Tachykinergic neurotransmission is enhanced in duodenum from dystrophic (mdx) mice

Abstract

1. Duodenal longitudinal muscle of mdx mice, an animal model for Duchenne muscular dystrophy, showed a decrease in the electrically evoked nonadrenergic, noncholinergic (NANC) inhibitory responses associated with a reduction of the participation of nitric oxide (NO). In this study, we investigated whether the impairment of NO could also lead to alterations in the NANC excitatory transmission.

2. Nerve-evoked responses consisted of an inhibitory phase followed, at the end of stimulation, by an excitatory response characterised by an increase in amplitude of the spontaneous contractions. In mdx mice, the amplitude of the nerve-evoked contractions was significantly higher than in normals.

3. N_ω-nitro-L-arginine methyl ester (L-NAME) or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylyl cyclase, increased the amplitude of the nerve-evoked contractions only in normals, being ineffective in mdx mice. Apamin, a blocker of Ca²⁺-dependent potassium channels, failed to affect the nerve-evoked contractions.

4. In both models, substance P and neurokinin A produced concentration-dependent contractions, reduced by tachykinin NK₁ and NK₂ receptor antagonists, respectively. Moreover, NK₁ and NK₂ receptor antagonists reduced the amplitude of the nerve-evoked contractions.

5. Sodium nitroprusside (SNP) reduced the amplitude of nerve-evoked contractions similarly in normal and mdx mice. ODQ, but not apamin, prevented the SNP-induced effects. SNP did not affect the contractions induced by exogenous tachykinins.

6. The results suggest that NO can exert an inhibitory modulatory role on tachykinergic excitatory transmission via activation of guanylyl cyclase in mouse duodenum. In mdx mice, the impairment of NO function leads to an increase in the nerve-evoked contractions.

Introduction

We are currently interested in studying the gastrointestinal motor dysfunctions in mdx mice, an animal model for Duchenne muscular dystrophy (DMD). Duchenne patients and mdx mice lack dystrophin, a membrane associated protein present in normal skeletal, cardiac and smooth muscle cells and in some neurones (Hoffman et al., 1987). Different hypotheses have been put forward to explain the mechanism by which the lack of dystrophin causes muscle dysfunction. In particular, a role for nitric oxide (NO) in the pathogenesis of muscular dystrophy has been proposed, because the lack of dystrophin in human DMD and in mdx mice causes neuronal nitric oxide synthase (nNOS) to disappear from its normal position at the sarcolemma. It becomes cytoplasmic, with a consequent decrease in its activity (Brenman et al., 1995). In mdx mice, changes in NO function have been reported to have a strong impact on motor and electrical patterns in different gastrointestinal segments, and alterations in the nNOS isoform have been found in the smooth muscle of the colon from dystrophic mice (Azzena & Mancinelli, 1999; Mulè et al., 1999, 2001; Baccari et al., 2000; Serio et al., 2001; Mulè & Serio, 2002; Zizzo et al., 2003). The duodenum of mdx mice showed functional motor alterations referable to a reduction in the nitrergic control associated to defects in the mechanisms necessary to transduce NO signals (Zizzo et al., 2003).

NO may act as an inhibitory neurotransmitter in the gastrointestinal tract either directly on the smooth muscle cells or indirectly by inhibiting the release of excitatory neurotransmitters such as acetylcholine and tachykinins. There are, however, tissue and species differences in the neuromodulatory effects of NO. NO-mediated inhibition of excitatory neurotransmitter release has been reported in the ileum and colon of guinea-pig (Wiklund et al., 1993; Kilbinger & Wolf, 1994; Hebeiss & Kilbinger, 1998; Mizhorkova et al., 2001), in dog ileum (Hryhorenko et al., 1994) and in mouse ileum (Mang et al., 2002). NO or nitric oxide synthase (NOS) inhibitors do not modify the electrically evoked acetylcholine release from the colon of guinea-pig and dog (Ward et al., 1996; Rae et al., 1998), and from stomach preparations of man (Leclere & Lefebvre, 2002), pig (Leclere & Lefebvre, 2001) and guinea-pig (Milenov & Kalfin, 1996).

