Department of Pharmacology, University of Mainz, D-55101 Mainz, Germany
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ABSTRACT |
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Nitric oxide (NO) synthesis was examined in intact longitudinal muscle-myenteric plexus preparations of the guinea pig ileum by determining the formation of [3H]citrulline during incubation with [3H]arginine. Spontaneous [3H]citrulline production after 30 min was 80-90 dpm/mg, which constituted ~1% of the tissue radioactivity. Electrical stimulation (10 Hz) led to a threefold increase in [3H]citrulline formation. Removal of calcium from the medium or addition of NG-nitro-L-arginine strongly inhibited both spontaneous and electrically induced production of [3H]citrulline. TTX reduced the electrically induced but not spontaneous [3H]citrulline formation. The electrically induced formation of [3H]citrulline was diminished by (+)-tubocurarine and mecamylamine and enhanced by scopolamine, which suggests that endogenous ACh inhibits, via muscarinic receptors, and stimulates, via nicotinic receptors, the NO synthesis in the myenteric plexus. The GABAA receptor agonist muscimol and GABA also reduced the electrically evoked formation of [3H]citrulline, whereas baclofen was without effect. Bicuculline antagonized the inhibitory effect of GABA. It is concluded that nitrergic myenteric neurons are equipped with GABAA receptors, which mediate inhibition of NO synthesis.
nitric oxide synthase; -aminobutyric acid; GABAA receptor; nicotinic
receptor
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INTRODUCTION |
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NITRIC OXIDE (NO) is a neurotransmitter of inhibitory nonadrenergic noncholinergic (NANC) nerves in the gastrointestinal tract of various species (for literature, see Ref. 17). Neuronal NO is synthesized by the isoform I of the enzyme NO synthase (6), which has been found in nerve cell bodies and nerve endings in the gastrointestinal tract of guinea pigs (7). The NO synthase-containing neurons constitute a prominent population of enteric neurons, and, in the guinea pig small intestine, ~20% of all myenteric neurons contain NO synthase immunoreactivity (4). The synthesis of neuronal NO is tightly regulated. NO is synthesized on demand from L-arginine when nerve stimulation leads to an increase in the intraneuronal calcium concentration to activate NO synthase. NO is not stored but instead simply diffuses from its site of production (29).
The activity-dependent synthesis and release of NO in the guinea pig small intestine has been studied in functional experiments in which electrical stimulation of the precontracted ileum caused smooth muscle relaxation (22, 27). Evidence to support NO as an inhibitory neurotransmitter was provided from the use of NO synthase inhibitors, which abolished or reduced the relaxations. Another approach to study NO synthase activity uses the citrulline assay, i.e., the conversion of [3H]arginine to [3H]citrulline. This reaction involves the oxidation of a guanidino nitrogen of L-arginine to NO together with the stoichiometric production of L-citrulline (for review, see Ref. 3). With the use of this method, NO synthase activity has been studied in the past in a variety of gastrointestinal preparations of the guinea pig, such as stomach (12), taenia coli (9), and isolated ganglia from the myenteric plexus (8). Surprisingly, no comparable studies have been performed on intact nerve-muscle preparations of the guinea pig small intestine, although this tissue has been widely used as a model system for the analysis of enteric inhibitory and excitatory neurotransmission.
The present investigation has two objectives. First, we aimed to investigate systematically NO synthesis in an intact myenteric plexus-longitudinal muscle preparation under a variety of different conditions using the citrulline assay. Second, we aimed to study whether [3H]citrulline formation is modulated by neurotransmitters of the enteric nervous system, such as ACh and GABA. GABA is colocalized with NO synthase in guinea pig myenteric plexus (28), but the functional significance of such colocalization is not known.
