Department of Internal Medicine II, Technical University Munich, 81675 Munich, Germany
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ABSTRACT |
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The effect of nitric oxide (NO) on the release
of bombesin-like immunoreactivity (BLI) was examined in synaptosomes of
rat small intestine. The NO donor
S-nitroso-N-acetylpenicillamine (SNAP; 107 to
10
4 M) significantly
stimulated BLI release. In the presence of the NO scavenger
oxyhemoglobin (10
3 M) or
the guanylate cyclase inhibitor ODQ
(10
5 M), SNAP-induced BLI
release was antagonized. In addition, SNAP increased the synaptosomal
cGMP content and elevation of cGMP levels by zaprinast (3 × 10
5 M), an inhibitor of the
cGMP-specific phosphodiesterase (PDE) type 5, and increased basal and
SNAP-induced BLI release. NO-induced BLI release was blocked by
Rp-adenosine 3',5'-cyclic
monophosphorothioate (3 × 10
5 M and
10
4 M), an inhibitor of the
cAMP-dependent protein kinase A, whereas KT-5823 (3 × 10
6 M) and
Rp-8-(4-chlorophenylthio)-cGMP
(5 × 10
5
M), inhibitors of the cGMP-dependent protein kinase G, had no effect.
Because cGMP inhibits the cAMP-specific PDE3, thereby increasing cAMP
levels, the role of PDE3 was investigated. Trequinsin (10
8 M), a specific blocker
of PDE3, stimulated basal BLI release but had no additive effect on
NO-induced release, suggesting a similar mechanism of action. These
data demonstrate that because of a cross-activation of cAMP-dependent
protein kinase A by endogenous cGMP BLI can be released by NO from
enteric synaptosomes.
nerve terminals; enteric nervous system; gastrin-releasing peptide; phosphodiesterase 3; phosphodiesterase 5; nitric oxide
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INTRODUCTION |
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THERE IS INCREASING evidence that nitric oxide (NO) relaxes smooth muscle by two possible modes of action. Besides acting directly on intestinal smooth muscle as an inhibitory nonadrenergic noncholinergic neurotransmitter, NO could affect intestinal motility indirectly by modulating the release of other neurotransmitters from enteric nerve terminals. The latter effect could be achieved by either inhibiting the release of excitatory transmitters such as ACh (17, 18) or stimulating inhibitory transmitters such as vasoactive intestinal peptide (VIP) (1, 15). Indeed, NO-generating compounds have been shown to modulate transmitter release in several neuronal preparations of the central (22), the autonomic, and the enteric nervous system (1, 15). However, the effects of NO or cGMP on isolated nerve terminals as the site of neurotransmitter release have been partially characterized in central nervous system preparations (22), whereas only sparse data exist from enteric neurons (1).
Bombesin-like immunoreactivity (BLI) comprises the mammalian counterparts gastrin-releasing peptide (GRP) and neuromedin B and C and represents a putative peptidergic neurotransmitter or neuromodulator of the enteric nervous system. BLI is considered to play a role as an excitatory neuropeptide stimulating smooth muscle contraction either directly (14) or indirectly by stimulating release of ACh (44) in vitro. BLI is present exclusively in intrinsic neurons and colocalizes with VIP and nitric oxide synthase (NOS) in a subpopulation of descending inhibitory interneurons within the myenteric plexus (9, 40). The release of BLI has been studied in isolated nerve terminals, which offer the unique opportunity to examine intracellular and subcellular mechanisms of neurotransmitter release without the interference of other local or systemic factors present in vivo or in the intact organ in vitro (26). The aim of the present study was 1) to characterize the effects of NO on BLI release from isolated nerve terminals of the enteric nervous system and 2) to study signal transduction pathways mediating the effects of NO.
