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Department of Cell and Molecular Physiology, University of North Carolina (X.Q., S.P., M.F.G.), Chapel Hill, North Carolina 27599; and Department of Molecular Reproduction, Development, and Genetics, Indian Institute of Science (A.N., S.S.V.), Bangalore 560012, India
Address all correspondence and requests for reprints to: Dr. Michael F. Goy, Department of Cell and Molecular Physiology, Campus Box 7545, University of North Carolina, Chapel Hill, North Carolina USA 27599-7545. E-mail: mgoy{at}med.unc.edu
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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One member of the rGC family, a peptide-sensitive cyclase called GC-C (9), controls cGMP synthesis in intestinal epithelial cells. GC-C was first recognized because it is pathologically targeted by a bacterially produced peptide toxin, the heat-stable enterotoxin (STa) (10). Binding of STa to an extracellular (intraluminal) domain of GC-C enhances cGMP synthesis in epithelial target cells. The subsequent increase in cGMP levels stimulates electrogenic chloride secretion via the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel (11) and also inhibits neutral sodium chloride uptake (12, 13). This leads to the luminal accumulation of sodium chloride and water (14) and thus to a severe form of diarrhea commonly referred to as traveler’s diarrhea. Gene knockout studies in rodents demonstrate that these effects on ion transport are mediated in small intestine primarily by the type II cGMP-dependent protein kinase (13, 15), whereas in colon they are mediated by an as yet unspecified pathway that is type II cGMP-dependent protein kinase independent (13).
Toxin-induced diarrhea promotes rapid transfer of pathogens from one host to another, thus providing toxigenic bacteria with a reproductive advantage. In contrast, the diarrhea has adverse health and reproductive consequences for the host; STa-induced diarrhea is a general health problem for all mammals, and for humans it is a leading world-wide cause of death among children under the age of 5 yr (16). Surprisingly, despite this strongly negative selective pressure, GC-C has been evolutionarily conserved in a wide variety of animal species, suggesting that it must play a critical role in some aspect of intestinal physiology. One suggestion consistent with this idea is that GC-C serves not only as a toxin receptor, but also, like other rGCs, as a receptor for one or more endogenous peptide hormones.
This hypothesis has been greatly strengthened by the identification of two candidate peptide ligands, uroguanylin and guanylin, both of which selectively activate GC-C, share significant structural homology with STa, and compete with STa for common binding sites (17, 18). Like GC-C, these putative ligands are expressed primarily in the intestine, although both the receptor and the peptides have been detected at very low levels in other epithelial tissues, such as kidney and lung (19, 20, 21, 22). In rodents, it appears from preliminary studies that uroguanylin levels are relatively high in proximal small intestine and much lower in colon, whereas guanylin shows the opposite pattern (19, 23, 24, 25, 26). Furthermore, intestinal uroguanylin and guanylin are made by distinct types of cells in the rat intestine; uroguanylin is made primarily by enterochromaffin cells (22, 24), whereas guanylin is made primarily by goblet cells (and perhaps also by a subset of columnar epithelial cells) (27, 28). These unique spatial and cellular distributions imply that the two peptides are likely to carry out distinct physiological functions.
Although initial studies have provided a partial assessment of guanylin and uroguanylin expression within the intestine, many regions of the bowel have not been evaluated. Similarly, expression of GC-C, the presumed guanylin/uroguanylin receptor, has not been well defined throughout the length of the intestine. This is particularly important in light of the already apparent lack of congruency in the distributions of its two putative ligands. Evaluation of the tissue expression patterns of these recently discovered peptides and their receptor is essential for understanding their physiological functions.
In this communication we survey uroguanylin, guanylin, and GC-C expression along the rostrocaudal axis of the intestine of the rat. Our measurements reveal that 1) uroguanylin is almost exclusively a small bowel peptide, with highest messenger RNA (mRNA) and propeptide expression in the jejunum, slightly lower levels in duodenum and ileum, and minimal levels in cecum and colon; 2) guanylin shows a nearly complementary pattern of expression, with mRNA and propeptide levels ascending to a peak in the cecum and declining slightly in the distal colon; 3) GC-C mRNA and STa-binding sites are both found at high and relatively constant levels in all regions of the small and large bowels, a distribution that is appropriate to mediate the effects of both uroguanylin and guanylin; and 4) STa-activated rGC activity is nonuniformly distributed, with relatively high levels of activity at the proximal and distal extremes of the intestine (duodenum and distal colon) and relatively low levels in the middle (ileum), suggesting that some form of posttranslational regulation of GC-C uncouples ligand binding from cyclase activity in specific parts of the intestine.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2 cm distal to
the duodenum, corresponding to segment 3), distal ileum (a 2- to 4-cm
length of small bowel just proximal to the cecum, corresponding to
segment 17), proximal colon [the proximal third (5 cm) of the large
bowel just distal to the cecum, corresponding to segment 19], and
distal colon [the distal third (5 cm) of the large bowel just
proximal to the rectum, corresponding to segment 21]. In all cases,
the luminal surface was exposed by cutting along the long axis of the
bowel, and the tissue was rinsed with cold physiological saline
solution (140 mM Na+, 120
mM Cl-, 25 mM
HCO3, 1.2 mM
Mg2+, 1.2 mM
Ca2+, 2.4 mM
K2HPO4, 0.4 mM
KH2PO4, and 10
mM glucose; bubbled with 95% O2-5%
CO2).