Previous investigations showed that the duodenum of mdx mice does not relax in response to electrical field stimulation (EFS). In fact, in place of relaxation observed in normal animals, EFS caused just a frequency-dependent inhibition of the spontaneous activity. Moreover, block of NOS with N_ω-nitro-L-arginine methyl ester (L-NAME) abolished the inhibitory response in normal animals, while it only partly reduced that in mdx preparations. Thus, in mdx mice there is a reduction in the nonadrenergic, noncholinergic (NANC) relaxation associated with a reduction of the participation of NO (Zizzo et al., 2003). In addition, in both preparations, after the initial inhibitory response termination ‘off' or rebound contractions could be observed, but these were not systematically analysed. Poststimulus excitatory responses have been described in a variety of the intestinal preparations and a role for tachykinins in their genesis has been assessed (Lecci et al., 2002). In particular, tachychinergic excitatory nerve-evoked responses have been described in mouse stomach (Mulè & Serio, 2002) and ileum (Goldhill et al., 1995; Saban et al., 1999).

Therefore, the present study was performed in mouse duodenum to investigate whether NANC excitatory motor transmission is modulated by NO and whether it can be affected in mdx mice due to the impairment of the NO function.

In particular, we analysed and compared the ‘off' contractile activity in response to NANC electrical nerve stimulation, the effects induced by L-NAME, sodium nitroprusside (SNP) and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) on NANC contraction and the role of tachykinins in normal and mdx mice.

Methods

Tissue preparation

Experiments, authorised by the Ministero della Sanità (Rome, Italy), were performed on dystrophic (mdx mutants; C57BL/10Sn-Dmd/J) adult (12 months old) male mice and their genetically related C57 control animals (Harlan, Italy) killed by cervical dislocation. The abdomen was immediately opened and segments of duodenum were removed and placed in Krebs solution (mM: NaCl 119; KCl 4.5; MgSO₄ 2.5; NaHCO₃ 25; KH₂PO₄ 1.2, CaCl₂ 2.5, glucose 11.1). The contents of the excised segments were gently flushed out and the segments (20 mm in length) were drawn over a glass rod and the mucosal layer was gently removed. The segments were suspended in 10 ml organ baths containing oxygenated (95% O₂ and 5% CO₂) Krebs solution maintained at 37°C. The distal end of each segment was tied to an organ holder and the proximal end was secured with a silk thread to an isometric force transducer (FORT 10, Ugo Basile, Biological Research Apparatus, Comerio VA, Italy). Longitudinal preparations were subjected to an initial tension of 200 mg and were allowed to equilibrate for at least 30 min in the Krebs solution before starting the experiments. Krebs solution contained atropine (1 μM) and guanethidine (1 μM), to establish NANC conditions, indomethacin (10 μM) to increase the reactivity of the muscle to contractile agents (Maggi et al., 1994), and phosphoramidon (1 μM) (N-(α-rhamnopyranosyloxyhydroxyphosphinyl)-Leu-Trp) and captopril (1 μM) ((2S)-1-(3-mercapto-2-methylpropionyl)-L-proline) to inhibit peptidases.

Mechanical activity was digitised on an A/D converter, visualised, recorded and analysed on a personal computer using the PowerLab/400 system (Ugo Basile. Italy). EFS was applied by a Grass S88 (Grass Instruments Co., Quincy, MA, U.S.A.) electrical stimulator through a stimulus isolation unit (SIU5) using direct coupling. Square-wave pulses (0.5 ms, 2–32 Hz, supramaximal voltage) were delivered in 10-s train via a pair of platinum plate electrodes placed in parallel with the tissue.

