Laboratory of Physiology, Institute for Environmental Sciences, University of Shizuoka, 52-1 Yada, Shizuoka, 422-8526, Japan
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ABSTRACT |
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Interaction between substance P (SP) and
PGE2 on Cl secretion in the guinea pig distal
colonic epithelia was investigated. A short-circuit current
(Isc) was measured as an index of ion transport.
Mucosa preparations deprived of muscle and submucosa of distal colon
were mounted in the Ussing flux chamber and treated with TTX and
piroxicam to remove the influences of neuronal activity and endogenous
PG synthesis, respectively. Although SP (10
7 M) itself
evoked little increase in Isc, exogenous
PGE2 concentration dependently enhanced the response of SP.
The effect of PGE2 on the SP-evoked response was mimicked
by forskolin and 8-bromoadenosine cAMP. Depletion of Ca2+
from the bathing solution reduced the PGE2-dependent
response of SP. Effects of PGE2, SP, and SP in the presence
of PGE2 on intracellular Ca2+ concentration
([Ca2+]i) in isolated crypt cells
were measured by the confocal microscope fluorescence imaging system.
SP, but not PGE2, temporally evoked an increase in
[Ca2+]i but declined to the baseline within 3 min. A return of the SP-evoked increase in
[Ca2+]i was slower in the presence of
PGE2 than SP alone. These results suggest that
PGE2 synergistically enhances SP-evoked Cl
secretion via an interaction between the intracellular cAMP and [Ca2+]i in the epithelial cells. In
conclusion, SP and PGE2 could cooperatively induce massive
Cl
secretion in guinea pig distal colon at epithelial levels.
colonic crypt; adenosine 3',5'-cyclic monophosphate; Ca2+, crosstalk; inflammation
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INTRODUCTION |
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SUBSTANCE P (SP) is a member of the tachykinin family widely distributed in the enteric nervous system (ENS) of small and large intestines (15, 16, 24, 25). The tachykinin family includes neurokinin A (NKA) and B (NKB) in addition to SP. These tachykinins are involved in smooth muscle contractions, blood flow, local immune functions, and epithelial transport. The effects of tachykinins are mediated by at least three different receptor subtypes: neurokinin-1 (NK1), NK2, and NK3. These receptors are coupled to G protein, and its stimulation induces an increase in intracellular Ca2+ concentration ([Ca2+]i) as a second messenger (25). In the intestine, SP and NKA, but not NKB, are mainly found in the intrinsic primary afferent neurons, interneurons, and motorneurons in the ENS (15). Grady et al. (17) reported the immunoreactivity of neurokinin receptors in the gastrointestinal tract of the rat and that NK1 receptors are distributed in myenteric and submucosal neurons and in interstitial cells of Cajal, NK2 receptors are localized to circular and longitudinal muscle cells and to nerve endings in the plexuses, and NK3 receptors are detected in numerous myenteric and submucosal neurons. Recently, Southwell and Furness (44) reported that NK1 receptor immunoreactivity was detected both on the muscle and on the mucosal epithelium of the guinea pig small and large intestines. It is known that endogenous SP and NKA in the intestine interact with other enteric transmitters, such as ACh, and regulate intestinal motility, and fluid and electrolyte transport (15).
Presence of SP in nerve fibers close to epithelial cells suggests a
role for SP in the regulation of epithelial transport. In a previous
study, Kuwahara and Cooke (23) reported that the exogenous
addition of SP evokes Cl secretion in the guinea pig
distal colon and that it is mediated by neurons and a nonneuronal
pathway. Involvement of ENS on SP-evoked Cl
secretion has
been intensively studied, but little is known about the nonneuronal
pathway of SP-induced Cl
secretion in the colon
(10, 14, 23).
