| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
ARTICLES |
Niedersächsisches Institut für Peptid-Forschung (S.M., K.A., W.-G.F.), 30625 Hannover; and Institut für Pharmakologie und Toxikologie (M.K.), Westfälische Wilhelms-Universität Münster, 48149 Münster, Germany
Address all correspondence and requests for reprints to: Michaela Kuhn, Institut für Pharmakologie und Toxikologie, Westfälische Wilhelms-Universität Münster, Domagkstrasse 12, 48149 Münster, Germany. E-mail: mkuhn{at}uni-muenster.de
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Both peptides are expressed predominantly within the intestinal mucosa, and it is postulated that they form a local system that modulates intestinal salt and water transport (5, 6, 7, 8). Their exact cellular source is still under debate because the immunoreactive products have been localized in several cell types, including enterochromaffin cells, goblet cells, and absorptive enterocytes (9, 10, 11, 12).
In immunohistochemical and Northern blot studies, low levels of guanylin, uroguanylin, and GC-C were also detected in extraintestinal tissues (such as stomach, kidney, heart, lung, and pancreas), but their physiological role in these tissues is largely unknown (7, 13, 14, 15). The observations that guanylin and uroguanylin circulate in blood and that uroguanylin and STa stimulate natriuresis in the isolated, perfused rat kidney have led to the speculation that an endocrine system links the intestine with the kidney (16, 17, 18, 19, 20).
In the intestine, GC-C is localized exclusively at the apical plasma membranes of epithelial cells. Accordingly, in vitro experiments with isolated intestinal mucosa mounted in Ussing chambers showed that STa and synthetic guanylin and uroguanylin stimulate epithelial ion transport only when added to the luminal side of the epithelium (21, 22). These observations suggest that, in vivo, the endogenous peptides are secreted in a side-directed, mainly luminal fashion. However, this peculiar secretion mode has not been definitively demonstrated, and what regulates the synthesis and release of the peptide hormones guanylin and uroguanylin has been largely unknown up to now. In the present study, we characterized the molecular form in which guanylin is secreted from the rat colonic mucosa, the direction of the release, and its modulation by neuronal mediators known to be involved in the regulation of intestinal ion and water transport.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Guanylin enzyme-linked immunosorbent assay (ELISA)
Antiserum. Synthetic human guanylin-99–115 (1 mg) was
conjugated to hemocyanin by glutaraldehyde cross-linking. Mice were
immunized intradermally with 6 µg guanylin conjugated to hemocyanin
in Freund’s incomplete adjuvant. The animals were boosted every third
day for 2 weeks. Popliteal and inguinal lymph node cells were fused to
X63-Ag.8.653 myeloma cells (no. ACC43, DSM, Braunschweig, Germany);
hybridomas were selected in hypoxanthine-aminopterin medium and
further subcloned by limiting dilution. The culture supernatant from
hybridoma L-G11 with the highest antibody production was concentrated
15-fold by ultrafiltration and dialyzed overnight against sodium
phosphate buffer (PBS, pH 7.4).
Buffers. ELISA buffer was PBS (50 mM, pH 7.0) containing 15 mM NaCl, 1% BSA, and 0.5% Tween. As washing buffer, PBS (10 mM, pH 7.2) with 60 mM NaCl and 0.05% Tween was used.
ELISA procedure. Determinations were always performed in duplicate using streptavidine-coated microtiter plates (Immundiagnostik, Bensheim, Germany). Guanylin-99–115 was N-terminally biotinylated by incubation with sulfosuccinimidyl-6-biotinamido-hexanoate (12 h, at 4 C). Each microplate was coated with 100 µl biotinylated guanylin-99–115 (5 ng/well) for 6 h at room temperature (RT). After washing, monoclonal antiserum (mAb-L-G11, 50 µl/well, final dilution 1:4000) and 50 µl sample containing various amounts of synthetic peptides or lyophilized HPLC fractions were added to each well and incubated for 16–20 h at 4 C. The plates were washed, and 100 µl of the second, horseradish peroxidase-conjugated rabbit antimouse IgG antibody (Sigma; final dilution 1:2000) were added for 2 h (at RT). Subsequently, tetramethylbenzidine was added as a substrate (100 µl/well, at RT). After 45 min, the reaction was stopped by the addition of 2 M sulfuric acid (100 µl/well). Absorbances were determined at 450-nm wavelength.
