator of renal function
(14).
secretion in T84 cell
monolayers (24, 27).
and
across the mucosa.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
/
) mice (5). All
experiments involving the animals were approved by the University
Institutional Animal Care and Use Committee.
Tissue Preparation
Before each experiment, the mice were fasted for a minimum of 1 h and only water was provided. The mice were killed by a brief exposure to a 100% CO2 gas atmosphere (to induce narcosis), followed immediately by a surgical pneumothorax. A midline abdominal incision was used to excise the gallbladder and the following intestinal segments: proximal duodenum (a portion from 2 mm distal to the pylorus to the sphincter of Oddi), midjejunum, ileum, cecum (a portion 1-2 cm proximal to the cecal apex), and distal colon. The excised segments were opened along the mesenteric border in ice-cold, oxygenated Krebs-Ringer-bicarbonate (KRB) solution and pinned mucosal-side down on a pliable silicone surface. The intestinal sections were stripped of their outer muscle layers by shallow dissection with a scalpel and fine forceps.Bioelectric Measurements
Each intestinal sheet (~1 cm in length) and the microdissected gallbladder (with a support of nylon gauze) were mounted in standard Ussing chambers with an exposed surface area of 0.25 cm2 for intestinal preparations (or 0.126 cm2 for ileum and colon) and 0.049 cm2 for the gallbladder as previously described (5). The tissue sheets were independently bathed on the serosal and mucosal surfaces with 4 ml of KRB solution containing (in mM) 115 NaCl, 4 K2HPO4, 0.4 KH2PO4, 25 NaHCO3, 1.2 MgCl2, and 1.2 CaCl2, pH 7.4. To facilitate pH adjustment of the medium in some experiments, a phosphate-free Ringer solution of the following composition was used (in mM): 115 NaCl, 5 KCl, 25 NaHCO3, 1.2 MgSO4, and 1.2 calcium gluconate, pH 7.4. In ion substitution experiments,Transmural Isc (µA/cm2 tissue surface area) was measured with the use of an automatic voltage clamp device (VCC-600; Physiologic Instruments, San Diego, CA) that compensates for electrode offset and the fluid resistance between the potential-measuring electrode bridges. Transepithelial potential difference (in mV) was measured via a pair of calomel half-cells connected to the serosal and mucosal baths by 4% agar-Ringer (wt/vol) bridges. Isc was applied across the tissue via a pair of Ag/AgCl electrodes that were kept in contact with the serosal and mucosal baths through 4% agar-Ringer bridges. All experiments were carried out under short-circuited conditions. Total tissue conductance (Gt, mS/cm2 tissue surface area) was calculated by applying Ohm's law to the current deflection resulting from a 5-mV bipolar pulse across the tissue every 5 min during the course of the experiment. In all cases, the serosal side served as ground and the Isc was conventionally referred to as negative when current flowed from the lumen to the serosa.
After the tissues achieved a stable
Isc (~20 min
post-TTX), a 20-min period was required to adjust the luminal bath pH.
The small intestine and gallbladder preparations were then sequentially exposed to a peptide (uroguanylin, 1.0 µM; guanylin, 1.0 µM; or STa, 0.02 µM) in the luminal bath for 30 min and then to bumetanide (0.1 mM) in the serosal bath for 10 min to inhibit the
Na+-K+-2
Cl cotransporter. After pH
adjustment, large intestinal preparations were first treated with
amiloride (0.1 mM) in the luminal bath for 20 min to inhibit
electrogenic Na+ absorption and
were then treated with peptide addition followed by bumetanide. In
studies in which the effects of acidic pH on the action of the peptides
were examined, the proximal duodenum, jejunum, and cecum were used, and
the pH of the luminal bath was decreased to pH 5.0-5.5 by addition
of 1 N HCl. An equal amount of 1 M NaCl was added simultaneously to the
serosal bath to prevent a transepithelial
Cl
diffusion potential. At
the end of an experiment, glucose (10 mM) was added to the luminal bath
of the small intestinal preparations and carbachol (CCh; 0.1 mM) was
added to the serosal bath of the large intestinal preparations as
measures of tissue viability.
