1 Laboratory of Environmental Physiology, Graduate School of Nutritional and Environmental Sciences, University of Shizuoka, Shizuoka 422-8526; and 2 Laboratory of Veterinary Anatomy, Hokkaido University, Sapporo 060-0818, Japan
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ABSTRACT |
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The effect
of ANG II on mucosal ion transport and localization of ANG type 1 receptor (AT1R) in the guinea pig distal colon was
investigated. Submucosal/mucosal segments were mounted in Ussing flux
chambers, and short-circuit current (Isc) was
measured as an index of ion transport. Serosal addition of ANG II
produced a concentration-dependent
(109-10
5
M) increase in Isc. The maximal response was
observed at 10
6 M; the increase in
Isc was 164.4 ± 11.8 µA/cm2. The
ANG II (10
6 M)-evoked response was
mainly due to Cl
secretion. Tetrodotoxin, atropine,
the neurokinin type 1 receptor antagonist FK-888, and piroxicam
significantly reduced the ANG II (10
6
M)-evoked response to 28, 45, 58, and 16% of control, respectively. Pretreatment with prostaglandin E2
(10
5 M) resulted in a threefold increase
in the ANG II-evoked response. The AT1R antagonist
FR-130739 completely blocked ANG II (10
6
M)-evoked responses, whereas the ANG type 2 receptor antagonist PD-123319 had no effect. Localization of AT1R was
determined by immunohistochemistry. In the immunohistochemical study,
AT1R-immunopositive cells were distributed clearly in
enteric nerves and moderately in surface epithelial cells. These
results suggest that ANG II-evoked electrogenic Cl
secretion may involve submucosal cholinergic and tachykinergic neurons
and prostanoid synthesis pathways through AT1R on the submucosal plexus and surface epithelial cells in guinea pig distal colon.
substance P; prostaglandin; local renin-angiotensin system; angiotensin type 1 receptor; neurokinin type 1 receptor
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INTRODUCTION |
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THE RENIN-ANGIOTENSIN SYSTEM is a major regulatory mechanism for plasma electrolyte concentrations and blood pressure control. The system is activated by extracellular volume depletion resulting from hyponatremia, dehydration, or hemorrhage. These stimuli trigger renin release from the kidneys, which cleaves the circulating angiotensinogen to ANG I. Angiotensin-converting enzyme (ACE) removes two COOH-terminal amino acids from ANG I, producing a biologically active octapeptide, ANG II. ANG II can be further degraded by an aminopeptidase-induced deletion of an NH2-terminal residue to yield ANG III.
The cloning of renin, angiotensinogen, ACE, and angiotensin receptor genes has demonstrated the widespread presence of the renin-angiotensin system in brain, heart, kidney, adrenal, and adipose tissue (3, 4, 14, 16, 27, 37). These results suggest other functions for ANG II in addition to those in the circulatory system. Two angiotensin receptors, the angiotensin type 1 receptor (AT1R) and the angiotensin type 2 receptor (AT2R), have been cloned (23, 24, 29, 39, 45). These reports suggest that AT1R is widely distributed throughout the body, predominantly regulating body fluid volume, whereas AT2R is found in minor locations and its function is not well known.
In the gastrointestinal tract, the distribution of ANG II receptors and ACE has been mapped by in vitro autoradiography, and ANG II has been shown to be present in rat intestine (15). Sechi et al. (41) reported that ANG II receptors are present in rat colonic mucosa and that the predominant receptor subtype is AT1R. Angiotensinogen mRNA was detected in rat mesentery and large intestines (3, 37). The existence of renin in the gastrointestinal tract has not been reported; however, a recent report has shown that serine proteinases, such as kallikrein, tonin, and cathepsin G, have renin activity and directly generate ANG II (37). Kallikrein and kallikrein-like enzyme immunoreactivity has been shown to be localized in goblet (or mucous) cells and mast cells of rat small and large intestines (40). Therefore, it is likely that ANG II is normally produced in intestinal tissues and may act as a local mediator in the control of intestinal functions.
