Department of Internal Medicine (K.H., T.T., C.O.), University of
Michigan Medical Center, Ann Arbor, Michigan and
First Department of
Medicine (K.H., M.F.), Yamanashi Medical University, Yamanashi, Japan
We investigated whether nitric oxide (NO) exerts an inhibition on its
own synthesis in the gastric myenteric plexus in rats. Nonadrenergic,
noncholinergic relaxations in response to transmural electrical
stimulation (TS) were markedly antagonized by
NG-nitro-L-arginine methyl ester,
(10
4 M) and abolished by tetrodotoxin (106
M). Pretreatment with various NO donors {3-morpholino-sydnonymide [SIN-1 (3 × 107 to 3 × 106
M)], S-nitroso-N-acetylpenicillamine (106 to
105 M), sodium nitroprusside (108 to 3 × 108 M) and 8-bromoquanosine 3',5'-cyclic monophosphate
[8-bromo-cGMP (106 to 3 × 106
M)]} significantly inhibited TS-evoked nonadrenergic, noncholinergic relaxations in a dose-dependent manner. In contrast, vasoactive intestinal polypeptide (108 M)-induced relaxations were
not affected by SIN-1 or 8-bromo-cGMP. TS evoked a significant increase
in 3H-citrulline formation, which was completely abolished
by calcium-free medium, NG-nitro-L-arginine
methyl ester, (104 M) and tetrodotoxin (106
M). 3H-citrulline formation evoked by TS was significantly
inhibited by SIN-1 (107 to 105 M) and
8-bromo-cGMP (107 to 105 M) in a
dose-dependent manner. The inhibitory effect of SIN-1 was partially
prevented by 1H-[1,2,4]oxadiazolo[3,4-a]quinoxalin-1-one (105 M), a guanylate cyclase inhibitor. We conclude that
NO synthesis in the gastric myenteric plexus is negatively regulated by
NO and cGMP. This suggests an autoregulatory feedback mechanism of NO
synthesis in the gastric myenteric plexus.
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Introduction |
NO
is a major NANC inhibitory neurotransmitter in the GI tract. NOS, a key
enzyme that mediates NO synthesis and release from the nerve terminals,
has been demonstrated in the myenteric plexus throughout the GI tract
(Costa et al., 1992
; Young et al., 1992; Aimi
et al., 1993). NO released by nerve stimulation of the
myenteric plexus causes rapid relaxation of the smooth muscle (Bredt
et al., 1990; Bult et al., 1990; Boeckxstaens
et al., 1991; D'Amato et al., 1992; Shimamura
et al., 1993; Takahashi and Owyang, 1995). A major action of
NO is to activate the soluble form of guanylate cyclase, resulting in
an accumulation of cGMP in the target tissues (Garthwaite, 1991).
The function of NO in neurotransmission, however, remains to be
investigated. L-NMMA, a NO biosynthesis inhibitor, markedly enhanced tonic responses to transmural stimulation in the presence of
atropine in the guinea pig ileum (Gustafsson et al., 1990). L-NMMA also increased sympathetic nerve activity in the
rabbit kidney (Harada et al., 1993). These observations
suggest that endogenous NO may have an inhibitory effect on the release
of excitatory neurotransmitters and cathecholamine in these tissues. In
cultured PC-12 cells after NGF treatment, K+ depolarization
elicited a marked enhancement of cGMP levels, accompanied by an
increase in ACh release. The release of both ACh and dopamine from
these cells was blocked by NOS inhibitors and reversed by the addition
of L-arginine (Hirsch et al., 1993). These
findings indicate that in certain tissues, NO enhances the release of
neurotransmitters through the activation of guanylate cyclase. Thus,
depending on the tissues, NO may stimulate or inhibit the release of
other neurotransmitters.
Feedback inhibition is an important autoregulatory mechanism of
neurotransmitter release in the central as well as the peripheral nervous system. Both ACh (Ito and Yoshitomi, 1988; Quirion, 1993; Sastry, 1995) and norepinephrine (Daly et al., 1989; Mermet
et al., 1990) had been shown to inhibit their own release.
This may be an important homeostatic mechanism to prevent
overstimulation of neural tissues. It is not clear whether NO regulates
its own synthesis and release in the nerve terminals. De Man et
al. demonstrated that prolonged exposure to NO donors inhibited
electrically induced nerve-mediated NANC relaxations without affecting
the postjunctional response to NO and VIP (De Man et al.,
1995). Exogenously applied NO inhibits activity of NOS in the rat
cerebellum (Rogers and Ingarro, 1992). These observations suggest that
there may be an autoregulatory mechanism modulating NO synthesis.
However, little is known about the feedback mechanism on nitrergic
transmission in the myenteric plexus of GI tract. The aims of the
present study are 1) to investigate whether NO regulates NOS activity
and its own synthesis in the gastric myenteric plexus and 2) if so, to investigate the intracellular mediator responsible for this phenomenon.
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Materials and Methods |
Recording of NANC relaxation.
