1 Laboratory of Physiology, School of Food and Nutritional Sciences, University of Shizuoka, Shizuoka 422-8526, Japan; and 2 Faculty of Nursing and Nutrition, Siebold University of Nagasaki, Nagasaki 851-2195, Japan
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ABSTRACT |
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Submucosal cholinergic and
noncholinergic neurons in intestines have been shown to be involved in
regulating epithelial transport functions, particularly stimulating
Cl secretion. This study investigates the role of
submucosal cholinergic neurons in regulating electrogenic
Na+ absorption in distal colon. Amiloride-sensitive
short-circuit current (Isc) and
22Na+ flux were measured in mucosal and
mucosal-submucosal preparations mounted in Ussing chambers. In the
mucosal preparation, carbachol (CCh) added to the serosal side
inhibited amiloride-sensitive Isc and
amiloride-sensitive 22Na+ absorption. The
inhibitory effect of CCh was observed at ~0.1 µM, and maximum
inhibition of ~70% was attained at ~30 µM (IC50 = ~1 µM). CCh-induced inhibition of amiloride-sensitive
Isc was almost totally abolished by 10 µM
atropine. Treatment of the tissue with ionomycin markedly reduced
amiloride-sensitive Isc, but a subsequent
addition of CCh further decreased it. Also, CCh still had an inhibitory
effect, although significantly attenuated, after the tissue had been
incubated with a low-Ca2+ solution containing ionomycin and
BAPTA-AM. Applying electrical field stimulation to submucosal neurons
in the mucosal-submucosal preparation resulted in inhibition of
amiloride-sensitive Isc, ~33% of this
inhibition being atropine sensitive. Physostigmine inhibited
amiloride-sensitive Isc, this effect being
abolished by atropine. In conclusion, submucosal cholinergic and
noncholinergic neurons were involved in inhibiting electrogenic
Na+ absorption in colon. This inhibition by cholinergic
neurons was mediated by muscarinic receptor activation.
enteric nerve; intracellular Ca2+; acetylcholine; intestinal secretion; epithelial Na+ channel
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INTRODUCTION |
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THE COLON, THE TERMINAL PART of the gastrointestinal tract, performs functions of both absorption and secretion of a variety of electrolytes by epithelial transport systems. Regulation of this absorption and secretion of electrolytes by neurocrine, paracrine, and endocrine systems probably plays an important role in maintaining the fluid and electrolyte homeostasis in the whole body as well as being involved in mucosal defensive functions (1, 25).
The colon, like other segments of the gastrointestinal tract, has an
intrinsic nervous system, the enteric nervous system, consisting of two
ganglionated plexi, namely the myenteric plexus and the submucosal
plexus (also called the submucous plexus). Myenteric neurons mainly
regulate contractile activity, whereas submucosal neurons are mainly
involved with epithelial transport functions (5, 12). It
has been shown that the stimulation of submucosal neurons by
electrical, mechanical, or pharmacological means led to enhanced
Cl secretion (5, 12). In addition, the
inhibition of electroneutral NaCl absorption (3, 23) and
stimulation of K+ secretion (6, 15, 16, 28)
are also likely to be induced by submucosal neurons. Cholinergic
neurons are predominantly responsible for these epithelial prosecretory
responses, although neurons containing vasoactive intestinal peptide
are probably also involved (5, 12-14, 22-24, 32, 33,
38-40, 51, 55).