The reported effects of ANG II on epithelial ion transport in the
intestine vary. It has been reported that intravenous injection of a
low dose of ANG II (7.0
ng · kg
1 · min
1)
stimulated Na+ and water absorption in rat small and large
intestines through extrinsic nerves, whereas a high dose of ANG II
(>7.0
ng · kg
1 · min
1)
inhibited Na+ absorption or promoted secretory processes by
the production of prostaglandin (33, 34). In an in vitro Ussing flux
chamber experiment, it was shown that serosal application of ANG II
(10
10-10
5
M) induced an increase in short-circuit current
(Isc) in rat jejunum by stimulating electrogenic
Cl
secretion, whereas the addition of the same
concentration of ANG II evoked a decrease in Isc in
rat colon (9). Hatch et al. (21, 22) showed that, in rat colon, serosal
application of ANG II
(10
9-10
5
M) induced net K+ secretion, whereas the concentration of
ANG II (10
4 M) additionally induced net
Cl
secretion, and that these secretory effects were
inhibited by AT1R antagonist. Jin et al. (26) reported that
a low dose of ANG II given in intravenous infusion (0.7 pmol · kg
1 · min
1)
stimulates net Na+ absorption through
AT2R-mediated cGMP activation, whereas a high dose of ANG
II (700 pmol · kg
1 · min
1)
inhibits Na+ absorption and induces prostaglandin
E2 (PGE2) synthesis in rat small intestine.
These results indicate regional differences, concentration dependency,
and different receptor-mediated actions of the ANG II effects on ion transport.
Various secretagogues are able to induce Cl
secretion in colonic mucosa. Secretagogue-induced Cl
secretion in the distal colon is important, because such
Cl
secretion provides an essential driving force for
lubrication or for the flushing of intestinal contents during host
defense against microbial invasion or artificial irritants. ACh and
substance P (SP) are involved in secretory reflexes (6, 8, 17, 30, 43).
Remarkably, a relationship between ANG II and SP in the central nervous
system was reported. In rat hypothalamus brain slices, it was observed
that ANG II significantly evokes SP release and mediates cardiovascular
effects by SP (11-13). Thus it is possible that an ANG II-evoked
response involves SP action in the enteric nervous system. However, the
effect of ANG II on the enteric nervous system in controlling
Cl
secretion has not been reported.
Therefore, the aim of this study was to examine whether the effect of
ANG II on mucosal Cl secretion includes an effect on
the enteric nervous system and prostanoid synthesis. The involvement of
cholinergic and/or tachykinergic pathways and ANG II receptors was also
tested. Furthermore, interaction between ANG II and PGE2
was examined. Immunohistochemistry was used to identify the
distribution of AT1Rs that may be involved in ANG II-evoked
Cl
secretion.
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MATERIALS AND METHODS |
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Ussing Flux Chamber Experiment
Male albino guinea pigs (Hartley-Hazleton, Nippon, Hamamatsu, Japan) ranging in weight from 414 to 840 g were allowed food and water ad libitum before the experiments. The animals were stunned and exsanguinated according to the method approved by the Guide for Animal Experimentation of the National Institute for Physiological Sciences of Japan. Segments of distal colon 5-10 cm proximal to the anus were removed, flushed with Krebs-Ringer solution, and cut along the mesenteric border. The tissues were pinned flat with the mucosal side down in a Sylgard-lined petri dish. The entire muscular layer including myenteric plexus was removed by blunt dissection, and the submucosal plexus was reserved intact. Four of these stripped mucosal preparations were obtained from one animal. Flat sheets of distal colon with intact submucosal ganglia were mounted between halves of Ussing flux chambers in which the total cross-sectional area was 0.64 cm2.The mucosal and serosal surfaces of tissues were bathed with 10 ml of
Krebs-Ringer solution by recirculation from a reservoir maintained at
37°C during the experiment. Tissues were left for 0.5-1 h
before the addition of drugs. Buffer solutions contained (in mM) 120 NaCl, 6 KCl, 1.2 MgCl2, 1.2 NaH2PO4, 14.4 NaHCO3, 2.5 CaCl2, and 11.5 glucose. The solution was gassed with 95% O2-5% CO2 and buffered at pH 7.2. For a
Cl-free solution, Cl
was replaced
by sulfate salts, and mannitol was added to make up the difference in
osmolarity. The Cl
-free solution contained (in mM)
2.7 K2SO4, 1.1 MgSO4, 1.2 NaH2PO4, 54.9 Na2SO4,
13 NaHCO3, 1.7 CaSO4, 60.4 mannitol, and 11.5 glucose (7). The chambers were equipped with a pair of Ringer-agar bridges and calomel half cells for the measurement of transmural electrical potential difference (PD). A pair of Ag-AgCl disk
electrodes was connected to an automatic voltage-clamp apparatus (model
SS-1335, Nihon-Kohden, Tokyo, Japan) that automatically compensated for solution resistance between PD-sensing bridges. Tissue
conductance (Gti) was calculated as the ratio of
Isc to open-circuit values of PD or by
determining the current necessary to change PD by 10 mV.