Guanylin bioassay
Cultured human colon carcinoma (T84) cells were used as the
detection system for activators of GC-C (16). T84 cells (passages
58–63, ATCC, Rockville, MD) were pretreated with 1
mM IBMX for 15 min and then incubated with the synthetic
peptides or lyophilized HPLC fractions for 60 min. The incubation
medium was aspirated, and intracellular cGMP content was measured by
RIA (25).
Ussing chamber experiments
Female Wistar rats (2–4 months old, 250–270 g) were used. The
rats had free access to water and food until the day of the experiment.
The animals were killed by cervical dislocation, and the proximal colon
was dissected, opened along the mesenteric border, and immersed
immediately in oxygenated ice-cold Krebs-Ringer bicarbonate solution
with the following composition (mM): 140 Na+,
123.4 Cl-, 21 HCO3-, 5.4
K+, 2.4 HPO42-, 0.6
H2PO4-, 1.2 Mg2+, 1.2
Ca2+, and 10 D-glucose (pH 7.4). Osmolarity was
adjusted to 300 mosmol/liter with mannitol. The specimens were then
prepared by stripping off the serosa and the muscularis propria to
obtain the mucosa-submucosa preparation of the colon. This was cut into
4–5 pieces of approximately 1.5 cm2, and the mucosal
sheets were mounted in modified Ussing chambers with an exposed surface
area of 1 cm2 (21). Tissues were bathed with Krebs-Ringer
solution in both the mucosal and serosal reservoirs (vol, 8 ml).
Short-circuit current (Isc) across the epithelia was recorded as
described (21). Isc values are given as maximal differences from the
former baseline (
Isc).
Guanylin secretion studies
Stripped colonic mucosa was mounted in Ussing chambers as
described. After 60 min, the incubation media of the basolateral and
apical reservoirs were collected separately and stored at -80 C.
Preliminary experiments showed that, to detect the basal release of
GC-C-stimulating material within a suitable range of the bioassay and
the guanylin ELISA, the incubation media of 4–5 chambers had to be
combined for HPLC purification (this is, 9–10 chambers, when both
guanylin assay systems were applied simultaneously). Thus, supernatants
were collected from 5–10 x 1 cm2 of intestinal
mucosa incubated under identical conditions and then subjected to HPLC
purification. To investigate the effect of agents on peptide secretion,
5 adjacent pieces of the same intestinal specimen were incubated in
parallel, with the following added to the serosal side: vehicle
(control), carbachol (0.1 mM), VIP (1 µM),
SNAP (1 mM), or 8-bromo-cGMP (1 mM).
Purification of guanylin by reversed-phase (RP) liquid
chromatography
The apical and basolateral incubation media collected from nine
Ussing chambers were combined, acidified with trifluoroacetic acid
(TFA) to pH 3.5, and centrifuged at 3000 x g for 15
min at 4 C. The supernatants were filtered and applied to an analytical
RP C18 HPLC column (300 Å, 5 µm, 4 x 250 mm) (Parcosil,
BioTek Heidelberg, Germany). The samples were eluted with
a flow rate of 0.7 ml/min and fractionated using a linear gradient from
0–100% eluent B in 60 min (eluent A, 0.1% TFA; eluent B, 80%
acetonitrile, 0.1% TFA; UV detection at 220 nm) (step 1). Fractions
were collected every 2 min, and aliquots of each fraction were tested
in the bioassay amd the ELISA. Synthetic guanylin-101–115,
uroguanylin-97–112, and native human guanylin-22–115 were analyzed
using the same chromatographic conditions.
