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Journal of Physiology (2001), 534.3, pp. 827-836
© Copyright 2001 The Physiological Society

Functional coupling between nitric oxide synthesis and VIP release within enteric nerve terminals of the rat: involvement of protein kinase G and phosphodiesterase 5

M. Kurjak, R. Fritsch, D. Saur, V. Schusdziarra and H. D. Allescher

	ABSTRACT

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1. The subcellular mechanisms involved in the effect of nitric oxide (NO) on the release of vasoactive intestinal polypeptide (VIP) were examined in synaptosomes isolated from rat small intestine.
2. VIP release was stimulated by the NO donor SNAP (10^-7-10^-4 M) in an oxyhaemoglobin-sensitive manner. The presence of the guanylate cyclase inhibitor ODQ (10^-5 M), or inhibition of protein kinase G (PKG) by KT 5823 (3 10^-6 M) or Rp-8Br-PET-cGMPS (5 10^-7 M), antagonized the SNAP-induced VIP release, suggesting a regulatory role of PKG, confirming previously published data from enteric ganglia. This finding was further supported by the fact that direct PKG activation by the stable cGMP analogue 8-pCPT-cGMP stimulated VIP secretion to the same extent as SNAP.
3. Basal VIP secretion was enhanced in the presence of zaprinast, an inhibitor of cGMP-dependent phosphodiesterase 5 (PDE 5), suggesting a functional role of PDE 5 in NO-cGMP signalling. Supportive evidence for this finding was obtained by demonstration of the presence of PDE 5 using RT-PCR.
4. Stimulation of endogenous NO production by L-arginine was also effective in releasing VIP. The effect was abolished in the presence of KT 5823, but was insensitive to oxyhaemoglobin (10^-3 M), suggesting that an interaction between NO and VIP is likely to occur within the same nerve terminal rather than between terminals.
5. NO synthesis was not affected by VIP (10^-8-10^-5 M), suggesting that there is no feedback regulation between the NO and the VIP pathways.
6. These findings support the notion that an anatomical and functional interrelationship exists between NO and VIP in enteric nerve terminals and that complex signalling mechanisms involving PKG and PDE 5 contribute to NO-induced VIP release.

	INTRODUCTION

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Nitric oxide (NO) is involved in relaxation of gastrointestinal smooth muscle either directly, by acting on smooth muscle cells, or indirectly, by modulating neuronal reflexes, e.g. by selectively regulating neurotransmitter release from enteric nerve terminals (Gustafsson et al. 1990; Kurjak et al. 1999a). Vasoactive intestinal polypeptide (VIP) is considered to be another important inhibitory transmitter in the enteric nervous system (Bryant et al. 1976; Lundberg, 1996), and is present in intrinsic neurons throughout the digestive tract (Furness et al. 1992; Bredkjaer et al. 1994). Colocalization with nitric oxide synthase (NOS) has been observed in a subpopulation of descending inhibitory interneurons within the myenteric plexus and in varicose nerve terminals of different species (Costa et al. 1992; Desai et al. 1994; Furness et al. 1995; Lefebvre et al. 1995; Wang et al. 1998). It is therefore likely that VIP and NO act in concert to exert relaxation or inhibition of intestinal contraction (Lefebvre et al. 1992; Keef et al. 1994) as well as regulation of blood flow and secretory processes (Lundberg, 1996; Mourad et al. 1999). Previous studies have shown that NO is capable of stimulating the release of VIP from enteric nerves (Grider & Jin, 1993; Daniel et al. 1994; Allescher et al. 1996). It has been demonstrated that, on the one hand, protein kinase G (PKG) contributes to NO-mediated VIP release and, on the other, VIP does not influence NO synthesis in guinea-pig myenteric ganglia (Grider & Jin, 1993). However, these data do not imply that the same mechanisms are also involved in isolated nerve terminals, especially those of motoneurons which are localized outside the ganglia within the muscle layers. In addition, species differences have been demonstrated in that, in guinea-pig all NOS-positive neurons were also immunoreactive for VIP (Costa et al. 1992), whereas this was not the case in the rat (Ekblad et al. 1994).

