1 Department of Anatomy, The effect of vasoactive intestinal polypeptide
(VIP), pituitary adenylate cyclase-activating peptide-38 (PACAP-38),
and PACAP-27 on the release of serotonin (5-HT) into the intestinal
lumen and the portal circulation was studied by using in vivo isolated
vascularly and luminally perfused rat duodenum. 5-HT levels were
determined by HPLC. VIP, PACAP-38, and PACAP-27 reduced
the luminal release of 5-HT but did not affect the vascular release of
5-HT. The inhibitory effect caused by VIP, PACAP-38, and PACAP-27 was
not affected by either atropine, hexamethonium, TTX, or TTX plus ACh,
but it was completely antagonized by the nitric oxide (NO) synthase
inhibitor NG-nitro-L-arginine
(L-NNA). The VIP receptor
antagonist VIP-(10
luminal release; isolated perfusion; vasoactive
intestinal polypeptide; pituitary adenylate cyclase-activating peptide
THE MECHANISM THAT REGULATES the release of serotonin
(5-HT) from the intestinal enterochromaffin (EC) cells has been widely investigated, using a variety of experimental models in different animals. Cholinergic and adrenergic neuronal mechanisms for regulation of 5-HT release have been most extensively examined in previous studies. In vitro experiments in isolated intestinal tissues of rats
and cats have shown that a Although several previous studies have shown cholinergic and adrenergic
neuronal involvement in 5-HT release, a relatively small number of
studies have shown that nonadrenergic, noncholinergic (NANC) mechanisms
regulate 5-HT release. In isolated vascularly perfused guinea pig
intestine, direct inhibitory action of vasoactive intestinal
polypeptide (VIP) on the release of 5-HT into the portal circulation
has been shown; therefore, it is possible that this peptide is the NANC
inhibitory neurotransmitter that regulates 5-HT release (32,
39). Other candidates for the NANC inhibitory neurotransmitter are ATP, which has been studied in isolated sheets of
rabbit intestine (24, 32), and GABA, which has been studied in isolated
vascularly perfused guinea pig intestine (40). In previous studies
investigating the role of NANC mechanisms on the release of 5-HT, only
the vascular release of 5-HT was examined. However, evidence has been
shown that EC cells can release 5-HT into the intestinal lumen by
demonstrating the intracellular immunoreactivities (17), and
furthermore the release into the vasculature and the lumen is known to
be mediated by independent mechanisms (20, 28). Therefore, the effects
of the NANC inhibitory mechanism on the luminal release of 5-HT should
be investigated in comparison to the vascular release of 5-HT.
In the present study, we aimed to examine the mechanism of action of
VIP, pituitary adenylate cyclase activating peptide-38 (PACAP-38), and
PACAP-27 on the release of 5-HT into the intestinal lumen as well as
into the portal circulation. PACAP-38 and its shorter form PACAP-27 are
newly isolated polypeptides and show structural homology with VIP (26,
27). Immunohistochemical localization of PACAP-27 (41) resembles that
of VIP (6) in the gut; however, the role of this peptide on the release
of 5-HT has not yet been examined. Furthermore, special attention has been paid to the relationship between VIP/PACAP and nitric oxide (NO),
which has been demonstrated to be another NANC inhibitory neurotransmitter in the gastrointestinal tract (36).
Male Wistar rats weighing 200-300 g were used. Care of animals was
conducted in accordance with the Guide
to
the
Care
and Use
of
Experimental
Animals (Shiga University of Medical
Science). Animals were housed in a light-controlled room
with free access to laboratory food and water but were fasted overnight
(16-18 h) before the operation. Each animal was anesthetized with
an intraperitoneal injection of pentobarbital sodium (60 mg/kg
Nembutal; Abbott Laboratories). The duodenum between the pylorus and
Treitz's ligament was prepared for both vascular and intraluminal
perfusion as described previously (15). Arterial perfusion was achieved through an aortic cannula with the tip lying adjacent to the celiac and
superior mesenteric arteries, and effluent perfusate was collected through a portal vein cannula. All vasculature apart from that leading
into the duodenal segment was cut between double ligatures. The
stomach, jejunum, ileum, colon, pancreas, and spleen were removed.
