Introduction

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An accumulating body of evidence has shown a role for nitric oxide (NO) and vasoactive intestinal polypeptide (VIP) in mediating nonadrenergic noncholinergic (NANC) relaxation in the gastrointestinal tract of a variety of species (Bitar et al., 1980; Goyal et al., 1980; Grider et al., 1985; Li and Rand, 1990). In the enteric nervous system, NO synthase (NOS) is colocalized in many neurons of the myenteric plexus, including that of human gastric fundus, that contain VIP (Furness et al., 1992; Tonini et al., 2000). These neurons project into the circular and longitudinal muscle layers and appear to be involved in muscle relaxations. Neural stimulation of smooth muscle is accompanied by a frequency-dependent increase in VIP release and NO production that is inhibited by the axonal conductance blocker tetrodotoxin (Grider et al., 1992). Also, evidence based on the use of specific VIP antiserum and selective VIP antagonists supports the idea that release of VIP from these neurones is partly responsible for relaxation of gastrointestinal smooth muscle (Grider et al., 1985; Grider and Rivier, 1990). The presence of NOS in VIP neurones suggests the possibility that VIP and a related peptide PACAP may be released from nerve terminals in parallel with NO or be linked to NO in serial pathways, such that NO regulates VIP release while VIP produces NO in target smooth muscle cells (Grider et al., 1992; Grider and Jin, 1993). Recent studies in rabbit, rat, and guinea pig gastric muscle strips reported that VIP-induced relaxations were inhibited by NOS inhibitors, suggesting the serial cascade model, in which VIP was proposed to be the primary neurotransmitter, inducing relaxation partially via activation of adenylyl cyclase and partially via stimulation of NO production (Grider et al., 1992; Jin et al., 1996). In contrast, the ineffectiveness of NOS inhibitors on the relaxations induced by VIP in many gastrointestinal tissues supports the idea that there is no interaction between NO and VIP in the gastrointestinal tract. NO is thought to induce relaxation via a guanosine 3'-5' cyclic monophosphate (cGMP)-dependent pathway and VIP via an adenosine 3'-5' cyclic monophosphate (cAMP)-dependent pathway (Lefebvre et al., 1995; Bayguinov et al., 1999; Dick et al., 2000). Although the localization of NOS to nerve structures in the smooth muscle wall and the demonstration of NO as a neurotransmitter in the gastrointestinal tract of the mouse were recently elucidated (Lefebvre, 1993; Ny and Andersson, 1998), little is known about the contribution of VIP to NANC relaxations in mouse gastric fundus. Recently, Mashimo et al. (1996) have shown an interaction between NO and VIP at the prejunctional site of the neuromuscular junction, where VIP was shown to be the primary neurotransmitter and caused the production of NO via activation of neuronal constitutive NOS. On the other hand, Ny et al. (2000) found that VIP-induced relaxations were not altered in mice lacking cGMP-dependent protein kinase I (cGKI), and the authors suggested that relaxant agents acting via the cAMP pathway can exert their effects independently of cGKI. It is clear that there is still controversy about the interaction between NO and VIP and that the underlying mechanisms of this interaction remain to be resolved. Therefore, the aim of the present study was to investigate the contribution of NO and VIP to NANC responses in the mouse gastric fundus and the possible interaction between NO and VIP. It has been reported that the ability of VIP to stimulate NO production is masked in muscle strips pretreated with carbachol, suggesting that the use of contractile agonists to raise basal tension leads to stimulation of protein kinase C (PKC) and inactivation of smooth muscle NOS (Murthy et al., 1994). To avoid the possible masking effect of contractile agonists on NOS activity, we used noncontracted tissue, i.e., basal tonus only. This present study provides evidence for participation of NO and does not exclude participation of VIP and related neuropeptides in nerve-mediated relaxation of mouse gastric fundal strips and supports the idea that the response to VIP is mediated largely via NO formation in smooth muscle.