Electrogenic Na+ absorption, which involves an apical
amiloride-sensitive Na+ channel and basolateral
Na+-K+-ATPase/K+ channel, is one of
the major pathways for Na+ absorption in the colon
(1, 25). This transport pathway is known to be stimulated
by aldosterone and thyroid hormones through genomic activation
(1, 25, 31). In addition, our recent studies have shown
that the -adrenergic agonist activates and vasopressin and ATP
inhibit electrogenic Na+ absorption (44, 49,
57). However, information concerning the regulation by enteric
neurons is very limited for electrogenic amiloride-sensitive
Na+ absorption in the colon, in contrast to that for
colonic Cl
secretion: chemical stimulation of intrinsic
cholinergic neurons and the application of the cholinergic agonist
carbachol (CCh) have been reported to inhibit electrogenic
Na+ absorption in the turtle colon (50, 53),
but there is no such report for mammalian colon. The purpose of this
study is, therefore, to elucidate the regulation of electrogenic
Na+ absorption by enteric submucosal neurons, particularly
the cholinergic type, in an isolated guinea pig distal colon mounted in
an Ussing chamber. We examined the effect of CCh on the
amiloride-sensitive short-circuit current (Isc)
and 22Na+ flux. We also investigated the effect
of stimulating submucosal cholinergic neurons on electrogenic
Na+ absorption by using electrical field stimulation (EFS).
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MATERIALS AND METHODS |
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Tissue preparation. Hartley-strain, male guinea pigs weighing 250-550 g were used in the experiments. All animals were injected twice with 375 µg/kg body wt of aldosterone (1.39 mM in saline) to enhance the electrogenic sodium absorption, once by a subcutaneous injection in the evening before the experiment and then by an intramuscular injection 4 h before the start of the experiment. The animals were stunned by a blow to the head and bled to death. The distal colon was excised and then opened along the root of the mesentery. A mucosal preparation consisting of the mucosal layer and a part of the muscularis mucosal layer was obtained with glass microscopic slides (55). The mucosal-submucosal preparation, consisting of the mucosal, muscularis mucosal, and submucosal layers was obtained with fine forceps. All procedures involving the animals were approved by the Institutional Animal Care Board at the University of Shizuoka.
Solutions. The standard bathing solution contained (in mM) 119 NaCl, 21 NaHCO3, 2.4 K2HPO4, 0.6 KH2PO4, 1.2 MgCl2, 1.2 CaCl2, and 10 glucose. A low-Ca2+, high-Mg2+ solution was prepared by omitting CaCl2 and adding 0.2 mM EGTA and 10 mM MgCl2. Each solution was gassed with 95% O2 and 5% CO2 (pH 7.3-7.4).
Isc measurements. The Isc and transmural tissue resistance (Gt) were measured in vitro in Ussing chambers as previously described (44). The mucosal or the mucosal-submucosal sheet was mounted vertically between acrylic resin chambers with an internal surface area of 0.5 cm2. The temperature of the 10-ml bathing solution in each chamber was maintained at 37°C by a water-jacketed reservoir. The tissue was continuously short-circuited, with compensation for the fluid resistance between the two potential-sensing bridges, by using a voltage-clamping amplifier (CEZ9100; Nihon Kohden, Tokyo, Japan). The transepithelial potential was measured through 1 M KCl-agar bridges connected to a pair of calomel half-cells, the transepithelial current being applied across the tissue through a pair of Ag-AgCl electrodes kept in contact with the mucosal and serosal bathing solutions through a pair of 1 M NaCl-agar bridges. The Isc value is expressed as positive when the current flowed from the mucosa to serosa. Gt was measured by recording the current resulting from short-duration, square, bipolar voltage pulses (±5 mV) imposed across the tissue and then was calculated according to Ohm's law.
EFS. Intramural neurons were electrically stimulated by passing a current parallel to the plane of the tissue via a pair of aluminum foil electrodes placed on the serosal surface of the tissue as reported by Cooke (5, 22). Bipolar rectangular 5-mA current pulses of 1-ms duration (0.5-ms duration in each direction) were delivered at 10 Hz.
22Na+ flux measurements. The unidirectional transmural flux of 22Na+ was measured under short-circuit conditions. The mucosal-to-serosal (Jms) and serosal-to-mucosal (Jsm) flux values were measured in adjacent tissues that had Gt values differing by <30%. Thirty minutes were allowed for the isotopic steady state to be reached after either the serosal or mucosal side of the bathing solution was labeled with 22Na+. Ten samples (0.5 ml each) were taken from the unlabeled side at 10-min intervals and replaced with an equal volume of the unlabeled solution. The medium samples were assayed for 22Na+ by the liquid scintillation procedure.
