Department of Internal Medicine II, Technical University of Munich, 81675 Munich, Germany
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ABSTRACT |
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The exact mechanisms controlling nitric oxide
synthase (NOS) activity within enteric neurons are largely unknown. In
this study, the effect of exogenous nitric oxide (NO) on NOS activity was investigated in enteric synaptosomes of rat ileum.
3-Morpholinosydnonimine (SIN-1;
104 M) and
S-nitroso-N-acetylpenicillamine
(SNAP; 10
4 M) significantly
inhibited NOS activity by 53% and 48%, respectively. However,
superoxide dismutase (SOD; 160 U/ml) as well as the NO scavenger
oxyhemoglobin (10
3 M) did
not influence NO donor-induced inhibition. In contrast, the inhibitory
effect was antagonized by diethyldithiocarbamate (3 × 10
4 M), an inhibitor of
endogenous Cu/Zn SOD. Inhibition of NOS by exogenous NO was dependent
on glutathione (GSH), since the inhibitory effect was augmented in the
presence of GSH (5 × 10
4 M) and antagonized by
the GSH-depletor
DL-buthionine-SR-sulfoximine (5 × 10
4 M),
suggesting that NO might be protected from extracellular breakdown by
reaction with GSH. The reaction product of SIN-1/SNAP and GSH was
identified as a nitrosothiol. In the presence of the Cu+-chelator neocuproine
(10
5 M), inhibition of NOS
by SNAP/SIN-1 was reversed, suggesting that nitrosothiol formation is
intermediary. These findings are indicative of a feedback inhibition of
enteric NOS, presumably via formation of a nitrosothiol intermediate.
nitric oxide synthase; enteric nervous system; rat ileum; S-nitroso-N-acetylpenicillamine; 3-morpholinosydnonimine
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INTRODUCTION |
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NITRIC OXIDE SYNTHASE (NOS) is localized in neuronal cell bodies and nerve fibers throughout the entire digestive tract of various species like rats (2, 6), guinea pigs (12), and humans (7). NOS immunoreactivity was demonstrated in inhibitory motoneurons and descending interneurons of the enteric nervous system (7, 12) and in synaptosomal preparations of the canine ileal myenteric plexus (27) and rat small intestine (21). Release of nitric oxide (NO) has been quantified in canine enteric neurons in vitro (42) and in rabbit intestinal tract in vivo (25). Endogenous NO mediates nonadrenergic noncholinergic (NANC) transmission in the gastrointestinal tract (10), causing tonic inhibition of intestinal smooth muscle (14). Exogenous NO mimics this relaxatory effect but, on the other hand, attenuates the relaxation induced by endogenous NO (15). Because the postjunctional response to authentic NO was not altered, it was suggested that NO exerts a prejunctional inhibitory effect on the nitrergic component of NANC innervation (15). Further evidence of an inhibitory effect of exogenous NO on NO synthesis comes from whole organ studies of the rat stomach (20). The subcellular mechanisms regulating neuronal NOS (nNOS) activity are not yet completely understood. An autoregulatory mechanism of the NO pathway has been postulated in subcellular preparations from the central nervous system (18), whereas no data on enteric neurons exist (36, 37, 45). Preparations of synaptosomes, pinched off nerve terminals, which exhibit the ability to reseal and form a discrete particle under special experimental conditions, have been shown to be a valuable tool for studying presynaptic cellular and subcellular mechanisms of transmitter release in the enteric nervous system without the neuronal network present in vivo or in the intact isolated organ (3, 28). In previous experiments, nNOS has been demonstrated and biochemically characterized in synaptosomes from rat small intestine (21). With respect to the above outlined functional data, suggesting an autoinhibitory pathway in the enteric nervous system (15, 20), this study was undertaken to investigate a possible inhibitory effect of exogenous NO on NOS activity in enteric synaptosomes and to further characterize the possible mechanisms of action.
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MATERIALS AND METHODS |
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Tissue handling and membrane preparation. Isolation of synaptosomes was performed as described previously (28). Briefly, male Wistar rats (250-350 g) were killed by cervical dislocation, and the small intestine was quickly removed and suspended in ice-cold buffer (20 mM MOPS, 10 mM MgCl2, and 8% wt/vol sucrose, pH 7.4). All further preparative steps were carried out at 0-4°C. The ileum was dissected, cleaned of mesenteric arcade and fat, and opened along the mesenteric attachment line. The mucosal layer was removed. For membrane preparation, the tissue was resuspended in isolation buffer consisting of 8% wt/vol sucrose, 20 mM MOPS, 10 mM MgCl2, 10 mM pepstatin A, 100 µM thiorphan, 50 µM phenylmethylsulfonyl fluoride (PMSF), 1 mM dithiothreitol, 500 µM trypsin inhibitor, and 1 mM captopril, minced with scissors, and homogenized with a Polytron PT20 homogenizer at an ~1,500 rpm setting for 15 s (3 × 5 s). The tissue homogenate was centrifuged in two steps of 800 g for 10 min. The supernatant was collected [postnuclear supernatant (PNS)] and recentrifuged at 3,500 g for 10 min to obtain the crude synaptosomal fraction. The supernatant after this spin was centrifuged at 100,000 g for 90 min. The pellet from this spin (named MIC1) was resuspended and centrifuged again at 10,000 g for 10 min. The resulting pellet and the supernatant from then on were referred to as the enriched synaptosomal fraction (P2) and MIC2, respectively.