The responses were continuously recorded on a chart recorder
(Recit-Horitz-8K, Nihon-Denki Sanei, Tokyo, Japan) and a Macintosh computer (MacLab/8 system, Analog Digital Systems, Castle Hill, Australia) for control and experimental tissues. The
Isc was calculated on the basis of the value
before and after stimulation. The tissues' currents were compared
before the stimulation was begun. The tissues were paired on the basis
of similar Gti. A concentration-response curve for
ANG II was established by addition of ANG II in a single concentration
to the serosal bathing solution. In further experiments, ANG II was
added in a single concentration (10
6 M)
to the serosal bathing solution in the presence or absence of
antagonists, blockers, or PGE2 to assess their effects on
baseline or stimulated Isc. The following
antagonists or blockers were used: the
Na+-K+-2Cl
cotransporter
inhibitor bumetanide (5 × 10
4 M),
atropine (10
6 M), tetrodotoxin (TTX,
10
7 M), the neurokinin type 1 (NK1) receptor antagonist FK-888
(10
8-10
6
M), the cyclooxygenase inhibitor piroxicam
(10
5 M), the EP1 receptor antagonist
SC-51089 (10
5 M), the AT1R
antagonist FR-130739
(10
8-10
5
M), and the AT2R antagonist PD-123319
(10
7-10
5
M). The blocking drugs were added
10 min before the addition of ANG
II (10
6 M). PGE2
(10
5 M) was added before or after the
addition of ANG II (10
6 M). Changes in
Isc during ANG II stimulation were measured as the
difference between the peak response and baseline
Isc before stimulation.
Immunohistochemistry
Male albino guinea pigs were used in the present immunostaining for AT1R. The animals were anesthetized with pentobarbital sodium and perfused via the aorta with physiological saline followed by 300 ml of 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Strips of the distal colon were removed and immersed in the same fixative for an additional 6 h. They were dipped in 30% sucrose solution overnight at 4°C and then rapidly frozen in liquid nitrogen. Frozen sections, ~20 µm thick, were prepared in a cryostat (Coldtome CD41, Sakura, Tokyo, Japan). Immunohistochemistry for AT1R was performed according to the avidin-biotin complex method. After treatment with a normal goat serum, frozen sections were incubated with a rabbit polyclonal antiserum (1:3,200 dilution) raised against amino acids 15-24 mapping at the NH2 terminus of the human AT1R. Recently, guinea pig AT1R cDNA has been cloned, and it has been confirmed that amino acids 15-24 of guinea pig and human AT1R are identical. The nucleotide sequence data of guinea pig AT1R have been recorded in the GenBank database (accession no. AF165888). The sites of antigen-antibody reaction were visualized using streptavidin and biotin-peroxidase complex Histfine. For whole mount preparations, the fixed distal colon was longitudinally opened, and the mucosal and outer muscle layers were separated into sheets under a dissecting microscope. They were then processed, as free-floating sections, with the avidin-biotin complex method, as mentioned above. The specificity of the immunoreaction was checked by preincubation of the antiserum with the corresponding antigen (10 µg/diluted antiserum).Chemicals
Ussing chamber experiments. ANG II was purchased from the Peptide Institute (Osaka, Japan); atropine sulfate, bumetanide, DMSO, and TTX from Sigma Chemical; PD-123319 from Research Biochemicals International; piroxicam and SC-51089 from Biomol Research Laboratories; and PGE2 from Cayman Chemical. FK-888 and FR-130739 were a gift from Fujisawa Pharmaceutical (Osaka, Japan). ANG II, atropine, TTX, SC-51089, FR-130739, PD-123319, and PGE2 were dissolved in distilled water or Krebs solution unless otherwise stated. Bumetanide, FK-888, and piroxicam were dissolved in DMSO. The volume of dissolved drugs in water or Krebs solution and DMSO added to the bath solutions did not exceed 100 µl and 10 µl/10 ml, respectively.
