5-HT activates nitric oxide-generating neurons to stimulate
chloride secretion in guinea pig distal colon
Atsukazu
Kuwahara1,
Hirofumi
Kuramoto2, and
Makoto
Kadowaki3
1 Laboratory of Environmental
Physiology, Institute for Environmental Sciences, University of
Shizuoka, Shizuoka 422-8526;
2 Department of Applied Biology,
Kyoto Institute of Technology, Matsugasaki, Sakyou-ku, Kyoto 606; and
3 Gastrointestinal Research Group,
Pharmacological Research Laboratories, Fujisawa Pharmaceutical Company,
Osaka 532, Japan
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ABSTRACT |
The participation of nitric oxide (NO) in
serotonin (5-hydroxytryptamine; 5-HT)-evoked chloride secretion in
guinea pig distal colon was examined. Submucosal/mucosal segments were
mounted in Ussing flux chambers, and an increase in short-circuit
current (Isc)
was used as an index of secretion. Addition of 5-HT to the serosal side
produced a concentration-dependent
(10
7-10
5
M) increase in
Isc caused by
chloride secretion.
NG-nitro-L-arginine
(L-NNA) significantly
reduced the 5-HT-evoked early (P-1) and late (P-2) responses to 61.1 and 70.6% of control, respectively. Neurally evoked response was also
inhibited by L-NNA. The NO donor
sodium nitroprusside (SNP,
10
4 M) increased basal
Isc mainly
because of chloride secretion. The SNP-evoked response was
significantly reduced by tetrodotoxin but was unchanged by atropine or
indomethacin. These results suggest that the 5-HT-evoked increase in
Isc is associated
with an NO-generating mechanism. Atropine significantly reduced the
5-HT (10
5 M)-evoked P-1 and
P-2 responses to 71.8 and 19.7% of control, respectively. Simultaneous
application of atropine and
L-NNA further decreased the
5-HT-evoked responses more than either drug alone; application of
L-NNA and atropine decreased the
5-HT-evoked P-1 and P-2 responses to 68.5 and 39.2% of
atropine-treated tissues, respectively. These results suggest that
noncholinergic components of P-1 and P-2 responses are 71.8 and 19.7%
of control, respectively, and that NO components of P-1 and P-2
responses are 32 and 61%, respectively, of the noncholinergic
component of the 5-HT-evoked responses. The results provide evidence
that NO may participate as a noncholinergic mediator of 5-HT-evoked
chloride secretion in guinea pig distal colon.
serotonin; ion transport; gastrointestinal; short-circuit
current
 |
INTRODUCTION |
SEROTONIN (5-hydroxytryptamine; 5-HT) is
contained within subsets of myenteric neurons as well as in endocrine
cells in the gastrointestinal tract (11, 13). The presence of 5-HT in
the neurons suggests that 5-HT functions as a neurotransmitter of the
enteric nervous system (ENS). Serotonergic myenteric neurons are
interneurons in the ENS, and they possess a single long axon that
projects aborally to neurons located in ganglia of either the submucous
or the myenteric plexus (13, 29). Several studies showed that exogenous
5-HT promotes the mucosal secretion of water and electrolytes in the
intestine of rats, guinea pigs, pigs, and humans (4, 5, 14, 15, 18,
24). In the guinea pig distal colon, 5-HT causes a biphasic increase in
short-circuit current
(Isc),
primarily due to chloride secretion (5, 18). The secretory response to
5-HT is partially inhibited by atropine and abolished by tetrodotoxin
(TTX) (5, 18), suggesting that 5-HT-evoked chloride secretion consists
of both cholinergic and noncholinergic components. 5-HT-evoked chloride
secretion is mediated in part by
5-HT3 and
5-HT4 receptors located on neurons
in guinea pig distal colon (19).