	Materials and Methods

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Preparation of Smooth Muscle Strips. Both sexes of Swiss albino mice (25-30 g) were fasted for 24 h with free access to water, and were killed by stunning and cervical dislocation. The stomach was excised immediately, and muscle strips (10-12 mm long) were prepared from the fundus by cutting in the direction of the circular muscle layer. The strips were suspended between two platinum plate electrodes under a load of 500 mg of tension in 5-ml organ baths containing Tyrode's solution (136.75 mM NaCl, 2.68 mM KCl, 1.8 mM CaCl₂, 0.95 mM MgCl₂6H₂O, 0.4166 mM NaH₂PO₄2H₂O, 11.904 mM NaHCO₃, 5.05 mM glucose, 0.05 mM EDTA, and 0.02 mM ascorbic acid). The Tyrode's solution always contained 1 µM atropine and 1 µM guanethidine to inhibit cholinergic and adrenergic responses. The solution was maintained at 37°C and gassed with a mixture of 95% O₂ and 5% CO₂. Muscle strips were allowed to equilibrate for a period of 60 min. At the end of this period, active tone rose to ~500 mg and remained at this level throughout the experiments without addition of exogenous contractile agonist. Changes in muscle tension were recorded isometrically via an isometric transducer (Ugo Basile 7003, Varese, Italy) connected to an ink-writer (Ugo Bassile 7070, Varese, Italy).

Experimental Protocols. Once a stable basal tone was obtained (see above), sodium nitroprusside (SNP; 10 µM) was applied and a maximal relaxation was produced. After rinsing SNP the tissue recovered the initial basal tone and then ES (0.5, 1, 2, 4, and 8 Hz; 25 V; 1 ms; 15-s trains) was applied to the tissue at 3-min intervals. After the relaxant responses to NANC nerve stimulation had been obtained, the strips were rinsed at 10-min intervals for at least 45 min, and the second series of responses were recorded in the same manner. To study the relaxant actions of 100 nM SNP, 50 nM VIP, and 10 nM isoproterenol, whose effects were shown to be submaximal in preliminary experiments, a similar protocol was used in separate experimental groups in different strips.

Drugs and Solutions. -Chymotrypsin, 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine, D-arginine hydrochloride, L-arginine hydrochloride, atropine sulfate, bovine serum albumin, guanethidine sulfate, hemoglobin, hydroxocobalamin, 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one, isoproterenol hydrochloride, N-nitro-L-arginine, sodium nitroprusside, and vasoactive intestinal poypeptide were all obtained from Sigma (St. Louis, MO). VIP antiserum 7913 from rabbit serum was provided by CURE/Gastroenteric Biology Center, Antibody/RIA Core, and National Institutes of Health Grant DK 41301 as a kind gift. All drugs were dissolved in distilled water, except for ODQ, which was dissolved in dimethylsulfoxide up to 1 mM; further dilutions were made in distilled water. The solvent, diluted in distilled water, had no effect on the tone of the strips or on the responses to various relaxant stimuli.

Presentation of Results and Statistical Analysis. Relaxations induced by ES, SNP, VIP, and isoproterenol were expressed as percentage of the relaxation induced by 10 µM SNP at the beginning of the experiment. Relaxations induced by SNP, VIP, and isoproterenol varied between experimental groups. Therefore, the responses obtained in the presence of inhibitor drugs have been expressed as a percentage of the initial control to control for differences in responsiveness between experimental groups. Results were expressed as mean ± S.E.M. and n refers to the number of animals used for each experiment. Results between tissues were analyzed by unpaired Student's t test and within tissues were analyzed by paired Student's t test. P values of less than 0.05 were considered statistically significant.

	Results

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Effects on the Responses to ES. ES (0.5, 1, 2, 4, and 8 Hz; 25 V; 1 ms) with 15-s trains at 3-min intervals induced transient and frequency-dependent relaxations (n = 10) that were fast in onset and returned rapidly to baseline after cessation of the stimulus (Figs. 1A and 2A). L-NOARG (100 µM), an inhibitor of NO biosynthesis, abolished the relaxations induced by ES after an incubation period of 15 min (n = 8) but had no effect on the basal tonus of the strips (Figs. 1B and 2A). When 1 mM L-arginine was administered 30 min before L-NOARG, the inhibitory effect of the NOS inhibitor was reduced (n = 8, data not shown). Incubation with 1 mM D-arginine before L-NOARG did not influence its effect (n = 4, data not shown). Furthermore, 20 µM hemoglobin was tested on the train stimulation of the strips, and it diminished the responses to a lesser extent than did L-NOARG (n = 6, Figs. 1C and 2A). Similarly, 100 µM hydroxocobalamin also inhibited the NANC relaxations to nerve stimulation (n = 6, Fig. 2A). Moreover, ODQ at a concentration of 10 µM, completely inhibited the relaxations induced by NANC nerves (n = 7, Figs. 1D and 2A). -Chymotrypsin (10 U/ml), a peptidase, did not affect the electrical responses (n = 6, Figs. 1E and 2B). Likewise, VIP antiserum (1/200 dilution) did not have any effect on these responses (n = 6, Figs. 1F and 2B). Nevertheless, VIP antiserum (1/200 dilution) did block VIP-induced relaxation (see Fig. 4).

