The spontaneous mechanical activity of the circular smooth muscle of the rabbit colon in vitro

Pharmacological Research 57 (2008) 132–141

The spontaneous mechanical activity of the circular smooth muscle of the rabbit colon in vitro Hassiba Benabdallah, Dalila Messaoudi, Kamel Gharzouli ∗ Department of Biology, Faculty of Science, University Ferhat Abbas, Setif, Algeria Accepted 8 January 2008

Abstract The spontaneous mechanical activity of the proximal, middle and distal colon of the rabbit shows in vitro two types of contractions: phasic contractions with low amplitude and high frequency, giant contractions (GCs) with high amplitude and low frequency. Both patterns of contractions did not present differences according to the region. Investigations on the neural control of giant contractions in the middle colon gave the following results. (1) GCs are insensitive to muscarinic antagonism by atropine and ganglionic blockade by hexamethonium; (2) GCs are converted into phasic contractions following the inhibition of acetylcholinesterase by neostigmine, and are abolished for a short period by dimethyl-phenylpiperazinium, a ganglionic nicotinic receptor agonist; (3) application of l-arginine, the substrate of nitric oxide (NO) synthase prolonged the duration of GCs without affecting their amplitude; sodium nitroprusside, a donor of NO, reduced both the amplitude and frequency of GCs; (4) inhibition of guanylate cyclase by methylene blue converted GCs into phasic contractions; (5) blockade of K+ channels with the non-selective blocker, tetraethylammonium, or with the more selective apamin-sensitive Ca2+ -dependent K+ channels blocker, dequaliniun, increased the resting tone and decreased the amplitude of contractions; whereas opening of ATP-sensitive K+ channels by diazoxide abolished any rhythmic contractile activity. These data taken together suggest that the amplitude and frequency of GCs are controlled by the endogenous release of NO which activates guanylate cyclase, the subsequent formation of cGMP activates in turn the opening of Ca2+ -dependent K+ channels. The cholinergic input seems to be responsible of the resting tone, and an increase of this tone is prone to impose the phasic contractions pattern to the tissue. © 2008 Elsevier Ltd. All rights reserved. Keywords: Colon; Spontaneous mechanical activity; Circular smooth muscle; Potassium channels; Nitrergic pathway

1. Introduction The circular smooth muscle in intact animals and human subjects generates three distinct types of contractions for distal propulsion of digest [1]: rhythmic phasic contractions, giant migrating contractions (GMCs) and tonic contractions. GMCs are high amplitude and low frequency contractions that migrate over long distances [2]. In vitro circular smooth muscle preparations from the colon of animals and humans present rhythmic giant contractions (GCs) and phasic contractions. The fundamental difference between GMCs and GCs reside in the neurogenic origin of the former and myogenic origin of the latter. In contrast to GCs, GMCs are susceptible to inhibition by ganglionic blockade with hexamethonium and muscarinic ∗ Corresponding author at: D´ epartement de Biologie, Facult´e des Sciences, Universit´e Ferhat Abbas, 19000 S´etif, Algeria. Tel.: +213 36 92 51 22; fax: +213 36 92 51 22. E-mail address: gharzouli [email protected] (K. Gharzouli).

1043-6618/$ – see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.phrs.2008.01.002

antagonism by atropine [2–4]. In vitro rhythmic contractions are controlled by an inhibitory tone exerted by NO which induces a cGMP-mediated opening of Ca2+ -dependent K+ channels [5–8]. Little attention has been paid to rabbit colon with respect to GCs. The first mention, to our knowledge, to rhythmic spontaneous contractions in the distal colon of rabbits was due to McKirdy [9]. This author reported the existence of both GCs and phasic contractions in distal colon used as flat sheets to record simultaneously longitudinal and circular mechanical activity. It is noteworthy to mention that GCs have been studied in detail in species other than the rabbit. The aims of this study were to investigate (1) the spontaneous contractile activity of the isolated circular colon of the rabbit which exhibits a remarkable difference in the interstitial cells of Cajal [10] which are important in controlling GCs and phasic contractions, (2) the role of the excitatory cholinergic transmission in the generation of spontaneous contractions, (3) the role of nitric oxide (NO) in the control of GCs. Since our results confirmed that NO abolishes GCs, we undertook experiments to throw some light on