Subsequent processing depended on the type of analysis being performed.
1) For prouroguanylin and proguanylin Western blots, mucosal tissue was
scraped away from the underlying smooth muscle with a microscope slide.
We observed comparable results using extracts prepared from whole
intestine, but the increase in specific activity obtained with mucosal
extracts greatly improves the signal to noise ratio. Scrapings were
immediately frozen on a metal plate chilled with dry ice and then
homogenized in buffer B [50 mM HEPES, pH 7.4, containing
the following protease inhibitor cocktail: 1 mM EDTA,
0.01% bacitracin, 2.5 mM 4-(2-aminoethyl)-benzenesulfonyl
fluoride, 38 µM pepstatin A, 35 µM
trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane,
100 µM bestatin, 55 µM
leupeptin, and 2 µM aprotinin]. Homogenates
were then cleared by centrifugation at 50,000 x g for
60 min at 4 C, and the pellet was discarded. 2) For STa binding assays,
mucosal scrapings were prepared as described above and homogenized in
buffer C (50 mM HEPES, pH 7.5, containing 1
µg/ml leupeptin, 1 µg/ml aprotinin, 2 mM
phenylmethylsulfonylfluoride, 2 mM
dithiothreitol, and 100 mM NaCl). The suspension
was first centrifuged for 10 min at 1,000 x g at 4 C,
and the resulting supernatant fraction was then centrifuged for 75 min
at 30,000 x g. The final pellet was resuspended in
buffer C without dithiothreitol at a protein concentration of 5 mg/ml.
Binding assays could not be performed reliably on membranes derived
from whole intestine due to excessive levels of nonspecific binding. 3)
For Northern blots, intact isolated tissues were frozen as quickly as
possible in liquid nitrogen (to prevent ribonuclease activity) and
stored at -80 C for subsequent RNA extraction. Although the specific
activities of uroguanylin, guanylin, and GCC mRNAs would undoubtedly be
higher in RNA isolated from mucosal scrapings, we found that scraping
results in an unacceptable level of RNA degradation. 4) For GC-C
bioassay experiments, isolated tissues were subdivided into smaller
pieces (3 x 3 mm) and stored briefly (<30 min) at 4 C in
physiological saline until assayed. These assays were performed with
intact tissues to minimize changes in activity that occur during
membrane purification; in our laboratories, GC-C-dependent cyclase
activity is always much higher in intact tissues than in isolated
mucosal membranes.
Northern blotting
Total RNA was purified from frozen tissues, blotted to
positively charged nylon membranes (Roche Molecular Biochemicals, Indianapolis, IN), and hybridized with
32P-labeled uroguanylin, guanylin, or GC-C
probes, as previously described (30). Templates for probe synthesis
were amplified by PCR from appropriate plasmids, then gel purified and
recovered from low melt agarose using the Wizard PCR Preps DNA
Purification System (Promega Corp., Madison, WI). Labeling
was performed with either a DECAprime II DNA labeling kit
(Ambion, Inc., Austin, TX) or a Strip-EZ DNA labeling kit
(Ambion, Inc.) using 32P-labeled
deoxy-ATP or deoxy-CTP (New England Nuclear Corp., Boston, MA). After
hybridization, an initial autoradiographic image was obtained on
Kodak X-OMAT Blue XB-1 film (Eastman Kodak Co., Rochester, NY), and probe binding was then quantitated on a
STORM 480 PhosphorImaging System (Molecular Dynamics, Inc., Sunnyvale, CA).
We tested several potential normalization standards (including ßactin and ubiquitin), hoping to use them to correct for sample to sample differences in RNA recovery. However, the relative expression levels of all standard mRNAs tested were themselves found to vary considerably from one region of the intestine to another (data not shown). Therefore, we normalized our results to the amount of RNA loaded in each lane (40 µg), as determined spectrophotometrically. We also monitored the intensity of the ethidium bromide-stained 18S and 28S ribosomal RNA bands (observed before transferring the samples to the blotting membrane) to confirm that the spectrophotometric measurements had allowed for uniform sample loading.
For each probe (uroguanylin, guanylin, and GC-C) we tested RNA isolated
from a set of contiguous tissue samples that had been obtained from a
single animal (white symbols). In addition, to evaluate
animal to animal variability, we measured uroguanylin, guanylin, and
GC-C mRNA levels in several independent animals, focusing on the five
standardized regions of the intestine described above. Because these
measurements were performed independently over a period of time, with
corresponding differences in exposure times and probe specific
activities, we normalized the set of data points obtained for each
animal so that they were distributed over a range of values that was
comparable for all animals. Specifically, for each animal that was
analyzed, we quantitated expression in the five standardized regions,
summed these five values to give a weighting factor, and then
normalized the data for each animal so that all of the weighting
factors would be brought to a common value. The normalized datasets
were then combined, and the mean values and SEs
(black symbols) were plotted at the appropriate
locations in Fig. 1
for uroguanylin, Fig. 2 for guanylin, and Fig. 8 for GC-C. The
results of our limited survey with multiple animals are fully
consistent with the more extensive study performed on the single animal
and confirm that the characteristic distributions of each mRNA species
are observed reproducibly.