Luminal perfusion was performed through a cannula inserted into the
pylorus; effluent perfusate was collected through a cannula placed into
the duodenal lumen at the level of Treitz's ligament.
The vascular perfusate consisted of Krebs solution containing 3%
dextran, 0.2% BSA (RIA grade; Sigma Chemical, St. Louis, MO), and 5 mM
glucose. The perfusate was saturated with 95%
O2-5% CO2 gas to maintain a pH of 7.4. We used 0.1 M PBS (pH 7.4) as a luminal perfusate. Both perfusates and
the preparation were kept at 37°C throughout the experiment by a
thermostatically controlled heating apparatus. The flow rates for
vascular and luminal perfusion were maintained at 3 and 1 ml/min,
respectively. After a 25-min equilibration period, both vascular and
luminal effluents were collected at 3-min intervals for 33 min into
ice-cold vials. Each vial contained 10 µl each of 57 mM ascorbic
acid, 10 mM EDTA-2 Na, 1 M perchloric acid, and 51 mM pargyline
hydrochloride in 1 ml of samples.
VIP (human and porcine), PACAP-38 (human), or PACAP-27 (human) (all
obtained from the Peptide Institute, Osaka, Japan) was introduced into
the vasculature via a side-arm infusion at a final concentration of 0.1 µM during perfusion periods
5-7.
In some experiments, 1 µM TTX (Sankyo, Tokyo, Japan), 1 µM atropine
sulfate (Sigma Chemical), 100 µM hexamethonium bromide (Sigma
Chemical), 100 µM
NG-nitro-L-arginine
(L-NNA; Peptide Institute), or 1 µM VIP-(10 We determined 5-HT levels by HPLC. Vascular effluents were filtrated
with Ultrafree-MC (30,000 NMWL, Nihon Millipore, Yonezawa, Japan) by
centrifuging for 30 min at 10,000 rpm at 4°C. Luminal effluents
were filtrated manually with a 0.22-µm pore disk filter (Millex-GV,
Nihon Millipore). We injected 100-µl aliquots of filtrates into HPLC
and measured the 5-HT content (14). Results were expressed as means ± SE (in ng/min in each fraction).
Statistical analysis of data was performed using single-factor ANOVA
for repeated measures followed by Scheffe's
F test. A paired
t-test (two-tail) was used to compare
the values of mean basal release (during
periods
1-3
or
1-4)
and mean 5-HT release during periods as indicated in the results. In
both cases, P < 0.05 was considered
statistically significant.
The basal release of 5-HT into the intestinal lumen as well as into the
vasculature was well maintained throughout the 33-min perfusion period.
Basal 5-HT release into the lumen in 32 different experiments was 5.38 ± 0.24 ng/min (range 4.02-7.73 ng/min) and that into the
vasculature was 2.28 ± 0.12 ng/min (range 1.52-3.81 ng/min).
When 1 µM TTX was infused, the release of 5-HT into the lumen was
decreased after 3 min of exposure to TTX and stayed at a significantly
lower level during perfusion periods
6-11
(Fig. 1A).
The mean level of luminal 5-HT release during
periods
6-11 (2.27 ± 0.23 ng/min, n = 3) was
significantly lower than the mean basal release of 5-HT into the lumen
(4.02 ± 0.19 ng/min), whereas the basal release of 5-HT into the
vasculature was not affected by TTX. When 1 µM atropine was infused,
the release of 5-HT into the lumen was decreased after 3 min of
exposure to atropine. This suppression continued during perfusion
periods
6-11,
as shown in Fig. 1B. The mean level of
luminal 5-HT release during periods 6-11
(2.41 ± 0.68 ng/min, n = 3) was
significantly lower compared with the mean basal release of 5-HT into
the lumen (7.21 ± 0.76 ng/min). The basal release of 5-HT into the
vasculature was not affected by atropine. Hexamethonium at a
concentration of 1 µM did not affect either luminal or vascular
release of 5-HT (data not shown). However, 100 µM hexamethonium
strongly inhibited luminal release of 5-HT but did not affect vascular
release of 5-HT, as shown in Fig. 1C.