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MATERIALS AND METHODS |
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Preparative Techniques
Tissue handling and membrane preparation. Synaptosomes were prepared as described previously (26). Briefly, five male Wistar rats 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. Approximately 6- to 8-cm pieces of small intestine were dissected, cleaned of mesenteric arcade and fat, and opened along the mesenteric attachment line. The mucosal layer was scraped off with a sharp razor blade, and the remaining muscle layers were put into cold buffer. The muscle tissue was blotted dry on filter paper and weighed. For membrane preparation, the tissue was resuspended in isolation buffer (8% wt/vol sucrose and 20 mM MOPS, pH 7.4), minced with scissors, and homogenized with a Polytron PT20 homogenizer at an ~1,500 rpm setting for 15 s (3 × 5 s).
Fractionation of tissue homogenate by differential centrifugation. The tissue homogenate was centrifuged in two steps of 800 g for 10 min to remove myofibrils and remaining nuclei. The supernatant was collected (postnuclear supernatant) and recentrifuged at 3,500 g for 10 min to obtain the mitochondrial 1 fraction. The supernatant was centrifuged again at 100,000 g for 90 min. The pellet from this centrifugation (microsomal 1) was resuspended and centrifuged again at 10,000 g for 10 min. The resulting pellet and the supernatant are referred to as mitochondrial 2 (P2) and microsomal 2 fractions, respectively.
Differential centrifugation led to a substantial enrichment of [3H]saxitoxin in the fraction P2 (8-fold, 44.9 ± 8 fmol/mg compared with the postnuclear supernatant at 5.5 ± 1.7 fmol/mg) and was paralleled by a fourfold increase in the content of BLI in the P2 fraction (1.2 ± 0.2 vs. 0.3 ± 0.04 ng/mg protein in the postnuclear supernatant, n = 14), as reported previously (26).Analytic Techniques
Protein assay.
Protein was measured spectrophotometrically according to the method of
Bradford (5). Bovine serum -globulin was used as standard.
Assay of occluded lactate dehydrogenase. Lactate dehydrogenase was determined using a commercially available assay kit [MPR1, Boeringer Mannheim, Mannheim, Germany (42)]. 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).
Spectrophotometric determination of nitrite production. Nitrite production of the superfused ileum was assayed using the Griess assay (16) as modified by Schmidt et al. (37). 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 KCl, gassed with 95% O2 and 5% CO2) maintained at 37°C. 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 absorbance was again determined, 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 cGMP content. The amount of cGMP in the synaptosomal fractions was determined with the use of RIA according to the method of Pradelles and Grassi (32) using a commercially available assay kit (Cayman Chemical, Ann Arbor, MI). 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 synaptosomes) and were supplemented by 0.1 mM zaprinast and various concentrations of other exogenous compounds, as indicated in the text. After incubation for 8 min at 37°C, the reaction was terminated by immediate heating of the samples (90°C, 5 min), followed by rapid cooling and centrifugation in a refrigerated centrifuge (10,000 g, 10 min). Fifty microliters of supernatant were then used for the RIA of cGMP following the procedure provided by the manufacturer. Triton X-100 pretreatment of the synaptosomal fractions resulted in a greater variance of responses but did not seem to alter the pattern of the response.
Assay of cAMP content. The amount of cAMP was measured by a commercially available cAMP enzyme immunoassay system (Biotrak, Amersham Pharmacia Biotech, Buckinghamshire, UK). Tissue preparation was carried out as described for the cGMP assay with the exception that no cAMP-specific phosphodiesterase (PDE) inhibitor was supplemented to increase the amount of detectable cAMP. One hundred microliters of supernatant were used for the nonacetylation enzyme immunoassay procedure provided by the manufacturer.
RIA. BLI was determined as described elsewhere (38) with an antibody raised against [Lys4]bombesin. The antibody was provided by Peninsula (Merseyside, UK). The antibody reacts equally well with GRP and neuromedin C but not with neuromedin B. [Tyr4]bombesin for the preparation of the labeled bombesin and synthetic bombesin as standard were purchased from Sigma (Munich, Germany).