Circular muscle strips of the
gastric body were surgically obtained from male Sprague-Dawley rats
(300-350 g). Muscle strips (15 mm in length and 3 mm in width) were
suspended between two platinum electrodes in a 30-ml organ bath filled
with Krebs-Henseleit Buffer (KHB) of the following composition; 118 mM
NaCl, 4.8 mM KCl, 2.5 mM CaCl2, 25 mM NaHCO3,
1.2 mM KH2PO4, 1.2 mM MgSO4 and 11 mM glucose. The solution was maintained at 37°C and aerated with a
mixture of 95% O2 and 5% CO2. Mechanical
activity was recorded on a polygraph using isometric transducers.
Muscle strips were stretched in 1-mm increments and repeatedly exposed
to 106 M carbachol to determine
L0, the length at which maximum active tension
developed. Dose-response curves were constructed on each muscle strip
for SNP (107 to 105 M), an activator of
soluble guanylate cyclase, and carbachol (108 to
105 M).
To study NANC relaxations, muscle strips were stimulated by TS (65 V, 2 msec, 1-20 Hz, for 30 sec) in the presence of atropine (106 M) and guanethidine (106 M).
Experiments were performed using muscle strips stimulated by 5-HT
(105 M) to evaluate the inhibitory effects of NO, as
previously described (Boeckxstaens et al., 1991). After the
study of NANC relaxation in response to TS (1-20 Hz; control
experiments), each muscle strip was pretreated with SIN-1
(107 to 105 M), SNAP (107 to
105 M), SNP (108 to 106 M)
and 8-bromo-cGMP (107 to 105 M) for 15 min.
The recordings of muscle relaxation studies were visually inspected,
and the NANC relaxation in response to TS was compared between the
experiments before and after pretreatment with SIN-1, SNAP, SNP and
8-bromo-cGMP. Each NANC relaxation was expressed as a percentage of the
maximum relaxation of control experiments. In most experiments, we
observed the maximum relaxation at 10 Hz in control experiments.
To examine the effects of NO donor on cholinergic neurotransmission,
muscle strips were stimulated by TS (65 V, 2 msec, 1-20 Hz, for 30 sec) with and without SIN-1 (3 × 107 to 3 × 106 M) or 8-bromo-cGMP (106 to 3 × 106 M). Previous studies had demonstrated that gastric
muscle contractions in response to electrical stimulation were
significantly reduced by atropine, a result that indicates mediation by
cholinergic pathways (Takahashi and Owyang, 1995). The muscle
contraction in response to TS was compared between control experiments
and pretreatment with SIN-1 (3 × 107 to 3 × 106 M) or 8-bromo-cGMP (106 to 3 × 106 M). Each contraction was expressed as a percentage of
the maximum contraction of control experiments. In most experiments, we
observed the maximum contraction at 20 Hz in control experiments.
It has been demonstrated that long duration of high-frequency
stimulation (16 Hz for 3 min) evokes release of VIP as well as NO (Li
and Rand, 1990; Boeckxstaens et al., 1992; De man et al., 1995). To investigate the effects of NO donor on VIP-mediated NANC relaxation, muscle strips were stimulated by TS (65 V, 2 msec, 16 Hz, for 3 min) with and without SIN-1 (3 × 107 to
3 × 106 M) or 8-bromo-cGMP (106 to
3 × 106 M) in the presence of atropine
(106 M) and guanethidine (106 M). To
eliminate NO-mediated relaxation, the muscle strips were incubated with
L-NAME before TS. In this manner, we compared the muscle
relaxation in response to TS between control experiments and
pretreatment with SIN-1 (3 × 107 to 3 × 106 M) and 8-bromo-cGMP (106 to 3 × 106 M).
NO formation study.
L-citrulline and NO are
produced in a 1:1 ratio from L-arginine by the action of
NOS. Production of NO was measured in gastric tissue preloaded with
L-[3H]-arginine and expressed as amount of
L-[3H]-citrulline formed in the tissue as
described by Bredt and Snyder (1989). After the removal of the mucosa,
gastric muscle strips were suspended between two platinum electrodes.
Muscle strips were incubated in a 1.5-ml organ bath for 30 min at
37°C in the presence of cofactors (1 mM NADPH, 10 µM FAD, 10 µM
FMN and 10 µM tetrahydrobiopterin) and were further incubated with
3H-arginine (3 µCi/ml) for 4 min at 37°C. Immediately
after TS (65 V, 2 msec, 1-5 Hz) for 1 min, the reaction was stopped by flash freezing in liquid nitrogen. The samples were stored at 
80°C
for subsequent measurement of
L-[3H]-citrulline. Muscle tissue was
homogenized with a 1-ml straight-wall grinder. After centrifugation at
3000 rpm for 10 min at 4°C, the supernatant in 10% trichloroacetic
acid was sonicated for 5 min. After washing with water-saturated ethyl
ether, the supernatant was applied to a Dowex AG50WX-8 resin column
(Na+ form), and L-[3H]-citrulline
was eluted with HEPES buffer (pH 5.5) and water, as previously reported
(Takahashi and Owyang, 1995).