Materials. Indomethacin, amiloride, benzamil, bumetanide, CCh, physostigmine, BAPTA-AM, 8-bromo-cAMP (8Br-cAMP), and TTX were purchased from Sigma (St. Louis, MO), and ionomycin was purchased from Calbiochem (San Diego, CA). All other chemicals were purchased from Wako Pure Chemicals (Osaka, Japan). Each drug was applied from a concentrated stock solution that had been dissolved in water or DMSO (bumetanide, ionomycin, and BAPTA-AM), the final volume of DMSO in an experimental solution always being <0.3%. 22Na+ was purchased from Dupont NEN (Boston, MA).
Statistical analyses. Each data value is presented as the mean ± SE of n guinea pigs. Statistical comparisons were performed by using the Student's t-test (paired or unpaired, as appropriate) or ANOVA for repeated measures (Dunnett post hoc test). Significance was accepted at P < 0.05.
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RESULTS |
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Effect of CCh on electrogenic Na+
absorption.
The experiments were performed in the mucosal preparation of the distal
colon obtained from the aldosterone-treated animals. An initial
indication that cholinergic agonists may inhibit electrogenic, amiloride-sensitive Na+ absorption in the colon was
observed in the first series of experiments (Fig.
1). Three measurements, each preceded by
a washing procedure, were consecutively performed on the same
preparation (Fig. 1A). The basal Isc
value after 20-30 min of equilibration gradually decreased from
the first to the third measurements (223 ± 46, 190 ± 53, and 118 ± 26 µA/cm2, respectively). The
Gt similarly decreased (18.9 ± 3.3, 14.1 ± 2.6, and 12.7 ± 2.6 mS/cm2,
respectively; n = 5). In the first measurements,
amiloride-sensitive Isc and
Gt under control conditions were estimated by
adding amiloride to the mucosal side (0.1 mM). In the second
measurements, the effect of the cholinergic agonist CCh on the
amiloride-sensitive Isc and
Gt values was examined. The addition CCh (1 mM)
to the serosal side induced an initial transient
Isc increase, which then decreased to below the
basal level (Fig. 1, A and B).
Gt also transiently increased and then decreased
to slightly above the basal level. Subsequent addition of amiloride
reduced Isc and Gt,
although the magnitude of these changes was smaller than that observed
in the first measurements. The possibility that these decreases in the
amiloride-induced Isc and
Gt values in the presence of CCh was mainly due
to a time-dependent decrease, rather than to the presence of CCh, can
be excluded, because, in the third measurements, the
amiloride-sensitive Isc and
Gt values under control conditions were both
larger than those observed in the second measurements, although they
were smaller than those observed in the first measurements. Thus, as
summarized in Fig. 1C, the amiloride-sensitive
Isc and Gt values were
significantly attenuated by CCh, indicating that CCh inhibited the
amiloride-sensitive, electrogenic Na+ absorption. It is
likely that the initial Isc and
Gt increases induced by CCh were due to the
stimulation of the electrogenic Cl secretion (6,
10, 24, 28, 34, 48, 56, 59).
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Role of Ca2+ in the CCh-induced
inhibition of electrogenic Na+
absorption.
We next examined whether the CCh-induced inhibition of amiloride
(benzamil)-sensitive Isc would be mediated by an
increase in intracellular Ca2+ concentration
([Ca2+]i) by using the mucosal preparation.