Peptide release.
Peptide release studies were carried out in Krebs-Ringer bicarbonate
solution (KRS) (in mM: 115.5 NaCl, 1.16 MgSO4, 1.16 NaH2PO4, 11.1 glucose, 21.9 NaHCO3, 2.5 CaCl2, and 4.16 KCl), which was gassed with 95% O2 and 5%
CO2. KRS (1,050 µl) and 150 µl
of drugs or KRS alone serving as a control (basal level) were incubated in separate test tubes at 37°C in a gently shaking water bath. The
reaction was started by adding 300 µl of synaptosomal membranes (300 µg protein) to each tube at timed intervals. The incubation lasted 5 min. To stop the reaction, the synaptosomal membranes were put on ice
and immediately sedimented by high-speed centrifugation in a
refrigerated centrifuge. The supernatant was withdrawn and immediately
frozen at 20°C until peptide determination by RIA.
Protein extraction. Extraction of proteins was performed as described previously (20). Tissue from bovine lung was suspended in an eightfold volume of MOPS buffer [200 ml MOPS containing 10 mM pepstatin A, 100 µM thiorphan, 50 µM phenylmethylsulfonyl fluoride (PMSF), 1 mM dithiotreitol, 0.5 µM trypsin inhibitor, and 1 mM captopril] and homogenized using a Polytron PT20 homogenizer (1,500 rpm, 15 s). The homogenate was centrifuged at 100,000 g for 60 min. To obtain the particulate protein fraction, the pellets were resuspended in buffer containing 1% Triton X-100 and recentrifuged at 100,000 g. The resulting supernatant was referred to as particulate protein fraction of bovine lung. The particulate protein fraction from the enteric synaptosomes was obtained by centrifugation of a synaptosomal P2 fraction at 100,000 g for 60 min after 1% Triton X-100 pretreatment. The resulting supernatant was the respective particulate fraction derived from synaptosomes.
Western blot analysis. The particulate protein fractions (synaptosomes: 250 µg protein, bovine lung: 80 ng protein) were separated by SDS-PAGE (7.5%) slab gels in a Bio-Rad minigel apparatus (27) 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, 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 for bovine PDE5 (antibody dilution 1:1,000; Chemicon, Hofheim, Germany). As secondary antibody, horseradish peroxidase-linked anti-rabbit IgG was used and detection was performed by enhanced chemiluminescence (Amersham, Braunschweig, Germany).
RNA isolation and RT-PCR.
Total RNA was extracted from liquid nitrogen frozen rat intestinal
longitudinal muscle/myenteric plexus (LM/MP) as described previously
(20). Tissues were homogenized and RNA was isolated using the guanidine
isothiocyanate-phenol-chloroform extraction method (12), followed by
DNase treatment for 15 min at room temperature (1 unit DNase I/µg
RNA; GIBCO BRL, Eggenstein, Germany). Three micrograms of total RNA
were reverse transcribed in a complementary DNA by using 100 ng random
hexamere primers (Boehringer Mannheim) and 200 units SuperScript II
RNase H reverse
transcriptase (GIBCO BRL). Incubation times were 20 min at 25°C and
1 h at 42°C. To determine mRNA expression of PDE3/IVd in LM/MP, we
subsequently performed a PCR using specific primers for the PDE3/IVd
isozyme (Table 1). Thirty-five rounds of
PCR amplification were carried out in a Biometra UNO I thermal cycler using 2.5 units Taq polymerase
(Promega, Mannheim, Germany) and 1 µl of the RT reaction mixture at
the following conditions. After a "hot start" with an initial
denaturation at 95°C for 3 min each, PCR cycle involved
denaturation at 94°C for 30 s, annealing at 62°C for 45 s, and
extension at 72°C for 45 s. The last cycle was followed by an
extension step at 72°C for 7 min. As negative controls, isolated
RNA amplified without reverse transcriptase or random hexameres were
used. The amplification product was separated by 1.5% agarose gel
electrophoresis and visualized by ethidium bromide staining. The band
was excised from the gel, purified by a gel extraction kit (Qiagen,
Hilden, Germany), and blunt-end cloned into pST blue vector (Novagen,
Bad Soden, Germany). The nucleotide sequence was deduced by cycle
sequencing of the isolated plasmid (QIAprep spin miniprep kit, Qiagen)
with T7 sequencing primer (GATC, Konstanz, Germany).