Bioassay for cGMP Accumulation in Mouse Intestine
Mucosal epithelium was prepared by scraping the intestinal segment from cftr(pH-Stat Titration
Proximal duodenum or cecum was mounted in a standard Ussing chamber bathed with 156.2 mM NaCl in the luminal bath and KRB in the serosal bath. In cecal experiments, 1.2 mM CaCl2 and MgCl2 were also added to the luminal bath. The luminal bath was gassed with 100% O2 and the serosal bath with 95% O2-5% CO2. To decrease the pH of the luminal bath, the luminal saline solution was gassed with 95% O2-5% CO2 and maintained at pH 5.15. The serosal-to-luminal flux ofReceptor Autoradiography
Dissected intestinal segments (proximal duodenum, midjejunum, ileum, cecum, and distal colon) from control and CFTR knockout mice were quickly frozen with dry ice and stored atMaterials
Opossum uroguanylin (QEDCELCINVACTGC) and E. coli ST (CCELCCNPACTGC), STa13, were synthesized by the solid-phase method, using an Applied Biosystems peptide synthesizer, as previously described (25). Opossum uroguanylin differs from murine uroguanylin (TDECELCINVACTGC) only in the sequence of the first three amino acids. Purified rat guanylin (PNTCEICAYAACTGC) was generously provided by Dr. Mark Currie (Searle Research and Development, St. Louis, MO). The iodination of E. coli ST (NSSNYCCELCCNPACTGCY) was performed using a lactoperoxidase method as previously described (15, 16). Membrane-permeable 8-bromo-cAMP (8-BrcAMP) and 8-bromo-cGMP (8-BrcGMP) were obtained from Research Biochemical International (Natick, MA). All other chemicals were purchased from either Sigma Chemical (St. Louis, MO) or Fisher Scientific (Springfield, IL). Uroguanylin, guanylin, and E. coli STa were dissolved in deionized water at a stock concentration (s.c.) of 1 mM. TTX was dissolved in 0.2% acetic acid at a stock concentration of 0.1 mM. Indomethacin (s.c., 10 mM), bumetanide (s.c., 0.1 M), methazolamide (s.c., 1.0 M), DIDS (s.c., 0.3 M), and amiloride (s.c., 0.1 M) were dissolved in DMSO.Data Analysis
Data are means ± SE. Student's t-test for paired or unpaired data or an ANOVA protected least-significant different test was used for comparisons of means among different intestinal segments and different treatment groups. In all cases, P < 0.05 was accepted as a statistically significant difference. ![]() |
RESULTS |
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Segmental Responses to Uroguanylin in the Mouse Intestine
Figure 1A shows the pattern of Isc responses to sequential treatment with specific agents on proximal duodenum. After the tissue was mounted, the addition of TTX (0.1 µM, serosal bath) resulted in a decrease in the baseline Isc to a stable value within 20 min. Uroguanylin (1.0 µM, luminal bath) caused a rapid increase in Isc, which was sustained for a 40-min period. Subsequent addition of STa (1.0 µM, luminal bath) elicited a further increase in Isc. Bumetanide (0.1 mM, serosal bath) treatment resulted in a decrease in the Isc but to a level that was elevated relative to the Isc before the uroguanylin/STa treatment. The inhibitory effect of bumetanide on Isc typically reached steady state by 10 min posttreatment, i.e., the percentage of bumetanide inhibition at 5 min was equal to 98 ± 1.1% of the Isc at 10 min postbumetanide (n = 15) in proximal duodenum. Glucose (10 mM, luminal bath) addition intended to stimulate Na+-coupled glucose transport caused a rapid increase in Isc. In contrast to apical treatment, the addition of either uroguanylin or STa to the serosal bath solution had no effect on the Isc (Fig. 1B).
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Cumulative data of the
Isc responses to
uroguanylin (1.0 µM) by different intestinal segments and the
gallbladder are shown in Fig. 2.
Uroguanylin elicited an
Isc response in
all intestinal segments but had no stimulatory effect on the
gallbladder preparations. Proximal duodenum and cecum had the greatest
mean Isc
responses to uroguanylin (1.0 µM) and were not significantly
different from each other. However, the
Isc response in
the proximal duodenum was significantly greater than the responses in
the other small intestinal segments (jejunum and ileum). Likewise, the
cecum had a greater
Isc response than
did the distal colon. The concentration of uroguanylin (1.0 µM) used
in these experiments was not a maximally stimulatory dose, since
subsequent addition of STa (1.0 µM) elicited further increases in the
Isc in all
intestinal segments that were tested
(Isc, in
µA/cm2): 95.5 ± 10.3 in
proximal duodenum (n = 7), 112.0 ± 18.5 in jejunum (n = 9),
64.5 ± 10.5 in ileum (n = 9), 77.5 ± 15.8 in cecum (n = 9), and 58.4 ± 13.5 in distal colon (n = 7).