It has been reported that SP-evoked Cl secretion is
inhibited in the porcine jejunum (46), dog
(35), guinea pig (23), and human colon
(38) by pretreatment with cyclooxigenase (COX) inhibitor,
and that SP induces PGE2 synthesis (46). These
effects are typically ascribed to agonist-stimulated release of PGs
from the subepithelium. These reports also suggest that SP evokes
Cl
secretion via activation of enteric neurons and/or
production of PG in mammalian colonic mucosa. PGs are well known as
inflammatory mediators and secretagogues in the gastrointestinal tract
(12, 13, 32, 41, 43). PGs are reported to act both on the
epithelium and on the submucosal plexus to evoke ion transport
(12, 13). Homaidan et al. (20) reported that
PGE2 binding sites are detected and cAMP levels are
increased by PGE2 in the crypt cells. An increasing body of
evidence indicates that SP is involved in the pathophysiology of
intestinal secretion and inflammation in animals and humans (6,
28, 29, 45). The administration of SP receptor antagonists reduces secretory and inflammatory changes in rat models of acute and
chronic intestinal inflammation (7, 34). Furthermore, SP
immunoreactivity and SP binding are increased in the colons of patients
with inflammatory bowel disease (22, 27). Thus it is
possible that SP and PGE2 interact with each other in
inflammatory conditions in the intestine. However, the interaction of
SP and PGE2 on epithelial cells and the cellular mechanism
of SP-evoked Cl
secretion have not been investigated in depth.
In the present study, we investigated the nonneuronal pathway of
SP-evoked Cl secretion in the guinea pig distal colon. In
particular, we focused on the interaction between SP and
PGE2 on Cl
secretion. Results show that
synergistic action of SP and PGE2 on Cl
secretion occurs at the epithelial cell level of guinea pig distal colon.
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MATERIALS AND METHODS |
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Ussing flux chamber experiment.
Male albino guinea pigs (Hartley-Hazleton; Nippon SLC, Hamamatsu,
Japan) ranging in weight from 400 to 900 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. In the present experiments, tissue was prepared
according to a previous study (4) to remove the neural
influence. Segments of distal colon were removed, flushed with
Krebs-Ringer solution, and cut along the mesenteric border. Tissues
were then laid flat on an acrylic board with the mucosal side up.
Mucosal preparation was made by longitudinally peeling off mucosa using
a pair of glass slides. This procedure removes submucosal ganglia, the
submucosal layer, and external muscle layers, including myenteric
plexus (4). Four sets of mucosal sheets were mounted
between halves of Ussing flux chambers in which the total
cross-sectional area was 0.64 cm2. Mucosal and serosal
surfaces of the 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 in the solution for 0.5-1 h before
the experiment. Krebs-Ringer solution contained (in mM) 120 NaCl, 6 KCl, 1.2 MgCl2, 1.2 NaH2PO4, 14.4 NaHCO3, 2.5 CaCl2, and 11.5 glucose. The Cl-free solution
contained (in mM) 2.7 K2SO4, 1.2 MgSO4, 1.2 NaH2PO4, 54.9 Na2SO4, 13 NaHCO3, 1.7 CaSO4, 60.4 mannitol, and 11.5 glucose. The solution was
gassed with 95% O2-5% CO2 and buffered at pH 7.2. For a Ca-free solution, CaCl2 was removed from the
Krebs-Ringer solution and 3 mM EDTA was added. The potential difference
(PD) across the tissue was measured by paired Ag-AgCl electrodes in Krebs-agar bridge and clamped to 0 mV by applying a short-circuit current (Isc) by Ag-AgCl electrodes with a
voltage-clamp apparatus (SS-1335; Nihon-Koden, Tokyo, Japan). Tissue
conductance (Gt) was calculated by determining
the current necessary to change PD by 10 mV. Responses were
continuously recorded on a chart recorder (Recti-Horitz-8K; Nihon-Denki
Sanei, Tokyo, Japan) and Mac/Lab8 system (ADInstruments; Cattle Hill,
Australia).
Isc was calculated on the basis
of the value before and after stimulation.
Isolation of crypts.
Distal colonic segments (~4 cm) were rinsed with cold Krebs-Ringer
solution with 3 mM dithiothreitol and then filled with PBS (),
including 25 mM EDTA and 3 mM dithiothreitol, until a moderate tension
was achieved by clamping both ends. Tissue was then incubated in
Krebs-Ringer solution for 3 min at 37°C. Then, the luminal solution
containing isolated crypts was collected by centrifugation (4°C;
1,000 rpm for 1 min). A portion of the supernatant was then removed,
and the tissue fragments containing crypts were rinsed twice with
Krebs-Ringer solution.