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Drugs. The reagents were purchased from the indicated sources: L-arginine, sodium nitroprusside (SNP), NG-nitro-L-arginine (L-NNA), VIP, isoproterenol, and forskolin were from Sigma (Munich, Germany); 8-(4-chlorophenylthio)-guanosine-3',5'-cyclic monophosphate (CPT-cGMP), Rp-CPT-cGMP, 8-bromo-cGMP (8-BrcGMP), and Rp-adenosine 3',5'-cyclic monophosphorothioate (Rp-cAMPS) were from Biolog Life Science Institute (Bremen, Germany); diethylamine-NO complex sodium (DEA-NO) was from Research Biochemicals International (Natick, MA); S-nitroso-N-acetylpenicillamine (SNAP), 9,10-dimethoxy-2-mesitylimino-3-methyl-2,3,6,7-tetrahydro-4H-pyrimido-(6,1-a)-isoquinolin-4-one-HCl (trequinsin), and KT-5823 were from Calbiochem (Bad Soden, Germany); 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and 2-(2-propyloxyphenyl)-8-azapurin-6-one (zaprinast) were from Tocris Cookson (Langford, UK); 3-morpholinosydnonimine (SIN-1) was from Casella-Riedel (Frankfurt, Germany); DL-thiorphan was from Fluka (Deisenhofen, Germany); pepstatin A, trypsin inhibitor, dithiotreitol, and captopril were from Sigma (Deisenhofen, Germany); and PMSF was from Serva (Heidelberg, 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 from separate preparations. For each value of a given drug of a single preparation, the release study was carried out in duplicate. The values of peptide release experiments showed considerable variation in separate experiments and were therefore expressed as the relative increase over basal levels (=100%). ANOVA, followed by Dunnett's post hoc test for multiple testings, was used to determine statistical significance. For comparisons of two means, paired or unpaired t-test was performed. Values of P < 0.05 were considered significant. ![]() |
RESULTS |
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NO Donor-Induced NO Formation and Synaptosomal Integrity
The amount of NO generated by SNAP (100 µM to 1 mM) in rat ileum 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. In comparison, other NO donors such as SIN-1 or SNP were less effective at generating nitrite under these assay conditions. Concentrations of SIN-1 and SNP, covering the dose range of 1-100 µM, corresponded to a NO concentration range of 0.15-1.5 µM and 0.2-5.0 µM, respectively.Lactate dehydrogenase release from SNAP-treated synaptosomes was 54.3 ± 3.8 U/l (93 ± 5%), which was not significantly changed compared with control [55.8 ± 4.0 U/l (100%), n = 4]. These data suggest that exposure of synaptosomes to SNAP does not result in membrane damage.
Effect of NO Donors on BLI Release
The NO donor SNAP (10
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In the presence of the NO scavenger oxyhemoglobin
(103 M), the SNAP-induced
BLI release was blocked [basal: 153.8 ± 16.6 pg/mg (= 100%),
SNAP at 10
4 M: 166.8 ± 17.2%, SNAP + oxyhemoglobin: 112.7 ± 7.0%;
P
0.01, n = 8] (Fig.
2).