[3H]saxitoxin binding assay. [3H]saxitoxin binding was performed as described previously (3) at 25°C for 15 min in a medium consisting of 50 mM Tris · HCl, pH 7.4, containing 0.2% BSA with 1.0 nM of the radioligand [3H]saxitoxin (17-46 Ci/mmol), which was approximately equivalent to the dissociation constant value. All experiments were carried out in triplicate. In the range from 10 to 200 µg of protein per assay tube, [3H]saxitoxin binding showed a linear relationship to the amount of protein used.
5' Nucleotidase and Mg2+-ATPase assay. The specific activity of 5' nucleotidase was assayed by incubation of the respective fractions (20-50 µg protein) for 30 min at 37°C in 50 mM imidazole buffer (pH 7.0) containing 5 mM AMP sodium salt and 5 mg MgCl2. The reaction was stopped by adding ice-cold TCA. The precipitated protein was sedimented by centrifugation, and the liberated phosphate was determined. The activity of Mg2+-ATPase was assayed by incubation in an imidazol buffer containing Na2ATP and 0.1 M MgCl2 at pH 7.2. The further steps were performed according to the above-described procedure for the 5' nucleotidase assay.
Protein assay.
Protein was measured spectrophotometrically according to the method of
Bradford (8). Bovine serum -globulin was used as the standard.
Assay of occluded lactate dehydrogenase. Lactate dehydrogenase (LDH) was determined by a commercially available assay kit (MPR1 Boeringer, Mannheim, Germany). After preincubation of membrane preparations for 5 min at 4°C with 0.1% Triton X-100 to allow excess of synaptosomal cytoplasm, the synaptosomes were centrifuged at 10,000 g for 10 min. Untreated synaptosomal fractions served as control. Extinction was measured in an Eppendorf photometer at 334 nm (37°C).
Smooth muscle relaxation and nitrite generation by NO donors in perfused ileum. A segment of ileum (2-3 cm length) was carefully dissected and placed in a 3-ml organ bath filled with Krebs buffer (in mM: 115.5 NaCl, 1.16 MgSO4, 1.16 NaH2PO4, 11.1 glucose, 21.9 NaHCO3, 2.5 CaCl2, and 4.16 mM KCl, gassed with 95% O2 and 5% CO2) maintained at 37°C. After an equilibration period of 30 min, the ileal segment was precontracted with 1 µM carbachol and concentration-response curves for the NO donors 3-morpholinosydnonimine (SIN-1) and S-nitroso-N-acetylpenicillamine (SNAP) were carried out. Results were expressed as percent relaxation of a carbachol-induced contraction of rat ileum.
Nitrite generation by the respective NO donors was assayed using the Griess assay as modified by Schmidt et al. (40). The superfusate was collected after addition of the respective NO donors. Diazotization was initiated by addition of sulfanilamide (1 mM) and HCl (0.1 N). The samples were centrifuged (1,000 g for 15 min at 4°C). The absorbance of the supernatant was measured at 548 nm. As a final step, samples were incubated with 1 mM N-(1-naphthyl)ethylenediamine dihydrochloride (NEDA) for 10 min at room temperature. The absorbances were determined again, and the difference between pre- and post-NEDA absorbance yielded the total NO2 present in the sample. Baseline absorbance was a mixture of buffer solution, HCl, sulfanilic acid, and NEDA.Assay of NOS activity. NOS activity was determined by monitoring the formation of L-citrulline from L-arginine by a modification of methods described previously (9). Enzymatic reactions were conducted at 37°C in 25 mM MOPS-8% sucrose buffer containing 1 mM NADPH, 0.1 µM tetrahydrobiopterin, 1 µM calmodulin, 1 mM CaCl2, 0.1 µM FAD, 0.1 µM flavin mononucleotide (FMN), and other test agents as specified later, in a final incubation volume of 750 µl. The L-[3H]arginine was purified by anionic exchange chromatography on columns of Dowex 1-X8, OH form, to remove traces of L-[3H]citrulline. After preincubation for 30 min at 4°C with synaptosomes (500 µl), the enzymatic reactions were started by adding ~500,000 dpm of L-[3H]arginine (63 Ci/mmol) and terminated after 15 min by immediately heating to 90°C for 6 min and adding 1 ml of distilled water containing 1 mM L-arginine and 1 mM L-citrulline. Samples were applied to columns containing 1 ml of Dowex AG50W-X8 resin, Na+ form, preequilibrated with sucrose MOPS buffer. The eluate (2 ml) was collected in a liquid scintillation vial. After addition of 1.5 ml of scintillation fluid, samples were counted in a Beckman LS 3801 spectrometer. The recovery of L-[3H]citrulline in the first 4 ml of the eluate was ~92%, and contamination with L-[3H]arginine did not exceed 2%. Basal values were obtained by heating samples to 100°C for 5 min before incubation.