Stimulation of NO production by VIP has been reported in dispersed gastric muscle cells (Murthy et al. 1993) and in isolated muscle strips (Jin et al. 1996), and in intracellular recordings from smooth muscle of neuronal NOS (nNOS)-knockout mice (Mashimo et al. 1996). It was concluded that both transmitters act synergistically, with NO potentially amplifying the biological effect of VIP. However, different control mechanisms have been demonstrated for the two transmitters (Daniel et al. 1994), indicating that enteric inhibitory neurons use NO and VIP as parallel transmitters, with NO as the primary inhibitory transmitter (Daniel et al. 1994; Bayguinov et al. 1999). Recent data support the view that NO and VIP act via independent mechanisms in postjunctional cells (Huizinga et al. 1992; Pfeifer et al. 1998; Bayguinov et al. 1999), e.g. NO stimulates a cGMP-dependent pathway and VIP a cAMP-dependent pathway, both of which result in smooth muscle relaxation. The question of which prejunctional interactions exist between NO production and VIP release has been adressed by studying isolated nerve terminals; these offer the unique opportunity of examining intracellular and subcellular mechanisms of neurotransmitter release without interference from other local or systemic factors present in vivo or in the intact organ in vitro (Kurjak et al. 1994). The aim of the present study was (1) to investigate whether a possible interaction exists between endogenous NO synthesis and VIP secretion within nerve terminals and (2) to study the subcellular signal transduction pathways mediating the effect of NO on VIP release in synaptosomes of rat small intestine.

	METHODS

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Preparative techniques

Tissue handling and membrane preparation. Synaptosomes were prepared as described previously (Kurjak et al. 1994). Briefly, five male Wistar rats were killed by cervical dislocation under permits granted by the Animal Ethics Commitee at the Technical University of Munich. The small intestine was quickly removed and suspended in ice-cold buffer consisting of 8 % (w/v) sucrose, 20 mM 3-(N-morpholino)propanesulfonic acid (Mops), 10 mM MgCl₂, 10 mM pepstatin A, 100 µM thiorphan, 50 µM PMSF, 1 mM dithiothreitol, 500 µM trypsin inhibitor and 1 mM captopril (pH 7.4). All further preparative steps were carried out at 0-4 °C. Approximately 6-8 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, consisting of circular muscle with deep muscular plexus and longitudinal muscle with adherent myenteric plexus, were placed into ice-cold buffer. The muscle tissue was blotted dry on filter paper and weighed. For membrane preparation the tissue was resuspended in isolation buffer, minced with scissors and homogenized with a Polytron PT20 homogenizer at a setting of approximately 1500 r.p.m. 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 (post-nuclear supernatant; PNS) and recentrifuged at 3500 g for 10 min to obtain the pellet (P1) fraction. The supernatant was centrifuged again at 100 000 g for 90 min. The pellet from this centrifugation (microsomal 1; MIC1) 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 (MIC2) fractions, respectively. We demonstrated previously that the P2 fraction shows a relative enrichment of neural elements compared with smooth muscle plasmalemma (Kurjak et al. 1994, 1999a,b) and a 7-fold enrichment of VIP content compared with crude tissue homogenate (PNS) (Allescher et al. 1996). Thus P2 is referred to as the synaptosomal fraction.

Analytical techniques

Protein assay. Protein was measured spectrophotometrically according to the method of Bradford (1976). Bovine serum -globulin was used as a standard.

Radioimmunoassay. VIP immunoreactivity was determined as described elsewhere (Mitchell & Bloom, 1978). The antibody used (Sigma, Munich, Germany) showed no interaction with NH₂-terminal fragments of VIP, secretin, the peptide histidine isoleucine, growth hormone-releasing factor, gastric inhibitory peptide, or pituitary adenylate cyclase-activating peptide (PACAP). [¹²⁵I] VIP, for the preparation of the labelled VIP, and synthetic VIP used as a standard were purchased from Amersham and Sigma, respectively.

Peptide release. Peptide release studies were carried out in Krebs-Ringer bicarbonate solution (KRS (mM): NaCl 115.5, MgSO₄ 1.16, NaH₂PO₄ 1.16, glucose 11.1, NaHCO₃ 21.9, CaCl₂ 2.5, KCl 4.16), gassed with 95 % O₂ and 5 % CO₂. KRS (1050 µl) and 150 µl of drugs, or KRS alone 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 the addition of 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 radioimmunoassay.

Assay of NO synthase activity. NOS activity was determined by monitoring the formation of L-citrulline from L-arginine using a modification of methods described previously (Bredt & Synder, 1989). Enzymatic reactions were conducted at 37 °C in 25 mM Mops-8 % sucrose buffer containing 1 mM NADPH, 0.1 µM tetrahydrobiopterin (H₄biopterin), 1 µM calmodulin, 1 mM CaCl₂, 0.1 µM flavin adenine dinucleotide (FAD), 0.1 µM flavin mononucleotide (FMN) and other test agents as specified later, in a final incubation volume of 750 µl. 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 the addition of approximately 500 000 d.p.m. of L-[³H]arginine (63 Ci mmol^-1) and terminated after 15 min by immediately heating to 90 °C for 6 min and addition of 1 ml distilled water containing 1 mM L-arginine and 1 mM L-citrulline. Samples were applied to columns containing 1 ml Dowex AG50W-X8 resin, Na⁺ form, pre-equilibrated with Mops-sucrose buffer. The eluate (2 ml) was collected in a liquid scintillation vial. After addition of 1.5 ml 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 about 92 %; contamination with L-[³H]arginine did not exceed 2 %. Basal values were obtained by heating samples to 100 °C for 5 min before incubation. Cofactor-substituted NOS activity was referred to as control (Kurjak et al. 1999b).