Recent immunohistochemical studies in the gastrointestinal tract
indicated various roles of nitric oxide (NO) in the ENS of mammalian
species, including monkeys and humans (1, 3, 6-9, 23). NO is a
neurotransmitter involved in the control of intestinal motility, and
recently it has been suggested that 5-HT-induced relaxation of
intestinal smooth muscle involves NO (2, 25). Like 5-HT, NO appears to
participate in mucosal function as well as in gastrointestinal
motility; for example, NO induces electrolyte secretion in the rat
intestine in vitro (21, 28, 30). Furthermore, the NO synthesis
inhibitor NG-nitro-L-arginine methyl
ester (L-NAME) prevents castor
oil-induced diarrhea (22). In the guinea pig colon, an
immunohistochemical study has shown that NO synthase (NOS)
immunoreactivity is found in myenteric neurons as well as in the
submucosa (23). A reasonable hypothesis based on these findings is that
5-HT-evoked chloride secretion might be linked to an NO pathway similar
to the serotonergic/nitrergic mechanisms involved in regulation of
intestinal motility. Support for such a hypothesis is the reported
evidence that 5-HT-induced diarrhea is inhibited by the NOS inhibitor
L-NAME in fasted mice (17).
The present study was therefore undertaken to test the idea that there
is a neuronal nitrergic component to 5-HT-evoked ion transport in the
distal colon of guinea pigs.
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MATERIALS AND METHODS |
Male albino guinea pigs (300-550 g; Hartley-Hazleton, Nippon SLC,
Hamamatsu, Japan) were allowed food and water ad libitum. The animals
were killed by a blow to the head and exsanguinated. Segments of distal
colon 5-10 cm proximal to the anus were removed, flushed with
Krebs-Ringer solution, and cut open along the mesenteric border. The
tissues were pinned flat with the mucosal side down in a Sylgard-lined
petri dish. The entire muscularis externa, including the myenteric
plexus, was removed by blunt dissection. Four of these stripped
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 experimental methods for studying mucosal transport were similar to
those previously described (20). Mucosal and serosal surfaces of
tissues were incubated with 10 ml of buffer solution by recirculation
from a reservoir maintained at 37°C for the duration of the
experiment. Mucosal and serosal Krebs-Ringer solutions were identical
and contained (in mM) 120 NaCl, 6 KCl, 1.2 MgCl2, 1.2 NaH2PO4,
14.4 NaHCO3, 2.5 CaCl2, and 11.5 glucose. The
solutions were gassed with 95%
O2-5%
CO2 and buffered to pH 7.2. The
chambers were equipped with a pair of Ringer-agar bridges and calomel
half-cells for the measurement of transmural electrical potential
differences (PD). A pair of Ag-AgCl electrodes was connected to a
voltage-clamp apparatus (SS-1335, Nihon-Kohden, Tokyo, Japan) that
automatically compensated for the solution resistance between
PD-sensing bridges. Tissue conductance was measured by calculating the
ratio of Isc to
open-circuit values of PD or by determining the current necessary to
change the transmural PD by 10 mV.
Submucosal neurons were electrically stimulated by passing current from
an electronic stimulator (Nihon-Kohden, SEN-7203) through a pair of
aluminum foil electrodes placed on the submucosal surface of the
tissue. Electrical stimuli consisting of bipolar, rectangular pulses
(0.5 ms, 10 V, 5 Hz) were applied for 30 s. The electrical stimulus was
followed by the addition of atropine (10
5 M) to inhibit the
cholinergic components of the response.
NG-nitro-L-arginine
(L-NNA;
10
4 and 3 × 10
4 M) and its vehicle were
added to the serosal bath, and the neurons were electrically stimulated
again. The tissue responses were continuously recorded on a chart
recorder (Recti-Horitz-8K, Nihon-Denki Sanei, Tokyo, Japan) or a
Macintosh computer (MacLab/8 system, Analog Digital Systems, Castle
Hill, Australia). Currents generated by the tissues both before and
after the electrical stimulation were compared for control (no drug)
and experimental tissues.
Tissues were paired on the basis of similar conductance. Noncumulative
concentration-response curves for 5-HT were established. These
concentration-response curves were constructed by adding 5-HT, at a
single concentration, to the serosal bathing solution in the absence or
presence of the NOS inhibitor
L-NNA.
L-NNA
(10
4 M) was added to the
serosal bathing solution 20-25 min before the addition of 5-HT.