L-[3H]-citrulline in the effluent was
measured by liquid scintillation spectroscopy, and the production of
L-[3H]-citrulline in the gastric tissue was
expressed as cpm/mg tissue.
Materials.
The following drugs and chemicals were used in
this study: atropine, guanethidine, 5-HT, NADPH, FAD, FMN, SNP and TTX
(Sigma Chemical Co., St. Louis, MO), 8-bromo-cGMP, SIN-1 and SNAP
(Biomol, Plymouth Meeting, PA), ODQ (Calbiochem, La Jolla, CA),
tetrahydrobiopterin (ICN Biomedicals, Costa Mesa, CA),
NG-nitro-L-arginine methyl ester
(L-NAME) (Research Biochemicals Inc., Natick, MA), VIP
(Peninsula, Belmont, CA), L-[3H]-arginine
(New England Nuclear, Boston, MA) and Dowex AG 50W-X8 (Biorad,
Richmond, CA).
Statistical analysis.
All data were expressed as the
mean ± S.E. Statistical analysis was performed using Student's
t test or analysis of variance (ANOVA). Significance was
accepted at the 5% level.
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Results |
NANC relaxation study.
In the 5HT-precontracted muscle strips,
TS (65 V, 2 msec, 1-20 Hz, for 30 sec) evoked NANC relaxations in a
frequency-dependent manner. The NANC relaxations were antagonized by
L-NAME (104 M) (fig.
1) and were completely abolished by TTX
(106 M) (data not shown). The effects of
L-NAME on NANC relaxations were frequency-dependent. NANC
relaxations evoked by low frequencies (<2.5 Hz) were almost completely
abolished by L-NAME (>90%).
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Fig. 1.
NANC relaxations in response to TS (65 V, 2 msec,
1-20 Hz, for 30 sec) in the absence and presence of L-NAME
(104 M). NANC relaxations were significantly antagonized
by L-NAME, which suggests mediation by NO release from the
gastric myenteric plexus. Results were obtained from four muscle strips
from three rats (mean ± S.E.).
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SIN-1 (3 × 107 to 106 M), a NO donor,
did not have any significant effects on basal tone and 5HT-induced
muscle contraction. The higher dose of SIN-1 (3 × 106 M), which slightly reduced the basal tone by 0.3 ± 0.09 g, also had no significant effect on 5HT-induced
contraction (95 ± 5.5% of control). However, SIN-1 (3 × 107 to 3 × 106 M) significantly
reduced TS-evoked NANC relaxations in a dose-dependent manner (fig.
2A; fig.
3A). The greatest effect was observed
with SIN-1 (3 × 106 M), which inhibited TS (5 Hz)-evoked NANC relaxations by 49.8% (dF = 1, 40, F = 60.1, P < .001) (fig. 3A). The
inhibitory effects of SIN-1 (3 × 107 to 3 × 106 M) on NANC relaxation were restored by washing (data
not shown). Higher concentrations of SIN-1 (105 to
104 M) caused significant inhibition of both basal tone
and 5HT-induced contraction in a dose-dependent manner (data not
shown).
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Fig. 2.
Effects of SIN-1 (3 × 105 M)
(panel a), SNAP (105 M) (panel b), SNP (3 × 108 M) (panel c) and 8-bromo-cGMP (3 × 106 M) (panel d) on NANC relaxations in response to TS
(1-10 Hz). 5HT (105 M)-induced contractions were not
affected by SIN-1 (3 × 105 M), SNAP
(105 M), SNP (3 × 108 M) or cGMP
(3 × 106 M). However, NANC relaxations were
significantly antagonized by SIN-1 (3 × 105 M),
SNAP (105 M), SNP (3 × 108 M) and
8-bromo-cGMP (3 × 106 M). The inhibitory effects of
SIN-1 (3 × 105 M), SNAP (105 M), SNP
(3 × 108 M) and 8-bromo-cGMP (3 × 106 M) were restored by washing (data not shown).
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Fig. 3.
Effects of SIN-1 (3 × 107 to
3 × 106 M) (panel a), SNAP (106 to
105 M) (panel b), SNP (108 to 3 × 108 M) (panel c) and 8-bromo-cGMP (106 to
3 × 106 M) (panel d) on NANC relaxations in
response to TS (1-10 Hz). The results were expressed as percent of
maximum relaxation of the control experiments. NANC relaxations were
significantly antagonized by SIN-1 (3 × 107 to
3 × 106 M), SNAP (106 to
105 M), SNP (108 to 3 × 108 M) and 8-bromo-cGMP (106 to 3 × 106 M) in a dose-dependent manner. Results were obtained
from 5 to 6 muscle strips from four rats (mean ± S.E.).
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A similar inhibitory effect was observed upon pretreatment with other
NO donors: SNAP (106 to 105 M) and SNP
(108 to 3 × 108 M) (fig. 2; fig. 3).