Measurement of [Ca2+]i in isolated crypt cells. Isolated crypts were suspended with Krebs-Ringer solution containing 0.1% BSA, 5 µM indo 1-acetoxymethyl ester (AM) and 0.05% Cremophore EL for 10 min in the dark at room temperature. Suspension of dye-loaded crypt was seeded on a specially designed glass perfusion vessel coated with cell adhesive Cell-Tak to fix the crypts for 20 min in a refrigerator. Then, the vessel was placed in a 5% CO2 incubator for 60-90 min at 37°C to allow indo 1-AM loading. After dye loading, the vessel was washed with Krebs-Ringer solution containing 0.1% BSA. The vessel was then placed on the stage of a laser-scanning confocal imaging system (ACAS Ultima 575 UVC; Meridian Instruments, Okemos, MI) with an inverted microscope (Axiovert 135; Zeiss) magnification ×40 (water immersion objective). The vessel was continuously perfused at 2 ml/min of flow rate with the oxygenated Krebs-Ringer solution containing 0.1% BSA at 32°C. Indo 1-AM was excited using the 350- to 360-nm line. Twin photomultiplier channels detected bands of fluorescence centered on 405 and 485 nm. Ratiometric images were collected every 10 s in the stimulated and unstimulated conditions for analysis of temporal change in [Ca2+]i.
Isolated crypts were perfused before stimulation with normal Krebs-Ringer solution containing 0.1% BSA. The crypts were then continuously stimulated by replacing the normal perfused Krebs-Ringer solution with Krebs-Ringer solution containing either SP (10Chemicals.
Substance P was purchased from Peptide Institute (Osaka, Japan);
bumetanide, DMSO, TTX, -conotoxins, and 8-br-cAMP were from Sigma
(St. Louis, MO); piroxicam and forskolin were from Biomol Research
Laboratories (Plymouth Meeting, PA); PGE2 was from Cayman (Ann Arbor, MI). Bumetanide and piroxicam were dissolved in dimethyl sulphoxide. The other drugs were dissolved in distilled water. Volume
of dissolved drugs in H2O or dimethyl sulphoxide added to
the bathing solutions did not exceed 100 and 10 µl, respectively.
Statistics.
All data are expressed as means ± SE. ANOVA was followed by the
Tukey test to determine significant differences between each experimental tissue. P < 0.05 was considered
statistically significant. Concentration-response curves were fitted to
Michaelis-Menten binding curves by the nonlinear-square procedure using
KyPlot, a data analysis and graph-creating software (50).
We considered the PGE2-evoked sustained phase (see
RESULTS) of Isc consisted of two
components of ion transport: K+ secretion as negative
Isc and Cl secretion as positive
Isc (19). Therefore, the equation
to fit the curve was calculated as the sum of the two following
Michaelis-Menten equations: I = IK · C/(C + EC50,K) + ICl · C/(C + EC50,Cl),
where I, IK, and
ICl are net K+ and Cl
Isc, respectively; C is PGE2
concentration, and EC50,K and EC50,Cl are the
half effective concentrations K+ Isc
and Cl
Isc, respectively,
constrained with IK
0 and
ICl, EC50,K, and EC50,Cl
0.
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RESULTS |
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Effects of TTX and piroxicam on SP-evoked increase in
Isc.
The present experiment was designed to examine the interaction between
SP and PGE2 on ion transport using a mucosal preparation of
guinea pig distal colon. The average PD, basal
Isc, and Gt just before
the addition of SP in the control, TTX-pretreated, piroxicam-pretreated, and both TTX- and piroxicam-pretreated groups were not significantly different, respectively. The PD,
Isc, and Gt of the
control group were 4.2 ± 0.2 mV, 55.5 ± 6.8 µA/cm2 and 12.0 ± 0.9 mS/cm2,
respectively (n = 4). In the mucosal preparations, SP
(10
7 M) evoked a biphasic increase in
Isc (1st phase: 21.9 ± 6.1 µA/cm2; 2nd phase: 105.9 ± 26.3 µA/cm2; n = 4) (Fig.
1). TTX (10
7 M)
pretreatment decreased the SP-evoked responses to 13.9 ± 5.4 µA/cm2 (1st phase, P = 0.56) and
28.3 ± 26.3 µA/cm2 (2nd phase, P < 0.05), respectively (n = 4) (Fig. 1B).