To this end, the effect of CCh was assessed under two experimental
conditions contrasting with each other, one with
[Ca2+]i having been presumably depleted and
the other with [Ca2+]i having been presumably
highly elevated. In the first series of experiments, tissues were
bathed with the low-Ca2+ (nominally Ca2+
free + 0.2 mM EGTA), high-Mg2+ (10 mM) solution. This
bathing solution also contained ionomycin (1 µM, mucosal and serosal
sides) to deplete the intracellular Ca2+ store
(35) and BAPTA-AM (50 µM, mucosal and serosal sides) to
buffer [Ca2+]i. Indomethacin, TTX, and
bumetanide were also included in this Ca2+-depletion
solution. Under these Ca2+-depleted conditions,
Gt gradually increased to 30-90
mS/cm2 30 min after the start of incubation, as shown in
Fig. 4A. The increase in
Gt is likely to have been due to the low
Ca2+-induced increase in tight-junction permeability, and
the increasing Mg2+ concentration in the solution
apparently failed to prevent this change. Therefore, the following
experimental values were obtained between 15 and 25 min after changing
to the Ca2+-depletion solution before the
Gt value became extremely elevated. The basal
Isc value was decreased from 230 ± 77 µA/cm2 under the control condition to 113 ± 26 µA/cm2 under the Ca2+-depleted condition
(Fig. 4, n = 5). The addition of CCh under the
Ca2+-depleted condition decreased
Isc, and subsequent addition of benzamil almost
totally abolished it (Fig. 4A). The percentage inhibition of
benzamil-sensitive Isc by CCh was significantly attenuated, but not abolished, under the Ca2+-depleted
condition compared with that under the control condition (Fig.
4B). We also examined the effect of Ca2+
depletion on the CCh-induced Cl and K+
secretions (Fig. 5). The measurements
were done in the absence of bumetanide and in the presence of benzamil.
Under these conditions, a submaximal concentration of CCh (10 µM)
caused a biphasic Isc response (Fig.
5A). The initial Isc increase, which
was probably due to the stimulation of electrogenic Cl
secretion, was followed by an Isc decrease to
below the basal level, probably due to the stimulation of electrogenic
K+ secretion. Both the increase and decrease in
Isc induced by CCh were almost totally abolished
under the Ca2+-depleted condition (Fig. 5, B and
C). Thus, in contrast to the CCh-induced inhibition of
amiloride-sensitive Isc, the CCh-induced stimulation of Cl
and K+ secretions, which
are putatively mediated by a Ca2+-signaling pathway, was,
in fact, strongly suppressed under the Ca2+-depleted
condition. We also examined the effect of the same
Ca2+-depleted condition on the 8Br-cAMP-induced stimulation
of electrogenic K+ secretion (Fig. 5,
D-E). The 8Br-cAMP-induced
Isc decrease, which was probably due to
K+ secretion, was significantly attenuated (by 51 ± 10%, n = 4) but still substantially preserved under
the Ca2+-depleted condition, indicating that general tissue
damage had not occurred under this Ca2+-depleted condition.
On the other hand, the large stimulation of Cl
secretion
induced by CCh added to the 8Br-cAMP-treated tissue (28,
56) was almost totally abolished under the
Ca2+-depleted condition, consistent with the above finding
(Fig. 5, D-E).
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Role of intramural cholinergic neurons.
We next investigated the role of submucosal cholinergic neurons in
regulating electrogenic Na+ absorption. The
mucosal-submucosal preparation, which contained the submucosal plexus
neurons, was used. As shown in Fig.