The mean basal release of 5-HT into the lumen as well as into the
vasculature was not changed from the mean level of luminal or vascular
release of 5-HT during infusion of 100 µM
L-NNA (Fig.
1D). When 1 µM VIP-(10
ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
28) blocked the effects of VIP, PACAP-38, and
PACAP-27. These results suggest that VIP and PACAP exert a direct
inhibitory effect on the luminal release of 5-HT from the
enterochromaffin (EC) cells via a common receptor site on the EC cells
and that this effect is mediated by NO but not by cholinergic pathways.
A single injection of TTX, atropine, or hexamethonium reduced the
luminal release of 5-HT, whereas a single injection of VIP-(10
28)
stimulated the luminal release of 5-HT and this effect was antagonized
by atropine, hexamethonium, or TTX. These results suggest that EC cells
may receive the direct innervation of cholinergic neurons as well as
VIP and/or PACAP neurons, with the former exerting a tonic
stimulatory influence and the latter exerting a tonic inhibitory
influence on 5-HT release into the intestinal lumen.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-adrenergic mechanism mediated the
release of 5-HT from EC cells measured by cytofluorometric methods (1,
29-31). In in vitro experiments in isolated sheets of
rabbit intestine, the vectoral release of 5-HT into the mucosal or
serosal side of the sheets has been shown to be mediated by a
cholinergic mechanism (11, 12, 22, 23). A number of in vivo experiments
have also been performed to investigate the mechanism for regulation of
5-HT release. In isolated vascularly perfused guinea pig intestine,
muscarinic and nicotinic cholinergic mechanisms mediated the release of
5-HT (37, 38), and in isolated vascularly perfused dog intestine
catecholamine stimulated the release of 5-HT (5). In isolated
vascularly and luminally perfused cat intestine, a cholinergic
mechanism stimulated the release of 5-HT into the intestinal lumen (2,
19, 20, 42); however, an adrenergic mechanism stimulated the release of
5-HT into the portal circulation (19, 20). Other mechanisms have also
been examined. Intraluminal pressure caused the release of 5-HT into the lumen from isolated luminally and vascularly perfused rat duodenum
(17) and from isolated loop of the guinea pig intestine (3, 4). A meal
stimulated the luminal release of 5-HT (10), and hypertonic glucose
stimulated the vascular release of 5-HT (7) from isolated perfused dog
intestine. The effect of luminal acidification on 5-HT release has also
been examined in both an in vivo study of rat intestine (34) or an in
vitro experiment of an isolated sheet of rabbit intestine (12, 22, 23).
In these experiments, low pH within the lumen stimulated the release of
5-HT into the intestinal lumen (22, 23, 34), and this effect was
mediated by muscarinic cholinergic and
-adrenergic mechanisms (22,
23).
MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
28) (Sigma Chemical) was infused either singly during
periods
5-7 or combined with each of the above-mentioned peptides during
periods 4-7.
In some experiments, 1 µM TTX plus 1 µM ACh or only 1 µM ACh was
infused combined with 0.1 µM VIP, 0.1 µM PACAP-38, or 0.1 µM
PACAP-27; TTX was infused during
periods
4-8,
ACh was infused during periods
5-8,
and each peptide was infused during
periods 6-8.
Furthermore, 10 mM L-arginine
(Nacalai Tesque, Kyoto, Japan) plus 100 µM
L-NNA were infused combined with
0.1 µM of VIP. L-Arginine was
infused during periods
1-8,
L-NNA was infused during
periods 4-8,
and VIP was infused during periods
5-8.