Piroxicam (10
6 M) pretreatment also decreased the
SP-evoked responses to 5.1 ± 1.6 µA/cm2 (1st phase,
P = 0.07) and 18.6 ± 14.7 µA/cm2
(2nd phase, P < 0.05), respectively (n = 4). Moreover, combination of TTX and piroxicam pretreatment decreased
the SP-evoked responses to 4.7 ± 1.4 µA/cm2 (1st
phase, P = 0.06) and 15.6 ± 10.6 µA/cm2 (2nd phase, P < 0.05),
respectively (n = 4). The SP response in the presence
of piroxicam and TTX was not significantly different from the response
with piroxicam or TTX alone. In the present experiments, the SP-evoked
biphasic increase in Isc was inhibited by TTX
and piroxicam, as mentioned above. This result indicated that the
effect of SP via neurons and endogenous PGs remained even in the
mucosal preparations. Moreover, the electrical field stimulation (25 V,
10 Hz, and 0.5 ms duration) for 2 min increased basal
Isc in the absence of TTX (105.9 ± 8.0 µA/cm2, n = 4), and the response was
completely abolished by pretreatment with TTX (10
7 M).
Thus for further experiments, all tissues were pretreated with TTX
(10
7 M) and piroxicam (10
6 M) to completely
remove the neuronal effect and production of endogenous PGs in tissues.
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Effect of PGE2 on basal Isc and SP-evoked
increase in Isc.
To investigate the interaction between SP and PGE2 on ion
transport, tissues were pretreated with PGE2
(109
10
4 M) 10 min before the
addition of SP (10
7 M). Figure
2A shows a representative
trace of the SP-evoked response 10 min after the addition of
PGE2 (10
5 M) in the presence of TTX and
piroxicam. The serosal addition of PGE2
(>10
7 M) concentration-dependently evoked biphasic
changes in Isc in both transient and sustained
phases (Fig. 2B). The maximal increase in
Isc was observed at 10
5 M
PGE2. The transient phase was observed ~1 min after the
addition of PGE2, and the sustained phase lasted for >20
min (Fig. 2A). Values of PGE2 (10
5
M)-evoked transient phase and sustained phase were 202.3 ± 24.2 and 61.7 ± 9.5 µA/cm2, respectively (Fig.
2B). EC50 of the transient phase of
PGE2 was 2.46 × 10
6 M. On the other
hand, low concentrations of PGE2 (<10
7 M)
evoked a decrease in Isc, and the responses
reached a plateau at 10 min or more. Maximal decrease was observed at
10
8 M PGE2 (
39.0 ± 0.7 µA/cm2, n = 4). Curve fitting of the
sustained phase was calculated by the sum of the two divided
components, including K+ and Cl
secretion
based on a previous study (19). EC50,A and
EC50,B were 1.42 × 10
9 M and 1.33 × 10
6 M, respectively.
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Effects of Cl-free solution and
bumetanide on PGE2- and PGE2-dependent
SP-evoked increase in Isc.
To determine the ionic basis for the increase in
Isc induced by PGE2- and
PGE2-dependent SP-evoked responses, bumetanide, an
inhibitor of the Na+-K+-2Cl
cotransporter and a Cl
-free solution were used.
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Effects of PGE2, forskolin, or 8-br-cAMP on SP-evoked
increase in Isc.
To determine whether the increase in intracellular cAMP mimics the
effect of PGE2 on the SP-evoked increase in
Isc, an adenylate cyclase activator forskolin or
a membrane-permeable cAMP analog 8-br-cAMP were used. Serosal addition
of forskolin (105 M) evoked an increase in basal
Isc (64.6 ± 13.2 µA/cm2,
n = 3). On the other hand, 8-br-cAMP (10
3
M) evoked a decrease in basal Isc (
16.8 ± 11.9 µA/cm2, n = 4), but the changes
were not statistically significant. The addition of SP
(10
7 M) 10 min after treatment with forskolin or
8-br-cAMP in the presence of TTX and piroxicam evoked the following
biphasic increases in Isc: forskolin
pretreatment, 1st phase 327.6 ± 7.3 µA/cm2, 2nd
phase 361.5 ± 19.1 µA/cm2, n = 3;
8-br-cAMP pretreatment, 1st phase 188.7 ± 33.4 µA/cm2, 2nd phase 317.6 ± 64.9 µA/cm2, n = 4 (Fig.
4).
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Effect of Ca2+ in bathing solution on PGE2- and PGE2-dependent SP-evoked increase in Isc. To investigate the affect of extracellular Ca2+ on PGE2- and PGE2-dependent SP-evoked increase in Isc, serosal, mucosal, or both sides bathing solution were replaced by a Ca2+-free solution before the addition of TTX and piroxicam. Depletion of serosal and both sides Ca2+ significantly changed in Gt from 13.3 ± 1.4 and 10.9 ± 0.9 mS/cm2 to 31.5 ± 3.1 and 34.8 ± 4.9 mS/cm2 (n = 4), respectively.