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Proximal Duodenum
Effect of luminal acidic pH on uroguanylin action.
To examine the effect of luminal acidity on uroguanylin bioactivity in
the native intestine, the pH of the luminal bath was reduced to
5.0-5.5 for 20 min before the tissue was treated with the peptide
agonists (pH adjustment period). The proximal duodenum was chosen
because this segment elicited pronounced increases in
uroguanylin-induced
Isc (Fig. 2) and
because this part of the small intestine has an acidic intraluminal
environment during digestion (1). The intraluminal pH reduction caused
a rapid and reproducible increase in the
Isc of 13.4 ± 2.7 µA/cm2, reaching a new
steady-state Isc
baseline in about 5 min (Fig. 3A, pH
adjustment period). A recent study suggests that the acid-induced current may represent stimulation of electrogenic, CFTR-dependent secretion in the murine duodenum
(30). Subsequent uroguanylin treatment at the acidic pH produced an approximately twofold greater (P < 0.05) increase in
Isc than was
observed at pH 7.4 (Fig. 3, A and
B). Again, the increased Isc elicited by
uroguanylin was only partially inhibited by bumetanide treatment: 18.7 ± 5.7% at pH 7.4 and 27.0 ± 8.0% at pH 5.0-5.5 (Fig.
3B). At both pH conditions,
uroguanylin treatment slightly increased
Gt, but the
changes were not statistically different from each other: 2.0 ± 0.4 mS/cm2 at pH 7.4 and 3.1 ± 0.3 mS/cm2 at acidic pH. The effect of
luminal acidity on uroguanylin bioactivity in jejunal tissue was also
examined. The reduction of intraluminal pH to 5.0-5.5 again
increased basal
Isc (19.6 ± 4.8 µA/cm2), and subsequent
treatment with 1.0 µM uroguanylin at acidic pH produced a
significantly larger increase in
Isc than was
observed at pH 7.4 (66 ± 4.2 vs. 44.6 ± 5.8 µA/cm2,
P < 0.05, n = 4). In a dose-response study,
uroguanylin was more active (P < 0.05) in acidic luminal conditions than at pH 7.4 within the tested
range of concentrations (Fig. 4). It was not possible to test higher concentrations of uroguanylin because the
supply of the peptide was inadequate for the experiment.
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Effect of luminal acidity on the actions of 8-BrcGMP and 8-BrcAMP. To test whether the effect of luminal acidity on uroguanylin action resulted from an effect of low extracellular pH on the bioelectric properties or intracellular signaling pathways of the epithelial cells, the Isc responses elicited by membrane-permeable cyclic nucleotides, 8-BrcGMP and 8-BrcAMP, were examined at acidic and physiological pH. In the proximal duodenum, membrane-permeable 8-BrcGMP (20 µM) stimulated the Isc more than did an equimolar concentration of 8-BrcAMP (P < 0.001) (Fig. 5). However, the Isc response elicited by 8-BrcGMP at pH 7.4 was similar to that observed under acidic luminal conditions. In contrast, 8-BrcAMP (20 µM) was significantly less effective under acidic conditions than at pH 7.4 (P < 0.05). The increased Isc elicited by either 8-BrcGMP or 8-BrcAMP was significantly inhibited (P < 0.05) by bumetanide (0.1 mM) treatment: 59.3 ± 1.8% at pH 7.4 and 65.7 ± 3.4% at pH 5.0-5.5, or 61.5 ± 5.9% at pH 7.4 and 90.8 ± 4.2% at pH 5.0-5.5, respectively.
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Effect of luminal acidity on guanylin and STa actions.
To investigate whether the pH dependence was specific for uroguanylin
action, we examined the effects of acidic pH on the secretagogue
actions of guanylin and STa (Fig. 6).
Whereas uroguanylin is more effective in stimulating the
Isc under acidic
luminal conditions (P < 0.01), the Isc
response to guanylin (1.0 µM) was significantly reduced at acidic pH
compared with pH 7.4 (P = 0.05). In
contrast, the Isc
responses to STa (20 nM) did not appear to be pH dependent. The
Isc responses
induced by guanylin or STa were incompletely inhibited by serosal
bumetanide treatment (25-35% at pH 7.4; 50% at acidic pH) (data
not shown). Gt in
STa-treated or guanylin-treated groups under acidic intraluminal
conditions was increased by 3-4
mS/cm2, whereas at pH 7.4, Gt in this
period was ~0.5-1.0 mS/cm2.