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the link between NO and Ca2+ -dependent K+ channels opening in this animal species. 2. Materials and methods 2.1. Recording of spontaneous contractile activity The care and handling of the rabbits and the research protocol were in accordance with the institutional guidelines for the use of experimental animals. Adult rabbits of both sex and 940–1150 g body weight were killed by bleeding from the carotid arteries. After a midline laparotomy the whole colon were removed and placed in a Petri dish containing HEPES buffered physiological solution (composition in mM: NaCl 126, KCl 6, MgCl2 1.2, CaCl2 2, EDTA 0.01, HEPES 10.5 and glucose 14, pH 7.4). The colon was divided into three different parts: proximal, middle and distal colon. Full thickness circular muscle strips of approximately 2 mm width and 10 mm length were prepared by cutting the tissue parallel to the circular axis. The strips were mounted vertically in organ bath chambers containing 25 ml of the physiological solution warmed at 37 ◦ C and continuously oxygenated. The thread anchoring the upper end of the strips was connected to the lever of a force–displacement transducer (FSG-01/3, Experimetria, Budapest, Hungary) connected to an amplifier (EXP-D, Experimetria, Budapest, Hungary). Muscle activities were stored in a PC and simultaneously visualized (WinDaqLite software, DATAQ Instruments, OH, USA) using an external analog/digital conversion card with a sampling rate of 2 Hz (DI 700-PGL USB, DATAQ Instruments, OH, USA). Before the start of the experiments, the resting tension was adjusted to 1 g and the tissue was allowed to equilibrate for 45 min during which time the solution was renewed every 15 min. To verify the viability and the responsiveness of the smooth muscle, the strips were challenged with 1 ␮M carbachol for 3 min, a period sufficient for the development of maximal tension. After washing the carbachol, a second period of stabilization (45 min) with periodical washing was started.

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a nicotinic antagonist [12], and dimethyl-phenyl-piperazinium (DMPP) 5 ␮M which acts as a nicotinic receptor agonist [13]. Neostigmine, a reversible inhibitor of acetylcholine esterase [14], was added to the medium cumulatively (0.03–10 ␮M) every 15 min to obtain a stable response [15]. The nitrergic pathway was explored using l-NAME 100 ␮M, a non-specific NOS inhibitor [16], l-arginine 4 mM the substrate of NOS [17]. SNP (0.1–100 ␮M), a NO donor [18] and methylene blue (10–100 ␮M), a guanyl cyclase inhibitor [19], were added cumulatively every 15 min. The activity of potassium channels was altered using TEA 5 mM, a non-selective blocker of K+ channels [11], dequalinium 10 ␮M, an apamin-sensitive K+ channel blocker, and diazoxide (1–100 ␮M with cumulative additions), an ATP-sensitive K+ channel opener [20]. 2.3. Drugs Drugs used were carbamylcholine chloride (carbachol), atropine sulfate, hexamethonium chloride, 1,1-dimethyl-4phenyl-piperazinium iodide, Nω-nitro-l-arginine methylester HCl, sodium nitroprusside dihydrate, neostigmine bromide, tetraethylammonium chloride, dequalinium chloride, diazoxide, methylene blue trihydrate (Sigma, St. Louis, USA) and l-arginine (Fluka, AG-Buchs, Switzerland). All drugs were prepared as stock solutions (carbachol 100 mM, atropine 100 mM, hexamethonium 100 mM, DMPP 50 mM, l-NAME 100 mM, SNP 100 mM, neostigmine 100 mM, TEA 500 mM, diazoxide 100 mM, methylene blue 100 mM, and l-arginine 300 mM) in distilled water except for diazoxide and dequalinium that were prepared in pure dimethyl sulphoxide (DMSO). Dilutions were made with the corresponding solvent of each drug and added in the bathing medium in volumes <0.5%. Preliminary experiments have shown that the vehicles (distilled water and

2.2. Experimental protocols In the initial experiments, both phasic and giant contractions (GCs) of the three parts of the colon were recorded and analyzed under control conditions. The concentration–dependent response of the strips from proximal, mid- and distal-colon to carbachol 10−9 –10−3 M was constructed. Each concentration of carbachol was added to the medium for a period of 3 min followed by repetitive washing and a 15 min period of stabilization before the addition of the next concentration. Having seen that there was no difference between the three parts of the colon (unstimulated and carbachol-stimulated strips), the mechanical effects of pharmacological agents were studied on the middle colon. The drugs were applied for 15 min to obtain a maximal and stable response, unless otherwise stated. The cholinergic transmission was altered by using atropine 1 ␮M, a muscarinic antagonist [11], hexamethonium 100 ␮M,

Fig. 1. Typical recordings of the spontaneous mechanical activity of the circular smooth muscle strips of the proximal, middle and distal colon of the rabbit. (a) high frequency phasic contractions; (b) low frequency giant contractions.