RNA isolation and RT-PCR

Total RNA was extracted from liquid nitrogen-frozen rat intestinal longitudinal muscle-myenteric plexus (LM-MP) as described previously (Huber et al. 1998b). Tissues were homogenized and RNA was isolated using the guanidine isothiocyanate-phenol-chloroform extraction method (Chomczynski & Sacchi, 1987), followed by DNase treatment for 15 min at room temperature (1 U Dnase I (µg RNA)^-1; Gibco BRL, Eggenstein, Germany). Three micrograms of total RNA were reverse transcribed to obtain complementary DNA using 50 ng random hexamer primers (Boehringer Mannheim, Mannheim, Germany) and 200 U SuperScript II RNase H^- reverse transcriptase (Gibco BRL). Incubation times were 20 min at 25 °C and 1 h at 42 °C. To determine expression of phosphodiesterase 5 (PDE 5) mRNA in LM-MP, we subsequently performed PCR using primers specific for the PDE 5 isozyme (Table 1). Thirty-five rounds of PCR amplification were carried out in a Biometra UNO I thermal cycler using 2.5 U Taq polymerase (Sigma), 1 µl of the RT reaction mixture and 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 60 °C for 60 s and extension at 72 °C for 45 s. The last cycle was followed by an extension step at 72 °C for 7 min. Isolated RNA amplified without reverse transcriptase or random hexamers was used as a negative control. 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 using a gel extraction kit (Qiagen, Hilden, Germany) and blunt-end cloned into pST Blue Vector (Novagen, Schwalbach, 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). Sequences were analysed using BLASTn homology search.

Drugs

L-[³H]Arginine (63 Ci mmol^-1) was purchased from Amersham. All other reagents were purchased from the indicated sources: L-arginine, sodium nitroprusside (SNP), N^G-nitro-L-arginine (L-NNA), NADPH, FAD, FMN, L-citrulline, calmodulin, superoxide dismutase (SOD), haemoglobin, pepstatin A, dithiothreitol, trypsin inhibitor, VIP and PACAP 1-14 (Sigma); tetrahydrobiopterin (H₄biopterin) (ICN Biomedicals, Eschwege, Germany); trequinsin, S-nitroso-N-acetylpenicillamine (SNAP) and 9-(tetrahydro-2'-furyl) adenine (SQ 22536) (Calbiochem, Bad Soden, Germany); (morpholino)sydnonimine (SIN-1) (Casella-Riedel, Frankfurt, Germany); 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and 2-(2-propyloxyphenyl)-8-azapurin-6-one (zaprinast) (Tocris Cookson, Bristol, UK); thiorphan (Fluka, Munich, Germany); diethylamine-nitric oxide complex sodium (DEA-NO) (Research Biochemicals Int., Natick, MA, USA); PMSF (Serva, Heidelberg, Germany); N-acetyl-L-cysteine (NAC), glutathione (GSH) and ascorbate (Merck, Darmstadt, Germany); Rp--phenyl-1,N ²-etheno-8-bromo-guanosine-3',5'-cyclic monophosphorothioate (Rp-8Br-PET-cGMPS), 8-(4-chlorophenylthio)-guanosine-3',5'-cyclic monophosphate (8-pCPT-cGMP) and R_P-adenosine-3'-5'-cyclic phosphorothioate (Rp-cAMPS) (Biolog Life Science Institute, Bremen, Germany); KT 5823 (Calbiochem, Bad Soden, Germany); and YC-1 (Alexis, San Diego, CA, USA). Adequate controls were performed with the vehicles used for solubilizing each reagent.

Statistics

Data are given as means ± S.E.M., with n indicating 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 showed considerable variation in separate experiments and were therefore expressed as the relative increase over basal levels (= 100 %). Analysis of variance, followed by Dunnett's post hoc test for multiple testings, was used to determine statistical significance. For comparison of two means, Student's paired or unpaired t test was performed. Values of P 0.05 were considered significant.

	RESULTS

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Synaptosomes isolated from rat intestine are physiologically active

We used fresh synaptosomes of rat intestine and first validated their responsiveness to physiological stimuli by monitoring depolarization-induced VIP release under various stimulation protocols. The results in Fig. 1 show that synaptosomes were sensitive to KCl-induced depolarization. Furthermore, VIP release was strictly dependent on Ca²⁺ since release was inhibited by both the presence of EGTA and absence of Ca²⁺ at 65 mM KCl. We concluded that the synaptosomes were suitable for the investigation of NO-mediated effects on VIP release.