The experiments using sodium nitroprusside (SNP) in the presence of
atropine, TTX, or indomethacin were carried out in the same way. In the
combination studies (atropine,
L-NNA, and atropine + L-NNA), the drugs were added
together to the serosal bathing solution 20-25 min before addition
of 5-HT (10
5 M).
All data are presented as means ± SE. Paired and unpaired
Student's t-test and one-way ANOVA
with pairwise comparisons by the Bonferroni method were used to
determine the statistical significance of differences between control
and experimental groups. Probability values <0.05 were considered
statistically significant.
Drugs.
The following chemicals were purchased from the suppliers indicated:
5-hydroxytryptamine creatinine sulfate (E. Merck, Darmstadt, Germany);
L-NNA, bumetanide, indomethacin,
and TTX (Sigma, St. Louis, MO);
L-arginine monohydrochloride
(L-Arg),
D-arginine monohydrochloride (D-Arg), and SNP (Nacalai
Tesque, Kyoto, Japan); and atropine sulfate (Wako, Osaka, Japan).
Bumetanide, indomethacin, and SNP were dissolved in dimethyl sulfoxide,
90% ethanol, and distilled H2O,
respectively. Other drugs were made up in Krebs-Ringer solution. The
volume of drug added to the bath solutions did not exceed 100 µl/10
ml. Control tests of vehicles demonstrated that neither dimethyl
sulfoxide nor ethanol (<0.5%) had an effect on baseline
Isc.
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RESULTS |
Effect of 5-HT on basal
Isc in presence of
L-NNA.
The effect of 5-HT on
Isc is shown in
Fig. 1. The control 5-HT-evoked response
consisted of two components, an early response and a late response,
designated P-1 and P-2, respectively (Fig. 1A). 5-HT added to the serosal
bath solution evoked a concentration-dependent increase in both P-1 and
P-2 components of the
Isc response
(Fig. 1, B and
C). At
10
5 M 5-HT, the maximum P-2
response averaged 364.9 ± 34.7 µA/cm2 (Fig.
1C, n = 7 animals) and was significantly greater than the maximum P-1
response (265.7 ± 37.8 µA/cm2,
P < 0.01, n = 7 animals, Fig.
1B).

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Fig. 1.
Effects of NG-nitro-L-arginine
(L-NNA) on serotonin
(5-hydroxytryptamine; 5-HT)-evoked changes ( ) in short-circuit
current (Isc)
in guinea pig distal colon. A:
representative traces to illustrate effects of
L-NNA and
L-arginine
(L-Arg) on 5-HT-evoked changes
in Isc. 5-HT
(10 5 M)-evoked change in
Isc (Control) is
made up of early (P-1) and late (P-2) phases (arrows) in guinea pig
distal colon. Vertical and horizontal bars are
Isc and time,
respectively. B and C: , control; ,
L-NNA; , L-NNA + L-Arg.
B: concentration-response curves for
5-HT-evoked P-1 responses in presence or absence of
L-NNA and/or
L-Arg.
C: concentration-response curves for
5-HT-evoked P-2 responses in presence or absence of
L-NNA and/or
L-Arg. Each tissue received a
single concentration of 5-HT. Noncumulative concentration-response
curves of 5-HT were established. Nitric oxide (NO) synthase (NOS)
inhibitor L-NNA
(10 4 M) reduced amplitude
of both P-1 and P-2 responses to 5-HT but did not alter basal
Isc. NOS
substrate L-Arg
(10 3 M) reversed inhibitory
action of L-NNA on 5-HT-evoked
responses but did not affect basal
Isc. Values are
means ± SE for 7 animals. * Significantly different
from control response to 5-HT (P < 0.05).
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To determine whether the epithelial response evoked by 5-HT was
mediated through the release of NO, the selective NOS inhibitor L-NNA was used. Addition of
L-NNA
(10
4 M) to the serosal
bathing solution significantly reduced the electrogenic secretory P-1
and P-2 responses to 5-HT
(10
5 M); the P-1 response
was reduced to 63.5% of control (Fig.
