Ileal segments prepared from M2-KO, M3-KO, M2/M3-double KO or wild-type mice were bathed in Tyrode solution containing guanethidine (1 μM) and L-NAME (100 μM) (see Methods), and tension changes in their longitudinal direction were recorded. All preparations showed spontaneous contractions and application of 70 mM KCl evoked phasic contractions (Figures 1, 2 and 3). The magnitude of contractions, as measured in grams, amounted to 0.73±0.06 g (n=19) in wild-type mice and 0.89±0.10 g (n=14), 0.63±0.09 g (n=11) and 0.63±0.05 g (n=20) in M2-KO, M3-KO, and M2/M3-KO mice, respectively. These four mean values did not statistically differ from one another.
Figure 1. Contractile responses to TE stimulation of an ileal segment from a wild-type mouse, before (a) and after (b) application of atropine. TE stimulation (50 V in strength, 0.5 ms in pulse duration) was applied for 5 s at the different frequencies and at the different time points, as indicated by closed triangles. Guanethidine (1 μM) and L-NAME (100 μM) were added in the bathing solution during all experiments (as in all other figures). As a standard, a K+-evoked contraction was obtained by the addition of 70 mM KCl (70 K, closed circle). Note that the initial fast contraction to TE stimulation was blocked by atropine (2 μM).
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TE stimuli, 50 V in strength and 0.5 ms in duration, were applied at an ascending series of frequencies of 2, 5, 10, 20 and 50 Hz (each for 5 s). In wild-type preparations, as shown in Figure 1, TE stimuli initiated a rapid, brief contraction followed by a slower, longer one on which spontaneous contractions were superimposed. Both the initial fast and later slow contractions increased as stimulus frequency was increased, and a maximal response was attained at 10–50 Hz for the initial contraction and at 20 or 50 Hz for the latter. In five out of twenty preparations, no appreciable contractile response was elicited at 2 Hz, but at higher frequencies, an initial fast contraction was invariably observed. Occasionally, a later slow contraction did not follow until the frequency was increased to 10 or 20 Hz. The initial fast contractions appeared immediately after the beginning of TE stimulation, reached a peak within ∼5 s and then rapidly declined. At relatively high frequencies (10 Hz or higher), during the declining phase of the initial contractions, a slow contraction often appeared. The later slow contraction did not only vary in amplitude but also in duration with different stimulus frequencies or among different preparations; at 20 Hz, it reached a peak after 30–90 s and then disappeared within 2–8 min. Tetrodotoxin (1 μM) totally blocked the contractile responses to TE stimulation (data not shown), whereas atropine (2 μM) inhibited only the initial fast contractions (Figure 1). Thus, although the initial fast and the later slow contractions were both neurogenic, only the initial contractions resulted from the activation of cholinergic nerves, involving the activation of muscarinic receptors in ileal smooth muscle by ACh released from these nerves.
Upon TE stimulation, preparations from either M2-KO or M3-KO mice behaved similar to wild-type preparations, as exemplified in Figure 2a and b. Briefly, all preparations responded with an initial fast and a later slow contraction, and only the initial contraction was abolished by atropine (Figure 2c). In preparations from M2/M3-double KO mice, TE stimulation produced frequency-dependent contractile responses, but these did not involve any initial fast contractions (Figure 3a). This was confirmed by faster recording of the responses to TE stimulation. Figure 3b and c show such responses at 20 Hz in M2/M3-double KO and wild-type preparations. It can be seen (left panels) that the M2/M3-double KO preparation did not display an initial fast response, unlike the wild-type response, but instead exhibited an initial inhibition of spontaneous activity. To rule out the possibility that an initial cholinergic contraction was masked by the observed initial inhibition, we examined whether atropine potentiated the inhibitory response by blocking the (potential) masked contraction. However, we found that atropine reduced the initial inhibitory response, while leaving the following contraction almost intact (see Figure 3b). Therefore, no appreciable cholinergic contraction occurred in the absence of both M2 and M3 receptors, strongly suggesting that the cholinergic contractions in M2-KO and M3-KO mice were mediated by M3 and M2 receptors, respectively.
Figure 4 shows averaged relationships between stimulus frequency and cholinergic contraction size. The contraction size, expressed as percentage of the contraction induced by 70 mM K+, was greater in wild-type than in M2-KO or M3-KO mice; differences were statistically significant at all frequencies except 2 Hz. The two KO strains showed similar responses at all frequencies. At 50 Hz, the averaged contraction sizes in the wild type, M2-KO and M3-KO strains were 91±3% (n=20), 74±9% (n=12) and 68±4% (n=11), respectively. The 50% effective frequencies determined by curve fitting of the data points in Figure 4 (see Methods) were 4.5, 6.6 and 4.8 Hz for the three mouse strains.
Injection of PTX to mice has been shown to prevent the muscarinic agonist carbachol from producing M2-mediated contractions in ileal muscle strips (Unno et al., 2005). We therefore investigated the effect of PTX on cholinergic contractions in ileal segments. Treatment with PTX (100 μg kg−1) did not affect the spontaneous activity and 70-mM K+-induced contractions in any mouse strain. The high-K+-induced contractions in preparations from PTX-treated wild-type, M2-KO and M3-KO mice were 0.66±0.07 g (n=12), 0.68±0.07 g (n=10) and 0.66±0.10 g (n=6), respectively. These values were comparable to the respective control values (0.73±0.06 g for wild type, 0.89±0.10 g for M2-KO and 0.63±0.09 g for M3-KO).