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Table 1 Characteristic parameters of the phasic and giant contractions in the colon Colon

Resting tone (g)

Amplitude (g)

Frequency (cpm)

Phasic contractions Proximal (n = 7) Middle (n = 5) Distal (n = 7) ANOVA Pooled data

0.84 ± 0.07 0.82 ± 0.05 0.87 ± 0.05 ns 0.84 ± 0.06

0.10 ± 0.02 0.13 ± 0.03 0.09 ± 0.02 ns 0.11 ± 0.03

15.4 ± 0.91 14.2 ± 0.63 14.5 ± 0.57 ns 14.7 ± 0.85

Giant contractions Proximal (n = 16) Middle (n = 19) Distal (n = 17) ANOVA Pooled data

0.62 ± 0.03 0.64 ± 0.04 0.64 ± 0.03 ns 0.63 ± 0.4

0.83 ± 0.06 0.78 ± 0.04 0.73 ± 0.07 ns 0.78 ± 0.07

1.11 ± 0.08 1.07 ± 0.07 1.08 ± 0.06 ns 1.08 ± 0.08

DMSO) were without effect on the mechanical activities of the tissues. 2.4. Statistical analyses The GCs were analyzed according to their amplitude (g), frequency (contractions min−1 , cpm), duration (s) and resting tone which corresponds to the average minimal tension developed between GCs. In general, the last 3 min of the incubation period were used to calculate the average of each of these parameters. The results are presented as mean values ± S.E.M. with n representing the number of animals. Statistical analyses were made using paired Student’s t-test or one-way analysis of variance followed by Dunnett’s test. A probability value of p ≤ 0.05 was regarded as significant. Emax and EC50 of the concentration–response curve of carbachol were obtained by fitting the data to the sigmoidal dose–response model (GraphPad Prism version 4.00 for Windows, GraphPad Software, San Diego, CA, USA). 3. Results 3.1. Spontaneous mechanical activity of the colon When stretched with an initial load of 1 g, the circular muscle strips of the colon showed two types of rhythmic contractions: phasic contractions (Fig. 1a) and giant contractions (Fig. 1b). Each type of contraction can be recorded in separate preparations taken from the same animal. All the three parts of the colon showed phasic contractions (incidence rate: 77/230 strips; 39 animals). The phasic contractions were characterized by high frequency contractions, a relatively high tone and small amplitude. The resting tone, the amplitude and the frequency of phasic contractions did not differ in the proximal, middle, and distal colon strips (Table 1). The giant contractions were observed in 153 out of 230 strips and did not differ along the colon with respect to their amplitude and frequency (Table 1). The GCs were superimposed in their falling phase with a very fluctuating number of phasic contractions (5–36 phasic contractions per GC; Fig. 1b). The GCs were characterized by low frequency (pooled mean: 1.08 ± 0.08 GC/min) and high amplitude con-

Duration (s)

53.8 ± 4.48 56.2 ± 5.72 51.9 ± 4.30 ns 54.0 ± 5.96

tractions (pooled data: 0.78 ± 0.07 g); corresponding to seven times of that of the phasic contractions (Table 1). The successive giant contractions were separated by a resting phase lasting for 17.8 ± 1.57 s (pooled mean). It is noteworthy that some strips started with phasic contractions and commuted to GCs after the viability test with carbachol 1 ␮M. 3.2. Cholinergic transmission Stimulation of the strips with carbachol induced a biphasic response: a phasic contraction followed by a tonic contraction which lasted as long as the tissue was in contact with the drug (90 min; Fig. 2a). Since the peak of the phasic contraction was reached in less than one minute, a non-cumulative concentration–response curve was constructed by adding carbachol in the medium for only 3 min. The amplitude of the phasic contraction was concentration–dependent (Fig. 2b,c). The results of the fitting of the data to a sigmoidal dose–response model are summarized in Table 2. The only difference between the three regions of the colon of the rabbit was noted in Emax decreased along the colon; the EC50 was not different from one region of the colon to the other. The treatment of the mid-colon strips by neostigmine converted the GCs into high frequency phasic contractions in a concentration–dependent manner (Fig. 3a). At low concentrations of neostigmine (0.03 and 0.3 ␮M), GCs were still observed but with a higher amplitude than basal conditions (p ≤ 0.05; Fig. 3b). The duration of the GCs remained unchanged (pooled mean: 57.8 ± 4.40 s). Increasing the conTable 2 Parameters of the response of proximal, middle and distal colon to carbachol Colon