Experimental protocols

At the beginning of each experiment, the preparation was challenged with 80 mM KCl for 2 min until reproducible responses were obtained. The amplitude of the KCl response was 387.0±63 mg (n=20) in normal and 382.5±72 mg (n=20) in mdx mice. Frequency–response curves to EFS were obtained first in control condition by stimulating the tissues with individual trains delivered at 15 min intervals. Subsequently, antagonists were added to the organ baths and, after a period of 20–30 min, curves to EFS were repeated. Tachykinergic receptor agonists were tested in a noncumulative manner (leaving 45 min between consecutive additions to avoid tachyphylaxis) and they were allowed to be in contact with the tissue for 3 min. Preliminary experiments showed that a second curve to the agonist obtained in these experimental conditions was reproducible. Tachykinergic receptor antagonists were added to the bath and a new curve to agonist was produced after 40 min contact time. Each preparation was tested with a single antagonist, except when otherwise stated.

Drugs

Drugs used were apamin, atropine sulphate, guanethidine monosulphate, ODQ, indomethacin, neurokinin A (NKA), L-NAME, (2S)-1-(3-mercapto-2-methylpropionyl)-L-proline (captopril), N-(α-rhamnopyranosyloxyhydroxyphosphinyl)-Leu-Trp (Phosphoramidon), SNP, substance P (SP), tetrodotoxin (TTX), all purchased from Sigma (Sigma-Aldrich, Inc., St Louis, U.S.A.). ((S)1-{2-[3-(3,4-dichlorophenyl)-1-(3-isopropoxy-phenylacetyl)-piperidin-3-yl]ethyl}-4-phenyl-1-azoniabicyclo[2.2.2]octane, chloride) (SR 140,333) and ((S)-N-methyl-N[4-(4-acetylamino-4-phenylpiperi-dino)-2-(3,4-dichloro-phenyl)butyl]benzamide (SR 48,968) were from Sanofi Recherche (Montpellier Cèdex, France). Indomethacin solution was prepared daily in 2% sodium carbonate solution, and the pH was adjusted to 7.4. ODQ, and SR 48,968 and SR 140,333 were dissolved in dimethylsulphoxide (DMSO). All the other drugs were dissolved in distilled water. The working solutions were prepared fresh on the day of the experiment by diluting the stock solutions in Krebs and were added to the organ bath. Control experiments using DMSO alone showed that it has no effect on the tissue responses studied.

Statistical analysis

All data are given as means±s.e. n indicates the number of animals from which the intestinal segment was taken. The excitatory responses to EFS were normalised to the amplitude of the spontaneous contractions set to 100%, being a nerve-evoked increase in the amplitude of the spontaneous contractions. The contractile responses to tachykinergic receptor agonists were expressed as per cent of the response to KCl (80 mM). Agonist responses were fitted to sigmoid curves (Prism 4.0, GraphPAD, San Diego, CA, U.S.A.), and EC₅₀ values (with 95% CLs) were determined from these curves. Moreover, antagonist potency was expressed as the negative logarithm of the concentration of the antagonist required to cause a two-fold rightward shift of the agonist dose–response curve (pA₂ value), calculated by nonlinear regression analysis of the individual dose–response curves using Prism 4.0. Statistical analysis was performed by means of Student's t-test or analysis of variance, followed by Bonferroni's test, when appropriate. A probability value of less than 0.05 was regarded as significant.

Results

Nerve-evoked excitatory responses: normal vs mdx mice

Duodenal segments, in the presence of atropine, guanethidine, indomethacin and peptidase inhibitors, exhibited spontaneous mechanical activity, consisting of rhythmic changes in isometric tension with a similar amplitude (Table 1).

Table 1.