For amino acid sequencing and mass spectrometric analysis, the mucosal
supernatants of 200 Ussing chambers were combined and separated into 5
batches by RP-HPLC as described above. The bioactive and/or
immunoreactive fractions from all 5 HPLC runs (see Fig. 2
, fractions 13
+ 14 and 19 + 20) were pooled and subjected to a second RP-HPLC step on
a Vydac C18 column (300 Å, 5 µm, 4.6 x 250 mm; The Separations
Group, Hesperia, CA); linear gradient from 10–80% eluent B in 120 min
(eluent A, 0.1% TFA; eluent B, 80% acetonitrile, 0.1% TFA; flow rate
0.7 ml/min, 30-sec collection fractions; UV detection at 220 nm) (step
2). All fractions were tested with the guanylin ELISA and the
immunoreactive fractions were pooled, lyophilized, and subjected to
liquid chromatography-mass spectrometry analysis.
|
Sequence analysis of rat proguanylin was carried out by automated Edman degradation on a 494 Procise Protein Sequencer (Perkin-Elmer Corp./ABI), according to standard procedures recommended by the manufacturer.
Statistics
Data are expressed as means ± SEM (N =
number of chambers combined for HPLC purification; n = number of
guanylin bioassays or ELISAs). The comparison between mean values was
performed using Student’s t test. P values of
less than 0.05 were considered statistically significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
). A comparable curve
was observed when guanylin-99–115 was replaced by equimolar amounts of
human or rat guanylin-101–115, clearly indicating a similar affinity
of the antibody mAb-L-G11 for all three peptides. In contrast,
proguanylin was recognized with much lower sensitivity in a linear
range from 50–800 pmol/well and with a lower detection limit of about
25 pmol guanylin-22–115/well (B50, 200 pmol/well) (Fig. 1). No cross-reactivity was observed with the other known activators of
GC-C, uroguanylin, and STa. In view of the different displacement
curves obtained with guanylin-101–115 and guanylin-22–115, either
synthetic guanylin-101–115 or native guanylin-22–115 was used as a
reference peptide in the corresponding ELISA to quantitate small,
bioactive guanylin and proguanylin in the colonic supernatants (HPLC
fractions 13/14 and 19/20) (see Figs. 2
and 4).
|
|
Bioassay. Figure 2 shows that a single bioactive,
GC-C-stimulating compound is released from the rat colonic mucosa,
eluting with 39–43% eluent B in fractions 13 and 14. This biological
material coeluted with synthetic guanylin-101–115 and guanylin-99–115
and was chromatographically clearly distinct from uroguanylin-97–112
(Fig. 2). Fractions 13 and 14, obtained from the apical incubation
media, led to 26 ± 9-fold and 5 ± 1-fold increases,
respectively, in T84-cell cGMP content. By comparison of these values
with the concentration-response curves obtained for synthetic
guanylin-101–115 in the same T84-bioassays, amounts of 4 ± 1.3
(fraction 13) and 1.5 ± 0.5 pmol guanylin (fraction 14) can be
calculated (Fig. 2A). Because we combined the supernatants of nine
chambers and half the amount was tested in the T84-bioassay and half in
the ELISA (in each assay, supernatant of 4.5 cm2
epithelium), a basal apical secretion rate of about 1.2
pmol/cm2 epithelium in 60 min was calculated. With the same
procedure, only a small amount of bioactive guanylin was detected in
fraction 13 of the basolateral supernatant, leading to a 3.5 ±
1-fold increase in T84 cell cGMP (calculated guanylin amount:
approximately 1 pmol; basolateral secretion rate, approximately 0.2
pmol guanylin/cm2 in 60 min) (Fig. 2B).
Guanylin ELISA. The GC-C-activating fractions were also found
to be positive in the guanylin ELISA. Applying this assay system, we
measured the following guanylin values: for the apical supernatants,
4.4 ± 1.5 pmol (fraction 13) and 2.4 ± 0.9 pmol (fraction
14) (Fig. 2A); for the basolateral supernatants, 0.6 ± 0.2
(fraction 13) and 0.5 ± 0.2 pmol (fraction 14) immunoreactive
guanylin (Fig. 2B). Again, considering that this material was obtained
from 4.5 cm2 epithelium, a basal guanylin secretion rate of
1.5 pmol/cm2·h to the apical, and 0.2
pmol/cm2·h to the basolateral compartment was estimated.
Thus, an excellent correlation was found between the two assay systems,
demonstrating that the amount of bioactive guanylin is about 6-fold
higher on the apical (compared with the basolateral) side of the
isolated rat colonic mucosa.