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Figure 1. VIP release in rat enteric synaptosomes Synaptosomes (300 µg) were assayed following depolarization with KCl (15-115 mM). When Ca2+ was eliminated by addition of EGTA or omission of Ca2+, release in the presence of 65 mM KCl was abolished (last two columns on the right). * P 0.05, compared with 65 mM KCl alone.

VIP release and NO synthesis - interplay between exogenous NO and VIP

The NO donor SNAP (10^-7-10^-4 M) increased VIP release in a concentration-dependent manner, with a potent stimulatory effect at a concentration of 10^-4 M (168.9 ± 10.6 %, P 0.01, n = 4; basal: 45.6 ± 2.4 pg mg^-1 (= 100 %); Fig. 2, Table 2). Other NO releasing compounds, such as DEA-NO, SNP or SIN-1, tested at a concentration of 10^-4 M as a comparison to SNAP, also significantly stimulated VIP release from synaptosomes (Table 2), but the degree of the response varied. We have shown previously that addition of 10^-4 M SNAP results in a concentration of NO in the incubation medium of approximately 10^-6 M, which is not associated with structural damage of the synaptosomes (Kurjak et al. 1999a, b).

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Figure 2. Effect of exogenous NO on VIP release The NO donor SNAP stimulated the release of VIP from synaptosomes of rat intestine at concentrations of 10-6 to 10-4 M. Data are means ± S.E.M. of 4 independent experiments with different preparations carried out in duplicate (*P 0.05, **P 0.01, compared with basal).

In the presence of the NO scavenger oxyhaemoglobin (oxy-Hb; 10^-3 M), SNAP-induced VIP release was blocked (basal: 57.5 ± 3.2 pg mg^-1 (= 100 %); 10^-4 M SNAP: 200.4 ± 22.5 %; SNAP + oxy-Hb: 112.4 ± 21.2 %; P 0.01, n = 7). Oxy-Hb (10^-3 M) alone did not alter basal VIP release (115.5 ± 13.4 %, n = 5). In a second series of experiments, the effect of oxy-Hb on SIN-1-induced release of VIP was investigated. The results obtained were similar to those with SNAP (basal: 110.5 ± 22.3 pg mg^-1 (= 100 %); 10^-4 M SIN-1: 145.5 ± 10.9 %; SIN-1 + 10^-3 M oxy-Hb: 104.2 ± 10.8 %; P 0.05, n = 9). Addition of SOD (100 U ml^-1) slightly, but not significantly, increased the net release of VIP evoked by SIN-1 (Table 2). These data suggest that VIP release is mediated by NO derived from NO donors, but not by by-products of NO catabolism. Evidence against a role for reactive oxygen species has also been obtained, as the antioxidants GSH, NAC and ascorbate did not influence SNAP-induced VIP release (basal: 37.0 ± 2.8 pg mg^-1 (= 100 %), 10^-4 M SNAP: 147.5 ± 5.7 %; SNAP + 10^-3 M GSH: 139.7 ± 13.9 %; SNAP + 10^-3 M NAC: 160.9 ± 17.5 %; SNAP + 10^-3 M ascorbate: 149.7 ± 9.5 %; n = 4, all treatments not significant compared with SNAP alone). None of the compounds influenced basal VIP release (data not shown). Inhibition of NOS by L-NNA (10^-4 M) in the presence of exogenous NO generated by SNAP did not significantly attenuate the evoked VIP release (basal: 33.2 ± 5.7 pg mg^-1 (= 100 %); 10^-4 M SNAP: 139.8 ± 11.4 %; SNAP + 10^-4 M L-NNA: 139.6 ± 15.0 %; n = 6).

VIP and pituitary adenylate cyclase-activating peptide 1-14 (PACAP 1-14), a peptide of the VIP family, at various concentrations had no effect on L-citrulline formation in enteric synaptosomes (basal: 20.0 ± 4.0 fmol mg^-1 min^-1, 10^-8 M VIP: 22.0 ± 4.0 fmol mg^-1 min^-1, 10^-7 M VIP: 18.0 ± 5.0 fmol mg^-1 min^-1; 10^-6 M VIP: 24.0 ± 13.0 fmol mg^-1 min^-1; 10^-5 M VIP: 20.0 ± 3.0 fmol mg^-1 min^-1; n.s., n = 3; basal: 251 ± 63 fmol mg^-1 min^-1, 10^-9 M PACAP: 185 ± 66 fmol mg^-1 min^-1, 10^-8 M PACAP: 182 ± 42 fmol mg^-1 min^-1, 10^-7 M PACAP: 189 ± 75 fmol mg^-1 min^-1; n.s., n = 4).