1B; 168.8 ± 8.0 µA/cm2,
n = 7 animals,
P < 0.05), and the P-2 response was
reduced to 68.4% of control (Fig. 1C;
249.5 ± 18.1 µA/cm2,
n = 7 animals,
P < 0.05). Furthermore, the addition
of L-Arg (10
3 M) to the serosal
bathing solution completely reversed the inhibitory action of
L-NNA
(10
4 M) on the 5-HT-evoked
responses (Fig. 1). On the other hand, D-Arg
(10
3 M) produced no change
in the inhibitory effect of
L-NNA;
L-NNA still reduced the maximum
5-HT-evoked increases in P-1 and P-2 Isc from 281.5 ± 35 to 199 ± 24 µA/cm2
(n = 9 animals,
P < 0.05) and from 340 ± 47 to
214.6 ± 29.2 µA/cm2
(n = 9 animals,
P < 0.05), respectively, in the
presence of D-Arg. Neither L-NNA nor
D-Arg altered the basal
Isc in any of the
preparations (data not shown).
To determine the ionic basis for the increase in
Isc induced by
5-HT, we treated the tissues with bumetanide (5 × 10
4 M). 5-HT
(10
5 M)-evoked increase in
Isc was greatly
reduced by the treatment with bumetanide; bumetanide reduced the
5-HT-evoked P-1 and P-2 responses from 296.5 ± 27.4 to 211.4 ± 22.9 µA/cm2
(n = 6 animals,
P < 0.05) and from 356.6 ± 44.3 to 29.2 ± 5.2 µA/cm2
(n = 6 animals,
P < 0.001), respectively. The
results confirmed that the 5-HT-evoked increase in
Isc is primarily
caused by chloride secretion, as had been previously reported (5, 15,
19).
Effects of NO donor SNP on basal
Isc in presence of
atropine, TTX, or indomethacin.
To test whether NO itself affects baseline
Isc, the NO donor
SNP was added to the serosal bathing solution. In preliminary experiments, we checked the effect of SNP on baseline
Isc, and at
concentrations <1 × 10
6 M, SNP did not have a
significant effect on
Isc. However, at higher concentrations (5 × 10
6-10
4
M), SNP evoked an increase in
Isc in a
concentration-dependent manner and SNP
(10
4M) reached maximum
response at 48.3 ± 15 µA/cm2
from baseline (n = 12 from 8 animals).
Thus we used 10
4 M SNP for
the subsequent experiments.
Addition of SNP (10
4 M) to
the serosal bathing solution produced a gradual transient increase in
baseline Isc
beginning within 1 min of the addition. SNP evoked an increase in
Isc of 24.7 ± 5.0 µA/cm2 from the
baseline
Isc (Fig.
2; n = 7 animals). Like 5-HT, chloride ion appears to be the major charge
carrier in this SNP-evoked current, because bumetanide (5 × 10
4 M) significantly reduced
the change in Isc
from 42.9 ± 6.5 to 17.5 ± 5.0 µA/cm2 in another experiment
(n = 8 animals,
P < 0.05).

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Fig. 2.
Effects of atropine, tetrodotoxin (TTX), or indomethacin on sodium
nitroprusside (SNP)-evoked increases in
Isc in guinea pig
distal colon. Tissues were treated with TTX (2 × 10 7 M), atropine
(10 6 M), or indomethacin
(10 4 M) 15 min before
addition of SNP (10 4 M).
TTX significantly reduced SNP-evoked increase in
Isc. Although
amplitudes of peak currents were reduced by both atropine and
indomethacin, neither effect was statistically significant. Values are
means ± SE for 6-7 animals. * Significantly different
from control response to SNP (P < 0.05).
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To determine whether the responses to SNP were caused by direct or
indirect actions on the epithelium, TTX, atropine, or the cyclooxygenase inhibitor indomethacin was added to the serosal bathing
solution at least 20 min before the addition of SNP. TTX (2 × 10
7 M) significantly
reduced the SNP-evoked response to 10.6 ± 1.7 µA/cm2 from 24.7 ± 5.0 µA/cm2 (Fig. 2;
n = 7 animals,
P < 0.05). Neither atropine
(10
6 M) nor indomethacin
(10
4 M) significantly
decreased SNP-evoked responses (from 24.7 ± 5 to 18.5 ± 4.3 and
16.3 ± 5.4 µA/cm2,
respectively, n = 7 animals; Fig. 2).