In other experiments, 1 µM sodium nitroprusside (SNP) was infused
singly during periods 5-7
or combined with 1 µM TTX, which was infused during
periods 4-7.
Finally, 1 µM atropine, 100 µM hexamethonium, or 1 µM TTX was
infused combined with 1 µM of VIP-(10
28). Atropine, hexamethonium, and TTX were infused during periods
4-7,
and VIP-(10
28) was infused during
periods
5-7.
RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
28) was
infused (Fig. 1E), the mean basal
release of 5-HT into the lumen (4.65 ± 0.46 ng/min,
n = 4) was significantly increased
during perfusion periods 6-7
(8.60 ± 1.05 ng/min, 213.96 ± 44.69% of basal release, see Fig. 6). However, the basal release of 5-HT into the vasculature was
not affected by VIP-(10
28) (Fig.
1E).
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Fig. 1.
Effect of TTX (A), atropine
(B), hexamethonium
(C),
NG-nitro-L-arginine
(L-NNA)
(D), and vasoactive intestinal
polypeptide (VIP) receptor antagonist VIP-(10 28)
(E) on luminal (
) and vascular
(
) release of serotonin (5-HT) from isolated perfused rat duodenum.
Values represent means ± SE of 3-min samples.
A-C:
n = 3. D and
E:
n = 4. Luminal release of
5-HT is significantly (* P < 0.05) decreased (A,
B,
C) and increased
(E) compared with period before drug
infusion (a).
When VIP was infused at a concentration of 0.1 µM, the basal release
of 5-HT into the lumen (4.87 ± 0.61 ng/min,
n = 9) was significantly decreased
during perfusion periods
7-8
(2.21 ± 0.32 ng/min, 51.46 ± 8.90% of basal release; Fig.
2). The vascular release of
5-HT was not affected by VIP (Fig.
2A). As shown in Fig. 1, luminal
release of 5-HT was influenced by a cholinergic mechanism. Therefore,
to determine whether the decrease in luminal 5-HT release caused by VIP
was mediated by a cholinergic mechanism, we tested the effect of
atropine (1 µM) as well as of hexamethonium (100 µM) on the VIP
(0.1 µM)-induced decrease of 5-HT by introducing them 3 min before
VIP administration. The basal release of 5-HT into the lumen before
atropine infusion (4.74 ± 0.69 ng/min,
n = 3) became significantly lower
during periods
7-10
(1.86 ± 0.12 ng/min, 40.26 ± 3.92% of basal release; Fig.
2B). Likewise, the basal release of
5-HT into the lumen before hexamethonium infusion (6.20 ± 0.72 ng/min, n = 3) was
significantly decreased during periods
6-8
(2.11 ± 0.60 ng/min, 35.58 ± 11.3% of basal release; Fig.
2B). There was no further reduction
in the VIP response in the presence of either atropine or
hexamethonium. A similar phenomenon was observed when TTX (1 µM) was
administered with VIP (0.1 µM). The basal release of 5-HT into the
lumen (5.24 ± 0.41 mg/min, n = 3) became significantly lower during
periods
6-11
(2.03 ± 0.41 ng/min, 39.01 ± 8.37% of basal release; Fig.
2B). When a lower concentration of
VIP (0.01 µM) was infused, basal release of 5-HT into the lumen
(4.78 ± 0.58 ng/min, n = 3) became lower during periods
7-8
(3.15 ± 0.43 ng/min, 67.01 ± 6.23% of basal release). Infusion
of VIP (0.01 µM) with TTX (1 µM) inhibited luminal 5-HT release
(38.72 ± 7.86% of basal release) more so than did VIP (0.01 µM)
alone. In other experiments, when both TTX (1 µM) and
ACh (1 µM) were infused with 0.1 µM VIP, the basal release of 5-HT
into the lumen (6.76 ± 0.79 ng/min,
n = 4) was significantly lower during
perfusion periods
7-11
(2.55 ± 0.36 ng/min, 41.04 ± 10.13% of basal
release; Fig. 2B). In
comparison, when ACh (1 µM) plus VIP (0.1 µM) were infused, ACh
stimulated the basal release of 5-HT (124.3 ± 2.8% of basal
release); however, the percent inhibition induced by VIP in the
presence of ACh (42.9 ± 8.2% of basal release) did not change from
that induced by TTX (1 µM) plus ACh (1 µM) plus VIP (0.1 µM).