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Oxyhemoglobin (103 M)
itself did not alter basal BLI release [110.1 ± 11.8%,
basal: 126.0 ± 20.8 pg/mg (= 100%);
n = 5] (Fig. 2). In a second
series of experiments, the effect of oxyhemoglobin on SIN-1-induced
release of BLI was investigated. The results obtained were similar to
those with SNAP [basal: 119.2 ± 13.9 pg/mg (= 100%), SIN-1
at 10
4 M: 134.7 ± 11.3%, SIN-1 + oxyhemoglobin at
10
3 M: 102.0 ± 10.5%;
P
0.05, n = 9]. The addition of
superoxide dismutase (100 U/ml) slightly but not significantly
increased the net release of BLI evoked by SIN-1 (Table 2).
Effect of L-NNA and L-Arginine
Inhibition of NOS by L-NNA (10Effect of NO Donors on cGMP Generation in Enteric Synaptosomes
Further experiments were directed toward the evaluation of whether exogenous NO is capable of increasing the soluble guanylate cyclase content in the synaptosomal fraction. The cGMP content in synaptosomes was stimulated fourfold in the presence of 100 µM SIN-1 from basal 20.7 ± 5.6 to 83.5 ± 29 fmol/mg (P
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Effect of ODQ and Zaprinast on BLI Release
Another series of experiments was conducted with respect to the characterization of the signal transduction pathways involved in the BLI release under the conditions employed. Because guanylate cyclase is the target enzyme of NO, its role was investigated by both specific inhibition of its activation by ODQ and alteration of the cGMP degradation by zaprinast, an inhibitor of the cGMP-specific PDE type 5. ODQ (10
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Effect of Inhibition of cGMP-Dependent Protein Kinase G
To test whether signal transduction involves activation of cGMP-dependent protein kinase G (PKG), the effects of various inhibitors of the cGMP-dependent PKG were studied. KT-5823 (3 × 10In addition, the effect of zaprinast on BLI release was not antagonized
in the presence of KT-5823 [163.5 ± 3.5%, not significant compared with 3 × 105
M zaprinast alone (143.4 ± 13.5%),
n = 3; basal: 93.0 ± 16.0 pg/mg (=
100%)]. To study the effect of direct activation of
cGMP-dependent PKG, the membrane-permeable and PDE-resistant cGMP
analogs CPT-cGMP and 8-BrcGMP were employed. Both compounds,
CPT-cGMP (3 × 10
5 M) and 8-BrcGMP
(10
7 to
10
4 M), did not influence
BLI release [basal: 109.3 ± 18.1 pg/mg (= 100%), CPT-cGMP:
95.8 ± 13.8%, 8-BrcGMP: 109.8 ± 12.1%, 102.8 ± 3.5%,
101.6 ± 6.5%, 93.8 ± 4.0% at
10
7 M,
10
6 M,
10
5 M, and
10
4 M, respectively].
Effect of Inhibition of cAMP-Dependent Protein Kinase A
Because the foregoing data suggest a more complex signal transduction for NO-induced BLI release than anticipated, the possibility of intracellular cross-talk via cAMP-dependent mechanisms was examined. Rp-cAMPS, an inhibitor of cAMP-dependent protein kinase A (PKA), significantly blocked BLI release evoked by SNAP (10
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With respect to the significant effect of inhibition of cAMP-dependent PKA on the release of BLI by exogenous NO, another series of experiments was conducted to further clarify the mechanism of cAMP-dependent PKA activation. Theoretically, elevated cGMP levels could either cross-activate cAMP-dependent PKA or modulate intracellular PDEs. Besides interaction with the cGMP-specific PDE5, cGMP inhibits PDE3, thereby increasing intracellular cAMP levels. In the context of the obtained data, it was of interest to examine the role of the latter isozyme in the BLI release by NO.
Effect of Inhibition of PDE3
Trequinsin 10
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Because the data suggest a functional role of PDE3 in the release
process, an additional series of experiments was conducted with respect
to the effect of trequinsin and zaprinast on cAMP levels in comparison
to the membrane-permeable cGMP analogs. cAMP levels were significantly
stimulated in the presence of 3 × 105 M zaprinast and
10
8 M trequinsin, whereas
8-BrcGMP (10
5 M) and
CPT-cGMP (5 × 10
5 M)
did not alter basal cAMP levels (Table 3).