Cumulative effect of L-NNA and
atropine on 5-HT-evoked response.
If NO contributes to the 5-HT-evoked responses as a
noncholinergic component, then the application of atropine and
L-NNA together would be expected
to decrease the 5-HT-evoked responses to a greater extent than would
pretreatment with atropine or
L-NNA alone. We therefore
examined the effects of atropine
(10
6 M) and
L-NNA
(10
4 M) together on
5-HT-evoked chloride secretion in guinea pig distal colon. 5-HT
(10
5 M)-evoked responses
were significantly decreased by pretreatment with atropine,
L-NNA, or atropine and
L-NNA together, as shown in Fig.
3. The 5-HT-evoked P-1 and P-2 responses in
the presence of atropine were decreased to 177.5 ± 35.3 µA/cm2 from the control value of
247.1 ± 37.8 µA/cm2 and to
53.1 ± 10.8 µA/cm2 from the
control value of 270.1 ± 59.4 µA/cm2, respectively [Fig.
3, B and
C (n = 5 animals); P < 0.05 and P < 0.01, respectively]. The
result showed that the noncholinergic components of 5-HT-evoked P-1 and
P-2 responses were 71.8 and 19.7%, respectively. A combination of
atropine and L-NNA further decreased the 5-HT-evoked P-1 and P-2 responses to 121.5 ± 24.5 and
20.8 ± 7.2 µA/cm2,
respectively [Fig. 3, B and
C
(n = 5 animals);
P < 0.05 and P < 0.01, respectively].
5-HT-evoked P-1 and P-2 responses in the presence of atropine and
L-NNA together were
statistically different from those of the pretreatment with atropine or
L-NNA alone (Fig. 3,
B and
C; P < 0.05). These results showed that NO components of P-1 and P-2
responses were 32 and 61% of the 5-HT-evoked noncholinergic responses,
respectively.

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Fig. 3.
Cumulative effects of atropine and
L-NNA on response to 5-HT in
guinea pig distal colon. A:
representative traces to illustrate effects of atropine,
L-NNA, and atropine + L-NNA on 5-HT-evoked changes in
Isc. 5-HT
(10 5 M)-evoked responses
(P-1 and P-2) were reduced by pretreatment with atropine or
L-NNA alone or atropine + L-NNA in guinea pig distal
colon. Simultaneous application of atropine and
L-NNA further decreased
5-HT-evoked responses. Vertical and horizontal bars are
Isc and time,
respectively. B: cumulative effects of
atropine and L-NNA on
5-HT-evoked P-1 responses. C:
cumulative effects of atropine and
L-NNA on 5-HT-evoked P-2
responses. 5-HT (10 5
M)-evoked P-1 (n = 5 animals,
P < 0.05;
B) and P-2
(n = 5 animals,
P < 0.01;
C) responses were significantly
reduced by pretreatment with
10 6 M atropine or
10 4 M
L-NNA alone. 5-HT-evoked P-1 and
P-2 responses in presence of atropine were further decreased by
presence of atropine + L-NNA
(B and
C; n = 5 animals, P < 0.05). Values are
means ± SE for 5 animals. Statistically different from control:
* P < 0.05, ** P < 0.01.
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Effect of L-NNA on
noncholinergically mediated changes in
Isc by electrical field
stimulation.
To determine whether NO was involved in noncholinergic secretory
response elicited by electrical field stimulation of submucosal neurons
of the guinea pig distal colon, submucosa/mucosa preparations were
electrically stimulated in the presence of atropine
(10
5 M). The addition of
atropine to the bath ensured that the neurally stimulated secretory
response did not contain a muscarinic component. L-NNA significantly reduced the
neurally evoked response in comparison to that of control, as shown in
Fig. 4. The
Isc response to
electrical field stimulation was decreased to 112.6 ± 16.4 from
132.8 ± 17.6 µA/cm2 and to
90.9 ± 10.8 from 116.1 ± 10.8 µA/cm2 in tissues exposed to 1 and
3 × 10
4 M
L-NNA, respectively (Fig. 4;
n = 7 animals, P < 0.05) However, the neurally evoked responses in different
concentrations of L-NNA were not
statistically different from each other.