When the NO synthase (NOS) inhibitor
L-NNA (100 µM) was introduced
with VIP, the basal release of 5-HT into the lumen (4.63 ± 1.02 ng/min, n = 3) was not changed from
the mean levels of luminal 5-HT release during periods
5-7
(3.86 ± 0.34 ng/min, 95.44 ± 28.11% of basal release; Fig.
2B). When
L-arginine (10 mM) and
L-NNA (100 µM) were infused concomitantly with VIP, the basal release of 5-HT into the lumen (6.23 ± 0.67 ng/min, n = 3) became
significantly lower during periods
5-7
(4.26 ± 0.47 ng/min, 68.62 ± 4.00% of basal release; Fig.
2B). When 1 µM VIP-(1028) was
introduced with VIP, the basal release of 5-HT into the lumen (6.03 ± 0.18 ng/min, n = 4) was not
changed from the mean level of luminal 5-HT release during periods
5-7
(7.26 ± 0.82 ng/min, 121.23 ± 15.73% of basal
release; Fig. 2B).
|
When 0.1 µM PACAP-38 was infused during perfusion
periods
5-7,
the basal release of 5-HT into the lumen (4.53 ± 0.28 ng/min, n = 4) was significantly decreased
during perfusion periods
7-9 (2.20 ± 0.23 ng/min, 48.42 ± 3.93% of basal release; Fig.
3), whereas the release of 5-HT into the
vasculature was not affected by PACAP-38 (Fig.
3A). The effect of atropine (1 µM), hexamethonium (100 µM), TTX (1 µM), TTX plus ACh (1 µM),
L-NNA (100 µM), or VIP-(1028) (1 µM) on the inhibitory effect of PACAP-38 on luminal 5-HT release was examined. The basal release of 5-HT into the lumen
before atropine infusion (4.25 ± 0.47 ng/min,
n = 3) became significantly lower
during periods
6-11
(0.97 ± 0.15 ng/min, 22.52 ± 1.28% of basal release; Fig.
3B). The basal release of 5-HT into
the lumen before hexamethonium infusion (6.78 ± 1.33 ng/min, n = 4) was decreased significantly
during periods
7-9
(1.52 ± 0.16 ng/min, 27.53 ± 9.45% of basal release; Fig.
3B). The basal release of 5-HT into
the lumen before TTX infusion (4.74 ± 0.36 ng/min,
n = 3) was lowered significantly
during periods
7-11
(1.55 ± 0.26 ng/min, 32.57 ± 4.55% of basal release; Fig.
3B). When TTX (1 µM) and ACh (1 µM) were infused concomitantly with 0.1 µM PACAP-38, the basal
release of 5-HT into the lumen (7.73 ± 0.57 ng/min,
n = 3) became significantly lower
during perfusion periods
7-11
(1.78 ± 0.61 ng/min, 25.28 ± 9.85% of basal release; Fig.
3B). When 100 µM
L-NNA was introduced with
PACAP-38, the basal release of 5-HT into the lumen (5.48 ± 0.42 ng/min, n = 3) was not changed from
the mean level of luminal 5-HT release during
periods
5-7
(5.77 ± 0.75 ng/min, 106.21 ± 15.56% of basal release; Fig.
3B). When 1 µM VIP-(10
28) was
introduced with PACAP-38, the basal release of 5-HT into the lumen
(5.40 ± 0.22 ng/min, n = 4) was
increased during periods
5-7
(7.84 ± 0.80 ng/min, 146.51 ± 17.23% of basal release; Fig.