8-Bromo-cGMP (106 M), a soluble analog of cGMP, had no
effect on the basal tone. At 3 × 106 M,
8-bromo-cGMP, which slightly reduced the basal tone by 0.17 ± 0.05 g (fig. 2D), also slightly reduced 5HT-induced muscle
contraction (87.4 ± 9.1% of control). 8-Bromo-cGMP
(106 to 3 × 106 M), however,
significantly reduced TS-evoked NANC relaxation in a dose-dependent
manner (fig. 2D; fig. 3D). The greatest effect was observed with
8-bromo-cGMP (3 × 106 M), which caused a 66.2%
inhibition on TS (5 Hz)-evoked NANC relaxations (dF = 1, 40, F = 395.4, P < .001) (fig. 3D). The inhibitory effects of 8-bromo-cGMP on NANC relaxation were reversible by washing
(data not shown). A higher dose of 8-bromo-cGMP (105 to
104 M) caused significant inhibition on both the basal
tone and 5HT-induced contraction (data not shown).
SIN-1 (3 × 107 to 106 M) had no
significant effect on basal tone or carbachol (106
M)-induced muscle contraction. The higher dose of SIN-1 (3 × 106 M), which slightly reduced the basal tone, had no
significant effect on carbachol (106 M)-induced
contraction (94.5 ± 6.7% of control). However, SIN-1 (3 × 107 to 3 × 106 M) significantly
reduced TS-evoked muscle contraction in a dose-dependent manner (figs.
4 and 5).
The largest effect was observed with SIN-1 (3 × 106
M), which inhibited TS (10 Hz)-evoked muscle contraction by 44.7% (dF = 1, 40, F = 55.1, P < .001) (fig. 5). A
similar inhibitory effect on TS-evoked muscle contraction was also
observed with 8-bromo-cGMP pretreatment (106 to 3 × 106 M). The greatest effect was observed with
8-bromo-cGMP (3 × 106 M), which inhibited TS (10 Hz)-evoked muscle contraction by 64.5% (P < .001).
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Fig. 4.
Effects of SIN-1 (3 × 106 M) on
muscle contraction in response to TS (1-20 Hz). SIN-1 (3 × 106 M), which slightly reduced the basal tone, had no
significant effect on carbachol (106 M)-induced
contraction (94 ± 6.7% of control) (data not shown on this
figure). However, SIN-1 significantly reduced TS-evoked muscle
contraction.
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Fig. 5.
Inhibitory effects of SIN-1 (3 × 107 to 3 × 106 M) on muscle
contraction in response to TS (1-20 Hz). The largest effect was
observed with SIN-1 (3 × 106 M), which inhibited TS
(10 Hz)-evoked muscle contraction by 44.7% (dF = 1, 40, F = 60.1, P < .001). Results were obtained from
four muscle strips from four rats (Mean ± S.E.).
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VIP (108 M) caused significant relaxation of the gastric
circular muscle strips. SIN-1 (3 × 107 to 3 × 106 M) or 8-bromo-cGMP (106 to 3 × 106 M), which caused significant inhibition of TS-evoked
NANC relaxation, had no effect on VIP (108 M)-induced
relaxation (fig. 6).
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Fig. 6.
Effects of SIN-1 (106 M) on VIP
(108 M)-induced muscular relaxations. VIP-induced
relaxations were not affected by SIN-1 (106 M). Similar
results were obtained with 8-bromo-cGMP (106 M)
pretreatment (data not shown). Results were reproducible from five
muscle strips from three rats.
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Pretreatment with L-NAME (104 M) had no
effect on VIP-induced relaxation of muscle strips in the basal state or
the precontracted state by 5-HT (fig. 7),
a result that suggests no interactions between NO and VIP action.
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Fig. 7.
Effects of L-NAME on VIP
(108 M)-induced muscular relaxations in basal state
(panel a) and precontracted state (panel b). VIP (108
M)-induced relaxations were not affected by L-NAME
(104 M) in either basal or the precontracted state.
Results were reproducible from five muscle strips from three rats.
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Prolonged electrical stimulation (16 Hz for 3 min) induced a rapid and
a sustained phase of NANC relaxation. The rapid phase, but not the
sustained phase, of NANC relaxations was abolished by
L-NAME (fig. 8).
L-NAME-insensitive NANC relaxations were significantly reduced by SIN-1 in a dose-dependent manner (3 × 107 to 3 × 106 M) (figs. 8 and
9). The greatest effect was observed with
SIN-1 (3 × 106 M), which inhibited TS (16 Hz)-evoked NANC relaxation by 72.8% (P < .001) (fig. 9). A
similar inhibitory effect on L-NAME-insensitive NANC
relaxations was observed with 8-bromo-cGMP pretreatment
(106 to 3 × 106 M). The greatest
effect was observed with 8-bromo-cGMP (3 × 106 M),
which inhibited TS (16 Hz)-evoked NANC relaxation by 79.5% (P < .001).