Immunohistochemistry. AT1 (N-10) antiserum (rabbit polyclonal IgG) and AT1 (N-10) were purchased from Santa Cruz Biotechnology; pentobarbital sodium from Abbot; and Histfine from Nichirei (Tokyo, Japan).
Statistics
Values are means ± SE. Unpaired Student's t-test was used to determine the statistical significance between control and experimental tissues. ANOVA in conjunction with the Bonferroni test was used to determine significant differences among multiple comparison groups. P < 0.05 was considered statistically significant. ![]() |
RESULTS |
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Effect of ANG II on Baseline Isc
The transmural PD in the guinea pig distal colon was 2.4 ± 0.2 mV (n = 220) and was oriented luminal side positive (with respect to the serosa). The corresponding Isc was negative and averagedAfter the addition of ANG II, Isc gradually
increased to a peak value within 1-2 min and then declined toward
baseline within 5-10 min. The increases in Isc
were concentration dependent (Fig. 1A). The maximal response for ANG
II was achieved at 106 M;
10
6 M ANG II evoked an increase in
Isc of 164.4 ± 11.8 µA/cm2 from
baseline (Fig. 1B), and this concentration was used in all subsequent studies.
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Effects of Bumetanide or Cl-Free Solution on ANG
II-Evoked Increase in Isc
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Effect of TTX on ANG II-Evoked Increase in Isc
To test whether the response to ANG II in the distal colon was mediated by the enteric nervous system, TTX was added to the serosal bath solution before the administration of ANG II (10Effects of NK1 Receptor Antagonist and Atropine on ANG II-Evoked Increase in Isc
To estimate the neural mediators on the ANG II-evoked response, FK-888 and atropine were used. Serosal addition of FK-888 did not significantly alter PD, baseline Isc, or Gti. Our preliminary experiment has shown that 90% of the maximal increase in Isc to SP (10
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The concentration of atropine (106 M)
was chosen on the basis of previous data (30). Serosal addition of
atropine did not significantly alter PD, baseline
Isc, or Gti. Atropine reduced the ANG II-evoked increase in Isc to 58.5 ± 12.4 µA/cm2 from the control value of 128.4 ± 11.6 µA/cm2 (Fig. 3B).
The combination of FK-888 and atropine further reduced the ANG II-evoked increase in Isc to 26.0 ± 6.5 µA/cm2 (Fig. 3B). This inhibition was significantly larger than that resulting from FK-888 alone (Fig. 3B), whereas there was no difference in the inhibiting effect between the FK-888-atropine combination and atropine alone.
Effects of Piroxicam and EP1 Receptor Antagonist on ANG II-Evoked Increase in Isc
To determine the involvement of prostanoid synthesis, the cyclooxygenase inhibitor piroxicam and the EP1 receptor antagonist SC-51089 were used. The concentration of piroxicam (10
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The concentration of SC-51089 (105 M)
was chosen on the basis of previous data (44). Serosal addition of
SC-51089 did not alter PD, baseline Isc, or
Gti. SC-51089 reduced the ANG II-evoked increase in
Isc to 66.9 ± 21.2 µA/cm2 from the
control value of 162.0 ± 18.9 µA/cm2 (n = 5, P < 0.05).
Effects of PGE2 on ANG II-Evoked Increase in Isc and ANG II on PGE2-Evoked Increase in Isc
To determine the involvement of prostaglandins, exogenous PGE2 (10Serosal addition of PGE2
(105 M) evoked a biphasic and
long-lasting increase in Isc (Fig.
5A). The increase in
Isc was 114.2 ± 2.1 µA/cm2 in the
first phase and 83.5 ± 16.3 µA/cm2 in the
second phase (Fig. 5B). Pretreatment with PGE2
enhanced the ANG II-evoked increase in Isc to 341.9 ± 33.2 µA/cm2 (Fig. 5, C and D). The
PGE2-evoked response was also enhanced by pretreatment with
ANG II (10
6 M; Fig. 5A). ANG II
enhanced the second phase of the PGE2-evoked increase in
Isc to 179.4 ± 23.0 µA/cm2 (Fig.
5B).