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GC-C bioassay
Small pieces of tissue isolated from each intestinal region were
placed in groups of three to six in glass shell vials, each vial
containing 1 ml oxygenated physiological saline solution with or
without STa at 1–4 µg/ml (equivalent to 100–400 U/ml), supplemented
with 0.5 mM isobutylmethylxanthine (IBMX) and in some
experiments additionally with 0.1 mM zaprinast. Tissues
were incubated for 30 min at 37 C and intermittently bubbled with 95%
O2-5% CO2. Reactions were
stopped by freezing tissue pieces on a metal plate chilled with dry
ice. Each individual piece was then homogenized in 6% trichloroacetic
acid (TCA) and centrifuged to separate TCA-insoluble protein from
TCA-soluble cGMP. The pellet was dissolved in 1 N NaOH and
assayed for protein content (Bio-Rad Laboratories, Inc.,
Hercules, CA; Bradford assay). The cGMP level was quantitated by RIA
using standard procedures and was normalized to protein to correct for
small differences in the sizes of the original pieces of tissue. In
some experiments, a portion of the incubation medium was also analyzed
by RIA to measure cGMP that was secreted from the tissue. Secreted cGMP
was normalized to total protein, taken as the sum of the protein
contributed by all of the pieces of tissue that had been coincubated in
the vial.
STa integrity assay
T84 cell membranes were prepared as described previously (31).
Membranes were diluted to a protein concentration of 1 mg/ml in buffer
B (see above), and stored at -80 C until used. To initiate cyclase
activity, a portion of the diluted membranes was thawed and incubated
with an equal volume of buffer A [50 mM HEPES, 8
mM MgCl2, 2 mM IBMX, 4
mM GTP, 60 mM phosphocreatine, 800 µg/ml
creatine phosphokinase (250 U/mg), and 1 mg/ml BSA] at 37 C, with or
without a sample of STa (or control medium) to be tested. At various
times thereafter, 80-µl samples were pipetted from the incubation
mixture into 200 µl 6% (wt/vol) TCA. Each sample was then extracted
four times with 1 ml diethyl ether to remove the acid, and the cGMP
content was determined by RIA. The rate of cGMP synthesis is given by
the slope of a line fit to 0, 5, and 10 min points by linear regression
analysis. Under subsaturating conditions the fold stimulation of cGMP
synthesis (stimulated rate/basal rate) is proportional to the
concentration of intact STa in the test solution.
STa binding assay
Binding was performed with freshly isolated membranes at pH 7.5
as described previously (32), using radiolabeled STY72F peptide as the
ligand and 100–200 µg protein for each assay point. Assays contained
an input radioactivity of approximately 100,000 dpm in either the
presence or absence of unlabeled STa peptide. Incubations were
continued for 1 h, after which the samples were filtered through
GF/C filters (Whatman, Clifton, NJ) and washed with
chilled PBS containing 0.2% BSA. Filters were dried and monitored for
radioactivity. Specific binding was always more than 80% of the total
binding and is taken as the difference between total and nonspecific
binding. Scatchard analysis was performed with the LIGAND program (33),
which allows direct comparison between single and multiple binding site
models; our data were best fit by assuming a single binding site.
Chemicals
Zaprinast (Calbiochem, La Jolla, CA) was made as a
100-fold concentrated stock solution (10 mM) in 1
M NaOH. IBMX (Sigma, St. Louis, MO) was
dissolved directly in saline solution at a final working concentration
of 0.5 mM. Protease inhibitor cocktail (for mammalian cell
extracts) and STa were purchased from Sigma. The anti-cGMP
antibody and protein A used for the RIA were obtained from Woods Assays
(Portland, OR), chemiluminescence reagent was obtained from NEN Life Science Products (Boston, MA), and protein assay reagent
was purchased from Bio-Rad Laboratories, Inc.
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Results |
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and 2a present results from a Northern blot analysis
of a single intestine, subdivided into 21 contiguous pieces that span
the full length from duodenum to distal colon. The RNA samples obtained
from each region have been hybridized with probes for uroguanylin (Fig. 1a) or guanylin (Fig. 2a), revealing the RNA expression
patterns for both peptides simultaneously within a single animal. Each
probe is specific, hybridizing exclusively (or, in the case of
uroguanylin, predominantly) to a single transcript (marked with the
arrows in Figs. 1a and 2a).