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Fig. 8.
NANC relaxation in response to prolonged TS (16 Hz
for 3 min) (panel a). The effects of L-NAME on TS-evoked
NANC relaxation (panel b) and the effects of SIN-1 on the
L-NAME-insensitive component of the NANC relaxation (panel
c). TS (16 Hz for 3 min) induced a rapid and a sustained phase of NANC
relaxation. Pretreatment with L-NAME abolished the rapid
phase of NANC relaxation. The L-NAME-insensitive component
of the NANC relaxation was significantly reduced by SIN-1 (3 × 106 M) (panel c).
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Fig. 9.
Effects of SIN-1 (3 × 107 to
3 × 106 M) on the L-NAME-insensitive
component of the NANC relaxation. The L-NAME-insensitive
NANC relaxation was significantly reduced by SIN-1 (3 × 107 to 3 × 106 M) in a dose-dependent
manner. The largest effect was observed with SIN-1 (3 × 106 M), which inhibited TS (16 Hz)-evoked NANC relaxation
by 72.8% (P < .001). Results were obtained from four muscle
strips from three rats (means ± S.E.).
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NO formation study.
The NANC relaxation study suggests that NO
donor and cGMP significantly inhibit NO synthesis in the gastric
myenteric plexus. To obtain direct evidence that NO donor and cGMP
inhibit NO synthesis in the gastric myenteric plexus, we examined NO
formation measured as 3H-citrulline formation in
neuromuscular preparations preloaded with 3H-arginine. The
basal level of 3H-citrulline in the muscle strips was
101 ± 10 cpm/mg tissue, which was significantly increased to
174 ± 4 cpm/mg tissue and 223 ± 42 cpm/mg tissue by 1 Hz
and 5 Hz of TS (65 V, 2 msec, for 30 sec), respectively.
3H-citrulline formation in response to TS (5 Hz) was not
affected by pretreatment with atropine (106 M), but it
was abolished by L-NAME (104 M), TTX
(106 M) or Ca++-free medium containing 2 mM
EDTA and 2 mM EGTA (fig. 10).
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Fig. 10.
Effects of atropine (106 M),
L-NAME (104 M), TTX (106 M) and
Ca++-free medium on 3H-citrulline formation in
response to TS (5 Hz). TS-evoked 3H-citrulline formation
was not affected by atropine (106 M) but was completely
abolished by L-NAME (104 M), TTX
(106 M) and Ca++-free medium. Results were
obtained from six muscle strips from four rats (mean ± S.E.,
*P < .05, **P < .01).
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Pretreatment with SIN-1 (107 to 105 M)
resulted in a significant reduction of 3H-citrulline
formation evoked by TS (5 Hz) in a dose-dependent manner (fig.
11A). The greatest effect was observed
with SIN-1 (105 M), which caused a 91.3% inhibition on
TS (5 Hz)-evoked 3H-citrulline formation. The inhibitory
effect of SIN-1 (105 M) was partially prevented by a
30-min pretreatment with ODQ (105 M), a guanylate cyclase
inhibitor (table 1). Pretreatment with 8-bromo-cGMP (107 to 105 M) also resulted
in a significant reduction of 3H-citrulline formation
evoked by TS (5 Hz) in a dose-dependent manner (fig. 11B).
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Fig. 11.
Effects of SIN-1 (107 to
105 M) (panel a) and 8-bromo-cGMP (107 to
105 M) (panel b) on 3H-citrulline formation
in response to TS (5 Hz). Results were expressed as percent increase
above basal of 3H-citrulline formation in response to TS (5 Hz). 3H-citrulline formation evoked by TS was significantly
antagonized by SIN-1 (107 to 105 M) and
8-bromo-cGMP (107 to 105 M) in a
dose-dependent manner. Results were obtained from six muscle strips
from four rats (mean ± S.E.).
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TABLE 1
Effects of ODQ, a guanylate cyclase inhibitor, on the inhibitory
effects of SIN-1 on TS-evoked 3H-citrulline formation of the
rat stomach
Results were expressed as percent increase above basal of
3H-citrulline formation in response to TS (5 Hz). The
inhibitory effect of SIN-1 was partially antagonized by ODQ. Results
were obtained from six muscle strips from four rats (mean ± S.E.,
*P < .05).
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Discussion |
Using neuromuscular preparations from rat stomach, we demonstrated
a reproducible, frequency-dependent relaxation in response to TS (65 V,
2 msec, 1-20 Hz, for 30 sec) in the presence of atropine and
guanethidine. TS-evoked NANC relaxations were abolished by pretreatment
with tetrodotoxin (106 M), which suggests mediation by
the release of inhibitory neurotransmitters. NANC relaxations induced
by 1 Hz and 2.5 Hz were markedly antagonized by L-NAME
(104 M), which indicates that NANC relaxations evoked by
lower frequencies were mediated mainly by NO release from the myenteric
plexus.