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Effects of AT1R and AT2R Antagonists on ANG II-Evoked Increase in Isc
To determine which ANG II receptor subtypes contribute to the ANG II-evoked increase in Isc, ANG II receptor antagonists were used. Serosal addition of the AT1R antagonist FR-130739 (10
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Immunohistochemistry
Immunostaining for the AT1R demonstrated the intense and specific immunoreactivity in neuronal soma of the submucous and myenteric nerve plexuses (Fig. 7, A and B). In whole mount preparations of the mucosal layer, less than half of all cell bodies in the submucous nerve plexus were positive in reaction and were intermingled with weakly positive or negative cell bodies. The intensely positive cell bodies tended to gather at the peripheral portion of the nerve plexus. Immunostaining of the outer muscle layer showed that only a limited number of nerve cells were immunoreactive in the myenteric nerve plexus and were dispersed throughout the nerve plexus. In both nerve plexuses, AT1R immunoreactivity appeared mainly in the cell bodies.
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Additional immunoreactivity to AT1R was observed on the surface epithelial cells of the distal colon, although it was less intense than that of the nerve plexuses (Fig. 7C). Furthermore, crypt cells lacked significant immunoreactivity to AT1R.
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DISCUSSION |
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It is known that the effect of ANG II on ion transport in the intestine
depends on its concentration. At picomolar concentrations, ANG II
stimulates Na+ and water absorption, whereas above
nanomolar concentrations, ANG II inhibits Na+ and water
absorption and stimulates secretory processes in rat jejunum and colon
(33). The present study has shown that ANG II
(109-10
5
M) evokes an increase in Isc in guinea pig distal
colon (Fig. 1). The ANG II (10
6
M)-evoked increase in Isc was reduced by the
Na+-K+-2Cl
cotransporter
blocker bumetanide or Cl
-free solution (Fig. 2).
These results suggest that the ANG II-evoked increase in
Isc is mainly due to Cl
secretion in guinea pig distal colon. However, Hatch et al. (21, 22)
reported that, in rat colon, ANG II
(10
9-10
6
M) evokes a decrease in Isc, which is due to
K+ secretion, whereas ANG II
(10
4 M) evokes an increase in
Isc by Cl
secretion. In the
present study we have used concentrations
(10
9-10
5
M) of ANG II similar to those previously reported, but ANG II did not
evoke the decrease in Isc. However, we did not
perform a flux experiment using isotopes, so we could not confirm the ANG II-evoked K+ secretion observed in the rat colon.
In the present study, TTX significantly reduced but did not abolish the
ANG II (106 M)-evoked increase in
Isc. This result suggests that ANG II-evoked Cl
secretion is partially mediated by submucosal
neurons. In contrast, Cox et al. (9) reported that ANG II
(10
10-10
5
M)-evoked changes in Isc are not affected by TTX in
rat small intestine. This report suggests that ANG II-evoked
Cl
secretion is not regulated by submucosal neurons
in rat small intestine. The discrepancy may reflect a segmental
difference between small and large intestines or a species difference.
In the present study we have further analyzed the contribution of
muscarinic and NK1 receptors to ANG II-evoked
Cl secretion. The muscarinic receptor antagonist
atropine and the NK1 receptor antagonist FK-888 reduced the
ANG II (10
6 M)-evoked increases in
Isc to 45 and 58% of control, respectively (Fig.
3). These results suggest that ANG II may activate cholinergic and
tachykinergic neurons to evoke Cl
secretion in
guinea pig distal colon. The results are supported by previous
observations that SP and ACh can evoke Cl
secretion
in the distal colon (6, 8, 17, 30, 43) and that SP-immunoreactive
neurons in the submucosal plexus in guinea pig small intestine are also
immunoreactive to choline acetyltransferase (18). The combination of
FK-888 and atropine further reduced the ANG II-evoked increase in
Isc to 20% of control, and the reduction was
significantly greater than with FK-888 alone. Thus another possible
pathway for ANG II-evoked Cl
secretion is ANG
II-induced SP release. SP may then evoke ACh release, which in turn
evokes Cl
secretion. This hypothesis is supported by
the observation that SP-evoked Cl
secretion is
partly inhibited by atropine in guinea pig distal colon (30).