S-nitrosothiol determinations.
The Saville method was used for the measurement of
S-nitrosothiols (38). Synaptosomes
were incubated for 5 min with SIN-1 (104 M) plus glutathione
(GSH; 5 × 10
4 M) or
SNAP (10
4 M) plus GSH
(5 × 10
4
M). The sample (50 µl) was reacted for 5 min with an equivalent volume of solution A (1%
sulfanilamide dissolved in 0.5 M HCl) or solution
B (solution A plus
0.2% HgCl2), allowing for the
formation of the diazonium salt. The formation of the azo dye product
was obtained by reacting the two samples for an additional 5 min with an equal volume of solution C (0.02%
NEDA dissolved in 0.5 M HCl), and the absorbance was subsequently read
at 540 nm with a Bio-Rad microplate reader. To counteract background
nitrite concentration, an equal volume of 0.5% ammonium sulfamate in
water was added to the samples 5 min before the addition of
sulfanilamide. S-nitrosothiols were
quantified as the difference of absorbance between
solutions B and
A.
S-nitrosoglutathione (GSNO) was used
as standard.
Assay of cGMP content. The amount of cGMP in the synaptosomal fractions was determined by RIA using a commercial kit (Amersham). The incubation mixtures consisted of 300 µl of synaptosomal fraction suspended in the isolation buffer (resulting in final concentrations of 15 mM MOPS, 188 mM sucrose, 0.67 mM MgCl2, and 300 µg of synaptosomes) and were supplemented by 0.1 mM zaprinast and amounts of other exogenous compounds as indicated in the text. After incubation for 8 min at 37°C, the reaction was terminated by instant heating of the samples (90°C, 5 min), followed by rapid cooling and centrifugation in a refrigerated centrifuge (10,000 g, 10 min); 50 µl of supernatant were then used for the RIA of cGMP following the procedure provided by the manufacturer.
Western blot analysis of superoxide dismutase. The synaptosomal protein fraction (200 µg protein) was separated by SDS-PAGE 7.5% gels in a Bio-Rad minigel apparatus (29) and blotted onto a polyvinylidene difluoride membrane (Bio-Rad, Munich, Germany) using Tris-glycine-SDS buffer (50 mM Tris, 380 mM glycine, 0.05% SDS, and 20% methanol). After the membrane was blocked overnight with 5% dry milk, blots were probed for 2 h at room temperature with a polyclonal antibody specific for human Cu/Zn superoxide dismutase (SOD; antibody dilution of 1:1,000, Biodesign International, Kennebunk, ME). As secondary antibody, horseradish peroxidase-linked donkey anti-sheep IgG was used and detection was performed by enhanced chemiluminescence (Amersham, Braunschweig, Germany).
Drugs. The labeled [3H]saxitoxin (17.1-46.0 Ci/mmol) and L-[3H]arginine (63 Ci/mmol) were purchased from Amersham. Sulfanilamide, sulfanilic acid, NEDA, NADPH, FAD, FMN, citrulline, calmodulin, SOD, pyrogallol, pepstatin A, dithiothreitol, trypsin inhibitor, 5,5'-dimethyl-1-pyrroline N-oxide (DMPO), DL-buthionine-SR-sulfoximine (BSO), and cuprizon were from Sigma (Munich, Germany). Tetrahydrobiopterin was from ICN Biomedicals (Eschwege, Germany). SNAP, Mn(III)tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP), carboxy 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO), and GSNO were from Calbiochem (Bad Soden, Germany); 8-(4-chlorophenylthio)cGMP (CPT-cGMP) was from Biolog Life Science Institute (Bremen, Germany); and SIN-1 was from Casella-Riedel (Frankfurt, Germany). 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and zaprinast were from Tocris Cookson (Langford, UK); thiorphan, ascorbate, and sodium bicarbonate were from Fluka (Munich, Germany); and PMSF was from Serva (Heidelberg, Germany). N-acetyl-L-cysteine (NAC), catalase, GSH, diethyldithiocarbamate (DETC), and neocuproine were from Merck (Darmstadt, Germany). Adequate controls were performed with the vehicles used for solubilizing each reagent.