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MATERIALS AND METHODS |
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Guinea pigs of either sex weighing 200-400 g were stunned by a
blow to the head and bled. A 30-cm section of ileum was taken, with the
distal end being 10 cm from the ileocecal junction. Longitudinal muscle-myenteric plexus strips (20-40 mg wet wt) were prepared as
described previously (23). The strips were suspended isometrically under a tension of 5 mN in a 2-ml organ bath and superfused (2 ml/min)
with a physiological salt solution (composition in
mmol/l: 149.3 Na+,
2.7 K+, 1.8 Ca2+, 1.05 Mg2+, 145.4 Cl, 11.9 HCO
3, 0.42 H2PO
4, and 5.6 D-glucose) of 37°C that was
bubbled with a mixture of 95% O2
and 5% CO2.
L-Arginine (10 µM) was added
to the solution, since it was shown that NO synthase activity in the
intestine increases with the
L-arginine concentration,
reaching a maximum at ~10 µM (18). After an equilibration period of
45 min, superfusion was stopped and the tissue was incubated for 30 min
(in a few experiments for 10 or 45 min) with
[3H]arginine (0.5 µCi/ml). During the incubation with
[3H]arginine, the
tissue was stimulated by electrical field stimulation using Grass S6
stimulators. Square wave pulses of 1 ms duration and 10 or 30 Hz were
applied intermittently (10-s stimulation periods at intervals of 30 s)
by two platinum electrodes that were positioned parallel to the strips
(distance of 0.6 cm). The applied current was 300-320 mA,
depending on the individual stimulator, and was controlled by
monitoring the potential drop over a 20
resistance. At the end of
the incubation, the strips were blotted, weighed, and homogenized with
an Ultraturrax in ice-cold 0.4 M HClO4 (1.0 ml). After
centrifugation at 1,000 g for 15 min,
aliquots of the supernatant (200 µl) were analyzed by HPLC according
to the method described recently (1). The
3H-labeled compounds
([3H]citrulline,
[3H]ornithine, and
[3H]arginine) in the
tissue extract were separated on a reverse-phase column (length of 250 mm, inner diameter of 4.6 mm, prepacked with 5 µm Shandon
ODS-Hypersil; Bischoff Chromatography, Leonberg, Germany) using as a
mobile phase 0.1 M phosphate buffer (adjusted to pH 1.8), which
contained octane sulfonic acid sodium salt (300 mg/l), sodium EDTA (0.3 mM), and methanol (4.2% vol/vol). The eluate was collected in 30-s
fractions into counting vials, and the radioactivity was determined by
liquid scintillation spectrometry (Packard Tricarb 4430). External
standardization was used to correct for counting efficiency.
[3H]citrulline eluted
from the column after ~8 min, and
[3H]ornithine and
[3H]arginine eluted
after ~10 and 80 min, respectively. The identity of the eluted
material as
[3H]citrulline was
established by regularly adding
[14C]citrulline to the
tissue extracts. The amount of
[3H]citrulline in the
sample was taken to represent the stoichiometric production of NO, and
the formation of
[3H]citrulline was
computed as dpm per milligram tissue. Drugs were added either 30 min
before addition of
[3H]arginine or
together with
[3H]arginine. The
effects of drugs on the electrically induced
[3H]citrulline
formation are given as percent increases above basal levels measured in
parallel under resting conditions.
Data analysis. Results are expressed as means ± SE. The significance of difference between two mean values was assessed using Student's t-test. For comparison of one control with several experimental groups, the significance of difference was estimated by one-way ANOVA followed by Dunnett's test.
Drugs.
L-[2,3-3H]arginine
HCl (38.5 Ci/mmol),
L-[ureido-14C]citrulline
(57.8 Ci/mmol),
L-[1-14C]ornithine
(40-60 Ci/mmol) were from NEN (Dreieich, Germany). ()-Bicuculline methiodide was from Research Biochemicals
International (Natick, MA).
L-Arginine hydrochloride,
(±)-baclofen,
NG-nitro-L-arginine,
mecamylamine hydrochloride, muscimol, oxotremorine sesquifumarate,
scopolamine hydrobromide, and TTX were from Sigma (Munich, Germany).
GABA and (+)-tubocurarine chloride pentahydrate were from Fluka (Buchs,
Switzerland). Drugs were dissolved in distilled water.
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RESULTS |
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Formation of [3H]citrulline.
Incubation of the longitudinal muscle-myenteric plexus preparation with
[3H]arginine led to a
spontaneous formation of
[3H]citrulline.