Note that an additional, low abundance 1.1-kb mRNA species was detected
with the uroguanylin probe after long exposure times (marked with the
asterisk in Fig. 1b). No such species was detected after
long exposure times with the guanylin probe (Fig. 2b). The significance
of this minor transcript is not clear. It is much larger than any of
the uroguanylin complementary DNA clones isolated to date, and its
tissue distribution (with highest expression in proximal large bowel)
does not match the distribution of the uroguanylin propeptide as
detected by Western blotting (see below). Further analysis is required
to determine whether it represents an alternately spliced product of
the uroguanylin gene or is the product of an independent gene with
sufficient homology to hybridize with the uroguanylin probe. One
possibility is that it encodes the rat form of lymphoguanylin (34), a
recently discovered peptide, to date identified only in opossums, that
has strong homology to uroguanylin.
We used a phosphorimager to quantify relative uroguanylin and guanylin
probe hybridization from each region of the intestine. These results
are presented in the lower portions of Figs. 1a and 2a, aligned beneath
the relevant lanes of the autoradiograms. The white symbols
indicate individual values obtained from the single animal shown in the
upper autoradiogram, and the black symbols indicate average
values for selected regions that were analyzed from multiple animals.
Comparison of uroguanylin and guanylin reveals significant differences
in abundance along the length of the intestine. Uroguanylin mRNA is low
in duodenum, increases rapidly to a high level that remains relatively
constant throughout the jejunum and most of the ileum, declines
somewhat in terminal ileum, and then declines rapidly to very low
levels in cecum and colon (detectable only after long exposure times;
see Fig. 1b). In contrast, guanylin mRNA increases relatively linearly
from very low levels in duodenum to a peak in terminal ileum, remaining
high in cecum, and declining to about half the maximal level along the
length of the colon. These results are fully consistent with the
previous, more limited Northern blot surveys of intestinal guanylin and
uroguanylin expression cited above.
Regional expression of uroguanylin and guanylin propeptides
We used Western blots to measure prouroguanylin and proguanylin
levels along the length of the intestine. Each propeptide was evaluated
with two different antipeptide antibodies. The two antiprouroguanylin
antibodies (6910 and 6912) were independently raised against short
sequences of amino acids taken from two regions of the prouroguanylin
molecule that have no homology with proguanylin. Similar considerations
apply to the two antiproguanylin antibodies (2538 and 6240). The
specificities of these antibodies have been extensively investigated
and described previously (24, 27).
Properties of the antibodies
Figure 3a shows
representative immunoblots obtained with the two antiprouroguanylin
antibodies. Two identical samples from a single protein extract were
run side by side on a 15% acrylamide gel; one lane was blotted with
antibody 6910, and the other with antibody 6912. Each antibody labels
what appears to be a single polypeptide in the 6- to 10-kDa range
(marked by the arrow). With either antibody, on occasional
blots this single band could be resolved into a closely migrating
doublet (not shown here, but see examples below). The two closely
migrating polypeptides in the doublet had comparable electrophoretic
mobilities when detected with either 6910 or 6912; as the two
antibodies were raised against synthetic peptides representing
different portions of the prouroguanylin sequence, this argues strongly
that both of the immunoreactive molecules must be derivatives of
prouroguanylin (perhaps representing intact prouroguanylin and a
lower mol wt metabolite).
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shows another pair of representative blots, in this
case obtained with the two proguanylin-specific antibodies (2538 and
6240). Again, two identical samples were run on the same gel, then
analyzed separately with each antibody. Both antibodies recognize a
pair of immunoreactive molecules (marked by the arrows)
that, on the basis of their mutual recognition by the two antibodies,
are probably derived from authentic proguanylin. Antibody 6240 also
recognizes a third, smaller molecule (marked by the
arrowhead), which may be a proguanylin cleavage product that
has lost the epitope recognized by antibody 2538. In addition, antibody
6240 recognizes 29- and 30-kDa polypeptides that are too large to be
derived from the proguanylin gene, are not recognized by antibody 2538,
and are therefore considered to represent nonspecifically
cross-reacting molecules. Additional arguments support the identification of the 6- to 10-kDa bands as authentic products of the uroguanylin and guanylin genes. 1) Polypeptides migrating in the 6–10 kDa range have appropriate molecular masses for prouroguanylin (predicted to be 9.4 kDa) and proguanylin (predicted to be 10.2 kDa), along with smaller processing fragments. 2) No immunoreactive 6- to 10-kDa polypeptides are detectable in tissues that lack uroguanylin or guanylin mRNA (Qian, X., D. T. Devries, and M. F. Goy, manuscript in preparation), whereas, as will be described below, the relative levels of the polypeptides detected by 6910/6912 and 2538/6240 along the rostrocaudal axis of the intestine agree reasonably well with the relative rostrocaudal abundance of the uroguanylin and guanylin mRNA transcripts, respectively. 3) Biological activity (the ability to activate GC-C) copurifies with the immunoreactive prouroguanylin- and proguanylin-like polypeptides on a reverse phase HPLC column (27, 35).