Statistics.
Data are given as means ± SE; n
indicates the number of independent observations in separate
experiments on separate preparations. For each value of a given drug of
a single preparation, the release study was carried out in duplicate.
When statistical difference of two means was determined, a paired
two-tailed Student's t-test was
performed. For multiple comparisons, ANOVA, followed by a post hoc test
with Bonferroni-Holm procedure for multiple testings, was carried out
to determine statistical differences. Values of P 0.05 were considered significant.
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RESULTS |
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NO donor-induced NO generation and smooth muscle relaxation.
The amount of NO generated by SNAP and SIN-1 in rat synaptosomes was
measured as nitrite production and was 1.8 ± 0.02 µM for 1 µM
SNAP, 2.4 ± 0.03 µM for 10 µM SNAP, and 8.2 ± 0.04 µM for 100 µM SNAP. Concentrations of SIN-1, covering the
range 1-100 µM, corresponded to a NO concentration range of
0.4-2.0 µM NO. With regard to smooth muscle contraction, no
effect of the respective NO donors was detectable up to the
concentration of 106 M. Thereafter, a significant response was observed with SIN-1, inducing a
30% relaxation of carbachol-induced precontraction at a concentration
of 10
4 M. The more
effective substance was SNAP, which elicited a significant response by
an almost 60% relaxation of carbachol-induced precontraction at a
concentration of 10
4 M.
Assessment of subcellular fractionation and synaptosomal integrity.
The relative enrichment of axonal varicosities in the synaptosomal
fraction (P2) was based on the assessment of an eightfold-enriched activity of
[3H]saxitoxin binding,
a marker of neuronal plasma membranes, over PNS. On the other hand, the
microsomal fraction (MIC2) showed threefold-enriched activities of the
5' nucleotidase and
Mg2+-ATPase over PNS, whereas
[3H]saxitoxin binding
was significantly less than in the P2 fraction (Table
1). The functional integrity
of the synaptosomes was assessed by the occluded LDH assay. LDH release
from SNAP- or SIN-1-treated synaptosomes was 54.3 ± 3.8 U/l (94 ± 6%) and 55.0 ± 3.2 U/l (95 ± 5%), respectively, which
was not significantly changed compared with control [55.8 ± 4.0 U/l (100%), n = 4],
suggesting that exposure to SNAP or SIN-1 does not result in membrane
damage.
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NOS activity in synaptosomal fractions.
In the present experiments, the rate of
L-[3H]citrulline
formation in the presence of cofactors (1 mM NADPH, 0.1 µM
tetrahydrobiopterin, 1 µM calmodulin, 1 mM
CaCl2, 0.1 µM FAD, and 0.1 µM
FMN) was 34.7 ± 6.1 fmol · mg1 · min
1,
equivalent to a 4.1 ± 0.5-fold stimulation over basal activity (8.4 ± 2.2 fmol · mg
1 · min
1;
n = 6, P < 0.05). In all further
experiments, cofactor-substituted NOS activity was referred to as
control. L-Citrulline formation was dependent on synaptosomal integrity, since net conversion was
markedly attenuated, when assays were conducted with osmotically shocked or ultrasound-treated synaptosomes before use.
Effect of NO donors on NOS activity in enteric synaptosomes.
In the presence of NO donors, the NOS activity was significantly
inhibited. SNAP inhibited
L-citrulline formation by 48%
at 104 M
(P < 0.05, n = 6) and by almost 83% at
10
3 M
(P < 0.01, n = 6) (Fig.
1). The inhibitory effect observed with
SIN-1 was comparable, resulting in a 53% inhibition at
10
4 M
(P < 0.05, n = 6) and a 58% inhibition at
10
3 M
(P < 0.05, n = 6) (Fig. 1). GSNO also
significantly inhibited NOS by 47% at a concentration of
10
4 M
(P < 0.05, n = 4) (Fig. 1).
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Effect of exogenous SOD or inhibition of endogenous Cu/Zn SOD. Because NO scavengers did not antagonize the inhibitory effect of SNAP on NOS, it was suggested that the effect might be mediated by a by-product of NO catabolism. Because NO combines rapidly with superoxide to form peroxynitrite, the effect of removal of superoxide anions by SOD was investigated in another series of experiments. Both the exogenous supply of SOD and the inhibition of a putative endogenous Cu/Zn SOD by the specific inhibitor DETC were studied.
SOD (160 U/ml) had no significant inhibitory effect on NOS activity (control: 36.8 ± 1.9 fmol · mg
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Western blot analysis of SOD.
With respect to the functional data suggesting a modulatory role of a
synaptosomal SOD, Western blot analysis was performed to demonstrate
the presence of this enzyme in rat enteric nerve terminals. The
positive control of Cu/Zn SOD comigrated with a band at ~16 kDa in
the synaptosomal fraction, specific for Cu/Zn SOD (Fig.