Surprisingly, application of the guanylin ELISA showed that colonic
explants release a second peak of immunoreactive material to the apical
side, which can be separated by HPLC (fractions 19 and 20, 55% eluent
B). This second peak was even more prominent but did not affect cGMP
levels in T84 cells. It was not detected in the serosal incubation
media. Interestingly, native human proguanylin-22–115 elutes with the
same retention time (Fig. 2A).
Sequence analysis and quantitation of released immunoreactive,
nonbioactive guanylin
The immunoreactive, biologically inactive material contained in
fractions 19/20 (Fig. 2A) was purified by three subsequent RP-HPLC
steps. Using Edman sequence analysis, the first 16 amino acids of this
product were unambiguously identified as VTVQDGDLSFPLESVK. This
sequence is identical with positions 22–37 of the guanylin
prepropeptide deduced from the rat cDNA sequence (4, 5). Further
structure information was obtained by mass spectrometry and resulted in
the identification of a major protein with a molecular mass of 10,396
Da (Fig. 3). This corresponds to the
calculated mass for guanylin-22–115 (10,400 Da). Taken together, these
analytical data demonstrate that rat proguanylin with 94 amino acid
residues is the apically released immunoreactive product contained in
fractions 19 and 20.
|
). Therefore, to quantitate the
amount of proguanylin in HPLC fractions 19 and 20 (Fig. 2A), native
human proguanylin (guanylin-22–115) was used as standard peptide for
ELISA. By this procedure, amounts of 30 ± 9 (fraction 19) and
50 ± 9 pmol (fraction 20) were calculated, corresponding to a
basal secretion rate of about 18 pmol proguanylin/cm2 in 60
min. Thus, surprisingly the concentration of proguanylin in the apical
compartment was about 15-fold higher than the concentration of the
smaller, bioactive peptide. As illustrated in Fig. 2B, we could not
detect immunoreactive proguanylin in the basolateral incubation media
under these experimental conditions.
Stability of bioactive guanylin
One potential drawback in our experimental set-up could be that a
substantial part of the endogenously released guanylin might have been
degraded either during the incubation time (e.g. because of
the activity of proteases in the epithelial brush-border membrane) or
during the HPLC purification step, thereby falsifying the final
measurements. The stability of small, bioactive guanylin in this
in vitro system was tested in incubation experiments, where
defined amounts (1, 3 and 6 pmol) of synthetic rat guanylin-101–115
were added to the apical or basolateral chamber solution for 60 min.
HPLC purification was performed as described (step 1), and the recovery
of synthetic guanylin was measured in the T84 cell bioassay, as well as
by ELISA. The results showed that, under these experimental conditions,
the recovery of the synthetic peptide was about 100%, suggesting that
the degradation of the endogenous peptide released during the 60-min
incubation time was negligible.
Modulation of guanylin release
Carbachol, VIP, SNAP, or 8-bromo-cGMP was added to the serosal
side of the isolated colonic epithelia for 60 min. These agents were
always tested in parallel tissue specimens obtained from the same
animal. One specimen remained untreated, as a control. To separate
small guanylin and proguanylin from each other, the basolateral and
apical supernatants from five incubation experiments (specimens
obtained from five rats) were pooled and submitted to HPLC
purification. Fractions 13+14 and 19+20, respectively, were combined
and tested in separate ELISAs. In total, four experiments were
performed under identical conditions.
As illustrated in Fig. 4A, 0.1
mM carbachol evoked an increase of about 8-fold in the
amount of both bioactive small guanylin and proguanylin on the apical
side of the epithelia: basal release, 17.7 ± 2 proguanylin and
1.6 ± 0.5 small guanylin; with carbachol, 124 ± 6
proguanylin and 14.8 ± 1.5 small guanylin (pmol/cm2
in 60 min). Similarly, treatment of epithelia with 1 mM
8-bromo-cGMP significantly increased the concentration of both peptides
on the apical side of the epithelia (to 154 ± 6 pmol
proguanylin/cm2 in 60 min and 8 ± 2 pmol small
guanylin/cm2 in 60 min). In contrast, VIP (1
µM) and SNAP (1 mM) had no effect.