Amplitude of the spontaneous contractions in control conditions and in the presence of L-NAME, ODQ, apamin or NK₁ and NK₂ receptor antagonists

Spontaneous activity (mg)
	Control (n=20)	L-NAME (n=7)	ODQ (n=7)	Apamin (n=5)	SR 140,333 (n=7)	SR 48,968 (n=7)	SR 140,333+SR 48,968 (n=7)
Normal	186.7±9.6	189.5±7.2	184±16.3	192.3±6.3	175.6±16.7	184.9±17.0	174.8±15.0
mdx	208.6±14.4	212.4±10.4	217.7±17.5	223.5±15.3	192.3±10.1	212.3±11.4	190.5±16.4

In normal mice, EFS produced a transient initial relaxation of longitudinal muscle (Zizzo et al., 2003), followed by TTX-sensitive, frequency-dependent, excitatory response, which consisted in an increase in amplitude of the spontaneous contractions (Figure 1). At 32 Hz, the maximal amplitude of the nerve-evoked contractions reached about 180% of the spontaneous contraction amplitude.

Figure 1.

Nonadrenergic, noncholinergic (NANC) responses evoked by electrical field stimulation (EFS) in normal and mdx longitudinal duodenal muscle preparations. Left: relationship between stimulation frequency and amplitude of the nerve-evoked contractions. All values are means±s.e. (n=20 for normal and for mdx mice) and are normalised to the amplitude of the spontaneous contractions set to 100%. ^*Significantly different from the normal animals. Right: typical tracings showing the NANC-evoked responses induced by EFS in duodenal segments from normal and mdx mice (0.5 ms, 32 Hz, supramaximal voltage for 10 s). Note that in mdx duodenum the nerve-evoked excitatory response is enhanced compared to normal animals.

As we have previously reported, in mdx mice, the inhibitory effects to EFS consisted just in the suppression of the phasic activity, and a frank relaxation was never observed (Zizzo et al., 2003). Instead, the excitatory response to EFS consisted in slowly developing increase in muscular tone with superimposed phasic contractions. The amplitude of the nerve-evoked contractions was significantly higher than in normal animals (Figure 1). At 32 Hz, the increase in the tone was 52.2±7.0% (n=20) of the response to KCl (80 mM) and the amplitude of the nerve-evoked contractions reached about 204% of the spontaneous contraction amplitude.

Nerve-evoked excitatory responses: effects of nitrergic drugs

L-NAME (100 μM), inhibitor of NOS, caused no change in the amplitude of the spontaneous contractions in both types of animals (Table 1). In normal animals, L-NAME (100 μM) increased the amplitude of the nerve-evoked contractions at all frequencies tested, but the tonic component of the response observed in mdx mice was not unmasked (Figure 2). In mdx mice, L-NAME (100 μM) did not significantly increase the response to EFS, leaving unaltered either the amplitude of the nerve-evoked contractions at all frequencies tested (Figure 2), and the underlying increase in the tone (at 32 Hz the increase in the tone was 61.3±5.0% (n=7) of the response to 80 mM KCl in the presence of L-NAME). The amplitude of the nerve-evoked contractions obtained in normal animals after L-NAME was not significantly different from that observed in mdx mice.

Figure 2.

Histograms showing the effects of L-NAME (100 μM) on the amplitude of the contractions evoked by EFS at different stimulation frequencies (0.5-ms pulse duration, supramaximal voltage for 10 s) in normal and mdx longitudinal duodenal muscle preparations. Data are means±s.e. (n=7 for normal and for mdx mice) and are normalised to the amplitude of the spontaneous contractions set to 100%. ^*P<0.05 when compared to the respective control.

Since we have previously demonstrated that in mouse duodenum, NO-induced inhibitory effects are mediated in part by activation of soluble guanylyl cyclase and in part by increasing the open probability of apamin-sensitive Ca²⁺-dependent potassium channels (Serio et al., 2003), we tested the effects of either ODQ, an inhibitor of soluble guanylate cyclase, or apamin on nerve-evoked responses. Both ODQ (1 μM) and apamin (0.1 μM) did not modify the amplitude of the spontaneous contraction in both types of animals (Table 1). In normal animals, ODQ and apamin had different effects on the excitatory responses. In fact, just ODQ increased the amplitude of nerve-evoked contractions, apamin was ineffective (Figure 3). The tonic component of the response to EFS observed in mdx mice was not unmasked by ODQ. In mdx mice, in contrast with normal animals, ODQ (1 μM) did not significantly increase the amplitude of nerve-evoked contractions and, once again, apamin was without any effect (Figure 3). The tonic component of the response to EFS was not affected by either ODQ or apamin (at 32 Hz the increase in the tone was 59.4±7.5% (n=7) and 51.9±6.4% (n=5) of the response to 80 mM KCl in the presence of ODQ or apamin, respectively).