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Representative traces showing the relaxation to ES (0.5-8 Hz, 25 V, 1 ms, 15-s trains; A) and the effects of L-NOARG (100 µM; B), Hb (20 µM; C), ODQ (10 µM; D), -CT (10 U/ml; E), and VIP antiserum (1/200 dilution; F) on the response to ES in the circular muscle strips of the mouse gastric fundus.

View larger version (15K): [in this window] [in a new window]   Fig. 2.   A, effects of L-NOARG (100 µM), ODQ (10 µM), Hb (20 µM), and hydroxocobalamin (HC; 100 µM). B, effects -CT (10 U/ml) and VIP antiserum (1/200 dilution) on the response to ES (0.5-8 Hz, 25 V, 1 ms, 15-s trains) in the circular muscle strips of the mouse gastric fundus. Values are mean ± S.E.M. from n = 4-10. *, P < 0.05 significantly different from control, paired Student's t test.

Effects on the Responses to SNP. The relaxations induced by SNP (100 nM) were fast in onset and sustained (n = 6). L-NOARG (100 µM) after an incubation period of 15 min, which abolished the responses to electrical stimuli, did not have any effect on these responses (n = 6, Fig. 3); whereas 10 µM ODQ attenuated these relaxations (n = 6, Fig. 3). However, 10 U/ml -chymotrypsin did not attenuate SNP-induced relaxations (n = 6, Fig. 3).

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Effect of L-NOARG (100 µM), ODQ (10 µM), and -CT (10 U/ml) on the response to SNP (100 nM) in the circular muscle strips of the mouse gastric fundus. Values are mean ± S.E.M. from n = 6. *, P < 0.05 significantly different from control, unpaired Student's t test.

Effects on the Responses to VIP. Responses to 50 nM VIP were developed at a slow rate, were sustained (n = 6), and were not potentiated in the presence of 0.1% bovine serum albumin (n = 4, data not shown). A peptidase, -chymotrypsin, in a concentration of 10 U/ml, abolished these responses (n = 6, Fig. 4). On the other hand, the inhibitory effect of VIP antiserum (1/200 dilution) was much less effective than that of -chymotrypsin (n = 6, Fig. 4). Addition of L-NOARG at a concentration of 100 µM significantly reduced the relaxations induced by VIP (n = 6, Fig. 4), and this inhibition was reversed by 1 mM L-arginine (n = 6, Fig. 4), but not by D-arginine (1 mM, n = 6, data not shown). The selective inducible NOS inhibitor AMT (5 µM) had no effect on these relaxations (n = 6, Fig. 4). As a corroboration of the results of L-NOARG, 10 µM ODQ clearly inhibited the responses to VIP to a similar extent as that of L-NOARG (n = 6, Fig. 4).

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Effects of -CT (10 U/ml), VIP antiserum (1/200 dilution), L-NOARG (100 µM), L-NOARG plus L-arginine (L-NOARG+L-ARG; 100 µM + 1 mM), AMT (5 µM), and ODQ (10 µM) on the response to VIP (50 nM) in the circular muscle strips of the mouse gastric fundus. Values are mean ± S.E.M. from n = 6. *, P < 0.05 significantly different from control; +, P < 0.05 significantly different from L-NOARG, unpaired Student's t test.

Effects on the Responses to Isoproterenol. Isoproterenol (10 nM) produced a slowly developing and sustained relaxation (n = 6). L-NOARG (100 µM) and ODQ (10 µM) had no effect on isproterenol-induced relaxation (n = 6, Fig. 5).

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Effects of L-NOARG (100 µM) and ODQ (10 µM) on the response to isoproterenol (10 nM) in the circular muscle strips of the mouse gastric fundus. Values are mean ± S.E.M. from n = 6. *, P < 0.05 significantly different from control, unpaired Student's t test.