4).
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Effect of superoxide anion generation and peroxynitrite scavengers.
Because the involvement of peroxynitrite in the inhibitory effect of
SNAP or SIN-1 could not be precluded, additional series of experiments
were undertaken. When pyrogallol, a superoxide anion
generator, was added together with SNAP, the inhibition of
NOS was not influenced (control: 35.9 ± 3.6 fmol · mg1 · min
1,
10
4 M SNAP: 27.8 ± 3.3 fmol · mg
1 · min
1,
10
5 M SNAP + pyrogallol:
22.6 ± 3.4 fmol · mg
1 · min
1,
n = 3). In addition, both the
peroxynitrite scavenger MnTBAP and sodium bicarbonate, which modifies
reactivity of peroxynitrite by rapid reaction with
CO2, failed to antagonize the
inhibitory effect of SNAP
(10
4 M SNAP: 29.2 ± 3.8 fmol · mg
1 · min
1,
SNAP + 10
4 M MnTBAP: 26.2 ± 10.8 fmol · mg
1 · min
1,
n = 5;
10
4 M SNAP: 18.2 ± 4.0 fmol · mg
1 · min
1,
SNAP + sodium bicarbonate: 15.6 ± 3.2 fmol · mg
1 · min
1,
n = 3), rendering the involvement of
peroxynitrite less likely. Comparable data were obtained with SIN-1
(data not shown). Because superoxide generation and concomitant
peroxynitrite formation have been proposed to occur in
tetrahydrobiopterin-depleted cell systems, the effect of omission of
exogenous tetrahydrobiopterin was investigated. No significant
difference could be obtained between synaptosomes supplemented by
tetrahydrobiopterin and those that were not (with tetrahydrobiopterin:
35.3 ± 5.6 fmol · mg
1 · min
1;
without tetrahydrobiopterin: 28.0 ± 6.4 fmol · mg
1 · min
1,
n = 3).
Effect of GSH and reducing agents.
Because peroxynitrite does not seem to be involved in the SNAP- or
SIN-1-induced inhibition of NOS, we inquired about the role of
S-nitrosothiols as an alternative
transport form of NO resistance to oxyhemoglobin inactivation. In the
presence of exogenous GSH, the inhibition of NOS induced by SNAP or
SIN-1 was further augmented (Fig. 5),
whereas ascorbate was ineffective (SNAP + 103 M ascorbate: 24.4 ± 4.6 fmol · mg
1 · min
1,
n = 4). GSH at a higher concentration
did not influence NO-induced inhibition (data not shown), and the
substance itself did not significantly attenuate basal NOS activity
(Fig. 5). However, when cofactor-stimulated NOS was incubated in the
presence
Cu(NO3)2 and GSH, a significant inhibition was observed compared with treatment with GSH or
Cu(NO3)2
alone [control: 37.8 ± 4.8 fmol · mg
1 · min
1,
with 10
5 M
Cu(NO3)2:
34.8 ± 2.5 fmol · mg
1 · min
1,
GSH + 10
5 M
Cu(NO3)2:
21.9 ± 6.7 fmol · mg
1 · min
1;
n = 3, P < 0.05].
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Formation of S-nitrosothiols in the
presence of SNAP/SIN-1 and GSH.
Because the foregoing data suggest formation of
S-nitrosothiols as the products of the
reaction between NO and GSH, it appeared to be necessary to establish
whether the conditions of our experiments were favorable for the
formation of these intermediates. In these experiments, the
concentration chosen for GSH is the same as that that resulted in a
marked increase of NOS inhibition in the presence of exogenous NO.
Synaptosomes showed S-nitrosothiol
formation following treatment with NO donors in the presence of GSH
according to the Saville method (control: 1.8 ± 0.5 µM,
104 M SNAP + 5 × 10
4 M GSH: 3.5 ± 0.4 µM, 10
4 M SIN-1 + 5 × 10
4 M GSH: 9.0 ± 1.0 µM; P < 0.05 compared
with basal, respectively, n = 4) after
a 5-min incubation period. The data obtained by SNAP require a
different interpretation, since SNAP is a
S-nitrosothiol and as such it is not
clear whether we measured SNAP or the thionitrite produced by the
reaction between the NO donor and GSH. When
Cu(NO3)2 was added, the amount of
S-nitrosothiols was significantly
reduced compared with NO donor plus GSH treatment
[10
4 M SNAP + 5 × 10
4 M GSH + Cu(NO3)2:
2.4 ± 0.4 µM, 10
4 M
SIN-1 + 5 × 10
4 M GSH + Cu(NO3)2:
6.7 ± 0.5 µM; P < 0.05, n = 4], indicating metal-catalyzed release of NO.
Effect of copper chelators.