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Fig. 4.
Effect of L-NNA on neurally
evoked secretion in presence of atropine. Submucosa/mucosa preparations
of guinea pig distal colon were electrically stimulated in presence of
atropine (10 5 M).
Repetitive-stimulus pulses of 0.5-ms length, 10-Hz strength, and 5-Hz
requency were used. Stimulus duration was 30 s.
L-NNA (1 and 3 × 10 4 M) reduced neurally
evoked noncholinergic responses. Filled bars, before addition of
vehicle or L-NNA; open bars,
after addition of vehicle or
L-NNA. Values are means ± SE; n = 7 animals.
* P < 0.05 significantly
different from control responses (filled bars). EFS, electrical field
stimulation.
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 |
DISCUSSION |
The submucous plexus regulates intestinal chloride secretion in
mammalian species. The idea that 5-HT is involved in the modulation of
electrolyte transport in the gastrointestinal tract is supported by a
considerable body of evidence. Functional postsynaptic 5-HT receptors
on submucosal neurons in the guinea pig small intestine and colon have
been electrophysiologically demonstrated by the intracellular recording
of depolarizing responses to the application of exogenous 5-HT (5, 27).
Morphological investigations have demonstrated that 5-HT
immunoreactivity is found in nerve fibers within submucous plexuses
(29). The mucosal response to exogenous 5-HT in guinea pig distal colon
is neurally mediated, because TTX abolishes this 5-HT-evoked response
(5, 18). Muscarinic-receptor blockade with atropine also reduces the
mucosal response to exogenous 5-HT, although a significant
atropine-insensitive component remains (5, 16, 18). Taken together,
these findings suggest that 5-HT stimulates mucosal electrolyte
transport in the guinea pig by acting at receptors located on
cholinergic as well as noncholinergic enteric neurons.
Among candidates as noncholinergic transmitters, NO has been shown to
be a neurotransmitter of intestinal motility (25). Recent evidence also
suggests a functional relationship between 5-HT and NO in the
regulation of intestinal motility (2). NO also induces
electrolyte secretion in the rat intestine in vitro (21,
28, 30).
5-HT-evoked increase in
Isc appears to be
a chloride secretory response. In the present studies, we have
confirmed this by demonstrating concentration-dependent 5-HT-evoked
increases in Isc
that were greatly reduced by pretreatment of tissues with bumetanide,
as reported previously (19). Pretreatment with the NOS inhibitory
substance L-NNA reduced the
5-HT-evoked biphasic increase in
Isc; moreover,
the reduction was reversed by the NOS substrate
L-Arg but not by its
D-enantiomer. Our data therefore support the idea that the 5-HT-evoked chloride secretion is, at least
in part, linked with the NO pathway. In addition, neither L-Arg nor
L-NNA altered the baseline
Isc in the guinea
pig distal colon. This result is consistent with a previous study in
rats in vivo (22), indicating that under normal conditions, NO is not
produced within the intestinal wall in an amount sufficient to exert a
tonic influence on the intestinal secretomotor function and that the
supply of L-Arg for NO synthesis
is not rate limiting.
We have examined whether NO itself can affect mucosal ion transport in
guinea pig distal colon using the NO-donating compound SNP. In the
present experiments, 10
4 M
SNP evoked an increase in baseline
Isc in the guinea
pig distal colon. This finding is consistent with the previous data
that NO donors stimulate secretion of electrolytes in guinea pig and rat small intestine and colon in vitro (21, 28, 30). We have also
determined that the ionic basis for SNP-evoked increase in
Isc is a chloride
conductance, because the response to SNP as well as to 5-HT was reduced
by bumetanide. In the present study, TTX significantly reduced the
SNP-evoked response but did not abolish it. This finding is in good
agreement with studies in the rat colon (28, 30), indicating that NO
can affect the crypt cells through both neural and nonneural pathways.