Figure 3.

Histograms showing the effects of ODQ (1 μM, n=7) or apamin (0.1 μM, n=5) on the excitatory responses evoked by EFS at different stimulation frequencies (0.5-ms pulse duration, supramaximal voltage for 10 s) in normal and mdx longitudinal duodenal muscle preparations. Data are means±s.e. and are normalised to the amplitude of the spontaneous contractions set to 100%. ^*P<0.05 when compared to the respective value obtained in the control.

Tachykinin-induced contractions: normal vs mdx mice

In normal animals, NK₁ receptor-preferring natural tachykinin, SP (10 nM–1 μM) produced concentration-dependent contractions with an EC₅₀ value of 45 nM (95% CL 21–97 nM), reaching an E_max value of 74.9±2.5% of response to KCl (n=7). NK₂ receptor-preferring natural tachykinin, NKA (10 nM–1 μM) produced concentration-dependent contractions with an EC₅₀ value of 28 nM (95% CL 11–76 nM), reaching an E_max value of 79.9±2.9% of response to KCl (n=6). There was no significant difference between EC₅₀ or E_max values between SP and NKA, or between their concentration–response curves (P>0.05). In mdx animals, addition of SP (10 nM–1 μM) produced concentration-dependent contractions with an EC₅₀ value of 91 nM (95% CL 34–241 nM), reaching an E_max value of 65.3±3.2% of response to KCl (n=7). NKA (10 nM–1 μM) produced concentration-dependent contractions with an EC₅₀ value of 24 nM (95% CL 8.8–65 nM), reaching an E_max value of 85.6±3.0% of response to KCl (n=6). Statistical analysis showed that there was no significant difference between EC₅₀ or E_max values of SP or NKA, or of their concentration–response curves (P>0.05) between normal and mdx mice.

Nerve-evoked excitatory responses: effects of NK₁ and NK₂ receptor antagonists

To assess the involvement of tachykinins in the excitatory transmission, we tested the effects of NK₁ and NK₂ receptor antagonists on the nerve-evoked contractions in response to EFS, both in normal and mdx mice. At first, in order to choose the antagonist concentration we analysed, in normal animals, the effects of the NK receptor antagonists on the concentration–response curves to the agonists. The selective NK₁ receptor antagonist, SR 140,333 (1 nM–0.1 μM), produced a concentration-dependent rightward shift of the dose–response curve to SP (pA₂=8.28±0.05) (Figure 4), whereas the selective NK₂ receptor antagonist, SR 48,968 (1 nM–0.1 μM), produced a concentration-dependent rightward shift of the dose–response curve to NKA (pA₂=8.31±0.02) (Figure 4). Conversely, SR 48,968 did not modify the response to SP (EC₅₀=64 nM (95% CL 19–211 nM) in the presence of SR 48,968, n=4, P>0.05) and SR 140,333 did not modify the contractions in response to NKA (EC₅₀=45 nM (95% CL 18–140 nM) in the presence of SR 140,333, n=4, P>0.05).

Figure 4.

Normal mice. Upper pannel: concentration–response curves to SP in the absence or in the presence of different concentrations of SR 140,333, NK₁ receptor antagonist. Lower panel: Concentration–response curves to NKA in the absence or in presence of different concentrations of SR 48,968, NK₂ receptor antagonist. Data are means±s.e. of at least four experiments and are expressed as per cent of the response to KCl (80 mM). ^*P<0.05 when the concentration–response curves were compared to that obtained in control condition.