Failure of TTX to completely block the response to ANG II suggests that
ANG II also acts directly on epithelial or subepithelial cells in the
mucosa. It has been reported that ANG II-evoked Cl
secretion is mediated by prostaglandin synthesis in rat small intestine
(9). In the present study, piroxicam reduced the ANG II-evoked increase
in Isc to 16% of control (Fig. 4). The result
suggests that the ANG II-evoked Cl
secretion is
linked with the prostaglandin synthesis pathway in guinea pig distal
colon as it is in rat small intestine. Furthermore, Jin et al. (26)
showed that ANG II (700 ng · kg
1 · min
1)
induces production of PGE2 in rat small intestine. EP1,
EP3, and EP4 receptor mRNAs are reported to be localized in rat small and large intestines (10). However, only the EP1 receptor antagonist was commercially available at the time of the experiment. The EP1
receptor antagonist SC-51089 reduced the ANG II-evoked response to 41%
of control. This result suggests that ANG II-evoked
Cl
secretion is partially mediated by EP1 receptor activation.
Prostaglandin synthesis is involved in the inflammatory process. Zipser
et al. (46) reported that the basal release of PGE2 is two
times greater in an artificial colitis condition than in normal tissues
and that ANG II increases the release of PGE2 in normal and
inflammatory conditions. It was reported that the colonic mucosal level
of ANG II was three times higher in Crohn's disease patients than in
healthy humans (25). These reports suggest that, in an inflammatory
state, ANG II may also play an important role in the modification of
electrolyte transport in colonic mucosa. In the present study we have
demonstrated the involvement of PGE2 in the ANG II
response. When the tissues were pretreated with PGE2
(105 M), the ANG II
(10
6 M)-evoked increase in
Isc was approximately three times higher and longer
than when the tissues were treated with ANG II alone (Fig. 5D).
Moreover, after application of ANG II
(10
6 M), the PGE2
(10
5 M)-evoked increase in
Isc in the second phase was approximately two times
higher than that with PGE2 alone (Fig. 5B). These
results suggest the possibility that the presence of PGE2
and ANG II in colonic mucosa may enhance Cl
secretion in the inflammatory state.
The ANG II-evoked Cl secretory mechanism may involve
increases in the concentration of intracellular cAMP and cytosolic free Ca2+ ([Ca2+]i). It has
been reported that PGE2 activates the apical cAMP-activated Cl
channels and basolateral cAMP-activated
K+ channels in colonic epithelial cells (35, 42). Strabel
and Diener (43) suggested that in rat colonic epithelia a
carbachol-evoked increase in Isc is enhanced by
pretreatment with cAMP-activating drugs [e.g., PGE2,
forskolin, or 8-(4-chlorophenylthio)-cAMP] and that the dominant
effect of carbachol is activation of basolateral Ca2+-activated K+ channels. It is known that
the activation of these channels induces Cl
secretion in colonic mucosa (19). Romero et al. (38) suggested that ANG
II induces an increase in [Ca2+]i
through AT1R in guinea pig ileum myocytes and activates the Ca2+-activated K+ channels. Jin et al. (26) and
Zipser et al. (46) showed that ANG II induces production of
PGE2 in rat small intestine and rabbit distal colon. In the
present study the ANG II-evoked response was blocked by inhibition of
the prostaglandin synthesis pathway. Thus these results suggest that
the ANG II response and the PGE2-enhanced ANG II
synergistic response may be regulated by the interaction of cAMP
concentration and [Ca2+]i in
colonic epithelial cells. However, the exact mechanism of the ANG
II-enhanced PGE2 response is not clear. Calderaro et al. (1) reported that PGE2-evoked Cl
secretion in rabbit distal colon is regulated by
[Ca2+]i, which regulates adenylate
cyclase and phosphodiesterase activities. Furthermore, they suggested
that a lower [Ca2+]i enhances a
PGE2-evoked sustained increase in Isc.
Therefore, one possible explanation for the ANG II-enhanced
PGE2 response is that ANG II may continually affect
[Ca2+]i and enhance cAMP metabolism
in the PGE2 response.
There are two major isoforms of the ANG II receptor: AT1R
and AT2R. Sechi et al. (41) showed that AT1R
and AT2R subtypes are present in the mucosa and the
muscularis mucosa of rat jejunum, ileum, and colon and that the
predominant ANG II receptor subtype is AT1R, but a small
proportion of AT2R is also present. Jin et al. (26)
reported that a low dose of ANG II given in an intravenous infusion
stimulates net Na+ absorption through AT2R,
whereas a high dose of ANG II inhibits Na+ absorption by
PGE2 synthesis through AT1R on rat small
intestine. The report has suggested that, in small and large
intestines, AT1R and AT2R regulate the ANG
II-evoked ion transport. In the present study the AT1R
antagonist FR-130739 (105 M) completely
blocked an ANG II (10
6 M)-evoked
increase in Isc, whereas the AT2R
antagonist PD-123319 had no effect (Fig. 6). We have also shown that
AT1R-immunoreactive neurons are located in the submucosal
plexus (Fig. 7A) and the ANG II-evoked increase in
Isc was partly blocked by pretreatment with TTX.