3B).
|
When PACAP-27 at a concentration of 0.1 µM was infused during
perfusion periods
5-7,
the basal release of 5-HT into the lumen (4.43 ± 0.80 ng/min,
n = 5) was significantly decreased
during periods
7-9
(1.39 ± 0.27 ng/min, 31.50 ± 2.51% of basal release; Fig.
4). However, the vascular release of 5-HT
was not affected by PACAP-27 (Fig.
4A). The effect of atropine (1 µM), hexamethonium (100 µM), TTX (1 µM), TTX plus ACh (1 µM),
L-NNA (100 µM), or VIP-(1028) (1 µM) on the inhibitory effect of PACAP-27 on luminal 5-HT release was examined. Again, the inhibitory response caused by
PACAP-27 was not altered by administration of either atropine, hexamethonium, TTX, or TTX plus ACh; however, it was completely antagonized by administration of
L-NNA and VIP-(10
28), as shown in Fig. 4B.
|
The effect of the NO donor SNP on the luminal and vascular release of 5-HT was examined (Fig. 5A). The mean basal release of 5-HT into the lumen (4.86 ± 0.33 ng/min, n = 3) was decreased significantly during periods 5-6 (2.47 ± 0.15 ng/min, 50.97 ± 0.40% of basal release; Fig. 5B) and then rapidly returned to the basal level. The vascular release of 5-HT was not affected by SNP (Fig. 5A). This inhibitory effect of SNP on the luminal release of 5-HT was not antagonized by TTX, as shown in Fig. 5B.
|
To determine the relationship between cholinergic mechanisms and VIP
neurons, we examined the effects of 1 µM VIP-(1028) plus 1 µM
atropine, 1 µM VIP-(10
28) plus 100 µM hexamethonium, and 1 µM
VIP-(10
28) plus 1 µM TTX on 5-HT release. The stimulatory effect of
VIP-(10
28) on the luminal release of 5-HT seen in Fig. 1E was completely blocked by the
presence of either atropine, hexamethonium, or TTX (Fig.
6).
|
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DISCUSSION |
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The present results show that a considerable amount of 5-HT was released from the isolated perfused rat duodenum into both the duodenal lumen and portal circulation and that the basal release of 5-HT into the lumen was always higher than that into the vasculature. Furthermore, the percent stimulation (or inhibition) of the basal release was much higher in the luminal release than in the vascular release in all experiments. Previously, the effects of cholinergic or noncholinergic mechanisms on the release of 5-HT have been studied in the guinea pig small intestine; however, only the vascular release of 5-HT was measured (32, 37-39, 40). These previous studies have shown that vascular release of 5-HT was much affected by several agents that showed little or no effect on the vascular 5-HT release in the present study. The discrepancy between the previous and present studies is not well understood. However, it may possibly be due to the difference in animal species or preparation. For example, in previous studies (13, 15, 16), the isolated loop of vascularly perfused guinea pig intestine was placed in the organ bath, but in the present study rat duodenum was treated with in vivo perfusion with intact extrinsic nerve supply into the intestinal segment. The release of 5-HT (2, 19, 42) or peptides such as somatostatin (15), gastrin (13), or peptide YY (16) into the intestinal lumen is widely accepted, and the mechanism for regulation is known to differ from that in vascular release. Recently, we have proposed morphological evidence for the luminal release of 5-HT from EC cells with demonstration of ultrastructural intracellular immunoreactivities (17). Results showed that EC cells without stimulation contained immunogold-labeled secretory granules in both apical and basal cytoplasm, while under stimulation of luminal release exocytotic granules were scarcely seen at the apical cell membrane, but immunogold label diffusely located over the apical cytoplasm and microvilli (17). In the present study, 5-HT detected in the lumen most likely originated from EC cells due to their localization in the epithelium as well as their higher 5-HT level compared with 5-HT-containing neurons in the gut. However, the contribution of 5-HT-containing nerves to luminal or at least to vascular release of 5-HT cannot be denied because a number of 5-HT-immunoreactive nerve fibers have been shown to distribute in the mucosal lamina propria or around the submucosal blood vessels in the rat duodenum (18).