Role of guanylate cyclase and protein kinase G in SNAP-induced VIP release

Another series of experiments was conducted in order to characterize the signal transduction pathways involved in VIP release under the conditions employed. As guanylate cyclase is the target enzyme of NO, its role was investigated by specific inhibition of its activation by ODQ. ODQ (10^-5 M) antagonized the release of VIP induced by SNAP (10^-4 M) (basal: 162.8 ± 79.7 pg mg^-1 (= 100 %); SNAP: 146.7 ± 8.4 %; SNAP + ODQ: 115.1 ± 7.9 %; P 0.05, n = 8; Fig. 3). ODQ alone had no effect on basal values (97.8 ± 9.9 %, n = 7). To test whether signal transduction involves activation of PKG, the effects of various inhibitors of PKG were studied. KT 5823 (3 10^-6 M) significantly attenuated VIP release evoked by SNAP (basal: 110.5 ± 22.1 pg mg^-1 (= 100 %); 10^-4 M SNAP: 200.4 ± 22.4 %; SNAP + KT 5823: 122.4 ± 6.2 %; P 0.05, n = 7; see Fig. 6). Rp-8Br-PET-cGMPS (5 10^-7 M), a specific inhibitor of the PKG I isoform, also blocked NO-stimulated VIP release (10^-4 M SNAP: 146.7 ± 8.4 %; SNAP + Rp-8Br-PET-cGMPS: 105.9 ± 8.8 %; P 0.05, n = 4). Neither KT 5823 nor Rp-8Br-PET-cGMPS alone influenced basal release.

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Figure 3. Effect of inhibition of guanylate cyclase The guanylate cyclase inhibitor ODQ antagonized SNAP (10-4 M)-induced VIP release without affecting basal VIP release. Means ± S.E.M. of n = 8 independent experiments carried out in duplicate (*P 0.05).

The role of PKG was further supported by the stimulatory effect of direct activators of this enzyme. The membrane-permeant and PDE-resistant cGMP analogue 8-pCPT-cGMP (3 10^-5 M) significantly stimulated VIP release (basal: 50.5 ± 11.2 pg mg^-1 (= 100 %); 8-pCPT-cGMP: 221.0 ± 25.7 %; n = 11, P 0.05) to a comparable extent to SNAP.

Role of phosphodiesterase type 5

Since cGMP degradation by cGMP-specific phosphodiesterase (PDE) type 5 appears to be another regulatory mechanism involved in cGMP-PKG signalling, the presence and functional role of PDE 5 were studied. The presence of a PDE 5 isozyme in the rat longitudinal muscle layer with attached myenteric plexus (LM-MP) preparation was determined by RT-PCR using specific primers and subsequent sequencing of the respective PCR product. A single band at the expected size for PDE 5 of ~800 bp was obtained (Fig. 4). Sequencing of the cloned RT-PCR product confirmed the cDNA sequence of this isozyme. Accordingly, zaprinast (3 10^-5 M), a specific inhibitor of PDE 5, stimulated basal VIP release (basal: 134.3 ± 22.8 pg mg^-1 (= 100 %); zaprinast: 134.9 ± 6.8 %; P 0.05, n = 8), but failed to augment the stimulatory effect of 10^-4 M SNAP (SNAP + zaprinast: 147.7 ± 12.4 %; compared with SNAP alone: 162.8 ± 20.7 %; n = 8; Fig. 5). Since the lack of potentiation of SNAP-induced VIP release by zaprinast was surprising, a new series of experiments was conducted with YC-1, a potent stimulator of guanylate cyclase, to investigate whether NO-independent stimulation of guanylate cyclase would enhance VIP output in the presence of exogenous NO. YC-1 (10^-6 M) induced significant VIP release, compared with basal; however, this compound was unable to further increase VIP release induced by SNAP (10^-4 M SNAP: 161.7 ± 29.8 %; YC-1: 154.3 ± 35.1 %; SNAP + YC-1: 150.2 ± 19.2 %; P 0.05, n = 3). The finding that activation of PKG by the PDE-resistant cGMP analogue 8-pCPT-cGMP and stimulation of guanylate cyclase by SNAP or YC-1 both resulted in comparable effects with respect to VIP release, suggests that following treatment with exogenous NO at the concentration used a rate-limiting stimulation of PKG, and concomitantly a maximum physiological response, was achieved. Under these circumstances, inhibition of cGMP breakdown by zaprinast would not be effective in further stimulating VIP release.

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Figure 4. Detection of PDE 5 by RT-PCR in the LM-MP preparation RT-PCR was performed with primers specific for PDE 5. A single band with the expected size of ~800 bp was obtained. The product was subsequently shown to be PDE 5 by sequencing. M, molecular weight markers.