Serosal and both sides, but not mucosal Ca2+-free solution, significantly increased the PGE2 (10
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Effects of PGE2, SP, and SP in the presence of
PGE2 on [Ca2+]i
in isolated colonic crypt cells.
Isolated crypt cells were used to investigate the involvement with
Ca2+ signaling pathway in PGE2-dependent
SP-evoked responses. Perfusion with a solution containing
PGE2 (105 M) did not affect
[Ca2+]i in isolated crypt cells. On the other
hand, SP (10
7 M) evoked a transient increase in
[Ca2+]i (peak value of the normalized ratio:
2.70 ± 0.19, n = 7) and returned to basal level
within 3 min (Fig. 6). The presence of PGE2 in the perfusate did not affect the peak values of the
SP-evoked increase in [Ca2+]i (2.89 ± 0.18, n = 6) (Fig. 6). However, the return of
[Ca2+]i to the basal level was slower in the
presence of PGE2 (Fig. 6A). The area
under the curves (AUCs; change in normalized ratio × s) for 3 min
were compared. Results show that the presence of PGE2
significantly increased the AUC of normalized ratio for 3 min by SP
from 97.5 ± 13.9 to 173.7 ± 9.6 (n = 6).
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DISCUSSION |
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In the present study, we have shown the direct action of SP and
the interaction with PGE2 on Cl secretion in
the guinea pig distal colonic epithelia. Previous studies have shown
that the SP-evoked nonneurally mediated secretion is much weaker than
neurally mediated secretion (10, 14, 23). We have also
obtained a similar result (Fig. 1A). However, in the present
study, we found that SP could induce massive Cl
secretion
at nonneural, perhaps epithelial cell levels, and in as large amounts
as the neurally mediated responses when a high concentration of
PGE2 was present. Moreover, we have shown that SP evokes a
direct and transient increase in [Ca2+]i, and
SP-evoked nonneural massive Cl
secretion is due to an
increase in intracellular cAMP level. Previous studies
(20) have indicated that PGE2 increases the intracellular cAMP level of colonic crypt cells via EP2
receptors. Therefore, it is suggested that SP may act in concert with
PGE2 to evoke Cl
secretion as a crosstalk
between Ca2+ and cAMP at the epithelial cell level and that
this massive Cl
secretion to flush out noxious agents
from the intestinal lumen is not mediated via neurons in the
inflammatory condition.
A previous study (23) has shown that the effect of SP on
ion transport in the colon is mediated by neurons and mast cell-derived mediators including histamine and PGE2. In the present
study, we chose to make aganglionated mucosal preparations to
investigate the direct action of SP and PGE2 on the
epithelium and found that the response of SP was comparatively smaller
than those of previous studies (14, 23) using
mucosa-submucosa preparations (Fig. 1). The present mucosal
preparations were histologically checked and there was no evidence that
submucosal ganglia remained (data not shown). However, the preparations
proved insufficient to remove ganglia, including nerve fibers, because
the SP-evoked response was still sensitive to TTX. It is reported that
SP stimulates NK1 and/or NK3 receptors on cell
somas and dendrites of secretomotor neurons (14). The
result raises the possibility that ganglia are located in the mucosal
plexus in the colon (30, 31). Moreover, Riegler et al.
(38) have reported that NK1 immunoreactivity on nerve cells is detected in mucosal lamina propria in the human colon. The SP-evoked response was also reduced by the inhibition of PG
synthesis using the COX-inhibitor, piroxicam (Fig. 1). The result
suggests that the SP-evoked response is due to endogenous PGs.
Moreover, the SP-evoked response in the combination of TTX and
piroxicam was not significantly different from TTX or piroxicam alone
(Fig. 1). Therefore, the results suggest that a part of the SP-evoked
response is dependent on both neuronal activity and PG synthesis.
Frieling et al. (12) have reported that
PGE2-evoked Cl secretion in guinea pig distal
colon is mediated by nerve-dependent and -independent mechanisms. Some
part of the SP-evoked response might be due to the interaction between
SP and endogenous PGE2 at the submucosal neuron level. In
the present study, we investigated the interaction between SP and
PGE2 on the epithelium. Therefore, the tissues were treated
with TTX and piroxicam together to avoid any effect of neuronal
activity and endogenous PG synthesis. Moreover, we considered that the
release of any neurotransmitters from nerve terminals is not involved,
because
-conotoxins do not affect any responses in the presence of TTX.