Quantitative analysis of prouroguanylin expression
Using antibodies 6910 and 6912, we analyzed uroguanylin propeptide
expression along the complete length of the intestines of four
independent animals. The upper portion of Fig. 4 shows the relevant portion of a
representative Western blot from one of these animals, performed with
antibody 6910 (comparable results are obtained with antibody 6912). A
barely resolved pair of uroguanylin-related polypeptides can be seen on
this blot in the 6–10 kDa range (marked by the arrow). For
quantitation, both of these bands were summed together to provide a
measure of total prouroguanylin-related polypeptide expression (note
that the ratio of the two bands appears to be fairly constant in all
segments). Similar densitometric analysis was performed on blots
obtained for each of the other three animals, all of which look similar
to the blot illustrated in the figure. Results for each animal were
then normalized so that the values would fall within a range that was
comparable for all animals (see Materials and Methods), and
the mean relative expression levels were calculated at each position
along the intestine and aligned beneath the appropriate lanes of the
autoradiogram in Fig. 4. Comparison of these quantitative polypeptide
measurements with the quantitative mRNA measurements in Fig. 1 reveals
very good correspondence overall, indicating that steady state
uroguanylin polypeptide levels are, in general, proportional to steady
state uroguanylin mRNA levels.
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). Using antibody 2538, two
guanylin-related polypeptides can be seen in the 6–10 kDa range in
some samples on every blot (marked by the arrows), but the
segment to segment expression level of these polypeptides fluctuates
greatly. Note also that the ratio of the two bands varies considerably
from segment to segment (as highlighted in Fig. 6 and described in further detail
below).
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, can be discerned
in every individual animal. However, the smooth, nearly linear proximal
to distal gradient of expression displayed by guanylin mRNA along the
length of the small bowel is not at all evident in the individual
proguanylin Western blots. Instead, each blot reveals a series of two
or three hot spots in the small bowel where propeptide expression is
high, separated by regions where propeptide expression is very low or
undetectable. Furthermore, in those regions where total propeptide
expression is low, the bulk of the immunoreactive material tends to be
shifted to lower molecular mass species (Fig. 6), suggesting a possible
precursor-product relationship between the larger and smaller
polypeptides.
Random proteolysis during sample processing would provide a
nonbiological mechanism for producing unexpectedly low guanylin
polypeptide expression in some regions but not others. We think this is
unlikely, however, for several reasons: 1) we routinely add a broad
spectrum cocktail of protease inhibitors to our homogenization and
sample processing buffers; 2) we always confirm bulk protein integrity
by Coomassie or Ponceau staining a portion of each sample after
electrophoresis (Fig. 7, left
panel); and 3) when we test
samples for expression of both guanylin and uroguanylin, we fail to see
significant uroguanylin degradation, even in samples in which guanylin
levels are very low (Fig. 7, center and right panels).
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As described above, we investigated GC-C expression along the full
length of the intestine of a single animal (Fig. 8, autoradiogram), using the same RNA
samples that had been assayed for uroguanylin and guanylin expression.
We quantitated GC-C mRNA levels at each point (Fig. 8, white
symbols), including five standardized regions where the results
were confirmed by obtaining measurements from multiple animals (Fig. 8, black symbols). Interestingly, the Northern blot
results demonstrate that, in marked contrast to its peptide ligands,
GC-C is expressed with comparable abundance in most regions of the
small and large bowels. This observation is in good agreement with
previous, less extensive regional Northern blot analyses of intestinal
GC-C mRNA expression in the mouse (36) and rat (26, 29).
Note that GC-C levels in duodenum (segment 1) are somewhat lower than the levels observed in other regions. Low GC-C expression in duodenum appears to correlate with the generally lower levels of ligand found at this location (almost no guanylin and somewhat reduced uroguanylin levels). This could reflect a restricted need for GC-C-mediated signaling at this location, or it may be an artifact resulting from the high levels of proteases and nucleases typically found in duodenum.
Regional distribution of STa-binding sites
The relatively uniform presence of GC-C mRNA throughout the
intestine does not necessarily imply that functional GC-C protein will
be equally well represented in all segments. As an indirect index of
GC-C protein expression, we used [125I]STa to
measure the numbers and affinities of ligand-binding sites present in
four standardized regions of the intestine (segments 1, 3, 17, and 20).
Representative Scatchard plots for each segment are presented in Fig. 9, and the average maximal binding
capacity (Bmax) and Km
values calculated from multiple assays are provided in Table 1. The Scatchard analyses demonstrate
that each segment expresses primarily, and perhaps exclusively, a
single class of binding site, and that the binding affinity of that
site does not differ significantly from one segment to another. The
relative Bmax for each segment (reflecting total
numbers of STa-binding sites per mg protein) is generally in good
agreement with the relative GC-C mRNA level measured in that segment
(compare to Fig. 8), although the slightly reduced levels of GC-C mRNA
observed in duodenum are not matched by a comparable decrease in
STa-binding sites. Overall, these data are consistent with the idea
that GC-C protein expression levels are approximately proportional to
GC-C mRNA levels. They also correlate well with previous regional STa
binding assays performed on mouse intestinal tissue at a single,
subsaturating dose of STa (36).