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Figure 5. Effect of inhibition of cGMP-specific phosphodiesterase 5 The PDE 5 inhibitor zaprinast (ZAP; 3 10-5 M) stimulated basal VIP release, whereas SNAP (10-4 M)-induced VIP release was not further potentiated. Means ± S.E.M. of n = 8 independent experiments carried out in duplicate (*P 0.05, compared with basal).

Role of protein kinase A, phosphodiesterase 3 and adenylate cyclase

Since cross-activation apparently occurs during cyclic nucleotide signalling and cGMP is known to inhibit PDE 3, thereby increasing cAMP levels, further experiments were conducted to study the role of cAMP-protein kinase A (PKA) signalling. Rp-cAMPS, an inhibitor of PKA, at a concentration of 10^-4 M, did not significantly influence VIP release evoked by 10^-4 M SNAP (basal: 33.2 ± 5.6 pg mg^-1 (= 100 %), 10^-4 M Rp-cAMPS + SNAP: 129.3 ± 15.5 %; n.s., n = 6, compared with SNAP alone: 139.8 ± 11.4 %, n = 6). Rp-cAMPS (10^-4 M) alone had no effect on basal VIP release. The combination of Rp-cAMPS with KT 5823 (3 10^-6 M) did not result in further inhibition of VIP release induced by SNAP, compared with KT 5823 alone (data not shown). Inhibition of PDE 3 by trequinsin and inhibition of adenylate cyclase by SQ 22536 failed to influence VIP release (basal: 63.2 ± 15.0 pg mg^-1 (= 100 %); 10^-8 M trequinsin: 107.0 ± 8.5 %; 10^-4 M SQ 22536: 109.5 ± 17.6 %; n.s., n = 5). These data suggest that cross-activation of PKA does not occur in the presence of exogenous NO.

Effect of endogenous NO synthesis on VIP release

When no exogenous NO was supplied, addition of L-NNA at a concentration of 10^-4 M did not reduce basal VIP release (basal: 33.2 ± 5.7 pg mg^-1 (= 100 %); L-NNA: 132.9 ± 34.4 %; n = 4), implying that basal NO production presumably is not sufficient to release VIP. However, supplementation with L-arginine reversed this condition resulting in significant VIP secretion (basal: 110.5 ± 22.1 pg mg^-1 (= 100 %); 10^-3 M L-arginine: 163.2 ± 22.3 %), which could be antagonized by both L-NNA and KT 5823 (L-arginine + 10^-4 M L-NNA: 109.5 ± 12.3 %; L-arginine + 3 10^-6 M KT 5823: 101.9 ± 11.8 %; P 0.05, n = 6 and n = 8, respectively; Fig. 6). In contrast to SNAP-induced VIP release, L-arginine-stimulated VIP secretion was insensitive to oxy-Hb (basal: 110.5 ± 22.2 pg mg^-1 (= 100 %); 10^-3 M L-arginine: 163.2 ± 22.3 %; L-arginine + 10^-3 M oxy-Hb: 141.0 ± 31.4 %; n.s., n = 8; Fig. 7). As oxy-Hb is not membrane permeant, this finding suggests that endogenously synthesized NO interacts with VIP-containing vesicles within the same membrane-enclosed compartment (i.e. single nerve terminal).

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Figure 6. Effect of inhibition of protein kinase G The effect of the PKG inhibitor KT 5823 (3 10-6 M) on basal VIP release and VIP release induced by SNAP (10-4 M) or L-arginine (10-3 M). Means ± S.E.M. of n = 7-8 independent experiments on rat ileal synaptosomes carried out in duplicate (*P 0.05).

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Figure 7. Effect of endogenous NO synthesis on VIP release Activation of endogenous NO production by L-arginine stimulates VIP release within the same nerve terminal, since addition of oxy-Hb does not antagonize the release effect. Means ± S.E.M. of n = 8 independent experiments (*P 0.05, compared with basal).