In the present experiment, pretreatment of the tissues with
PGE2 concentration dependently enhanced the SP-evoked
responses although SP itself also evoked a small increase in
Isc (Fig. 2, A and C). The
SP-evoked response in the presence of PGE2 was inhibited by
bumetanide and a Cl-free bathing solution (Fig.
3B). These results indicate that the SP-evoked increase in
Isc in the presence of PGE2 is
mainly due to Cl
secretion. Homaidan et al.
(20) reported that PGE2 increases cAMP level
in isolated rabbit colonic crypt cells via the EP2 subtype
of PGE receptors. Therefore, the results suggest that SP can evoke
Cl
secretion in the colonic epithelial cells when
intracellular cAMP level increases. We have further tested whether
activators of cAMP mimic the effect of PGE2 on the
SP-evoked response using an adenylate cyclase activator, forskolin, or
a membrane-permeable cAMP analog, 8-br-cAMP. Data showed that
pretreatment with forskolin and 8-br-cAMP could mimic the effect of
PGE2 pretreatment on the SP-evoked increase in
Isc (Fig. 4). The SP-evoked increase in Isc in the presence of forskolin also resulted
in Cl
secretion, because the response was bumetanide
sensitive. Thus the effect of PGE2 on the SP-evoked
Cl
secretion is probably due to the increase in
intracellular cAMP level in the guinea pig distal colonic epithelial
cells. Yajima et al. (49) showed similar results, namely
that bethanechol-evoked Cl
secretion is enhanced when the
tissue is pretreated with PGE2, VIP, and 8-br-cAMP.
It has been suggested that all tachykinin receptors are coupled to
Gq/11 protein, and tachykinins evoke an increase in
[Ca2+]i by the SP > NKA NKB potency
order in isolated guinea pig colonic crypt cells (unpublished
observations by K. Shiokawa, Y. Hosoda, Y. Shimoda, M. Suzuki, S. Karaki, M. Ceregrzyn, and A. Kuwahara). Cooke et al.
(10) showed that the NK1 receptor mRNA is
expressed and binding of SP is inhibited by the NK1
receptor antagonist GR-82334 on guinea pig colonic crypt cells. It has been reported that NK1 receptor immunoreactivity is located
on the mucosal epithelium of the guinea pig distal colon
(44), but NK2 receptors are rare
(33). These reports suggest that SP stimulates
NK1 receptors and increases in
[Ca2+]i in the distal colonic crypt cells.
Therefore, we investigated the role of Ca2+ on
Cl
secretion by SP in the presence of PGE2 on
the colonic epithelia. Removal of Ca2+ from serosal or both
sides bathing solution significantly reduced the SP-evoked increase in
Isc in the presence of PGE2 (Fig.
5B). The result indicates that the SP-evoked increase in
Isc in the presence of PGE2 depends
on serosal-side extracellular Ca2+. It has been
reported that an increase in [Ca2+]i
opens 1) the calcium-activated chloride channel on the
apical membrane and 2) the basolateral K+
channel. It also enhances the driving force for Cl
secretion (2, 3, 18). In the present study, we measured the change in [Ca2+]i in isolated guinea pig
distal colonic crypt cells when the crypts were perfused with
PGE2, SP, or SP in the presence of PGE2 using a
confocal laser-microscope and the calcium imaging system. Results
showed that SP evoked a transient increase in
[Ca2+]i both in the presence and absence of
PGE2. However, SP in the absence of PGE2 evoked
little electrogenic ion transport in the mucosal preparations (Fig.