As mentioned before, under resting conditions, small amounts of
bioactive guanylin (but no proguanylin) were detected on the
basolateral side (Fig. 2B and 4B). In contrast, after incubation with
carbachol and 8-bromo-cGMP, a clear basolateral release of proguanylin
was detected at a rate of 12.6 ± 3 and 10.9 ± 2
pmol/cm2 in 60 min, respectively (basolateral release about
10-fold less, compared with the stimulated, apical release).
Interestingly, the concentration of bioactive short guanylin in the
serosal chamber solution was not changed by carbachol or by
8-bromo-cGMP (Fig. 4B). Again, VIP and SNAP did not affect the
basolateral release of proguanylin and bioactive guanylin.
To analyze the effect of these test agents on colonic electrogenic
electrolyte transport, all incubation experiments were performed under
short-circuited conditions. The maximal changes in Isc were (Isc, in
µA/cm2): 183 ± 9 for carbachol, 109 ± 7 for
VIP, 66 ± 5 for 8-bromo-cGMP, and 13 ± 1 for SNAP. Separate
experiments showed that these effects on Isc were significantly
attenuated in the presence of 0.1 mM bumetanide, indicating
stimulation of chloride secretion (data not shown).
Biological activity of proguanylin and small guanylin
The biological activity of native human guanylin-22–115 and rat
guanylin-101–115 was compared by evaluating the effects on cGMP
content in T84 cells, as well as on electrolyte transport in isolated
rat colonic mucosa mounted in Ussing chambers. The peptides were always
added to the apical side of the epithelia. As shown in Fig. 5, proguanylin increased both cyclic GMP
content in T84 cells and Isc across colonic epithelia but with markedly
lower potency and efficacy, compared with guanylin-101–115. This
indicates that proguanylin is not completely biologically inactive but
far less active than the C-terminal fragment. The threshold
concentration for bioactivity (
1 µM) is much higher
than the concentration of proguanylin in the HPLC fractions derived
from the colonic supernatants, explaining why these fractions did not
affect cGMP content in T84 cells.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Combination of the T84-cell bioassay and the guanylin ELISA demonstrates that two different molecular forms of guanylin are released to the apical side of the colonic epithelia. One is bioactive and immunoreactive and coelutes with synthetic guanylin-101–115. The second, more-hydrophobic material, is recognized by the antiguanylin-antibody, does not affect cGMP synthesis in T84 cells, and has a retention time corresponding to that of native human guanylin-22–115. After HPLC purification, mass spectrometric and amino acid analyses demonstrated unambiguously that this material corresponds to the 94-amino acid, 10.4-kDa proguanylin, guanylin-22–115. Unfortunately, the earlier eluting bioactive guanylin molecule could not be characterized by mass spectrometry and sequencing because of the low quantity remaining after purification. The initial studies that identified guanylin revealed that the propeptide contains an acid-labile Asp-Pro amide bond (2, 4), raising the question of whether the small bioactive peptide was artifactually cleaved from the precursor within the acidic HPLC environment that we used for the purification. However, this possibility was definitively excluded by chromatographic and mass spectrometric analyses (28). Several native, C-terminal forms of rat guanylin have been described in the literature: guanylin-16 and guanylin-15, purified from intestinal extracts, and guanylin-14, an N-terminal truncation of guanylin-15 that was subsequently isolated from intestinal perfusates (2, 29, 30, 31, 32). The elution pattern of the bioactive peptide released from isolated rat colonic mucosa is consistent with this peptide being one of these forms, but our HPLC protocol does not distinguish among the three.