	DISCUSSION

Top Abstract Introduction Methods Results Discussion References

NO and VIP are considered to be inhibitory gastrointestinal neurotransmitters. Whereas NO fulfills tissue and cellular criteria for being the inhibitory non-adrenergic, non-cholinergic (NANC) mediator in the gastrointestinal tract (Sanders & Ward, 1992), evidence has been presented against a central role for VIP as a NANC transmitter at the cellular level (Daniel et al. 1994; Bayguinov et al. 1999). However, there is a possibility that both compounds collaborate in inhibitory NANC transmission by inducing different cellular mechanisms in smooth muscle or controlling the release of each other from presynaptic sites. The sequence of events constituting inhibitory transmission in the gut remains controversial. It is still not clear whether NO and VIP act as enteric cotransmitters in parallel (Li & Rand, 1990; Desai et al. 1994; Bayguinov et al. 1999) or by means of a 'serial cascade' (Grider & Jin, 1993; Mashimo et al. 1996; Jin et al. 1996). The latter hypothesis would imply that either NO induces VIP release (Grider & Jin, 1993; Jin et al. 1996), or presynaptic NO production is activated by VIP release (Mashimo et al. 1996). Also, VIP could stimulate NO synthesis in postjunctional cells, which consecutively enhances VIP release from prejunctional sites (Jin et al. 1996). NO has previously been shown to stimulate VIP release from enteric nerve terminals (Allescher et al. 1996) and from isolated ganglia (Grider & Jin, 1993). The latter authors have demonstrated that PKG contributes to NO-mediated VIP release in these myenteric ganglia. However, in both experimental settings the release most probably occurs from nerve terminals and, as ganglia do not contain nerve terminals of motoneurons relevant for VIP release at the smooth muscle site, it appeared of physiological relevance to characterize whether the same or different subcellular mechanisms are involved in the effect of NO on VIP release from this more isolated anatomical site. The methodological approach chosen in the present study eliminates both interference from a ganglionic network and contamination by interstitial cells of Cajal (ICCs) as a potential source of NO and represents a mixture of nerve terminals from all layers of the intestine including those from motoneurons. Furthermore, in the study of Grider & Jin (1993) no data were presented showing that the observed effect was specifically related to NO, since no NO-scavenger was used, and the fact that by-products of NO metabolism and reactive oxygen species might play a role was not taken into consideration. In our study, oxy-Hb completely reversed the SNAP-induced VIP release, suggesting that the effect is clearly NO mediated. Additional data were presented which indicate that reactive oxygen species presumably are not involved.

Activation of soluble guanylate cyclase and the subsequent elevation in intracellular cGMP levels is considered to be the primary mode of action of NO. We showed recently that enteric synaptosomes are capable of accumulating cGMP following stimulation by NO donors (Kurjak et al. 1999a). This is in agreement with previous findings from canine enteric synaptosomes (Kostka et al. 1993). Subsequently, we examined whether NO-induced cGMP elevation contributes to VIP release. The data obtained with ODQ, a selective inhibitor of soluble guanylate cyclase, together with the accumulation of neuronal cGMP by exogenous NO, indicate an involvement of cGMP in the release process. Another objective of the present study was to further characterize the signalling pathway downstream of the activation of cGMP. cGMP elicits its physiological effects by acting directly on PKG, on phosphodiesterases (PDEs) or by cross-activating PKA (Wang & Robinson, 1997).

Firstly, the role of PKG was examined. The presence of both PKG (Huber et al. 1998b) and soluble guanylate cyclase (Schmidt et al. 1992) has been demonstrated in enteric neurons of the rat gastrointestinal tract. Thus the necessary elements of the cGMP-PKG-pathway appear to be present in the enteric nervous system. The PKG blocker KT 5823 significantly inhibited VIP release induced by exogenous NO. With respect to the predominance of the PKG I-subtype in enteric nerves (Huber et al. 1998b), we studied the effect of Rp-8BrPET-cGMPS, a specific inhibitor of the PKG I isoform, which also blocked VIP release induced by NO. Thus the data presented confirm that, in isolated nerve terminals also, the cGMP-PKG pathway is involved in NO-mediated VIP release, as shown previously by Grider & Jin (1993) in myenteric ganglia. The finding that VIP does not influence NO synthesis in nerve terminals was also in agreement with the data reported by these authors.