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Figure 10 shows intracellular and
extracellular cGMP levels measured under control and STa-stimulated
conditions at standardized points along the rat intestine. Initial
studies (Fig. 10a) focused on intracellular cGMP levels in tissue
treated with IBMX, a general phosphodiesterase inhibitor that blocks
most of the known cGMP-selective phosphodiesterase isoforms (8). Under
these conditions, STa responsiveness was found to be greatest in
duodenum and colon and to drop off dramatically in other regions of the
bowel. The most striking feature of these results is the barely
detectable response to STa in distal portions of the small intestine.
In terminal ileum, for example, the increment in cGMP synthesis induced
by STa (stimulated level minus basal level) is approximately 6% of the
increment induced in duodenum. The STa-dependent increment in cGMP
synthesis has been calculated for selected intestinal segments and is
plotted in Fig. 11 (gray
symbols) to provide a direct comparison with GC-C mRNA
levels (replotted from Fig. 8 using black symbols) and
STa-binding site densities (Bmax values obtained
from Table 1, plotted as white symbols). The mismatch is
evident; STa-dependent cyclase activity varies markedly from region to
region, whereas GC-C mRNA and STa-binding sites remain relatively
constant.
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, right side, white
bars), whereas duodenum responded robustly (left side, white
bars). In fact, zaprinast actually potentiated the duodenal
response; the basal and stimulated levels in these particular animals
in the absence of zaprinast were 0.98 ± 0.18 and 1.73 ±
0.29, respectively (P < 0.03), whereas they were
1.45 ± 0.29 and 6.07 ± 1.03 (P < 0.00001)
in its presence. We also measured extracellular (secreted) cGMP levels
in these studies (hatched bars) and again observed strong
responses to STa in duodenum, whereas terminal ileum was essentially
insensitive. Indeed, no matter which combination of phosphodiesterase
inhibitors was employed, extracellular cGMP was found to closely
parallel intracellular cGMP (although at substantially lower levels),
with STa sensitivity again peaking at the proximal and distal extremes
of the bowel and essentially undetectable in terminal small intestine
(Fig. 10c).
The apparent lack of STa sensitivity in terminal ileum could also
potentially be explained by a local mechanism that inactivates STa, for
example through a degradative enzyme or a clearance receptor. To
evaluate this possibility we collected STa-containing test medium after
exposing it for 30 min to various intestinal tissues and measured how
much biological activity remained in the medium. We also collected
medium that had been exposed to tissues in the absence of STa to
evaluate whether endogenous GC-C ligands, such as uroguanylin or
guanylin, might have been secreted by the tissue during the incubation.
In each case, activity was assayed by applying the conditioned medium
to GC-Ccontaining membranes isolated from T84 cells. No
significant difference was observed between STa-containing test media
that had been exposed to duodenum or to terminal ileum (Fig. 12). Thus, tissue-specific depletion of
STa cannot account for the region to region differences observed in
Fig. 10. Figure 12 also shows that tissue-specific secretion of
endogenous agonists or antagonists (if any such exist) into the
incubation medium is unlikely to account for the differences.
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Discussion |
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We have begun to investigate this latter possibility by analyzing the intestinal expression of GC-C, the only rGC identified to date that responds to either uroguanylin or guanylin. Although GC-C responds to both ligands, it is actually about 10 times more sensitive to uroguanylin than to guanylin (18), implying that it might serve preferentially as a uroguanylin receptor. Our Northern blot measurements, however, demonstrate that GC-C mRNA is expressed relatively uniformly in all regions of the intestine, rather than matching the distribution of one or the other peptide. In addition, binding assays reveal relatively constant numbers of STa-binding sites along the length of the bowel, suggesting that GC-C protein expression parallels GC-C mRNA expression.
Direct measurement of STa-activated cyclase activity, however, yields the surprising finding that ligand sensitivity is not constant along the length of the rat intestine. It is most pronounced at the proximal and distal extremes (corresponding to regions with high uroguanylin and guanylin levels, respectively) and is low or undetectable throughout the terminal regions of the small intestine. A strikingly similar rostrocaudal distribution of STa-dependent cGMP synthesis has previously been noted after luminal introduction of STa into ligated segments of rat intestine (26). This in vivo result not only provides support for our current in vitro observations, but also indicates that the observed segment to segment variability is not an artifact that occurs as a consequence of removing the intestine from the animal. In addition, comparable regional patterns of STa sensitivity have been reported along the length of the raccoon (39), opossum (39), duck (40), turkey (40), skink (41), and alligator (41) intestine. Although parallel mRNA measurements were not performed in these studies, it seems possible that a similar mismatch between RNA levels and enzyme activity may be a general property of GC-C expression in many animals.