As shown, the affinity of the monoclonal antibody L-G11 for proguanylin is markedly lower than for the C-terminal fragment guanylin-101–115. Recent NMR-spectroscopical studies of our group revealed that proguanylin adopts a structure in which the N terminus is in close proximity to the C-terminus containing the bioactive, GC-C-activating fragment (28). This three-dimensional relation of the termini may explain both the minor bioactivity of proguanylin by shielding the C-terminal domain from the receptor (GC-C), as well as the minor immunoreactivity by impeding the binding of the mAB L-G11 to its C-terminal epitope. In view of this different immunoreactivity, to quantitate both peptide forms in the supernatants from colonic explants, either native human guanylin-22–115 or synthetic rat guanylin-101–115 was used as standard peptide in respective ELISAs. Because the mAB L-G11 has similar affinity for rat and human guanylin-101–115, we assume that this is also true for the respective precursors (rat and human proguanylin). However, the N-terminal sequence of the propeptides differs in approximately 40 residues, and this could potentially influence the binding of the mAB-L-G11 to the C-terminal epitope. Thus, the affinity of the mAB-L-G11 for rat proguanylin may differ slightly from that determined for human proguanylin; and therefore, we cannot completely exclude that the use of human proguanylin as standard peptide for the ELISA decreases the accuracy of our calculations. Taking into account this potential limitation, our results indicate that, at the apical side of the colonic epithelia, the amount of proguanylin is about 15-fold higher than the amount of the smaller, bioactive peptide. This suggests that intestinal guanylin is secreted as 94-residue proguanylin and then extracellularly processed to the smaller, bioactive peptide at the apical side.
In contrast, on the basolateral side of the isolated epithelia, only the smaller, bioactive guanylin (and no immunoreactive proguanylin) was detected, at least under resting conditions. Also, the concentration of the bioactive peptide was about 6-fold lower in the basolateral (compared with the apical) compartment. The possibility that these small amounts of bioactive guanylin derive from transepithelial, passive diffusion of the apically released peptide was excluded with 125I-labeled guanylin-101–115, added to the mucosal or serosal side (n = 3; data not shown). In each experiment, after a 60-min incubation, less than 1% of the iodinated peptide was detected on the opposite (basolateral or apical) so-called cold side.
If bioactive, small guanylin derives from extracellular processing of proguanylin, how can the presence of the former in the absence of the latter on the basolateral side be explained? In view of the low immunoreactivity of proguanylin, this discrepancy might be related to the inability of the ELISA to detect low amounts of proguanylin. Another possibility is that small, bioactive guanylin represents a second tissue storage form being actively secreted to both sides of the epithelia, because it has been described that rat intestinal mucosa contains not only the 94-residue precursor but also lower amounts of the C-terminal fragments, guanylin-16 and guanylin-15 (29, 30). Published studies revealed that guanylin is expressed by goblet cells and superficial epithelial cells of the rat proximal colon (10, 11, 33). Thus, one could even speculate that one cell type secretes unprocessed proguanylin, whereas another cell type could secrete the processed, biologically active peptide.
The observation that the concentrations of both proguanylin and bioactive, small guanylin are markedly higher at the apical (compared with the basolateral) side of isolated epithelia suggests a vectorial secretion of the peptide into the intestinal lumen (luminocrine system). The identification of guanylin at the basolateral side is consistent with previous findings showing that guanylin and uroguanylin circulate in blood (16, 17). Although guanylin expression was also found in low amounts in other tissues (13, 34), Northern blot analysis indicates that the peptide is present preferentially in the intestine, indicating that this is the tissue which predominantly contributes to the circulating levels (1, 5, 8). In the context of the natriuretic responses to guanylin and uroguanylin, it has been suggested that the peptides may serve as an endocrine axis that links the intestine with the kidney, to regulate urinary salt excretion during postprandial periods of salt absorption by the digestive tract (18, 19, 20).
Whereas there is convincing evidence that guanylin regulates intestinal salt and water transport, the stimuli that trigger the synthesis and release of the peptide are largely unknown. In a recent study, Li and co-workers (35) showed that intestinal guanylin is down-regulated as an adaptive response to salt restriction. However, few data have been reported on a short-term, i.e. neuronal, regulation of release. In the present study, the muscarinic agonist carbachol significantly increased the release of proguanylin to both sides of the colonic mucosa, the apical concentration remaining about 10-fold higher than the basolateral concentration. This observation is in good agreement with a study in isolated vascularly perfused rat colon, in which intraarterial infusion of betanechol also stimulated the secretion of immunoreactive guanylin both into the intestinal lumen and into the circulation, suggesting that the synthesis and/or release of the peptide is under vagal control (36). Surprisingly, in our study a concomitant increase in the amount of the smaller, bioactive guanylin form was observed only on the apical side of the colonic mucosa; whereas the basolateral concentration did not change, in spite of the increase in proguanylin. At the present time, we have no explanation for this observation. One possibility is a proteolytic cleavage of proguanylin, only on the apical side, by enzymes at the brush border membrane of the epithelium. If so, the source for the small amounts of bioactive guanylin detected on the basolateral side remains an intriguing question.