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Effect of cAMP-Dependent Mechanisms
Release of BLI was significantly stimulated by isoproterenol 10Western Blot Analysis of PDE5
With respect to the functional data suggesting a modulatory role of PDE5, Western blot analysis was performed to demonstrate the presence of this isozyme in rat enteric synaptosomes. The particulate protein fraction of bovine lung comigrated with a band at ~99 kDa in the synaptosomal fraction, specific for PDE5 (Fig. 8).
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Detection of PDE3 mRNA by RT-PCR
The presence of a PDE3/IVd isozyme in the rat LM/MP preparation was determined by RT-PCR with specific primers and subsequent sequencing of the respective PCR product. A single band at the expected size of ~700 bp was obtained for PDE3/IVd (Fig. 9). Sequencing of the cloned RT-PCR product confirmed the cDNA sequence of this isozyme.
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DISCUSSION |
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This study demonstrates that NO acts presynaptically to stimulate BLI release from enteric nerve terminals. This work extends previous reports that NO modulates neuropeptide and neurotransmitter release in the gastrointestinal tract in preparations of isolated ganglia (15), synaptosomes (1), longitudinal muscle with adherent myenteric plexus (17, 18), and circular muscle (19). The release of BLI in the present work is mediated by NO and not by by-products of the NO catabolism, since it was demonstrated that the BLI release induced by exogenous NO was antagonized by oxyhemoglobin, which rapidly inactivates NO due to high-affinity binding (13).
In the LM/MP preparation, Hebeiss and Kilbinger (18) showed that the effects of NO on the release of ACh are mediated via activation of the soluble guanylyl cyclase (18). However, the mechanism whereby guanylyl cyclase activation inhibits ACh release has not yet been studied. Furthermore, no information about the signaling pathway involved in the modulatory action of NO on the release of any other neurotransmitter or neuropeptide in the gut is available to date.
Activation of soluble guanylate cyclase and subsequent elevation in intracellular cGMP levels are considered to be the primary modes of action of NO (23, 36). In the present work, it could be demonstrated that enteric synaptosomes are capable of accumulating cGMP following stimulation by NO donors. This confirms previous findings of enteric synaptosomes of canine ileum (24). Subsequently, we have examined whether NO-induced cGMP elevation contributes to BLI release. The approaches employed were both reduction and elevation of intracellular cGMP levels by addition of ODQ, a selective inhibitor of soluble guanylate cyclase (12), and zaprinast, an inhibitor of the cGMP-specific PDE5 (2), respectively. The data obtained by using both compounds, together with the accumulation of neuronal cGMP by exogenous NO, were highly indicative of an involvement of cGMP in the release process. Another objective of the present study was to further characterize the signaling pathway downstream of the activation of cGMP. cGMP can elicit its physiological effects in different ways, acting directly on cGMP-dependent PKG or on PDEs, thereby increasing or decreasing cAMP levels (for reviews, see Refs. 3 and 41).
First, the role of cGMP-dependent PKG was examined. It has been
reported that guanylate cyclase is present in enteric neurons of the
rat but does not colocalize with NOS (35). We previously demonstrated
the presence of cGMP-dependent PKG I and I
in
enteric neurons and nerve processes of the rat ileum (20). Thus the necessary elements of the cGMP-dependent PKG pathway seem to be present
in the enteric nervous system. However, pretreatment of synaptosomes
with the cell-permeable cGMP analogs 8-BrcGMP and CPT-cGMP,
which selectively activate cGMP-dependent PKG, failed to increase BLI
release at doses reported to elicit a physiological response in other
systems (17). The cGMP-dependent PKG blocker KT-5823, which has been
demonstrated to specifically interact with cGMP-dependent PKG in
various smooth muscle preparations, including gastrointestinal muscle
(30), did not significantly inhibit BLI release induced by exogenous
NO. With respect to the predominance of the cGMP-dependent PKG I
subtype in enteric nerves (20), we studied the effect of
Rp-CPT-cGMP, a specific inhibitor of
cGMP-dependent PKG I isoform (7).