	Discussion

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This study provides evidence for participation of NO but cannot exclude participation of VIP and related neuropeptides in nerve-mediated relaxation of mouse gastric fundal strips. This study also supports the idea that the response to VIP is mediated largely via NO formation in smooth muscle strips. It has been well-established that both NO and VIP serve as inhibitory NANC mediators in the gastrointestinal tract (Bitar et al., 1980; Goyal et al., 1980; Grider et al., 1985; Li and Rand, 1990). In our study, the inhibition of the NANC nerve-induced relaxations by L-NOARG show that NO is a mediator in the mouse gastric fundus. Furthermore, hemoglobin and hydroxocobalamin, which bind and inactivate NO extracellularly (Li and Rand, 1993; Rajanayagam et al., 1993), effectively inhibited electrical relaxations, suggesting that the transmitter released from the nerve terminals is probably free NO. Moreover, ODQ, the soluble guanylyl cyclase inhibitor, similarly abolished the relaxations induced by NANC nerves and revealed further evidence for NO being a mediator, because the relaxant action of NO in the smooth muscle cells is due to the activation of the cytosolic guanylyl cyclase (Ignarro, 1991). In addition, we investigated the involvement of VIP, which has relaxant effects on smooth muscle cells through activation of adenylyl cyclase, increase of cAMP, and subsequent activation of protein kinase A (Bitar and Makhlouf, 1982; Murthy and Makhlouf, 1995), in NANC relaxations. For these studies, we used -chymotrypsin, a peptidase that is thought to diffuse to neuromuscular junctions and is capable of degrading released peptides during ES (De Beurme and Lefebvre, 1987). However, the involvement of VIP is not likely to be the case because -chymotrypsin in a concentration 10 U/ml, which abolished the relaxations induced by VIP, did not affect the NANC nerve induced relaxations. The lack of effect of -chymotrypsin in our studies may be due to the limited ability of this large molecule to penetrate the neuromuscular junction in this tissue. VIP antiserum, which neutralizes endogenous VIP, in a dilution of 1/200 also did not influenced the relaxations to nerve stimulation. Several investigators have reported that VIP antiserum and VIP antagonists can block effectively the response to nerve stimulation (Grider et al., 1985; Grider and Rivier, 1990). It is possible that the ineffectiveness of VIP antiserum in our study may be due to the dilution of this agent, because the same dilution of VIP antiserum used in ES studies was only partially capable of blocking the effect of a very low concentration (50 nM) of exogenous VIP and did not produce sufficient immunoneutralization in our study. Recent studies in muscle strips of guinea pig, rat, and rabbit stomach have shown by direct measurement that relaxation induced by ES is accompanied by a frequency-dependent increase in VIP release and NO production, and NOS inhibitors abolish both VIP release and NO production (Grider et al., 1992; Jin et al., 1996). In the present study, blocked ES responses by the NOS inhibitor L-NOARG may reflect inhibition of VIP (and related peptides, such as PACAP; Katsoulis et al., 1996) release from nerve terminals and NO formation in nerve terminals and muscle strips. Furthermore, the excitatory tachykinins, substance P and neurokinin A, are also co-released with VIP and PACAP during electrical stimulation of the gastrointestinal tract (Jin et al., 1993a; Zagorodnyuk et al., 1995; Smid et al., 1998), and in the present study the excitatory effects of tachykinins could mask the effect of VIP or PACAP. Consequently, our results provide evidence for participation of NO but do not exclude participation of VIP and related peptides.