NO and VIP are neuronal mediators with an important role in the local control of intestinal motility and secretion (37, 38). In contrast to the muscarinic system, the effects of NO and VIP are mediated via increases of intracellular cGMP and cAMP. Indeed, both the NO-donor SNAP, and VIP significantly increased chloride secretion in the rat colon; the release of guanylin, however, was not affected.
To further investigate the role of the second-messenger cGMP in guanylin secretion, 8-bromo-cGMP was tested. This compound significantly increased the apical and basolateral release of proguanylin. Again, as with carbachol, only at the apical side of the epithelia, a concomitant increase in the amount of shorter, bioactive guanylin was observed. In future experiments, we will investigate which of the cGMP-linked systems in the intestine (guanylin itself or natriuretic peptides) is involved in the control of guanylin secretion.
In conclusion, our results suggest that, in the intestine, guanylin is secreted as 94-residue prohormone. Proguanylin is secreted mainly to the apical side, where it is processed to the smaller, much more bioactive peptide that stimulates epithelial GC-C and thereby modulates intestinal electrolyte transport. Secretion of guanylin to the basolateral side of the intestinal epithelium may form an endocrine axis to the kidney and distant cells of other organs. Stimulation of apical and basolateral proguanylin release by carbachol and 8-bromo-cGMP suggests that the synthesis and/or secretion of the peptide are under the control of cholinergic/muscarinic, as well as cGMP-dependent mechanisms. Short-term regulation of the local guanylin/GC-C system may mediate, in part, the effect of other (i.e. cholinergic) modulators of intestinal salt and water transport.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Received March 12, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  N. G. Moss, D. A. Riguera, R. M. Solinga, M. M. Kessler, D. P. Zimmer, W. J. Arendshorst, M. G. Currie, and M. F. Goy The Natriuretic Peptide Uroguanylin Elicits Physiologic Actions Through 2 Distinct Topoisomers Hypertension, May 1, 2009; 53(5): 867 - 876. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. A. Rudolph, J. A. Hawkins, and M. B. Cohen Proguanylin secretion and the role of negative-feedback inhibition in a villous epithelial cell line Am J Physiol Gastrointest Liver Physiol, September 1, 2002; 283(3): G695 - G702. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Kulaksiz, E. Rehberg, W. Stremmel, and Y. Cetin Guanylin and Functional Coupling Proteins in the Human Salivary Glands and Gland Tumors : Expression, Cellular Localization, and Target Membrane Domains Am. J. Pathol., August 1, 2002; 161(2): 655 - 664. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Kulaksiz, A. Schmid, M. Honscheid, A. Ramaswamy, and Y. Cetin Clara cell impact in air-side activation of CFTR in small pulmonary airways PNAS, May 14, 2002; 99(10): 6796 - 6801. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Kunzelmann and M. Mall Electrolyte Transport in the Mammalian Colon: Mechanisms and Implications for Disease Physiol Rev, January 1, 2002; 82(1): 245 - 289. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Potthast, E. Ehler, L. A. Scheving, A. Sindic, E. Schlatter, and M. Kuhn High Salt Intake Increases Uroguanylin Expression in Mouse Kidney Endocrinology, July 1, 2001; 142(7): 3087 - 3097. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Qian, S. Prabhakar, A. Nandi, S. S. Visweswariah, and M. F. Goy Expression of GC-C, a Receptor-Guanylate Cyclase, and Its Endogenous Ligands Uroguanylin and Guanylin along the Rostrocaudal Axis of the Intestine Endocrinology, September 1, 2000; 141(9): 3210 - 3224. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Moro, F. Levenez, E. Nemoz-Gaillard, S. Pellissier, P. Plaisancie, and J. C. Cuber Release of Guanylin Immunoreactivity from the Isolated Vascularly Perfused Rat Colon Endocrinology, July 1, 2000; 141(7): 2594 - 2599. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