Emax (g)

Proximal (n = 36) Middle (n = 36) Distal (n = 36)

2.65 ± 0.09 [2.42–2.88] 2.38 ± 0.06* [2.19–2.57] 2.15 ± 0.07* [1.95–2.35]

pD2 6.67 ± 0.18 [7.00–6.29] 6.45 ± 0.17 [7.01–6.21] 6.63 ± 0.22 [7.08–6.18]

Values between brackets represent 95% confidence interval. * p ≤ 0.05 With respect to proximal colon.

EC50 (M) 2.13 × 10−7 3.54 × 10−7 2.32 × 10−7

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Fig. 2. Contractile effect of carbachol on circular smooth muscle of the rabbit. (a) Typical recording of the biphasic contraction induced by carbachol (CCh). (b) Typical recording of the effect of increasing concentrations of carbachol. The strips were incubated with carbachol for 3 min ( ), just the time to record the phasic contraction before the washout. (c) Dose-response curve of circular smooth muscle of the proximal, middle and distal colon for carbachol added non-cumulatively. Values are mean ± S.E.M. (n = 6);.

centration to 1 and 10 ␮M induced a net increase of the resting tone and a decrease of both amplitude and frequency of the phasic contractions (p ≤ 0.05; Fig. 3b–d), changes that are characteristic of the phasic contractions pattern. Atropine 1 ␮M was without effect on the characteristic parameters of the GCs except a slight decrease of the resting tone (p ≤ 0.05; Table 3). Similarly, hexamethonium 100 ␮M was

without effect on the resting tone and the amplitude of the GCs (p > 0.05; Table 3). The effect of DMPP 5 ␮M on the spontaneous mechanical activity of the mid-colon showed a biphasic pattern (Fig. 4). During the first minutes (148 ± 41 s, n = 8), the contractile activity was abolished (incidence ratio: 15 out of 19 strips) without affecting the resting tone (0.73 ± 0.06 g, n = 8; p > 0.05 with respect to basal condition). After this latency, the GCs resumed

Fig. 3. Effects of cumulative addition of neostigmine on the giant contractions of the middle colon. (a) Typical recording of the effect of neostigmine. (b) Expanded scale of typical recordings of GCs under basal condition (upper trace) and neostigimine (lower trace) during 15 min. (c) Effects of neostigmine on the resting tone and amplitude of the giant contractions. (d) Modification of the frequency of the contractions by neostigmine. Values are mean ± S.E.M. (n = 5); *: p ≤ 0.05 with respect to basal control (B); ns: non significant.

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Table 3 Effect of atropine, hexamethonium and DMPP on the spontaneous mechanical activity of the mid-colon of the rabbit Treatment

Resting tone (g)

Amplitude (g)

Frequency (cpm)

Duration (s)

Basal (n = 8) Atropine 1 ␮M

0.85 ± 0.06 0.76 ± 0.04

0.72 ± 0.07 0.76 ± 0.06 ns 0.84 ± 0.07 0.82 ± 0.09 ns 0.67 ± 0.07 0.69 ± 0.07 ns

0.80 ± 0.03 0.82 ± 0.05 ns 0.94 ± 0.05 0.97 ± 0.07 ns 0.90 ± 0.08 0.90 ± 0.06 ns

81.9 ± 3.63 76.9 ± 6.13 ns 68.6 ± 3.27 66.4 ± 4.57 ns 73.0 ± 6.36 59.2 ± 4.45 ns

*

Basal (n = 9) Hexamethonium 100 ␮M Basal (n = 8) DMPP 5 ␮M

0.77 ± 0.05 0.81 ± 0.07 ns 0.88 ± 0.06 0.69 ± 0.04 *

Values correspond to measurements made during the last 3 min of the incubation period (15 min). * p ≤ 0.05 With respect to the corresponding basal period; ns: not significant.

with a lower resting tone (p ≤ 0.05) but with the same amplitude as in basal condition (Table 3).

n = 6), but the resting tone was maintained at a high level (1.13 ± 0.09 g, n = 6; Fig. 6b).