[3H]ornithine was not
detected. Spontaneous
[3H]citrulline
production after 30 min was 89 ± 4 dpm/mg
(n = 13), which accounted for 1.1 ± 0.05% of the total
[3H]radioactivity in
the tissue. Intermittent field stimulation at 10 Hz for 30 min led to a
threefold increase in the
[3H]citrulline content
(276 ± 21 dpm/mg; n = 13), which
accounted for 3.0 ± 0.26% of the tritium content of the tissue.
Increasing the stimulation period to 45 min did not further
significantly enhance the
[3H]citrulline content
(Fig. 1). Moreover, there was no further increase of
[3H]citrulline in
response to stimulation at 30 Hz (30 min). Therefore, in all subsequent
experiments, the strips were stimulated at 10 Hz for 30 min.
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Effects of cholinergic agonists and antagonists.
[3H]citrulline
formation under resting conditions was not significantly changed by the
muscarinic agonist oxotremorine or the antagonist scopolamine (Table
1). However, scopolamine
significantly enhanced
[3H]citrulline
production during electrical stimulation (Fig.
3, top),
whereas oxotremorine did not modify the stimulation-evoked [3H]citrulline
formation.
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GABAergic inhibition of
[3H]citrulline formation.
GABA (10 and 100 µM) inhibited the electrically induced formation of
[3H]citrulline by 43 and 78%, respectively (Fig. 4). Similarly, the GABAA agonist muscimol (10 µM) reduced the stimulation-evoked production of
[3H]citrulline,
whereas the GABAB agonist baclofen
(10 µM) was without effect. Neither drug had an effect on spontaneous
[3H]citrulline
production (Table 2).
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DISCUSSION |
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Our study shows that NO synthase can easily be measured in the myenteric plexus-longitudinal muscle preparation of the guinea pig ileum by the conversion of [3H]arginine to [3H]citrulline. This method has recently been used to measure NO synthase activity in the rat myenteric plexus-longitudinal muscle preparation (1). The [3H]citrulline formation under resting conditions and during field stimulation was nearly prevented by the NO synthase inhibitor NG-nitro-L-arginine, which indicates that this [3H]citrulline originates specifically from the NO synthase reaction. Moreover, spontaneous and electrically induced formation of [3H]citrulline was largely inhibited by removal of extracellular calcium, suggesting that the NO synthase was of the constitutive form, as neuronal NO synthase is known to be.
The electrically induced formation of [3H]citrulline was significantly reduced but not abolished by TTX, which implies that a component of the [3H]citrulline formation is not due to the sodium-dependent neuronal action potential propagation. A similar result has been reported in studies on canine enteric neurons, in which the NO release evoked by nicotinic receptor stimulation was only partly TTX sensitive (24). We suppose that strong electrical currents of 300 mA depolarize the entire neuron well beyond its threshold even in the presence of TTX. This leads to increased intracellular calcium concentrations and hence increased NO synthesis in all parts of the neuron. NO synthase is present in the whole nerve cell of guinea pig myenteric neurons (19), and electrical stimulation releases NO not only from the nerve terminal but also from the axon and soma (26). Such direct effects of electrical field stimulation at high current strength on nerves of the autonomic nervous system are well known. Illes et al. (11), for instance, showed that field stimulation of sufficient strength caused a TTX-resistant but calcium-dependent release of norepinephrine from the mouse vas deferens. A complete inhibition of the electrically induced NO synthesis and release was reported for other isolated gastrointestinal preparations, but in these studies both current strength and pulse duration were submaximal compared with the present experiments (in Ref. 1, rat myenteric plexus-longitudinal muscle strips were 240 mA and 0.4 ms; in Ref. 5, rat gastric fundus strips were 120 mA and 1 ms, respectively). The spontaneous [3H]citrulline production was not modified by TTX. Thus there is probably a spontaneous, stimulus-independent activity of NO synthase in guinea pig myenteric neurons similar to that in the rat small intestine (1).