Although we do not understand the basis for the RNA/cyclase activity
mismatch in rats, we believe that it does not simply reflect region to
region differences in GC-C protein levels, because this would not be
consistent with the simplest interpretation of our STa binding studies
or with available GC-C Western blot data (29, 42). Several alternative
explanations should therefore be considered. 1) One hypothesis, which
we favor, is that some form of posttranslational modification
differentially regulates GC-C’s catalytic activity in specific regions
of the intestine without otherwise affecting its RNA or protein
expression levels or STa binding capacity. Possible mechanisms include
dephosphorylation, which has been shown for other members of the rGC
family to produce a desensitized receptor that still retains an active
binding site but no longer synthesizes cGMP in response to ligand
binding (43, 44), and limited proteolysis, which has been shown to
release the intracellular (catalytic) portion of GC-C while leaving the
membrane-anchored ligand-binding domain intact and able to interact
with agonists (45). Indeed, a recently published regional Western blot
analysis of GC-C expression within the rat intestine lends support to
this latter possibility (29). In this study the total amount of
material that was detected by the GC-C antibody appears fairly constant
from segment to segment (consistent with the constant RNA expression
levels observed in our current work), but the molecular masses of the
immunoreactive species varied considerably. In fact, the largest
species (150 kDa) was seen almost exclusively in those segments
where we find biological activity (duodenum, cecum, and colon), whereas
the smallest fragment (85 kDa) was seen most abundantly in those
segments where activity is low (jejunum and ileum). A third species
(140 kDa) was relatively uniformly distributed in all segments. It
is tempting to speculate that the 150-kDa species may represent intact,
fully functional GC-C, whereas the 140-kDa species may represent an
inactive (perhaps incompletely glycosylated) form, and the 85-kDa
species may represent a completely inactivated C-terminal proteolytic
fragment. 2) A second hypothesis to explain the RNA/activity mismatch
could be that one or more additional STa-sensitive rGCs (distinct from
GC-C) is expressed in duodenum and distal colon, leading to elevated
rates of STa-activated cGMP synthesis in these tissues. Several
observations, however, argue against this idea. First, STa-dependent
cGMP synthesis is essentially eliminated in the GC-C knockout mouse
(with the caveat that not every region of the intestine has been
carefully evaluated in these mice) (37). Second, multiple components
were not observed in Scatchard analysis of our STa binding data. 3) A
third mechanism that could generate differential toxin sensitivity
would be a region-specific diffusion barrier (for example, an
excessively thick mucus coating) that restricts access to GC-C in some
segments but not in others. If so, this barrier must be consistently
present in the terminal small bowel of every animal tested and must be
difficult to detect by visual inspection, as we failed to observe any
structural correlate in live or histologically prepared tissues. 4) A
final possibility is that some regions of the intestine may degrade or
eliminate cGMP more effectively than other regions. The conventional
routes for elimination of intracellular cGMP are degradation by
phosphodiesterase enzymes (8) and active export into the extracellular
fluid compartment (46). In our studies we have employed a pair of
phosphodiesterase inhibitors (IBMX and zaprinast) that, in combination,
are effective at inhibiting all known cGMP-hydrolyzing enzymes (8).
This suggests that differential phosphodiesterase activity is not a
likely explanation for the observed region to region variability,
although we cannot rule out the unexplored possibility that a novel
IBMX- and zaprinast-resistant phosphodiesterase is preferentially
expressed in ileum and terminal jejunum. With regard to cGMP export,
direct measurement of extracellular cGMP levels after STa stimulation
fails to reveal differential rates of cGMP export at any point in the
intestine.
Interestingly, a mismatch between protein and RNA expression is also observed for guanylin, occurring most markedly in the small intestine. One possible contributor to such a mismatch might be recently documented circadian variations in guanylin expression (29), especially if RNA and protein levels were to vary out of phase with one another. Arguing against this idea, however, is the observation that circadian variability is actually much more striking for uroguanylin than for guanylin (29), and yet uroguanylin RNA and protein expression follow each other rather closely. Furthermore, we killed all of our test animals within a relatively narrow time window, thereby limiting circadian fluctuations. An alternate possibility is that the proguanylin molecule is particularly susceptible to proteolysis by some as yet unidentified protease that is nonuniformly distributed within the small bowel. If so, then this proteolytic activity must be highly selective for proguanylin, arguing that it is likely to be a processing event that occurs as a natural step in the proguanylin life cycle. A final possibility is that variability could reflect local differences in guanylin secretion. For example, low steady state levels could be characteristic of regions with high secretory activity followed by rapid degradation or loss of the secreted materials, or high steady state levels could be characteristic of regions with high secretory activity followed by trapping and accumulation of the secreted products within the mucous layer that coats the apical surface of the epithelium. Whatever the explanation, the segment to segment variability suggests that some aspect of guanylin metabolism is highly dynamic, leading to conspicuous fluctuations in the protein to RNA ratio along the length of the small bowel. This contrasts markedly with the relatively constant protein to RNA ratio observed for uroguanylin in the same tissue segments.