To clarify the findings indicative of an involvement of a nitrosothiol
in the inhibition of NOS by NO donors, another series of experiments
was carried out with the
Cu+-specific chelator neocuproine
and the Cu2+-specific chelator
cuprizon. Neocuproine antagonized the inhibition of NOS by SNAP/SIN-1
(control: 33.8 ± 0.7 fmol · mg1 · min
1,
10
4 M SNAP: 21.9 ± 1.5 fmol · mg
1 · min
1,
SNAP + 10
5 M neocuproine:
33.2 ± 2.5 fmol · mg
1 · min
1;
P < 0.05, n = 6), whereas cuprizon
(10
5 M) was ineffective
(data not shown). These data suggest that nitrosothiol formation is
intermediary and Cu+-induced
liberation of NO in the presence of thiols presumably accounts for the effect.
Effect of NO donors on cGMP generation in enteric synaptosomes. In view of the quantitative NO production by the respective NO donors, it appeared to be of substantial interest to examine whether cGMP formation is stimulated by addition of exogenous NO. These additional experiments were carried out in enteric synaptosomes.
The cGMP content in synaptosomes was stimulated by increasing concentrations of all NO donors tested. A maximal stimulatory effect of all compounds occurred at a concentration of 10Effect of the cGMP analog CPT-cGMP and ODQ.
Additional series of experiments were carried out to determine whether
the SNAP-induced inhibition of NOS activity was mediated by a direct or
indirect mechanism. The membrane-permeable cGMP analog CPT-cGMP failed
to significantly inhibit NOS activity even at a submaximal
concentration of 103 M
(Fig. 7).
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DISCUSSION |
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The present data provide direct evidence that NO from exogenous sources can inhibit endogenous synthesis of the putative neurotransmitter NO within enteric synaptosomes by the nNOS. These results extend earlier findings in functional studies that suggest a prejunctional modulatory effect of NO on NANC nerves in the rat gastrointestinal tract, which could be either stimulatory (3, 17) or inhibitory (15, 20). Due to the observation that a reproducible quantitative isolation of nerve terminals from rat stomach could not be established in earlier experiments compared with ileal tissue (28), we used ileal synaptosomes to further characterize the effect of exogenous NO on the nNOS activity. In a first set of experiments, both ileal smooth muscle relaxation and NO production by the respective NO donors SIN-1 and SNAP were studied in isolated ileum. A positive correlation between the ability to generate NO and to relax smooth muscle was observed with both compounds. Because SIN-1 was less potent in relaxing smooth muscle and generating NO than SNAP, but similarly effective in inhibiting NOS, it was suggested that even small amounts of NO present elicit significant NOS inhibition or that additional mechanisms not related to free NO might play a role (23).
The NO donor-induced inhibition was not reversed in the presence of the NO scavengers carboxy-PTIO or oxyhemoglobin, suggesting that the effect was not mediated by free NO. Because SOD acts by removing superoxide anions from the medium, thereby sustaining NO-mediated effects, both exogenous SOD and DETC, an inhibitor of the endogenous Cu/Zn SOD, were studied. Our data demonstrate that exogenous supply of SOD did not significantly influence basal NOS activity and inhibition of NOS by exogenous NO. However, the finding that DETC, an inhibitor of the endogenous SOD, antagonized the SNAP-induced inhibition of NOS activity in our experiments supports the view that an intracellularly located Cu/Zn SOD is operating. DETC irreversibly inhibits Cu/Zn SOD; thus the observation that the effect could not be antagonized by addition of SOD in our experiments suggests that SOD presumably does not enter the nerve terminal. It has also been shown that DETC exerts additional effects and for instance induces decomposition of nitrosothiols (4), blockade of thiol groups, or GSH depletion (26). Because DETC elicits comparable results with SIN-1 treatment, decomposition of nitrosothiol appears not to be responsible for the effect. Additional series of experiments were carried out to investigate the alternative modes of action of DETC. The SNAP-induced inhibition of NOS was augmented in the presence of 0.5 mM GSH, suggesting a modulatory role of this thiol. Thus GSH depletion or blockade of thiol groups might participate in the overall response to exogenous NO. According to this presumption, we investigated whether Cu/Zn SOD is present at all in our synaptosomal preparation. Indeed the presence of Cu/Zn SOD could be confirmed by Western blot analysis in enteric synaptosomes. This finding is in good agreement with recent reports demonstrating the presence and colocalization of NOS with Cu/Zn SOD in enteric nerves (31). Further indirect supportive evidence for a role of endogenous SOD comes from experiments with catalase. SOD converts superoxide to molecular oxygen and hydrogen peroxide; the latter is converted to water by either GSH peroxidase or catalase. Addition of catalase significantly augmented the inhibitory effect of SNAP on NOS in our experiments and inhibited basal NOS activity. The notable effect of catalase could be explained as follows. Hydrogen peroxide, formed by the action of an endogenous SOD as by-product of the reaction between NO and GSH (16), might simply counteract the inhibitory effect of exogenous NO, or free NO is generated by catalase in the presence of hydrogen peroxide (33) and as such augments inhibition of NOS. The use of SOD mimetics like Mn(III)porphyrin analogs or similar agents to further clarify the role of SOD is limited by the inhibitory effect on NOS activity exerted by these compounds, as shown previously (35).