Either SR 140,333 (0.01 μM) or SR 48,968 (0.01 μM), alone or in combination, had no effect on the amplitude of the spontaneous contractions (Table 1). Indeed, SR 140,333 (0.01 μM) or SR 48,968 (0.01 μM) significantly reduced the amplitude of the nerve-evoked contractions in responses to EFS at all stimulus frequencies tested in both normal and mdx mice in the presence or in the absence of L-NAME (Figure 5). Moreover, in mdx mice, the tonic component of the response to EFS was slightly affected by either SR 140,333 or SR 48,968 (0.01 μM) (at 32 Hz the increase in the tone was 39.7±6.5% (n=7) and 43.9±7.2% (n=7) of the response to 80 mM KCl in the presence of SR 140,333 or SR 48,968, respectively). The blockade of both NK₁ and NK₂ receptors by co-administration of the selective antagonists resulted in a further reduction of the contractile responses evoked by EFS (Figure 5). Moreover, the increase in the tone observed in the NANC responses in mdx mice was abolished by co-administration of the NK₁ and NK₂ receptor antagonists.

Figure 5.

Histograms showing the effects of SR 140,333 (0.01 μM, n=7), NK₁ receptor antagonist, and SR 48968 (0.01 μM, n=7), NK₂ receptor antagonist, alone or in combination (n=5) on the responses evoked by EFS at different stimulation frequencies (0.5-ms pulse duration, supramaximal voltage for 10 s) in the presence or in the absence of L-NAME (100 μM) in normal and mdx longitudinal duodenal muscle preparations. Data are means±s.e. and are expressed as a percentage of the respective control, taken as 100%. ^*P<0.05 when compared to the respective control. ^§P<0.05 when compared to SR 140,333 or SR 48,968 alone. ^#P<0.05 when compared to the respective values without L-NAME.

Influence of SNP on nerve-evoked excitatory responses and on exogenous tachykinin contractions

To study the influence of exogenous NO on the excitatory responses to EFS and on tachykinin-induced contractions, we analysed the effects of SNP at submaximal dose (Zizzo et al., 2003). In normal mice, SNP (10 μM) significantly reduced the amplitude of the nerve-evoked contractions (Figure 6). A low concentration of ODQ (0.1 μM), which per se did not appreciably increase the amplitude of the nerve-evoked contractions, prevented the effects induced by SNP (Figure 6). In mdx mice, in the presence of SNP (10 μM), the amplitude of nerve-evoked contractions was reduced as in the normal animals, and pretreatment with ODQ (0.1 μM) prevented the effects induced by SNP on the nerve-evoked contractions. Lastly, the inhibitory effect induced by SNP was not prevented by apamin both in normal and mdx mice (Figure 6).

Figure 6.

Histograms showing the effects of SNP alone (10 μM), and after pretreatment with ODQ (0.1 μM) or apamin (0.1 μM) on the NANC excitatory responses evoked by EFS at different stimulation frequencies (0.5-ms pulse duration, supramaximal voltage for 10 s) in normal and mdx longitudinal duodenal muscle preparations. Data are means±s.e. (n=5 for normal and for mdx mice) and are calculated taking into account the amplitude of the phasic contractions, which is normalised to the amplitude of the spontaneous contractions set to 100%. ^*P<0.05 when compared to the respective value obtained in the control.

Moreover, we tested the responses to SP and NKA in the presence of SNP (10 μM). In both animals, the responses to SP (1 μM) and to NKA (1 μM) were not significantly different from those observed before addition of SNP (Figure 7).

Figure 7.

Histogram showing the effects of SNP (10 μM) on the contractions evoked by SP (1 μM) and by NKA (1 μM) in normal and mdx longitudinal duodenal muscle preparations. Data are means±s.e. (n=4 for normal and for mdx mice) and are expressed as per cent of the response to KCl (80 mM).

Discussion

The results of the present study indicate that, in mouse duodenum, NO exerts an inhibitory modulatory role on tachykinergic excitatory transmission, via activation of guanylyl cyclase, and in mdx mice the impairment of NO pathways leads to increased tachykinergic muscular contractions in response to EFS.