2C), although [Ca2+]i increased
considerably (Fig. 6). Thus these results suggest that a transient
increase in [Ca2+]i itself in colonic crypt
cells cannot evoke Cl
secretion; however, SP can evoke
Cl
secretion through a transient increase in
[Ca2+]i with an increase in intracellular
cAMP level in the epithelia. Mall et al. (26) reported
similar results that carbachol-induced increase in
[Ca2+]i can induce Cl
secretion
only in the presence of cAMP. Carew and Thorn (5) have
also reported that autocrine release of PGs from epithelial cells is
sufficient to support the carbachol-induced Cl
secretion
and that carbachol-evoked Cl
secretion is dependent on
continuous basal production of cAMP in the epithelium. This evidence
and our present results suggest that Ca2+-dependent
Cl
secretion is also dependent on cAMP. The mechanism of
cAMP-dependent Cl
secretion is considered to be that an
increase in cAMP in the epithelial cell opens the cAMP-dependent
Cl
channel and the K+ channel on the apical
and basolateral membranes, respectively (18). Therefore,
it is suggested that the SP-induced increase in
[Ca2+]i may open the
Ca2+-dependent K+ channel (mentioned above) and
produce an electrical driving force for anion secretion. In addition,
although there was no significant difference between peak values of the
SP-induced increase in [Ca2+]i in the
presence and absence of PGE2, the return to the basal level
was significantly slower in the presence of SP and PGE2 than with SP alone (Fig. 6). Therefore, it is suggested that the effect
of PGE2 on the SP-evoked increase in
[Ca2+]i may contribute to the massive
Cl
secretion. However, the SP-evoked increase in
Isc in the Ussing flux chamber experiments
was comparatively long-lasting although the SP-evoked increase in
[Ca2+]i in isolated crypt cells was
transient. Therefore, we have hypothesized that the transient increase
in [Ca2+]i in colonic epithelial cells might
have a role as a trigger of change in intracellular pathways affecting
the cAMP regulation and evoking massive Cl
secretion
although there is no firm evidence yet. Moreover, Carew and Thorn
(5) showed that PGE2 secretion in the
nanomolar range (1 nM) is sufficient to support the carbachol-induced
Cl
secretion. In the case of SP, >100 times higher
concentrations of PGE2 (>10
7 M) were
necessary to evoke massive Cl
secretion (Fig.
2C). It is hypothesized that the difference in sensitivity
to the PGE2 level between SP and carbachol might be due to
the role of the respective chemical transmitters. Thus in the normal
condition with a low concentration of PGE2, acetylcholine affects the epithelium to evoke Cl
secretion as a
physiological function, whereas in an inflammatory condition with a
high concentration of PGE2, SP might affect the epithelium
to evoke massive Cl
secretion as a pathophysiological action.
In the present study, PGE2 itself concentration dependently
evoked a transient increase and a sustained response in
Isc (Fig. 2, A and B).
Pretreatment of the tissues with bumetanide did not alter the
PGE2-evoked response, but a Cl-free solution
decreased the transient phase (Fig. 3A). Rechkemmer et al.
(36) suggested that the PGE2-evoked net
Isc is consistent with the sum of the
electrogenic K+ and Cl
secretion and was
bumetanide insensitive. Recently, Halm and Halm (19)
reported more detailed experiments to describe prostanoids-evoked K+ and Cl
secretion in guinea pig distal
colon. They suggested that at low concentrations (<30 nM) and high
concentrations (>100 nM), PGE2 stimulates K+
secretion via EP2 receptors and Cl
secretion
via DP receptors, respectively. In the present experiment, we had the
same results of Isc response, and bumetanide was
also insensitive to the PGE2-evoked response. It has been
reported that bumetanide completely blocks PGE2-evoked
K+ secretion but not Cl
secretion
(36). From our results, the ionic basis of the
PGE2-evoked increase in Isc could
not be defined, but some part of the transient phase may be due to
Cl
secretion. Furthermore, HCO3
might also contribute to an increase in Isc
evoked by PGE2, especially in the Cl
-free
condition (36).
Removal of Ca2+ from serosal or both sides bathing solution
significantly enhanced the sustained phase of the
PGE2-evoked increase in Isc but not
the transient phase (Fig. 5A). Calderaro et al. (3) reported that a Ca2+-free solution
increases the intracellular cAMP concentration and
PGE2-evoked Cl secretion in rabbit distal
colonic epithelia. They suggest that an increase in Cl
secretion in a Ca2+-free solution is due to lower cyclic
nucleotide phosphodiesterase activity and higher adenylate cyclase
activity than in a Ca2+ containing solution. Thus serosal
Ca2+ may continuously inhibit the PGE2-evoked
sustained phase by decreasing the cAMP level in guinea pig colonic epithelia.
Although the cellular sources of PGs in the present study cannot be
defined, it is well established that they can be released from lamina
propria cells, including basophils, fibroblasts, macrophages, and mast
cells (9). Sharon and Stenson (42) reported
that levels of PGs markedly rise in inflammatory bowel disease. Singer et al. (43) also reported that COX-2 protein is not
detected in normal human colonic epithelial cells but is detected in
Crohn's disease and ulcerative colitis epithelial cells. In general,
COX-1 is thought to be responsible for production of the PGs associated with the maintenance of gastrointestinal integrity, whereas COX-2 is
believed to be responsible for the production of PGs associated with
the mediation of inflammation (40). Furthermore, Mantyh et
al. (27) reported that high concentrations of
NK1 receptor binding sites are expressed in the colon of
inflammatory bowel disease. From their results, it is suggested that SP
may also be involved in the pathophysiology of intestinal inflammation. Mast cells have been implicated in the pathophysiology of intestinal inflammation. Wang et al. (47) reported that mast
cell-deficient mice exhibit a reduced ileal secretory response to SP.