TS-evoked NANC relaxations were significantly reduced, in a
dose-dependent manner, by pretreatment with the NO donors SIN-1 (3 × 107 to 3 × 106 M), SNAP
(106 to 105 M) and SNP (108
to 3 × 108 M). A similar phenomenon was observed
upon pretreatment with 8-bromo-cGMP (106 to 3 × 106 M). However, those NO donors and 8-bromo-cGMP
(106 to 3 × 106 M) had little or no
effect on 5HT-induced contractions. These results suggest that in the
concentrations tested, NO donors and 8-bromo-cGMP preferentially
inhibit NANC relaxations without affecting the myogenic contraction.
In contrast, higher doses of SIN-1 (105 to
104 M) and 8-bromo-cGMP (105 M) had
significant inhibitory effects on basal tone as well as 5HT-induced
contractions. Our present studies suggest that lower concentrations of
SIN-1 (3 × 107 to 3 × 106 M) or
8-bromo-cGMP (106 to 3 × 106 M)
preferentially inhibit NOS activation prejunctionally and that higher
doses of SIN-1 (105 to 104 M) or
8-bromo-cGMP (105 to 104 M) have direct
inhibitory effect on smooth muscle cells as well. De Man et
al. previously demonstrated that NO donor significantly antagonized TS-evoked NANC relaxation in the rat gastric fundus (De Man
et al., 1995). They also demonstrated that pretreatment with
SIN-1 had no effect on the relaxations induced by authentic NO, which
suggests that the action of NO was prejunctional, rather than affecting
the sensitivity of the smooth muscle to NO (De Man et al.,
1995).
To confirm further that NO regulates its own synthesis, we examined NO
production, measured as 3H-citrulline formation in
neuromuscular preparations preloaded with 3H-arginine. The
3H-citrulline formation was significantly increased by TS
(1-5 Hz) in a frequency-dependent manner, and this effect was
abolished by the pretreatment with L-NAME
(104 M) and Ca++-free medium. This result
suggests that the constitutive type of NOS
(Ca++/calmodulin-dependent), but not the inducible type of
NOS (Ca++/calmodulin-independent), is activated by TS. The
constitutive type of NOS is present in the vascular endothelium as well
as in the myenteric plexus of the stomach wall. Endothelial NOS of the
gastric vascular bed is activated by stimulation of peripheral muscarinic receptors (Saperas et al., 1995). In our present
study, 3H-citrulline formation in response to TS was not
affected by the pretreatment with atropine (106 M) but
was completely abolished by TTX (106 M), which indicates
that 3H-citrulline formation during TS is of neural origin.
Pretreatment with SIN-1 (107 to 105 M)
resulted in a concentration-dependent reduction of
3H-citrulline formation in response to TS (5 Hz). We
further demonstrated that 8-bromo-cGMP (107 to
105 M) itself also inhibited 3H-citrulline
formation in response to TS (5 Hz) in a concentration-dependent manner.
These results suggest that cGMP inhibits nitrergic transmission in the
gastric myenteric plexus, resulting in the reduction of NANC
relaxation.
The inhibitory effect of SIN-1 on 3H-citrulline formation
in response to TS was partially prevented by ODQ. ODQ has been shown to
be a potent inhibitor of soluble guanylate cyclase (Southam et
al., 1996; Williams and Parsons, 1997). Therefore, it is likely that the inhibitory action of SIN-1 is mediated, at least in part, by
the activation of guanylate cyclase. This results in an increase of
intracellular cGMP in the myenteric plexus, which inhibits NO
synthesis. On the other hand, it has been proposed that NO may act
independently of a cGMP-dependent mechanism (Lipton et al.,
1993; Ravichandran et al., 1995). For example, it has been demonstrated that NO can inhibit NOS activity by binding to the heme
group of NOS (Wang et al., 1994; Griscavage et
al., 1994). Inhibition of NO synthase by NO and other heme ligands
supports the view that heme is involved in the catalytic activity of NO synthase (Wang et al., 1994; Griscavage et al.,
1994). It is therefore conceivable that besides cGMP, NO itself
(Ravichandran et al., 1995; Wang et al., 1994;
Griscavage et al., 1994) or related nitroso-compounds (peroxynitrite) (Lipton et al., 1993) may act via
other mechanisms to inhibit NOS activation in the gastric myenteric
plexus.
A lower concentration of SIN-1 (107 M) or cGMP
(107 M), which had no inhibitory effect on NANC
relaxation, significantly reduced NO synthesis in response to TS (5 Hz). SIN-1 (3 × 106 M) and 8-bromo-cGMP (3 × 106 M), which nearly abolished 3H-citrulline
formation evoked by TS (5 Hz), caused only a 75% inhibition of NANC
relaxations evoked by TS (5 Hz). It is conceivable that
3H-citrulline assay may be a more sensitive method for
detecting NO synthesis than measurement of NANC relaxation. Mechanical
responses are the sum of the stimulatory and inhibitory effects in
response to TS and depend on the balance of these opposing activations in the myenteric plexus. NANC relaxation in response to TS may also
involve some other inhibitory neurotransmitters.