NO is an inhibitory neurotransmitter that causes inhibition of intestinal motility. This action is not confined just to its direct action on the smooth muscle cells, but NO may decrease gastrointestinal motility also acting indirectly by inhibiting the release of excitatory neurotransmitters such as acetylcholine (Wiklund et al., 1993; Hryhorenko et al., 1994; Kilbinger & Wolf, 1994; Hebeiss & Kilbinger, 1998; Mizhorkova et al., 2001) and tachykinins (Wiklund et al., 1993; Yunker & Galligan, 1996). In particular, in mouse ileum, NO inhibits cholinergic transmission and nNOS is the enzymatic source of NO-mediating inhibitory action (Mang et al., 2002).

Different regions of the gastrointestinal tract of mdx mice show motor alterations referable to a reduction in the nitrergic control (Mulè et al., 1999; Serio et al., 2001; Mulè & Serio, 2002; Zizzo et al., 2003), and the expression of nNOS isoform is altered in the smooth muscle of colon from dystrophic mice (Mulè et al., 2001). In mdx mice duodenum, there is a reduction of NANC relaxation associated with a reduced participation of NO (Zizzo et al., 2003). Therefore, our working hypothesis was that the impairment of NO system could interfere with the function of NANC excitatory transmission. Consequently, in the present study we analysed and compared the contractile responses to NANC nerve stimulation in normal vs mdx duodenal segments. In both tissues, EFS evoked a neurogenic excitatory response, significantly higher in mdx mice than in normal mice. Interestingly, in mdx mice, NOS inhibition with L-NAME failed to affect the evoked contractions, while in normal animals it increased significantly the EFS-induced contractions, whose amplitude reached that of the mdx tissue. Therefore, endogenous NO, released concurrently by field stimulation, markedly interfere with excitatory NANC neurotransmission in normal animals. We have previously showed that, in mouse duodenum, relaxations to NO released endogenously by enteric nerves or to exogenous SNP are mediated in part by cGMP-dependent mechanisms and in part by activation of apamin-sensitive Ca²⁺-dependent potassium channels (Serio et al., 2003). Indeed, the NO modulation of NANC excitatory response is likely mediated via activation of guanylyl cyclase. In fact, although both ODQ and apamin significantly antagonised the EFS-induced relaxation (Serio et al., 2003), only ODQ enhanced the amplitude of nerve-evoked contractions, being apamin ineffective. In support of this hypothesis, there is the observation that exogenous SNP decreased the evoked contractions acting through the activation of guanylyl cyclase, because its effect was antagonised by ODQ, but not apamin. A similar mechanism is involved in NO inhibition of acetylcholine release and cholinergic contractions in mouse ileum (Mang et al., 2002) and in NO inhibition of cholinergic and tachykinergic components of nerve-mediated contraction in guinea-pig ileum (Yunker & Galligan, 1996; Hebeiss & Kilbinger, 1998).

In mdx mice, neither ODQ nor apamin increased the amplitude of nerve-evoked duodenal contractions indicating that, as L-NAME, they had no facilitating role on the NANC-evoked contractions. However, when in mdx mice, NO is supplied exogenously is able to exert its inhibitory effects on the NANC excitatory system, as indicated by the observation that SNP decreased the evoked contractions even in mdx mice. We suggested that, in mdx mice, there is loss of the nitrergic modulation of NANC excitatory transmission, which in turn leads to an increase in the contractile responses to EFS.