The production of [3H]citrulline was strongly impaired by (+)-tubocurarine and mecamylamine. This finding was not unexpected, since previous studies showed that the nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium iodide stimulated [3H]citrulline formation in myenteric ganglia from guinea pig small intestine (8) and increased neuronal NO synthase mRNA expression in rat gastric myenteric ganglia (20). Nicotinic receptors have recently been detected on the soma of NO synthase-immunoreactive neurons in the myenteric plexus of guinea pig small intestine (13). These findings together with those in this study suggest that nitrergic neurons are equipped with nicotinic receptors whose stimulation by endogenous ACh enhances NO synthesis.
Scopolamine increased the formation of [3H]citrulline, which indicates that NO synthesis is inhibited by endogenous ACh via muscarinic receptors. Oxotremorine caused only a slight but statistically not significant reduction in [3H]citrulline production. It is possible that the inhibitory muscarinic receptors were already maximally activated by endogenous ACh released during field stimulation so that oxotremorine could not exert the expected inhibition. A similar muscarinic receptor-mediated inhibition of [3H]citrulline synthesis has been reported in studies on rat myenteric plexus preparation (1). Thus ACh may enhance (via nicotinic receptors) and diminish (via muscarinic receptors) NO synthesis and release in the small intestine.
The most interesting finding of the present study is the inhibition of NO synthesis through GABA. The effect of GABA was mimicked by muscimol, but not by baclofen, and was antagonized by bicuculline, which indicates that the inhibition was mediated by GABAA receptors. Another neurotransmitter whose release from guinea pig myenteric neurons is inhibited via GABAA receptors is GABA itself (25). A prejunctional location of these GABAA autoreceptors is suggested by the finding that the GABAA agonist muscimol inhibited the release of [3H]GABA evoked by high potassium in the presence of TTX, i.e., when axonal conduction was blocked (25). Although our study gives no information on the location of the GABAA receptors inhibiting NO synthesis, it seems likely that they are located on nerve terminals of nitrergic neurons. In experiments on the rat colon, GABAA receptor immunoreactivity was found on cell somata of NO synthase-positive neurons but not on nerve fibers (16). There might thus be a species difference, since the above-mentioned [3H]GABA release experiments suggest the occurrence of prejunctional GABAA receptors on guinea pig myenteric neurons (25). GABA is costored with NO in a subpopulation (~20%) of nitrergic neurons, which are inhibitory motoneurons supplying the longitudinal and circular muscles of the guinea pig small intestine (28). If the GABAA receptors are located at these neurons, they may be regarded as autoreceptors, which inhibit not only the synthesis and subsequent release of NO but also the release of the cotransmitter GABA.
The actions of exogenous GABA on gastrointestinal motility are complex and comprise stimulation and inhibition of the release of both ACh and NO. GABA, via presynaptic GABAB receptors, reduces motility through inhibition of the electrically evoked ACh release (14). In contrast, GABA, via GABAA receptors, may stimulate motility, since GABAA receptor blockade reduces peristalsis (21). This excitatory effect can be attributed to the stimulation of cholinergic neurons by GABAA receptors located on the neuronal cell somata (14). A similar biphasic GABAergic modulation probably exists for the release of NO from myenteric neurons. On the one hand, GABA causes NANC relaxations in the ileum of dogs (2) and guinea pigs (15) via activation of GABAA receptors, which presumably are located on the cell soma of nitrergic neurons. On the other hand, the present study shows that GABA inhibits NO formation through GABAA receptors, which may be localized prejunctionally on nitrergic neurons. The inhibition of NO synthesis and release may, in turn, lead to an increase of intestinal motility.
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ACKNOWLEDGEMENTS |
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We thank M. Doris Erbelding for expert technical assistance.
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FOOTNOTES |
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This study was supported by the Deutsche Forschungsgemeinschaft (Ki 210/8).
Part of this work has been communicated to the German Pharmacological Society (10).
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 and other correspondence: H. Kilbinger, Dept. of Pharmacology, Univ. of Mainz, D-55101 Mainz, Germany (E-mail: KILBINGE{at}mail.uni-mainz.de).
Received 15 September 1998; accepted in final form 7 December 1998.
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