What can we conclude about the function(s) of uroguanylin and guanylin from an analysis of their distributions at the cell and tissue levels? In the case of guanylin, our current observations reinforce and extend the proposal that guanylin-mediated fluid and electrolyte transport plays a role in the hydration of mucin (required for the proper formation of mucus) (24). This hypothesis is consistent with several observations. First, mucin and guanylin are both produced by goblet cells (27, 28, 47) and are both vectorially secreted into the lumen (47, 48), a compartment that is monitored by apically targeted receptors such as GC-C. Second, successful hydration of secreted mucin requires luminal sodium ions and water (49), both of which are provided when GC-C is activated (50). Third, guanylin expression is prominent throughout the colon, a tissue that is otherwise designed for efficient withdrawal of water and electrolytes from the lumen. Interestingly, despite the paucity of these essential components, the production of well hydrated mucus actually becomes increasingly important for lubrication and protection as the luminal contents of the colon solidify. One mechanism that could allow successful production of mucus and, at the same time, dehydration of the lumen would be cosecretion of guanylin and mucin. Spatial and temporal linkage between these molecules would provide a restricted local mechanism capable of supplying calibrated amounts of fluid only on demand. Note that GC-C activity is also high in all regions of colon, providing a route by which guanylin can actively control ion transport in this tissue compartment.
Our observations about uroguanylin are also relevant to previously formulated hypotheses concerning its physiological function(s) (51, 52, 53). Uroguanylin is a small bowel peptide, made by enterochromaffin (EC) cells (22, 24). Intestinal EC cells form an epithelial sensory system that responds to noxious luminal stimuli (54). For example, when gastric acid enters the intestine, it triggers the secretion of alkali by a reflex mediated in part by EC cells (54, 55). Unlike many other types of enteroendocrine cell, whose secretory products are released exclusively basolaterally, EC cells discharge a portion of their secretory products into the lumen of the intestine (55, 56). Thus, when activated by acid-related stimuli, small bowel EC cells are very likely capable of delivering uroguanylin to luminally oriented GC-C, thereby activating CFTR via the pathway outlined above. Interestingly, this would contribute directly to the alkaline reflex, because CFTR, along with its widely recognized permeability to chloride ions, also has high permeability to bicarbonate ions. Indeed, recent experiments with isolated duodenal explants have shown that apical application of GC-C ligands produces marked CFTR-dependent alkalinization of the apical fluid compartment (52, 53). In this context, restriction of uroguanylin primarily to small bowel makes sense, because the intestinal phase of digestion (with its requirement for a slightly alkaline pH) occurs within this tissue compartment. Further, it is intriguing to note that GC-C activity appears to be down-regulated in distal regions of the small intestine; this could provide a reserve population of receptors for rapid up-regulation if gastric acid delivery becomes excessive.
It is also noteworthy that small amounts of uroguanylin are produced by enterochromaffin-like (ECL) cells in the stomach (57). ECL cells play an important role in gastric acid production by releasing histamine, a locally acting hormone that triggers acid secretion by parietal cells. If ECL cells also release uroguanylin into the gastric lumen, then the uroguanylin would flow into the intestine along with the acidic contents of the stomach and provide an additional stimulus for alkali secretion. In addition, as GC-C is expressed at low levels within the stomach itself (and is particularly abundant in a subpopulation of nonparietal cells within the gastric glands) (26), it also appears likely that uroguanylin plays an as yet undefined local role in gastric physiology.
In addition to the foregoing analysis of uroguanylin’s potential functions within the digestive system, there are convincing reasons to consider targets of uroguanylin action that might occur outside of the intestine. 1) Although EC and ECL cells can deliver secretory products to the lumen, it is generally acknowledged that they secrete preferentially into the basolateral fluid compartment, making their secretory products available to the blood. 2) Consistent with this, uroguanylin has been detected in both plasma (58, 59, 60) and urine (18, 22). Plasma levels of uroguanylin increase further in patients with renal disease or in normal subjects after prolonged periods of elevated salt intake (61). 3) High affinity STa-binding sites are found outside of the intestine, most prominently in kidney (62). 4) Natriuresis, kaliuresis, and diuresis are observed when uroguanylin is introduced into the renal artery of an isolated, perfused kidney (63). Taken together, these observations have led to the hypothesis that uroguanylin is the hormonal mediator of a gut/kidney endocrine axis that serves to couple increased salt intake (detected by the intestine) to increased salt excretion (accomplished by the kidney) (51). Because dietary salt is completely absorbed within the small intestine, this hypothesis is consistent with our observation that high levels of uroguanylin expression are observed only in this part of the bowel.
Whether uroguanylin actually participates in salt and/or pH homeostasis, as outlined above, remains to be established by rigorous experimental analysis. In particular, confirmation that uroguanylin secretion is triggered by salt or acid entry into the small intestine remains a critical experimental objective. Similarly, the postulated role for guanylin in mucus hydration has not yet been validated by direct evidence that guanylin and mucin are cosecreted. Thus, at present, our physiological speculations are not overly constrained by an excess of experimental data. However, even at this stage, it is apparent that whatever physiological roles are ultimately established for this new peptide family and its guanylate cyclase-coupled receptor, they will need to be consistent with the elaborate and well defined tissue distribution that is described by our current studies.
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2 Current address: Southwestern Medical Center, Dallas, Texas
75235.
Received January 21, 2000.
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