Rp-CPT-cGMP also failed to
antagonize BLI release induced by NO, suggesting an alternative mode of
action of cGMP on BLI release from nerve terminals.
Indeed, the signaling pathway seems to involve the activation of the cAMP-dependent PKA, as Rp-cAMPS, a specific inhibitor of the cAMP-dependent PKA (11), completely abolished the release of BLI in the presence of exogenous NO. In this context, it is noteworthy that considerable cross-activation of cAMP-dependent PKA at high concentrations of cGMP can occur in various tissues (21) and that there is a considerable overlap of proteins serving as substrates for both cAMP-dependent PKA and cGMP-dependent PKG (36, 41), providing a mechanism for convergence in the nervous system.
In a second series of experiments, we studied whether cGMP modulates cellular signaling by interaction with PDE enzymes present in synaptosomes. The cGMP-specific PDE5 preferably hydrolyzes cGMP rather than cAMP (4, 6). Zaprinast, a selective inhibitor of this enzyme, significantly enhanced the basal and NO-stimulated BLI release, suggesting that PDE5 is a possible target for the cGMP response in enteric synaptosomes. Recently, it has been shown that PDE5 is expressed in the central nervous system of humans (29) and rats (25), whereas to date no information is available about its presence in the peripheral or autonomic nervous system. Western blot analyses of PDE5 in enteric synaptosomes in this study show the presence and expression of PDE type 5 in enteric nerves. Because rat and bovine PDE5 share ~93% of amino acid homology (25, 29), a specific antibody against PDE5 from bovine lung was used for Western blot analysis in rat enteric synaptosomes. PDE5 was detected in particulate protein fractions of bovine lung and rat enteric synaptosomes.
With respect to the demonstrated stimulatory effect of cGMP and the involvement of cAMP-dependent PKA in the release of BLI, another series of experiments was carried out to clarify whether the cGMP-inhibited PDE3 is operational in synaptosomes. This isozyme exerts its action by preferentially breaking down cAMP. Because cGMP binds tightly to the enzyme, but is hydrolyzed poorly, cGMP is supposed to act as an inhibitor of cAMP hydrolytic activity by this PDE (3). Trequinsin, which is a highly specific inhibitor of this enzyme (34), was found to mimic the effect of endogenous cGMP by stimulating basal BLI release. Under conditions with prestimulated cGMP levels in the presence of zaprinast or SNAP, no additive effect of trequinsin was observed, suggesting that their mechanism of action is similar. Thus it might be speculated that basal guanylate cyclase activity is usually sufficient to produce maximal interaction with trequinsin to induce PDE3 inhibition. On the other hand, it could be hypothesized that a rapid accumulation of cGMP in the presence of zaprinast or SNAP by unknown mechanisms (e.g., additional direct cross-activation of cAMP-dependent PKA) induces an upregulation of PDE3 activity. Immediate phosphorylation and activation of PDE3 presumably by cAMP-dependent PKA have been shown to occur in various tissues (10). Alternatively, activation of cAMP-dependent PKA might in turn stimulate PDE5 and thereby terminate the NO-induced cGMP signal (33). Whether these effects take place in neurons as well has not yet been determined. The presence of a rat PDE3 mRNA, the PDE3/IVd, in neuronal tissue has been demonstrated recently (39). With respect to the functional data suggesting a modulatory role of PDE3 in the release of BLI and because a specific antibody for PDE3 is not available, RT-PCR of PDE3 was performed in LM/MP preparations of rat small intestine. The primers used were specific for PDE3/IVd, which has been shown to contain a cAMP-dependent PKA consensus sequence (39), thus representing a putative target of cAMP-dependent PKA. The cDNA detected in LM/MP showed sequence homology for PDE3/IVd, adding supportive evidence for a modulatory role of this PDE3 isozyme.