Figure 5 shows typical responses produced by TE stimulation in preparations from PTX-treated wild-type, M2-KO and M3-KO mice. In PTX-treated M3-KO mice, TE stimulation failed to initiate an appreciable fast contraction at any frequency, but did produce slow contractions normally (Figure 5e), indicating that M2-mediated cholinergic contractions were blocked by PTX treatment. In PTX-treated M2-KO or wild-type mice, TE stimulation continued to evoke the initial fast contraction as well as the later slow contraction (Figure 5a and c). In preparations from PTX-treated M2-KO mice, the sizes of the initial fast contractions closely resembled the corresponding control values at 10–50 Hz, but were significantly greater at 2 or 5 Hz (Figure 5d). In preparations from PTX-treated wild-type mice, the contraction sizes were significantly reduced at 10–50 Hz, little altered at 5 Hz, and significantly increased at 2 Hz compared to the corresponding control values (Figure 5b). Consequently, following PTX treatment, the averaged relationships between stimulus frequency and contraction size were very similar for wild-type and M2-KO preparations (cf. the curves with closed circles in Figure 5b and d). The contraction sizes at 50 Hz for the PTX-treated wild-type and M2-KO groups were 65±5% (n=11) and 67±5% (n=7), respectively. The 50% effective frequencies were ∼1 and 2 Hz, respectively.
As mentioned earlier, TE stimulation evoked atropine-resistant contractile responses in ileal segments from all mouse strains studied. The contractile responses were completely blocked by tetrodotoxin (1 μM) (Figure 6a), indicating that they resulted from the activation of noncholinergic nerves. To characterize the noncholinergic contractions, we carried out TE stimulation experiments in the presence of atropine (2 μM).
Averaged relationships between stimulus frequency and noncholinergic contraction size for the individual mouse strains are shown in Figure 6b. Contraction sizes, normalized by 70-mM K+-induced contractions, increased as the stimulus frequency was increased. Responses were greater in M2/M3-double KO than in wild-type, M2-KO and M3-KO mice; differences were statistically significant at 10 Hz and higher frequencies. The latter three strains showed similar responses at all frequencies. The mean contraction size at 50 Hz in the M2/M3-double KO strain (108±16%, n=16) was about twice as great as the corresponding values for the wild-type (67±17%, n=6), M2-KO (42±4%, n=5) and M3-KO strains (54±6%, n=6). The 50% effective frequencies estimated from the data in Figure 6b were 7.7 Hz for the M2/M3-double KO mice and 15.7, 12.5 and 11.8 Hz for the wild-type, M2-KO and M3-KO strains, respectively. The latter three values were greater than the corresponding frequencies for cholinergic contractions (4.5, 6.6 and 4.8 Hz).
The noncholinergic contractions in all mouse strains studied were insensitive to PTX. Their amplitudes at 20 Hz in PTX-treated M2/M3-double KO mice (101±15%, n=4) did not significantly differ from the corresponding control value (87±14%; see 20 Hz in Figure 6b), and neither did the responses at 20 Hz in PTX-treated wild-type preparations (48±9%, n=4) differ from the control value (41±11%; Figure 6b).
Tachykinin (NK)-releasing nerves are known to be involved in the generation of neurogenic atropine-resistant contractions in various gut smooth muscles including the mouse ileal circular muscle (De Schepper et al., 2005). We therefore wanted to examine whether TE stimulation-evoked noncholinergic contractions in ileal segments involved the activation of tachykininergic nerves. Experiments were carried out in the presence of atropine (2 μM), unless otherwise stated. Data were pooled without distinction of the mouse strain, because atropine was continued to be present throughout the experiments and there was no notable difference in data obtained among the different strains used. As a tool, we used capsaicin, desensitization to which is known to block the effect of a subsequent stimulus in releasing NKs from enteric sensory nerves (Maggi, 2000; Barthó et al., 2004). Application of capsaicin (10 μM) to wild-type or M2-KO preparations produced a contraction, and 8–12 min later, the contractile response disappeared owing to desensitization (Figure 6c). Under these conditions, TE stimulation evoked a reduced contraction response (by 47±15% (n=3) at 10 Hz and by 43±7% (n=4) at 20 Hz), compared with the respective control responses. When similar experiments were carried out in the absence of atropine, desensitization to capsaicin did not affect the initial fast contraction but did reduce the following slow contraction in wild-type preparations (n=2, data not shown).
We carried out additional studies with NK receptor antagonists in atropine (2 μM)-treated, M2/M3-double KO preparations. The NK1 receptor-preferring antagonist spantide (1 or 5 μM; Beaujouan et al., 1993) failed to reduce the contractions to TE stimulation at 10 or 20 Hz but rather increased contraction amplitudes by 10–40% (n=4). In contrast, the NK2 receptor-preferring antagonist GR159897 (10 μM; Beresford et al., 1995) significantly reduced the contraction sizes at 10, 20 and 50 Hz by 47±8% (n=4), 42±6% (n=6) and 45±10 (n=3), respectively (Figure 6d). The percent reduction in contraction amplitudes at 20 and 50 Hz in M2/M3-double KO preparations was similar to that in wild-type preparations (44±12% at 20 Hz and 47±5% at 50 Hz, n=4 each), suggesting that the proportion of NK2 antagonist-sensitive component to the whole noncholinergic contraction was similar between the two mouse strains. The different effects of spantide and GR159897 on the noncholinergic contractions would be consistent with the view that NK1 receptors are located mainly on myenteric nerves, whereas NK2 receptors are largely confined to smooth muscle cells (Grady et al., 1996; Portbury et al., 1996a, 1996b).