7A, the activation of the
intramural neurons by an EFS protocol evoked an initial
Isc peak that was followed by an
Isc decrease to below the basal level. When EFS
was terminated, the Isc value further decreased
slightly and then gradually increased toward the basal level. The
Gt value (Fig. 7A) increased
transiently but returned to the basal level during EFS, and, after
terminating EFS, it fell to below the basal level and then gradually
increased. To estimate the changes in Isc and
Gt derived from electrogenic Na+
absorption, EFS was also applied in the presence of amiloride (0.1 mM,
mucosal; Fig. 7B). The initial Isc
peak induced by EFS in the presence of amiloride had a similar value to
that in its absence but was followed by a sustained
Isc increase instead of the decrease. The
Gt value increased during EFS. Both the
increased Isc and Gt
levels rapidly returned to the basal level after terminating EFS. It is
clear from the difference in electrical response in the presence and
absence of amiloride that the amiloride-sensitive Isc and Gt values were
attenuated by EFS (Table 2). In
particular, the lowest levels of Isc and
Gt observed soon after terminating EFS in the
absence of amiloride would provide a good estimate for the inhibition
of amiloride-sensitive Isc and
Gt, since, at that point, the effect of EFS on
the amiloride-insensitive components of electrical parameters had
almost disappeared (Fig. 7B). The electrical response
induced by EFS was likely to have been mediated by the submucosal
neurons, because it was almost totally abolished in the presence of TTX
(300 nM, serosal side; data not shown). We next examined the
involvement of cholinergic neurons in the EFS-induced inhibition of
amiloride-sensitive Isc by applying EFS in the
presence of atropine (10 µM, serosal; Fig. 7). The Isc decrease during EFS and the lowest level of
Isc soon after terminating EFS were both found
to be significantly attenuated in the presence of atropine, in addition
to the significant inhibition of the initial Isc
peak (Table 2). In the amiloride-treated tissue, the secondary,
sustained Isc increase and the
Gt increase were suppressed by atropine,
although the effect was not significant. Thus atropine presumably
attenuated the EFS-induced inhibition of amiloride-sensitive
Isc by approximately one-third (Table 2), suggesting that submucosal cholinergic neurons were involved via muscarinic receptor activation in the inhibition of electrogenic Na+ absorption. The EFS-induced increase in
Isc that was observed in the presence of
amiloride might have been due to electrogenic anion secretion that had
not been completely suppressed by serosal bumetanide.
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Effect of cholinesterase inhibitor.
To further investigate the role of intramural ACh, we examined the
effect of applying the cholinesterase inhibitor physostigmine (Fig.
8). Physostigmine added to the serosal
side of the mucosal-submucosal preparation (10 µM) induced a slow
decrease in Isc, which reached its lowest level
~15 min after the physostigmine treatment, with slight recovery
thereafter (maximum Isc change, 123 ± 40 µA · cm2 · h
1;
n = 4). The Gt value was hardly
affected by the physostigmine treatment (Gt
change,
1.0 ± 0.7 mS/cm2). When atropine (10 µM,
serosal) was administered after 20-25 min of the physostigmine
treatment, the Isc value rapidly returned to its
basal level (Fig. 8A). In addition, the atropine
pretreatment completely abolished the physostigmine-induced
Isc response (data not shown, n = 4). The Isc response to physostigmine was
almost totally abolished when the tissue had been pretreated with
amiloride (0.1 mM, mucosal; Fig. 8B, n = 4).
Therefore, after its degradation had been inhibited, the tissue ACh
concentration could increase enough to inhibit, via muscarinic receptor
activation, the electrogenic Na+ absorption. The
Isc decrease induced by physostigmine was not affected when the tissue had been pretreated with tetrodotoxin (300 nM,
serosal; maximum Isc decrease: control,
79 ± 26; +tetrodotoxin,
89 ± 40 µA · cm2 · h
1;
n = 3). Therefore, ACh released by ongoing submucosal
cholinergic nerve activity is unlikely to have been mainly responsible
for the physostigmine-induced Isc response.
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DISCUSSION |
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It is well established that submucosal neurons are mainly involved
in the activation of Cl secretion in the colon. The
results of this study show for the first time that submucosal neurons
also inhibited electrogenic, amiloride-sensitive Na+
absorption and that the cholinergic pathway is one of the major components of this mechanism. The amiloride-sensitive
Isc value was decreased by the electrical
stimulation of submucosal neurons, the response being partially blocked
by atropine. In addition, externally applied CCh caused a reduction of
both amiloride-sensitive Isc and
22Na+ absorption by a similar magnitude.
Inhibition of electrogenic Na+ absorption during the
stimulation of intrinsic cholinergic neurons has previously been
reported in the turtle colon (50) but not in the mammalian colon.