VIP (108 M)-evoked relaxations were not significantly
affected by SIN-1 (3 × 107 to 3 × 106 M) or 8-bromo-cGMP (106 to 3 × 106 M). VIP causes relaxation in various regions of the
GI tract via cAMP formation in the smooth muscle cells. It
has been demonstrated that the effect of VIP is mediated by direct
action on the VIP receptors located on the muscle cells (Bitar and
Makhlouf, 1982). SIN-1 and 8-bromo-cGMP had no effect on VIP-induced
relaxation, so it appears that these agents do not directly affect the
intracellular pathways that mediate muscular relaxation induced by VIP.
Grider et al. demonstrated that VIP released from the
myenteric plexus is capable of stimulating NO formation in the smooth muscle cells of the guinea pig stomach and that this effect is mediated
by activation of the constitutive type of NOS of the smooth muscle
cells (Grider et al., 1992). This conclusion is supported by
evidence that VIP-induced relaxation is antagonized by pretreatment
with L-NNA in the stomach of various species (Grider et al., 1992, Jin et al., 1996). However, the
inhibitory effect of L-NNA on VIP-induced relaxation was
observed in the guinea pig stomach but not in the guinea pig tenia coli
(Grider et al., 1992), which suggests possible regional
differences. Recently, investigators using eNOS-specific primers and
reverse transcription polymerase chain reaction (RT-PCR) have shown NOS
isoform expressed in the smooth muscle cells of the GI tract to be the
endotherial type (eNOS) (Teng et al., 1996). However, the
antibody raised against the constitutive type of NOS (eNOS and cNOS)
failed to stain smooth muscle cells throughout the GI tract (Costa
et al., 1992; Young et al., 1992; Ward et
al., 1994).
Others have shown that VIP-induced muscular relaxation was not
antagonized by L-NNA or L-NAME in the rat
stomach (Li and Rand, 1990; Boeckxstaens et al., 1992;
D'Amato et al., 1992; McLaren et al., 1993;
Takahashi and Owyang, 1995), which suggests that the action of VIP is
not mediated by NO in this tissue. The reason for these controversies
is not clear. Murthy et al. (1994) demonstrated that PKC
activated by 5-HT in precontracted tissues inhibits NOS activity in the
smooth muscle cells. This may mask the action of VIP on NO synthesis in
the smooth muscle cells (Grider et al., 1992). In our
present studies, L-NAME failed to inhibit VIP-induced relaxation both in the basal and the precontracted state. It appears that the increased 3H-citrulline formation evoked by TS was
due mainly to the activation of NOS in the gastric myenteric plexus in
rats.
We have previously shown that lower frequencies of stimulation (<2.5
Hz) of the vagus nerve preferentially stimulate NO release, whereas
higher frequencies of stimulation (>5 Hz) provoke VIP release from the
vascularly isolated perfused rat stomach (Takahashi and Owyang, 1995).
We proposed that the L-NAME-insensitive component of the
NANC relaxation evoked by 5 to 20 Hz is mediated, at least in part, by
VIP release from the gastric myenteric plexus (Takahashi and Owyang,
1995). In the present study using the circular muscle strips of rat
stomach, NANC relaxations induced by 5 to 20 Hz were only partially
antagonized by L-NAME, a result that indicates partial
mediation by NO release at higher frequencies. It has been proposed
that lower frequencies of stimulation preferentially stimulate NO
release, whereas higher frequencies stimulate VIP release as well as NO
release in the rat stomach (Li and Rand, 1990; Boeckxstaens et
al., 1992; De Man et al., 1995). Prolonged electrical
stimulation (16 Hz for 3 min) induced a rapid phase and a sustained
phase of NANC relaxation. The rapid phase, but not the sustained phase,
of relaxation was abolished by L-NAME, and the
L-NAME-insensitive NANC relaxation was significantly
antagonized by trypsin or by VIP antibody, which suggests mediation by
VIP release from the gastric myenteric plexus (Li and Rand, 1990; Boeckxstaens et al., 1992; De Man et al., 1995).
In our current studies, the L-NAME-insensitive component of
the NANC relaxation in response to TS (16 Hz for 3 min) was
significantly reduced by SIN-1 in a dose-dependent manner. Similar
inhibitory effects on the L-NAME-insensitive relaxation
were observed upon treatment with 8-bromo-cGMP. Because SIN-1 has no
effect on VIP-induced muscle relaxation, the inhibitory effects of
SIN-1 should be prejunctional, which suggests that NO is also capable
of inhibiting VIP release. In contrast to our results, De Man et
al. (1995) previously demonstrated that NO donor has no effect on
the electrical stimulation (16 Hz for 3 min)-induced NANC relaxation of
the longitudinal muscle in the rat fundus. In their experiments,
pretreatment with a high concentration of NO donor (105
to 104 M) was used, and the preparations were washed
thoroughly before precontracting with 5HT. In our experiments, circular
muscle strips obtained from gastric body were treated with lower
concentrations of NO donor (3 × 107 M to 3 × 106 M) and were left in the bath during contraction with
5HT. The differences in experimental design may explain the different
results obtained in the two studies. It is conceivable that circular
and longitudinal muscle may react differently to feedback by NO.