With respect to the presented data, two questions have to be addressed. First, how can NO released from SIN-1 or SNAP escape complete destruction by extracellular oxyhemoglobin? Second, if free NO is not the mediator, what else mediates the effect and how is this related to the effect of endogenous Cu/Zn SOD? Two explanations might be possible: either small amounts of NO remaining after scavenging by oxyhemoglobin are sufficient to elicit NOS inhibition or NO exists in a form that cannot be scavenged by oxyhemoglobin. A rapid diffusion into the intraterminal compartment as an explanation for the lacking effect of exogenous SOD and oxyhemoglobin is very unlikely because NO does not diffuse directionally (30) and thus, due to a first-order reaction kinetic between oxyhemoglobin and NO (5), any free NO would be scavenged. Therefore, it could be speculated that NO exists in a storage form resistant to attack by NO scavengers. Because it can be assumed that any cell system produces relevant amounts of superoxide anions (24) and NO reacts with superoxide anion to form peroxynitrite, the latter is a likely candidate mediating inhibition of NOS in response to NO donors. Peroxynitrite is cytotoxic but has been reported to mediate biological responses like nitrosation of tyrosine residues (5, 24).
In another series of experiments, the peroxynitrite scavenger MnTBAP did not influence the SNAP- and SIN-1-mediated inhibition. Addition of sodium bicarbonate, leading to increased levels of CO2 that rapidly traps peroxynitrite in physiological fluids (47), also did not modify the SNAP- and SIN-1-induced inhibition of NOS. Furthermore, under conditions of expected high concentrations of superoxide anions in the presence of pyrogallol, the inhibition of NOS by exogenous NO was not influenced. These data render the involvement of peroxynitrite as an intermediate NO storage form less likely.
Alternatively, nitrosation of protein thiols (43) could be involved. Recently, it has been shown that nitrosation of thiol groups by NO could mediate signaling functions similar to free NO and that the nitrosation reaction occurs faster than reaction of NO with metal centers of enzymes (44). Furthermore, a NO/cGMP signaling pathway involving GSNO as intermediate has been recently described (32, 41).
To test for the second hypothesis, the influences of GSH and compounds either increasing or decreasing intracellular GSH content were studied. Exogenous supply of GSH did not influence basal NOS activity but augmented SNAP- and SIN-1-induced inhibition of NOS. In the presence of NAC, a precursor of endogenous GSH synthesis with antioxidant properties, the SNAP-induced NOS inhibition was slightly but not significantly increased, whereas depletion of endogenous GSH by BSO completely antagonized the effect of SNAP and SIN-1. These data suggest that the effect of SNAP and SIN-1 on NOS activity is strongly dependent on GSH. On the other hand, it could be speculated that the striking effect is mediated by an increased activity of Cu/Zn SOD, which is reconstituted by a transfer of copper from a Cu(I)-GSH complex formed in the presence of GSH (11, 32).
To reach its pharmacological targets, NO has to diffuse through environments with GSH levels in the range of 5-10 mM (19). However, little is known about the reaction of NO with GSH in vivo and evidence for the formation of S-nitrosothiols from endogenous NO remains scarce. The direct reaction of NO with thiols to form an S-nitrosothiol is strongly dependent on an electron acceptor, which could be molecular oxygen or trace metals under physiological conditions (13, 16). However, the oxygen-dependent pathways for nitrosothiol formation are quite slow and inefficient due to their requirement for thiol autooxidation or nitrite formation. The rate constants for the reaction of NO with oxygen or GSH indicate that the reaction with GSH will proceed 40 times faster than with oxygen (19). It has also been shown that transition metals in the presence of thiols readily catalyze nitrosothiol formation by oxidizing thiol to thyil radical or by forming a reduced metal-NO+ species that attacks the thiol directly (13). Both reactions with oxygen or transition metals concomitantly generate thyil radicals as by-products (13, 32). Indirect evidence for the intermediate formation of thyil radicals was assessed by the significant blockade of the SNAP- and SIN-1-induced NOS inhibition in the presence of the thyil radical scavenger DMPO.
Interestingly, under conditions of low oxygen tension, which occur in isolated membranes, transient association of NO and GSH has been postulated, allowing for diffusion of NO through membranes and subsequent release into intracellular environments (19).