The inhibitory effect of CCh on the amiloride-sensitive
Isc value was mainly mediated by the muscarinic
receptor, because the CCh-induced inhibition of amiloride-sensitive
Isc was almost totally suppressed by atropine.
The CCh-induced inhibition of Isc was preserved
in the presence of TTX, suggesting that CCh may have activated the
muscarinic receptor on the epithelial cells. The muscarinic receptor
that was coupled to the inhibition of electrogenic Na+
absorption in the colon might exist on the surface epithelial cells,
since electrogenic Na+ absorption has been suggested to
occur mainly in surface epithelial cells (11, 20, 27).
Muscarinic receptors have been shown to be present in the colonic
mucosa or epithelial cells from the specific binding of the muscarinic
antagonist [3H]quinuclidinyl benzilate (41, 42,
58). In addition, it has been demonstrated that functional
cholinergic receptors are present in colonic crypt cells and colonic
carcinoma cell lines (2, 8, 9, 16, 21, 26). However, it
remains to be determined whether or not surface epithelial cells in the
colon actually express a muscarinic receptor(s). The inhibition of
electrogenic Na+ absorption by CCh has previously been
observed only in the turtle colon (50). In addition to the
inhibition of electrogenic Na+ absorption, muscarinic
agonists have previously been reported to affect a variety of transport
pathways in the colon, including the stimulation of electrogenic
Cl and K+ secretions and the inhibition of
electroneutral NaCl absorption (6, 15, 16, 24, 28, 34, 48, 56,
59).
The inhibition of amiloride-sensitive electrogenic Na+ absorption by cholinergic receptor agonists has been observed not only in the turtle colon (50, 53) but also in the toad bladder (43, 52) and in frog skin (7). Electrogenic Na+ absorption involves the apical amiloride-sensitive Na channel and basolateral Na+-K+-ATPase/K+ channel (1, 25). The decrease in Gt value in association with the inhibition of amiloride-sensitive Isc induced by CCh that has been demonstrated in this study (Fig. 2) is at least consistent with the involvement of the inhibition of apical Na+ channel activity. The inhibition of both the apical Na+ channel and basolateral K+ channel have been suggested to be involved in the CCh-induced inhibition of Na+ absorption in the turtle colon (50, 53). Clearly, regulation of the transport processes responsible for the CCh-induced inhibition of electrogenic Na+ transport in the guinea pig distal colon remain to be determined.
We investigated in this study the role of the
Ca2+-signaling pathway in the CCh-induced inhibition of
electrogenic Na+ absorption. It was found that the
CCh-induced inhibition of Na+ absorption was not markedly
attenuated when the tissue was incubated with a low-Ca2+,
high-Mg2+ solution containing ionomycin and BAPTA-AM, a
condition whereby an agonist-induced increase in
[Ca2+]i was presumably largely attenuated
(35). This condition, however, almost completely
suppressed the CCh-induced stimulation of Cl and
K+ secretion, consistent with the notion that these
responses were mediated by an increase in
[Ca2+]i. In the additional experiments, we
have shown that ionomycin markedly decreased the amiloride-sensitive
Isc value that was probably mediated by the
[Ca2+]i increase and that, in the presence of
ionomycin, CCh still induced the inhibition of residual
amiloride-sensitive Isc. These findings suggest
that a [Ca2+]i -independent signaling pathway
leading to the inhibition of electrogenic Na+ absorption
was activated by CCh at least under certain conditions. The
Ca2+-independent signaling pathway as well as the
Ca2+-dependent one is known for muscarinic receptors. Five
different G protein-linked muscarinic receptor subtypes, M1-M5,
have been identified (4). Activation of the M1, M3, and M5
receptors is known to be coupled to the inositol
phospholipid/Ca2+-signaling pathway, whereas that of M2 and
M4 is coupled to the inhibition of cAMP production. A direct action of
G protein or the activation of adenylyl cyclase has also been reported
to mediate the response to M2, M4, and M5 activation in certain
tissues. Clearly, it remains to be determined whether the
Ca2+-signaling pathway is responsible and whether this
putative Ca2+-independent pathway plays a role in the
inhibition of electrogenic Na+ absorption induced by CCh
under normal conditions.