Previous studies have shown that the relaxant effect of exogenous VIP on gastric muscle strips or isolated muscle cells is accompanied by an increase in NO production and is partly inhibited by NOS inhibitors implying that NO could be produced in muscle cells as a result of the action of VIP (Grider et al., 1992; Jin et al., 1993b, 1996; Murthy et al., 1994). The present study confirmed this hypothesis in gastric muscle strips of mouse. The relaxations induced by VIP were diminished partially but significantly by L-NOARG, and this inhibition was reversed by addition of L-arginine suggesting that at least part of the relaxation by VIP is due to NO synthesis in the smooth muscle strips. This was corroborated by the results with the selective guanylyl cyclase inhibitor ODQ (Garthwaite et al., 1995), which inhibited the relaxant effect of VIP. In several studies, investigators have measured not only NO but also cAMP and cGMP and protein kinases in response to exogenous VIP. They have shown that both cAMP-dependent protein kinase and cGMP-dependent protein kinase inhibitors (R)-p-adenosine 3,5-cyclic phosphorothioate and KT5823, respectively, inhibit VIP stimulated relaxation, and a combination of these inhibitors abolish relaxation (Grider, 1993; Jin et al., 1993b; Murthy and Makhlouf, 1998). These results support the possibility that VIP-induced relaxation may be mediated partly by NO-dependent stimulation of cGMP. In our study, the same concentrations of L-NOARG and ODQ did not affect the relaxations induced by isoprotorenol, which activates adenylyl cyclase via -adrenoceptors (Honeyman et al., 1977). These results are in agreement with those of Grider et al. (1992), who showed that relaxation induced by exogenous VIP, but not isoproterenol and forskolin, was inhibited by an NOS inhibitor in guinea pig stomach, implying that NO was produced by the direct action of VIP in muscle strips of mouse gastric fundus. These results are in contrast to those of Rekik et al. (1996), in which NOS inhibitors reduced the responses to both VIP and forskolin, which directly stimulate adenylyl cyclase activity, and 8-bromo-cAMP, an analog of cAMP. These authors suggested that VIP may activate the inducible NOS-NO-cGMP pathway in the intestinal muscle cells of the guinea pig via a cAMP and protein kinase A pathway. The data obtained in the present study supports the hypothesis that there is an interaction between NO and VIP, in which VIP directly induces the production of NO by stimulating NOS activity and thereby increasing cGMP levels. The presence of a membrane-bound NOS was described in plasma membranes of the rabbit stomach (Murthy and Makhlouf, 1994), and a similar observation was reported recently for the canine lower esophageal sphincter (Salapatek et al., 1998). It was also suggested that the site of NO release in response to VIP may be neurons rather than target smooth muscle cells (Chakder and Rattan, 1993; Mashimo et al., 1996). Further studies are needed to clarify the site of NO release in response to VIP in mouse gastric fundus.

In contrast to our findings, some investigators could not find any inhibitory effect of NOS inhibitors on the relaxant effect of VIP in the gastric fundal strips of a variety of species (Boeckxstaens et al., 1992; Lefebvre, 1993; Lefebvre et al., 1995; Bayguinov et al., 1999; Baccari and Calamai, 2001) suggesting that NO is not involved in these relaxations. Also, relaxations to VIP and forskolin were not affected in cGKI-deficient mice, suggesting that NO and VIP work via independent mechanisms in postjunctional cells of the circular muscle strips of mice (Ny et al., 2000). Conflicting results related to VIP stimulation of NO formation in smooth muscle can often be traced to the use of contractile agonists, such as carbachol or histamine, to enhance basal tension. These contractile agonists stimulate Ca²⁺ release and PKC. Recently, Murthy et al. (1994) suggested that agents that stimulate PKC may mask the ability of VIP to activate NOS in dispersed gastric muscle cells and isolated muscle strips of rabbit stomach. To avoid the possible masking effect of contractile agonists on NOS activity, we used a noncontracted tissue, i.e., basal tonus only. The contradictory findings may be due to the different experimental protocols used.

In recent studies, the relaxant effect of VIP was not antagonized by the nonselective NOS inhibitor L-NOARG, the selective inducible NOS inhibitor 1400W, and the selective guanylate cyclase inhibitor ODQ in smooth muscle strips, but antagonized in smooth muscle cells (Dick et al., 2000). The authors suggested that inductible NOS, possibly induced by the procedure used to prepare the smooth muscle cells, is involved in the relaxant effect of VIP in isolated smooth muscle cells but not in smooth muscle strips. To understand whether NO produced by the activation of iNOS could contribute to this relaxation, the effect of AMT, the selective inductible NOS inhibitor (Nakane et al., 1995), was investigated. However, in the concentration used, AMT did not attenuate the relaxations induced by VIP, ruling out the proposal mentioned above.

In conclusion, our results provide evidence for the involvement of NO in relaxation to short-term ES of NANC nerves and possibly participation of VIP (and related neuropeptides) in relaxation of mouse gastric fundal strips. In addition, these findings also support the idea that VIP directly induces the formation of NO by stimulating NOS activity and thereby activating soluble guanylyl cyclase in smooth muscle.