Alternatively, given that NO is a gas with a very short half-life, it
is possible that the continued presence of NO is required to inhibit
VIP release.
TS-evoked muscle contraction was significantly reduced by SIN-1 in a
dose-dependent manner. SIN-1 has no effect on carbachol-induced muscle
contraction, so the inhibitory effects of SIN-1 on muscle contraction
should be prejunctional. It is well known that TS-evoked muscle
contraction is atropine-sensitive, and this suggests mediation by ACh
release. Although we did not measure the release of ACh and VIP in
these studies, the functional studies indicate that NO exerts
prejunctional inhibitory effects on cholinergic and VIP-ergic
neurotransmission as well as NO formation in the gastric myenteric
plexus. Our results agree with the observations of Hryhorenko et
al., who have shown that inhibition of NO synthesis enhanced ACh
release during field stimulation and that NO inhibited field-stimulated ACh release and circular muscle contractility of the canine ileum (Hryhorenko et al., 1994).
It is not known how cGMP inhibits NOS activation, resulting in reduced
NO formation in the gastric myenteric plexus. Depolarization-induced release of neurotransmitters from nerve terminals is dependent on the
influx of extracellular Ca++ ions through voltage-sensitive
channels (Reichardt and Kelly, 1983; Augustine et al., 1987;
Miller, 1987; Smith and Augustine, 1988). Ca++ current of
embryonic chick dorsal root ganglia neurons is inhibited by NO,
possibly by a cGMP-dependent mechanism (Ward et al., 1994). On the basis of electrophysiological studies, voltage-sensitive Ca++ channels have been divided into at least three
different types: T, L and N (Nowycky et al., 1985; Fox
et al., 1987; Miller, 1987). In addition to differences in
electrophysiological properties, these three types of voltage-sensitive
Ca++ channels also exhibit different tissue distributions
and pharmacological properties (Tsien et al., 1988;
Yoshikami et al., 1989). Recent experimental evidence
suggests that the predominant voltage-sensitive Ca++
channels associated with the release of neurotransmitters are of the N
type (Nowycky et al., 1985; McClessky et al.,
1987; Hirning et al., 1988; Russel et al., 1990;
Wessler et al., 1990). Boeckxstaens et al., 1993
demonstrated that the release of NO in response to NANC nerve
stimulation is mediated by N-type Ca++ channels in the
canine ileocolic junction. It has been demonstrated that cGMP inhibits
L-type Ca++ channel current through a mechanism that
involves cGMP-dependent protein kinase in rat pinealocytes (Chik
et al., 1995) and rabbit heart cells (Tohse et
al., 1995). NO selectively inhibits voltage-dependent calcium
influx in neuronal cells through a cGMP-dependent mechanism (Desole
et al., 1994). However, it is not clear whether cGMP affects N-type Ca++ channels in the gastric myenteric plexus.
Further studies are needed to sort out the mechanism(s) by which NO
inhibits its own synthesis.
In conclusion, our present studies indicate that NO synthesis in the
gastric myenteric plexus is regulated by NO donors and cGMP. This
suggests an autoregulatory feedback mechanism of NO synthesis. We
propose that NO synthesis and release are prejunctionally inhibited by
NO and that this inhibition is mediated, at least in part, by a cGMP
pathway in the gastric myenteric plexus.
NO, through the cGMP pathway or other additional mechanism(s),
negatively regulates nerve terminal calcium channels, especially the
N-type calcium channels. Inhibition of calcium channels would reduce
calcium entry and thereby reduced NO synthesis, which is calcium-dependent. This inhibitory mechanism is unlikely to be specific
for NO synthesis and is probably applicable to other neurotransmission
systems, such as cholinergic and VIP-ergic transmission in the
myenteric plexus.
8-bromo-cGMP, 8-bromoguanosine 3', 5'-cyclic
monophosphate;
5HT, 5-hydroxytryptamine;
L-NAME, NG-nitro-L-arginine methyl ester;
NANC, nonadrenergic, noncholinergic;
NO, nitric oxide;
NOS, NO synthase;
SIN-1, 3-morpholino-sydnonymide;
SNAP, S-nitroso-N-acetylpenicillamine;
SNP, sodium nitroprusside;
TS, transmural electrical stimulation;
TTX, tetrodotoxin;
VIP, vasoactive intestinal polypeptide;
L-NMMA, NG-monomethyl-L-arginine;
NGF, nerve growth factor;
ODQ, 1H-[1,2,4]oxadiazolo
[3,4-a]quinoxalin-1-one.
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Abstract
Introduction
-Conotoxin: Direct and persistent blockade of specific types of calcium channels in neurons but not muscle.
Proc Natl Acad Sci USA
84:
4327-4331