The inhibition of amiloride-sensitive Isc
induced by EFS was partly, but not totally, suppressed by atropine,
indicating that both cholinergic and noncholinergic submucosal neurons
were involved in inhibiting electrogenic Na+ absorption. It
has previously been shown that stimulation of the submucosal neurons
activated anion secretion by activating both the cholinergic and
noncholinergic pathways in the colon (12, 18, 22, 23, 32, 33,
51). Approximately half of the submucosal plexus neurons in the
colon have been reported to contain ACh from choline acetyltransferase
immunoreactivity and other morphological studies (13, 14, 30, 32,
33, 38, 39). Further evidence for the presence of cholinergic submucosal neurons has been the [3H]ACh release induced
by nerve stimulation (19, 29, 54, 58). However, a
morphological investigation of choline acetyltransferase immunoreactivity has failed to clearly demonstrate cholinergic nerve
fibers in the lamina propria of colonic mucosa (38, 39), although the concentration of choline acetyltransferase in the mucosal
nerve fiber may not be high enough to be detected. Vasoactive intestinal peptide and substance P have been suggested to be released and to be responsible for the noncholinergic component of
Cl secretion (17, 32, 40, 51). Whether these
substances can mediate the noncholinergic component of the inhibition
of electrogenic Na+ absorption remains to be determined.
The cholinesterase inhibitor physostigmine inhibited the
amiloride-sensitive Isc, the effect being
abolished by atropine. Cholinesterase is known to be present in the
intestinal mucosa (45, 46). This finding suggests that
tissue ACh can reach a sufficiently high concentration to activate the
epithelial muscarinic receptor, leading to the regulation of
electrogenic Na+ absorption at least under certain
conditions. The cell types from which tissue ACh was released are not
clear at present. ACh release from the nerve terminal of ongoing
submucosal cholinergic neurons is unlikely, because TTX failed to
inhibit the physostigmine-induced response. The possibility cannot be
excluded, however, that a small amount of ACh was constitutively
released without action potential from the nerve terminals.
Alternatively, epithelial and nonepithelial cells exhibiting
immunoreactivity to choline acetyltransferase might have been the
source of tissue ACh (30, 38, 39). A role of tissue ACh
that was independent of the cholinergic nerve activity has been
previously suggested from the finding that TTX inhibited the luminal
propionate-induced Cl secretion in the colon by 40%,
whereas atropine inhibited it by 90% (55).
In summary, the submucosal neurons not only activated Cl
secretion but also inhibited electrogenic Na+ absorption,
thereby leading to a prosecretory state in the colon. In support of
this, the enteric neuron has previously been suggested to inhibit NaCl
absorption and to stimulate mucus secretion (36, 37, 47).
In addition, K+ secretion may be enhanced by cholinergic
neurons, as suggested from the results of this and previous works.
Collectively, the submucosal neurons may function to lubricate the
luminal surface so as to facilitate the movement of fecal pellets and,
when excessively activated, to protect the mucosa and to flush the
colonic lumen of noxious agents. Cholinergic neurons may play an
important role in these activities.
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ACKNOWLEDGEMENTS |
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We thank T. Innes for helping us to edit the English text.
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FOOTNOTES |
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This work was supported in part a Grant-in-Aid for Scientific Research on Priority Areas of Channel-Transporter Correlation from the Ministry of Education, Science, Sports, and Culture of Japan and by the Salt Science Research Foundation (9435 and 9537).
Address for reprint requests and other correspondence: Y. Suzuki, Laboratory of Physiology, School of Food and Nutritional Sciences, Univ. of Shizuoka, Shizuoka 422-8526, Japan (E-mail: yuichi{at}smail.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.
First published November 20, 2002;10.1152/ajpgi.00201.2002
Received 28 May 2002; accepted in final form 31 October 2002.
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