We examined the effects of the axonal blocker TTX, the cholinergic muscarinic blocker atropine, and the cholinergic nicotinic blocker hexamethonium on luminal and vascular release of 5-HT from the duodenum. The results show that TTX, atropine, and hexamethonium reduced the basal luminal release of 5-HT. Therefore, this suggests that the basal luminal release of 5-HT is tonically controlled by the neuronal pathways and both cholinergic muscarinic and nicotinic receptors were involved in this mechanism. The effect of cholinergic mechanisms on the luminal release of 5-HT was examined previously in an in vivo experiment in dog jejunum (10) in which meal-induced stimulation of luminal release of 5-HT was blocked by intravenous injection of atropine. In in vivo experiments in cat jejunum (19), vagally mediated release of 5-HT into the lumen was blocked by atropine and hexamethonium administered intravenously. In an in vitro experiment (22) with isolated sheets of rabbit duodenum, acid-induced release of 5-HT into the luminal side of the sheet was blocked by both 1 µM atropine and 100 µM hexamethonium. Furthermore, from the findings in isolated vascularly perfused guinea pig ileum, the presence of muscarinic and nicotinic receptors on the EC cells has been indicated (37).
VIP inhibited the release of 5-HT into the lumen but did not affect the vascular release of 5-HT. This inhibitory effect of VIP was not affected by the presence of atropine, hexamethonium, or TTX. These results imply that VIP exerts a direct inhibitory effect on the luminal release of 5-HT from the EC cells and that other neuronal mechanisms, including cholinergic neurons, do not mediate this mechanism. This hypothesis was further confirmed by the finding that the luminal release of 5-HT was inhibited by VIP even in the presence of both TTX and ACh. VIP plus TTX had no greater effect than did VIP alone, except when a lower concentration of VIP was used. Similarly, VIP plus TTX plus ACh had no greater effect than did VIP plus ACh. These data suggest that the luminal release of 5-HT is neuronally mediated and that VIP and cholinergic neurons might be arranged in parallel but not in series. The effect of VIP on the release of 5-HT has previously been examined (39) in the isolated guinea pig small intestine, but only the vascular release of 5-HT was examined, in which VIP inhibited the vascular release of 5-HT and this effect was not affected by TTX. Although the direction of the release of 5-HT from the EC cells caused by VIP is different between the previous and present studies, it might be concluded that VIP exerts a direct inhibitory action on the luminal release of 5-HT from the EC cells. Endogenous VIP is involved in the neuronal cell bodies in both the myenteric and submucosal plexuses, and the number of VIP-positive neurons was much higher in the submucosal plexus than in the myenteric plexus (6). Furthermore, it has been reported that VIP-containing nerve fibers are located closely to the EC cells in the rat and guinea pig intestine (21). These morphological findings may suggest the direct action of VIP neurons on the EC cells.
The inhibitory effect of VIP on the luminal release of 5-HT was antagonized by the NOS inhibitor L-NNA. This effect of L-NNA was reversed by the pretreatment of the NOS substrate L-arginine. These data suggest that the inhibitory action of VIP on the luminal release of 5-HT from the EC cells is mediated by the NO pathway. Because a single injection of the NO donor SNP caused the reduction of luminal 5-HT release and this effect was not antagonized by TTX, it was suggested that NO exerts a direct inhibitory action on the EC cells and that no other neuronal mechanisms mediate this action. It is known that NO and VIP are functionally linked cotransmitters; for example, VIP release from myenteric neurons is regulated by NO production (25). Despite their functional cooperation, NOS-containing neurons were not always colocalized with VIP in both the myenteric and submucosal plexus (8, 9). The presence of NOS-containing neurons in the submucosal plexus has been demonstrated in the rat small intestine (8, 9). Therefore, these neurons are likely to mediate the inhibitory action of submucosal VIP neurons on the release of 5-HT from EC cells, although the detail of the interaction between NO and VIP neuronal cell bodies or nerve fibers in the submucosa or mucosa is not known at present.