In addition, the nature of the neurotransmitter/s involved in the neurogenic-evoked contractions in duodenal segments from normal animals and possible difference in respect to mdx mice were analysed. A role for acetylcholine, noradrenaline or prostanoids in the evoked response was ruled out because the experiments were conducted in the presence of atropine, guanethidine and indomethacin. On the other hand, in our experiments, the sensitivity to tachykinergic receptor antagonists indicates a major role for tachykinins in the genesis of nerve-evoked contractions in duodenal muscle. The tachykinins are important excitatory neurotransmitters in the enteric nervous system and are involved in the coordination of gastrointestinal motility. Three tachykinin receptors have been identified, NK₁, NK₂ and NK₃ receptors differently located on smooth muscle, interstitial cells of Cajal and neurones depending on the region and on the species considered (Holzer & Holzer-Petsche, 2001). In particular, NK₁ and NK₂ receptors are reported to mediate excitatory neuromuscular transmission in the gut (Holzer & Holzer-Petsche, 2001). Results from our experiments indicate the presence of functional NK₁ and NK₂ receptors in mouse duodenum. In fact, NK₁ receptor antagonist significantly inhibited the SP-evoked responses, without interfering with the NKA-induced contractions, and NK₂ receptor antagonist significantly inhibited the NKA responses, without interfering with the SP-induced contractions. No difference has been found in the potency of the two tachykinins. Moreover, either NK₁ receptor antagonist or NK₂ receptor antagonist is able to inhibit the contractions evoked by EFS. These effects were more evident when the modulatory role of NO on the tachykinergic component had been removed by L-NAME. In addition, coadministration of both receptor antagonists induced additive effects causing a further inhibition of the nerve-evoked contractions. Thus, these observations confirm our conclusion that at our parameters of stimulations, both tachykinergic receptors were recruited. These observations indicate that in mouse duodenum both NK₁ and NK₂ receptors play a fundamental role as NANC excitatory transmitters. It has been reported that NK₁ and NK₂ receptors modulate the responses to exogenously applied agonists also in mouse ileum, while in this region NK₁ are the primary tachykinin receptors involved in the noncholinergic excitatory neurotransmission (Saban et al., 1999).

In mdx mice, both tachykinergic receptor antagonists were able to inhibit the contractions evoked by EFS and coadministration of both receptor antagonists induced additive effects causing also the inhibition of the tonic component of the nerve-evoked responses. Thus, in mdx duodenum, the increase in the contractile response to EFS is due to an increase of participation of the tachykinins as excitatory neurotransmitters. The observation that there is no change in the muscular responsiveness to tachykinins in mdx mice suggests that the increased contractile response to EFS is not the consequence of changes in muscular sensitivity to tachykinins; instead, it can be ascribable to the reduction of nitrergic control on the tachykinin-mediated responses. Alteration in tachykinergic transmission due to the loss of the interaction with other transmitters, one of these being NO, has been reported in other pathophysiological conditions such as inflammation (Goldhill et al., 1997).

Lastly, the modulator influence of NO might be on the NANC excitatory nerves or on the smooth muscle cells. From our studies, it is not possible to assess the site(s) of action of NO (i.e. pre- vs postjunctional) for a certainty. However, the observation that exogenous NO does not influence the response to tachykinins rules out the possibility of a functional antagonism strongly supporting the idea that the interaction is at the prejunctional level.

In conclusion, in mdx mouse duodenum, the impairment of NO pathway leads not only to a reduction of the muscular relaxation but also to an increase in the muscular contractions evoked by noncholinergic nerve stimulation due to alteration of the modulation of tachykinergic neurotransmission. Such mechanisms can explain gastrointestinal clinical manifestations observed in DMD patients, such as chronic constipation.

Abbreviations

DMD

Duchenne muscular dystrophy

EFS

electrical field stimulation

L-NAME

N_ω-nitro-L-arginine methyl ester

NANC

nonadrenergic, noncholinergic

NKA

neurokinin A

ODQ

1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one

NO

nitric oxide

NOS

nitric oxide synthase

SNP

sodium nitroprusside

SP

substance P

SR 140,333

((S)1-{2-[3-(3,4-dichlorophenyl)-1-(3-isopropoxy-phenylacetyl)-piperidin-3-yl]ethyl}-4-phenyl-1-azoniabicyclo[2.2.2]octane, chloride)

SR 48,968

((S)-N-methyl-N[4-(4-acetylamino-4-phenylpiperi-dino)-2-(3,4-dichloro-phenyl)butyl]benzamide

TTX

tetrodotoxin