3.3. Role of the nitrergic pathway

3.4. Role of potassium channels

Incubation of the strips with l-NAME 100 ␮M was without significant effect on the GCs (resting tone, amplitude, frequency and duration). Addition of l-arginine 4 mM to the bathing solution did not affect both the amplitude and the resting tone of GCs. In contrast, the frequency of the GCs was reduced and their duration was increased progressively to reach a significant level at the end of the incubation, i.e. 20 min (p ≤ 0.05; Table 4). SNP, added cumulatively to the medium, had not significant effect on the resting tone (basal: 0.68 ± 0.04 g (n = 7); SNP 100 ␮M: 0.58 ± 0.04 g (n = 7); p > 0.05). However, the amplitude of the contractions was significantly decreased in a concentration–dependent manner (n = 7; p ≤ 0.05; Fig. 5a,b). The significant decrease of the frequency of the GCs (Fig. 5c) was accompanied by that of the duration of the GCs (Fig. 4d) and by an increase of the duration of the resting period. The cumulative addition of methylene blue to the medium converted the GCs into phasic contractions (Fig. 6a). At 10 ␮M, the resting tone was increased from 0.54 ± 0.03 to 0.92 ± 0.08 g (n = 6; p ≤ 0.05; Fig. 5b). From 30 to 100 ␮M, the rhythmic contractile activity was almost abolished (amplitude: 0.08 ± 0.3 g,

Treatment of the strips from the mid-colon with TEA 5 mM induced a complex response. In 13 out of 21 animals, the GCs were converted progressively into phasic contractions (Fig. 7a); in this case the resting tone was significantly increased (p < 0.05) while the amplitude of the contractions was reduced (p < 0.05; Fig. 7c). Whereas in 8 out 21 animals, the profile of the contractions was altered during the first five minutes and then returned to normal GCs (Fig. 7b). In this last case, the frequency of the contractions increased during the first 5 min from 0.79 ± 0.05 to 3.56 ± 0.26 (n = 8, p ≤ 0.05) and then decreased to 0.97 ± 0.12 cpm (n = 8; p ≤ 0.05) during the last 5 min of the incubation period (Fig. 7d); these changes were accompanied by changes in the duration and amplitude of the contractions at the end of the incubation (from 0.89 ± 0.06 to 1.16 ± 0.12 g, n = 8; p ≤ 0.05). Dequalinium 10 ␮M (apamin-sensitive K+ channel blocker) converted progressively the GCs into phasic contractions (Fig. 8a). The resting tone was increased whereas the amplitude of the contractions was significantly reduced (p ≤ 0.05; Fig. 8b). Diazoxide 10 ␮M (ATP-sensitive K+ channel opener) was without effect on the amplitude, the frequency and the duration of the GCs. Increasing the concentration to 30 ␮M reduced the amplitude and the frequency of the contractions. A further increase of diazoxide to 100 ␮M abolished any rhythmic mechanical activity (Fig. 9a,b). The resting tone remained unchanged for all the test concentrations of diazoxide (Fig. 9b). 4. Discussion

Fig. 4. Typical recording of the effect of DMPP 5 ␮M on the giant contractions. Note the short lasting inhibition of the contractions upon the addition of the drug.

In the present study, two types of spontaneous mechanical activities were recorded along the isolated circular preparations of the colon. The most frequent pattern of motility, termed giant contractions, is characterized by low frequency and high amplitude contractions. The second pattern, termed phasic contractions, is less frequent and characterized by high frequency and low amplitude contractions. This latter type of contraction is superimposed on the GCs during the falling phase. These pat-

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Table 4 Effect of l-NAME and l-arginine on the spontaneous mechanical activity of the middle colon of the rabbit Treatment

Resting tone (g)

Amplitude (g)

Frequency (cpm)

Duration (s)

Basal (n = 8) l-NAME 100 ␮M (n = 8) Basal (n = 5) l-Arg 4 mM (n = 5)