With respect to our data suggesting a possible formation of S-nitrosothiols in NO signaling, we inquired about nitrosation reactions in the synaptosomal fraction. Indeed, we were able to identify the product of SIN-1/SNAP and GSH incubation as a nitrosothiol by the Saville assay. Interestingly, SIN-1 by an unknown mechanism was more effective at generating nitrosothiols than SNAP, which might explain why SIN-1 is less effective than SNAP at generating NO or relaxing smooth muscle but equipotent at inhibiting NOS. Furthermore, the reaction product decomposed to release NO on addition of Cu(NO3)2, as shown in a similar manner by others (32, 41), and, in the presence of neocuproine, a Cu+-chelator, no significant decomposition was detected, suggesting that Cu+ is required for the reaction.
These data suggest that, on addition of SIN-1 or SNAP in the presence of GSH, a nitrosothiol, presumably GSNO, is formed, which is able to protect NO from scavenging and delivers NO by a trace metal-catalyzed reaction.
However, our data suggest that formation of nitrosothiols is intermediary because endogenous Cu/Zn SOD and catalase appear to potently influence the effect of SNAP and SIN-1. This concept is further supported by the finding that neocuproine is able to antagonize the inhibitory effect of SNAP and SIN-1, suggesting that the effect is dependent on release of NO by a Cu+-catalyzed nitrosothiol decomposition as reported in other systems (32, 41).
Because nitrosothiols do not readily cross membranes, release of NO from nitrosothiols might occur in close relationship to a predominantly membrane-associated nNOS (21), resulting in a subsequent inhibition of the enzyme. However, transnitrosation reactions with membrane-bound thiols (43) cannot be precluded. NO, once it enters the nerve terminal, might be protected from reactions with superoxide anion by endogenous Cu/Zn SOD and as such inhibits NOS activity. However, at present, this is speculative and deserves further elucidation.
The precise mechanism by which NO inhibits NOS activity is at present unknown. NO might directly bind to the heme prosthetic group of the NOS catalytically active center (1, 18).
An alternative explanation for NOS inhibition by NO is the reaction of NO with a redox-sensitive cofactor (34). Recent studies have shown that tetrahydrobiopterin is able to protect against NO inhibition of NOS (22). In the absence of tetrahydrobiopterin (39) or in response to L-arginine deficiency (46), nNOS could generate stoichiometric amounts of NO and superoxide anions, both of which combine rapidly to form peroxynitrite (5, 39). In the presence of tetrahydrobiopterin, this uncoupling is suppressed, but superoxide generation also takes place as result of tetrahydrobiopterin autooxidation (39). In our preparation, omission of tetrahydrobiopterin did not significantly influence basal NOS activity, suggesting indirect inhibition of NOS by affecting cofactor levels is presumably of minor relevance.
However, it is difficult to assess to what extent NO produced by NOS in the basal state is capable of inhibiting NOS activity and whether all the mechanisms outlined above for exogenously supplied NO are involved as well. Recently, it has been shown that cofactor-activated NOS does not produce detectable amounts of NO unless stimulated with copper and GSH (32), which suggests that NOS activation by cofactors also leads to nitrosothiol formation. Our data showing a remarkable inhibition of cofactor-activated NOS activity in the presence of copper and GSH compared with either substance alone support this view and add further evidence for a feedback inhibition of NOS by NO.
Because NO elicits its biological effect by concomitant activation of soluble guanylate cyclase, the amount of generated cGMP by these NO donors was investigated in enteric synaptosomes. All NO donors were capable of stimulating endogenous cGMP levels; however, activation of guanylate cyclase and concomitant formation of cGMP do not seem to be involved in the inhibition of NOS by exogenous NO, since the membrane-permeable cGMP analog CPT-cGMP showed no effect on NOS activity and the selective guanylate cyclase inhibitor ODQ did not influence the NO-induced inhibition of the enzyme.
In conclusion, both the functional and biochemical findings of the present study are highly indicative of an inhibition of the NO pathway in the enteric nervous system by exogenous NO, presumably via the intermediate formation of a nitrosothiol.
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ACKNOWLEDGEMENTS |
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We acknowledge the cooperation of Prof. Dr. B. Gänsbacher, Prof. W. Erhardt, and their collaborators, Department of Experimental Surgery, Technical University of Munich. We thank H. Paeghe and S. Anton for expert technical assistance and L. Kots for proofreading the manuscript.
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FOOTNOTES |
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This study was supported by Deutsche Forschungsgemeinschaft SFB-391-C5, and parts of this work have been presented in abstract form at the annual meeting of the American Gastroenterological Association in Washington, DC, 1997.
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. D. Allescher, Dept. of Internal Medicine II, Technical Univ. of Munich, Ismaningerstr. 22, 81675 München, Germany (E-mail: hans.allescher{at}lrz.tu-muenchen.de).
Received 30 March 1999; accepted in final form 19 July 1999.
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