Thus mast cells may be responsible as one source of PGE2
release. Taken together, the present results suggest that in
pathophysiological states, an increased level of PGE2
enhances SP-evoked Cl secretion to ensure the secretory
responses induced by SP.
In the gastrointestinal tract, SP as a neurotransmitter is involved in the physiological control of several digestive functions, including blood flow, intestinal motility, and fluid and ion transport (15). In addition to these effects, many experimental results suggest that SP acts as a mediator for the regulation of intestinal inflammation, as mentioned above. Watanabe et al. (48) showed that SP-immunoreactive nerve fibers are increased in the colonic mucosa of ulcerative colitis patients. A recent publication by Renzi et al. (37) showed that mRNA expression and immunoreactivity for the NK1 receptor are dramatically increased in the crypt cells of both Crohn's disease and ulcerative colitis patients. These reports suggest that the SP and NK1 receptor may be involved in inflammatory reactions in the human distal colon. Moreover, Stucchi et al. (45) suggested that the NK1 receptor antagonist can have a therapeutic effect in the treatment of chronic ulcerative colitis. Our present findings provide evidence that SP can be a strong secretagogue when the intestinal PGE2 level is increased. It has been reported that COX (PG synthesizing enzyme) activity level increases in the inflammatory condition (40, 43).
What is the functional role of the synergistic action between SP and
PGE2 observed in the present study? SP is well known as a
neurotransmitter in ENS (11, 15). The neural pathways involved in secretory reflexes have not been clearly defined. Classical
reflexes contribute to the control of ion transport. In addition to the
classical reflexes, axonal reflexes must also be considered potential
regulatory mechanisms of ion transport. SP is a key transmitter both in
axonal and classical reflexes. In the present experiment, SP still
evoked Cl secretion by direct action on the epithelium,
although the response was smaller than that induced by the classical
reflex (23). Therefore, in the physiological state,
classical reflexes involving SP are probably important for flushing
secretory IgA into the lumen continuously and for maintenance of mucous
fluidity necessary to lubricate the luminal contents during their
propulsion along the gastrointestinal tract. On the other hand, in the
pathophysiological state, axonal reflexes where SP is directly released
to the epithelium may be important for flushing out deleterious
antigens or microorganisms. Inflammation is characterized by the
presence of increased numbers of immune cells, including mast cells and
macrophages, etc. Antigen challenge of sensitized tissue causes
Cl
secretion that is mediated, in part, by PGs (1,
39). The release of PGE2 during anaphylaxis has also
been reported (8). In the guinea pig distal colon,
PGE2 and PGD2 evoke Cl
secretion
by both acting on the epithelial cells directly and through mediation
by neurons (12, 13). Much experimental data suggest that
SP also acts as a mediator in the regulation of intestinal inflammation, as mentioned above. Therefore, in the inflammatory condition, excessive secretion caused by the synergistic action between
SP and PGE2 may participate to protect mucosal lining by
flushing the crypts of potentially deleterious antigens or microorganisms. In the present study, we have shown one example of the
neuroimmune interaction on Cl
secretion by SP and
PGE2. Although details of the mechanism of Cl
secretion in interaction between neruotransmitters and immunomediators are not yet clear, this interaction may have an important role for host
defense mechanisms.
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ACKNOWLEDGEMENTS |
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This work was partly supported by a Monbusho International Research Grant 11694302 and by a Salt Foundation grant (to A. Kuwahara).
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FOOTNOTES |
---|
* Y. Hosoda and S.-I. Karaki, contributed equally to this work.
Address for reprint requests and other correspondence: A. Kuwahara, Laboratory of Physiology, Institute for Environmental Sciences, University of Shizuoka, 52-1 Yada, Shizuoka, 422-8526, Japan (E-mail: kuwahara{at}sea.u-shizuoka-ken.ac.jp).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajpgi.00504.2001
Received 27 November 2001; accepted in final form 21 March 2002.
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