Both PACAP-38 and PACAP-27 inhibited the release of 5-HT into the lumen but did not affect the vascular release of 5-HT. These inhibitory effects exerted by PACAP-38 or PACAP-27 on luminal 5-HT release were not changed by the presence of atropine, hexamethonium, or TTX. Furthermore, PACAP-38 and PACAP-27 reduced the luminal release of 5-HT even in the presence of both TTX and ACh, suggesting that PACAP-38 and PACAP-27 exert a direct inhibitory effect on the luminal release of 5-HT from the EC cells, but no other neuronal mechanisms, including cholinergic neurons, involved this mechanism. The effects observed in PACAP-38 and PACAP-27 were quite similar to those observed in VIP. PACAP-27-containing neurons have been shown to distribute in the myenteric and submucosal plexus, and the number of cell bodies was numerous in the submucosal plexus (41). Such distribution was quite similar to that of VIP-containing neurons in the intestine (6). Furthermore, some PACAP-27 neurons are known to colocalize with VIP neurons (41). The inhibitory effects seen in PACAP-38 and PACAP-27 were antagonized by L-NNA. This suggests that functional interaction exists both between VIP and NO and between PACAP and NO.
The VIP receptor antagonist VIP-(1028) completely antagonized the
inhibitory effect of VIP, PACAP-38, and PACAP-27 on the luminal release
of 5-HT. The inhibitory effects of VIP, PACAP-38, and PACAP-27 were all
abolished by the same concentration of VIP-(10
28). These data suggest
that VIP and PACAP share a common receptor site on the EC cells of rat
intestine. The common VIP/PACAP receptor, which has been characterized
in human small intestine, has equal affinity to VIP, PACAP-38 and
PACAP-27 (type II) and therefore is clearly differentiated from type I
in the central nervous system (33, 35). The common VIP/PACAP receptor
on the EC cells seems to possess the same characteristics as type II
sites.
Finally, the results showed that the single injection of the VIP
receptor antagonist VIP-(1028) stimulated the luminal release of
5-HT, while the single injection of the NOS inhibitor
L-NNA did not affect the basal
release of 5-HT into the lumen. Furthermore, the stimulatory effect of
VIP-(10
28) was completely antagonized by atropine, hexamethonium, or
TTX. These results suggest that the luminal release of 5-HT may be
caused by both tonic stimulatory influence from cholinergic mechanisms
and tonic inhibitory influence from VIP/PACAP pathways. Once the
inhibitory effect is blocked by VIP-(10
28), the basal release of 5-HT
seems to become abnormally high. NO-generating pathways, on the other
hand, may not participate to sustain the basal release of 5-HT.
In conclusion, the present study provides evidence that VIP- and PACAP-generating pathways directly inhibit the luminal release of 5-HT from EC cells of rat duodenum and that they share a common receptor site, probably type II, on the EC cells. NO-generating pathways also directly inhibit luminal 5-HT release, and NO mediates the inhibitory action of VIP/PACAP. The results suggest that EC cells may receive the direct innervation of cholinergic neurons as well as VIP and/or PACAP neurons, with the former exerting a tonic stimulatory effect and the latter exerting a tonic inhibitory effect on the luminal release of 5-HT. Furthermore, VIP/PACAP are functionally linked with NO in this action.
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ACKNOWLEDGEMENTS |
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We thank Noboru Urushiyama in the Central Research Laboratory of Shiga University of Medical Science for technical support in the HPLC analysis.
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FOOTNOTES |
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This work was supported by a grant from the Joint Research Program of the National Institute for Physiological Sciences (M. Fujimiya and A. Kuwahara).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: M. Fujimiya, Dept. of Anatomy, Shiga Univ. of Medical Science, Seta, Otsu, Shiga 520-21, Japan.
Received 11 March 1998; accepted in final form 12 June 1998.
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