In contrast, TTX has no effect on SNP-evoked increase in
Isc in guinea pig small intestine (21). Possible explanations for these conflicting data
may be the regional differences between the small and large intestine,
or to species differences. Indeed, we have previously demonstrated that
5-HT-evoked chloride secretion is entirely linked with the enteric
nervous system through 5-HT3 and
5-HT4 receptors in the guinea pig
distal colon (19), whereas in the guinea pig ileum, the 5-HT-evoked
chloride secretion contains a TTX-insensitive component mediated
through 5-HT4 receptors (26).
Atropine did not reduce the SNP-evoked chloride secretion, but TTX
significantly reduced it in the present experiments. These results
suggest that NO also acts within the neural microcircuitry to alter
epithelial transport. It is well known that prostaglandins can
stimulate colonic fluid secretion by direct action on enterocytes and
by the indirect activation of submucosal neurons (10, 12). In the
present study, indomethacin tended to inhibit SNP-evoked responses, but
the decreases in our experiments were not significant. However, Tamai
and Gaginella (28) have reported that piroxicam, another cyclooxygenase
inhibitor, greatly inhibited the SNP response in rat colon. This
discrepancy probably reflects the inherent difficulties encountered in
comparing functional effects of different preparations. Therefore, NO
can directly stimulate the epithelium and, moreover, activate some
neuronal pathways to secrete the chloride ion, but neither
cyclooxygenase nor acetylcholine dependent pathways appear to be
involved in the action of NO in guinea pig distal colon.
The present study has shown that 5-HT-induced chloride secretion was
reduced by L-NNA and that the
SNP-evoked response was not significantly blocked by atropine in guinea
pig distal colon. These results support the idea that NO may be a
noncholinergic transmitter in an enteric neural circuit that is
activated by 5-HT. To further define the relationship between
cholinergic and nitrergic neurons in 5-HT-evoked chloride secretion,
additive effects were tested by the simultaneous administration of
L-NNA and atropine. In the
present experiments, the inhibition of 5-HT-evoked chloride secretion
was greater when L-NNA and atropine were
administered simultaneously than that observed when either atropine or
L-NNA was used alone;
simultaneous application of
L-NNA and atropine further
decreased the 5-HT-evoked P-1 and P-2 responses to 68 and 39% of
atropine treatment tissues, respectively. In the present experiments, atropine significantly reduced the 5-HT
(10
5 M)-evoked P-1 and P-2
responses to 71.8 and 19.7% of control (no drug), respectively. Taken
together, the results suggest that noncholinergic components of P-1 and
P-2 responses are 71.8 and 19.7% of control, respectively, and NO
components of P-1 and P-2 responses are 32 and 61%, respectively, of
the noncholinergic component of the 5-HT-evoked responses. The
simultaneous application of
L-NNA and atropine did not
completely block the 5-HT-evoked responses, also suggesting that other
mediators might be involved in 5-HT-evoked chloride secretion.
Finally, in the present experiments,
L-NNA reduced electrical field
stimulation-evoked response in the presence of atropine. The results clearly indicate that NO can function as a noncholinergic neurotransmitter. This is supported by morphological data that NOS-positive neurons are located in submucous plexus (8, 9).
In conclusion, the results provide strong evidence that 5-HT-induced
chloride secretion in the guinea pig distal colon is, at least in part,
mediated by an NO pathway and that NO may function as a noncholinergic
transmitter in the submucosal plexus.
 |
ACKNOWLEDGEMENTS |
This work was supported by a Monbusyo International research grant,
the Salt Foundation, and Health Science Foundations (A. Kuwahara). Part
of this study was presented at the Third IUPHAR Satellite Meeting on
Serotonin in Chicago, IL, in 1994, and was published in the proceedings
(Behav. Brain Res. 73: 293-296,
1996).
 |
FOOTNOTES |
Address for reprint requests: A. Kuwahara, Lab. of Environmental
Physiology, Inst. for Environmental Sciences, Univ. of Shizuoka,
Shizuoka 422-8526, Japan.
Received 4 September 1997; accepted in final form 11 May 1998.
 |
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