In a second series of experiments we studied whether cGMP modulates cellular signalling by interaction with phosphodiesterase (PDE) enzymes present in synaptosomes. cGMP-specific PDE 5 preferably hydrolyses cGMP rather than cAMP. Zaprinast, a selective inhibitor of this enzyme, significantly enhanced basal VIP release, suggesting that PDE 5 plays a role in termination of the cGMP response in enteric synaptosomes. PDE 5 is expressed in the central nervous system of human (Loughney et al. 1998) and rat (Kotera et al. 1997). We demonstrated recently the presence of PDE 5 in the enteric nervous system of the rat by Western blot analysis (Kurjak et al. 1999a). As the only antibody against bovine PDE 5 available shows only about ~90 % homology with rat PDE 5, a complementary RT-PCR of PDE 5 was performed in LM-MP preparations of rat small intestine. The primer used was specific for PDE 5. The cDNA detected in LM-MP showed sequence homology with PDE 5, adding supportive evidence for a regulatory role of this PDE 5 isozyme. Interestingly, the NO-stimulated VIP release was not augmented in the presence of zaprinast, a finding which has been reported by others in a different system (Weimann et al. 2000). This could be due to a nearly maximal stimulation of guanylate cyclase by exogenous NO, since pretreatment of synaptosomes with the PDE-resistant PKG activator 8-pCPT-cGMP increased VIP release to the same extent as SNAP. In addition, it has been shown that selective activation of guanylate cyclase by YC-1 (Friebe et al. 1998) could not potentiate the SNAP effect on VIP release, which supports the notion that at the concentration of exogenous NO used a maximum physiological response is achieved. A 4-fold stimulation of cGMP accumulation, as shown in the presence of zaprinast and SNAP in synaptosomes (Kurjak et al. 1999a), is generally accepted as inducing a maximum physiological response (Corbin & Francis, 1999). Theoretically, allosteric binding of cGMP to PDE 5 can lead to a conformational change in the structure of PDE 5, causing it to become a substrate for phosphorylation by PKG. In the phosphorylated state, the enzyme is activated and breaks down cGMP, thereby exerting a regulatory feedback mechanism (Corbin et al. 2000), which could concomitantly result in a reduced VIP output. However, this is speculative and deserves further elucidation. In contrast, no evidence for cross-activation of PKA could be obtained. Rp-cAMPS, a specific inhibitor of PKA, failed to antagonize the release of VIP in the presence of exogenous NO. Theoretically, cGMP could also inhibit PDE 3, thereby increasing intracellular cAMP levels, which have been shown to be responsible for the release of bombesin from NO-stimulated enteric neurons (Kurjak et al. 1999a). However, no biochemical evidence for a role of PDE 3 in the release of VIP from VIPergic nerves has been found.

As shown in our previous paper (Allescher et al. 1996), L-arginine stimulates VIP release from nerve terminals, thereby offering the opportunity of investigating the role of endogenous NO production in VIP release, in order to determine the functional role of NO within the nerve ending. Neuronal nitric oxide synthase (nNOS) has been shown to be present in enteric nerve terminals (Huber et al. 1998a). With respect to endogenous NO synthesis interesting and functionally relevant observations have been made. L-Arginine-stimulated NO synthesis was sensitive to inhibition by KT 5823, indicating an identical signal transduction pathway for NO from endogenous sources. However, the L-arginine-induced VIP release could not be reversed by oxy-Hb. As oxy-Hb is not membrane permeant, these findings suggest that endogenously produced NO interacts with VIP-containing vesicles within the same nerve terminal rather than between terminals. At the ultrastructural level, NO synthase does not appear to be located in the same domain as the VIP-containing vesicles (Berezin et al. 1994), raising the possibility that both transmitters are regulated independently. Taken together, the biochemical findings suggest that NO synthesized by nNOS might be crucial for an optimal VIP effect. This has been demonstrated in intracellular recordings in ileal smooth muscle cells of guinea-pig (He & Goyal, 1993) or nNOS-knockout mice (Mashimo et al. 1996), as well as in parasympathetic nerves (Modin et al. 1994). The finding that both blockade of NO synthesis or sequestration of NO does not alter the postjunctional responses to VIP (Bayguinov et al. 1999) does not preclude a presynaptic stimulatory effect of NO on VIP release, since other mechanisms might stimulate VIP release (Allescher et al. 1996). In contrast, this observation strengthens the functional importance of VIP for smooth muscle relaxation. In this context, it is of interest that in diabetic rats impaired VIP release and defective NANC inhibition have been demonstrated (Belai et al. 1987) and that this impairment is presumably due to a selective decrease in nNOS expression, leaving VIPergic innervation intact (Watkins et al. 2000). The associated dysfunction in smooth muscle relaxation could be effectively treated by application of insulin or a PDE 5 inhibitor, the latter of which restores NO signalling (Watkins et al. 2000).

Therefore our findings could provide a possible functional link between the in vitro and in vivo observations and might be of importance for potential therapeutic concepts designed to interfere with the signalling pathway described.

In conclusion, we have demonstrated that an anatomical and functional interrelationship exists between VIP and NO within isolated nerve terminals and that PKG and PDE 5 are involved in NO-mediated VIP release. The presence and expression of PDE 5 in the LM-MP preparation adds supportive evidence for its putative functional role in neuronal NO-cGMP-PKG signalling and neuropeptide secretion.

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Acknowledgements

The authors acknowledge the co-operation of Professor Dr Gänsbacher, Professor Erhardt and their collaborators, Department of Experimental Surgery, Technical University of Munich. We would like to thank H. Paeghe and S. Herda for their expert technical assistance. The study was supported by Deutsche Forschungsgemeinschaft SFB 391/C5.

Corresponding author

H. D. Allescher: Department of Internal Medicine II, Technical University of Munich, Ismaninger Strasse 22, 81675 Munich, Germany.

Email: hans.allescher{at}lrz.tu-muenchen.de

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