Because the presented data suggest a high degree of intracellular cross-talk, the effect of an increase of cAMP levels was investigated. Adenylate cyclase was activated either via receptor activation by VIP and isoproterenol or via direct stimulation by forskolin. As demonstrated by our data, all of these compounds were capable of releasing BLI from synaptosomes. In the presence of the cAMP-dependent PKA inhibitor Rp-cAMPS, the isoproterenol-mediated effect was antagonized. The finding that cAMP levels were significantly stimulated in the presence of zaprinast and trequinsin supports the view that PDE3 is functionally operative in synaptosomes. Because the membrane-permeable cGMP analogs did not alter basal cAMP levels, it was suggested that their incapability of releasing BLI might be due to a lack of PDE3 inhibition.
These data suggest that a rather indirect signaling pathway mediates
NO-induced BLI release. The relative contribution of both mechanisms
responsible for cAMP-dependent PKA activation, cross-activation by a
direct mechanism or by increased cAMP levels via cGMP-mediated
inhibition of PDE3, cannot be assessed in our experimental system.
However, the data presented strongly suggest a functional role of PDE3
in the release process. Whether cyclic nucleotide signaling may be
influenced by autophosphorylation processes of cAMP-dependent PKA
resulting in an increased cAMP/cGMP selectivity and concomitantly
facilitating cross-activation as demonstrated for the cGMP-dependent
PKG I (28) or by phosphorylation of PDE3 by cAMP-dependent PKA
remains speculative and cannot be answered by the present study.
Furthermore, our data demonstrate that
L-NNA inhibits basal BLI
release, suggesting that there is some basal NOS activity modulating
BLI release. The fact that both
Rp-cAMPS and ODQ do not affect basal
BLI release and that trequinsin is able to stimulate BLI release might
suggest that the influence of NO on basal BLI release is mediated by a
different signal transduction pathway. The observation that
L-arginine stimulates BLI
release seems to confirm the suggestion of a stimulatory role of
endogenous NO. However, the effect of
L-arginine might also indicate
that synaptosomes become
L-arginine deficient during membrane preparation. L-Arginine
deficiency has been shown to alter NOS activity in the sense that
equimolar amounts of NO and superoxide anion are generated, both of
which combine rapidly to form peroxynitrite (43), which by itself is
able to induce transmitter release (31). Thus it can be hypothesized
that under basal conditions free NO might not be responsible for BLI
release in our experimental system. The lacking effect of oxyhemoglobin on basal release might support this view. However, at present, this is
speculative and deserves further elucidation.
In conclusion, we demonstrated that BLI can be released by NO acting presynaptically and this is presumably due to cross-activation of cAMP-dependent PKA via modulation of neuronal PDE3 by intracellular cGMP. A modulatory role in signal transduction is also demonstrable for PDE5. The presence and expression of both PDE isozymes, PDE3 and PDE5 in LM/MP preparation and synaptosomes, respectively, add supportive evidence of their putative functional role in neuronal NO-cGMP signaling.
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ACKNOWLEDGEMENTS |
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We acknowledge the cooperation of Prof. Dr. Gäsbacher and Prof. Erhardt and their collaborators at the Department of Experimental Surgery, Technical University of Munich. We thank H. Paeghe and S. Herda for expert technical assistance.
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FOOTNOTES |
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This study was supported by Deutsche Forschungsgemeinschaft SFB 391/C5.
Parts of this work have been presented in abstract form at the annual meeting of the American Gastroenterological Association in New Orleans 1998.
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, Ismaninger Str. 22, 81675 Munich, Germany (E-mail: hans.allescher{at}lrz.tu-muenchen.de).
Received 28 December 1998; accepted in final form 12 March 1999.
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