Moreover, the moderate immunoreactivity of AT1Rs was also
found in surface epithelial cells (Fig. 7C). It has been shown
that Cl
secretion is restricted to the crypts (19,
20). However, Kockerling and Fromm (28) showed that in rat distal colon
the cAMP-dependent Cl
secretion is not confined to
crypts but is also performed by surface epithelial cells. Thus the
results of the present study suggest that AT1R regulates
ANG II-evoked Cl
secretion and that AT1R
in the submucosal plexus and surface epithelial cells most likely
contributes to the secretory effect. In the present study the ANG II
response was reduced to 16% of control by pretreatment with piroxicam.
Therefore, ANG II-evoked Cl
secretion is partially
linked with prostaglandin synthesis. Many subepithelial cells,
including mast cells, fibroblasts, and also crypt epithelial cells are
able to produce prostaglandins (2, 5, 36). However, we did not observe
AT1R immunoreactivity in these cells. Thus it is unlikely
that ANG II directly induces prostaglandin synthesis through
AT1R. Therefore, further study is needed to identify an ANG
II-induced prostaglandin synthesis mechanism in guinea pig distal
colonic mucosa.
We have also shown that AT1R-immunoreactive nerves are located in the colonic myenteric plexus (Fig. 7B). Leung et al. (32) showed that ANG II induces smooth muscle contractions in guinea pig small and large intestines and that these responses are inhibited by TTX and AT1R antagonist but are not affected by AT2R antagonist. These results suggest that ANG II regulates colonic motility as well as electrolyte transport through AT1R in the enteric nervous system.
In the present study we have used a concentration of ANG II much higher
than the concentration of circulatory ANG II. Hatch et al. (22)
reported that the calculated extracellular tissue space concentration
of ANG II was 2.7 × 106 M when the
bath concentration was 1 × 10
4 M
and suggested that the active concentration of ANG II at the tissue
level might be much lower than the concentration in the bath solution.
Using the rate of ANG II tissue hydrolysis reported, we have estimated
the tissue concentration of ANG II in guinea pig distal colon when the
ANG II concentration of the bathing solution was
10
6 M. The estimated tissue
concentration of ANG II was two orders of magnitude less than the bath
concentration. These results indicate that relatively high bath
concentrations of ANG II are necessary to evoke Cl
secretion under experimental conditions.
Some reports have shown the possibility that ANG II exists as the tissue renin-angiotensin system in the gastrointestinal tract. The hypothesis is supported by the following observations: 1) angiotensinogen mRNA has been detected in rat mesentery and large intestine (3, 37); 2) kallikrein was observed in rat colonic goblet cells (40); and 3) ANG II receptors and ACE have been shown to be present in rat colon by in vitro autoradiography (15, 41).. These findings suggest that ANG II may be produced and may regulate local intestinal functions in the gastrointestinal tract.
In conclusion, the results provide evidence for the involvement of ANG
II in the local regulation of Cl secretion in guinea
pig distal colon. ANG II-evoked Cl
secretion may
involve submucosal cholinergic and tachykinergic neurons and prostanoid
synthesis pathways. These responses may be regulated by
AT1R on the submucosal plexus and surface epithelial cells.
Furthermore, ANG II and PGE2 synergistically evoke ion transport. Thus the present study indicates that ANG II functions as a
local mediator in colonic mucosa in physiological and
pathophysiological states.
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ACKNOWLEDGEMENTS |
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The authors thank Dr. M. Ceregrzyn for kind and helpful discussion.
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FOOTNOTES |
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This work was supported by a Monbusho International Research Grant to A. Kuwahara.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: A. Kuwahara, Laboratory of Environmental Physiology, Institute for Environmental Sciences, University of Shizuoka, Shizuoka 422-8526, Japan (E-mail: kuwahara{at}sea.u-shizuoka-ken.ac.jp).
Received 28 May 1999; accepted in final form 24 November 1999.
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