0.76 ± 0.07 0.74 ± 0.04 ns 0.77 ± 0.05 0.73 ± 0.07 ns

1.03 ± 0.10 1.04 ± 0.12 ns 0.61 ± 0.15 0.62 ± 0.17 ns

0.76 ± 0.07 0.95 ± 0.13 ns 0.79 ± 0.13 0.46 ± 0.08

83.0 ± 10.8 73.9 ± 9.52 ns 80.6 ± 10.1 148.6 ± 26.2

*

*

Values correspond to measurements made during the last 3 min of the incubation period (15 min). * p ≤ 0.05 With respect to the corresponding basal period; ns: not significant.

terns of contractions have been described in both longitudinal and circular distal colon of the rabbit used as stretched flat sheet [9]. The presence of phasic contractions and GCs have also been reported in the rat, mouse and human colon with the same range of frequencies as in the present study [3,21–26]. The lack of a significant difference in amplitude and frequency according to the region (proximal, middle and distal) of the rabbit colon in the present study is contrasting with that reported in the rat where the amplitude of the GCs decreases along the colon [21]. Phasic contractions and GCs are still observed in NANC condition without changes [27] and are driven in the rat colon by two types of pacemakers [21]. However, the pacemaker activ-

ity shows significant difference between species [26,28,29]. It is now widely accepted that the interstitial cells of Cajal (ICC) constitute the pacemaker cells associated with Auerbach’s plexus. It is then reasonable to postulate that the characteristics of the rhythmic contraction depend on the organization and density of ICCs. It is noteworthy that a dense network of neurites with abundant interstitial cells of Cajal [10] was found at the circular muscle-submucosal interface in several animal species and human except the rabbit. Stimulation of the circular smooth muscle of rabbit colon by carbachol induced a phasic contraction followed by a tonic contraction, a response already reported in the rat

Fig. 5. Effects of cumulative addition of SNP on the giant contractions of the middle colon. (a) Typical recording of the effect of SNP. (b–d) Concentration–response relationship for amplitude, frequency and duration of the giant contractions in response to SNP. Values are mean ± S.E.M. (n = 7); *: p ≤ 0.05 with respect to basal control (B).

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Fig. 6. Effects of cumulative addition of methylene blue on the giant contractions of the middle colon. (a) Typical recording of the effect of methylene blue. (b) Effects of methylene blue on the resting tone and amplitude of the giant contractions. Values are mean ± S.E.M. (n = 6); *: p ≤ 0.05 with respect to basal control (B); ns: non significant.

[30]. The sensitivity of the circular smooth muscle to carbachol (pD2) is constant along the three regions of the colon; but the potency (Emax ) was greater in proximal than in distal colon. Numerous data suggest regional heterogene-

ity in myogenic properties of smooth muscle throughout the gastrointestinal tract. The neurohumoral influence on circular smooth muscle may account for this difference since isolated smooth cells of the rabbit colon do not

Fig. 7. Effects of TEA 5 mM on the giant contractions of the middle colon. (a) Recording showing a typical conversion of the giant contractions into phasic contractions. (b) Recording showing altered giant contractions pattern. (c) Effects of TEA on the resting tone and amplitude of the contractions in the case of the phasic contraction pattern (n = 13). (d) Biphasic effect of TEA on the frequency and duration of the conserved giant contractions pattern; Ph1, Ph2: first 5 min and last 5 min of the incubation period, respectively (n = 8). Values are mean ± S.E.M.; *: p ≤ 0.05 with respect to basal control (B); ns: non significant.

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Fig. 8. Effects of dequalinium 10 ␮M on the giant contractions of the middle colon. (a) Recording showing a typical conversion of the giant contractions into phasic contractions. (b) Effects of dequalinium (Deq.) on the resting tone and amplitude of the giant contractions. Values are mean ± S.E.M. (n = 9); *: p ≤ 0.05 with respect to basal control (B).

present regional difference in maximal response to carbachol [31]. Having seen the absence of differences between the different regions of the colon, we focused our study on the neural control of the GCs in the middle colon. The addition of atropine to the organ bath had no effect on the amplitude and frequency of the GCs, showing the lack of muscarinic regulation of GCs at the level of the smooth cell. Activation of muscarinic receptors could be partially involved in maintaining a resting tone since a decrease of 11% of the resting tone was observed after treatment with atropine. Application of the nicotinic antagonist, hexamethonium, to the circular strips was without effect on amplitude and frequency of the GCs, showing a lack of control by ganglionic neurons. The spontaneous generation of GCs in the rat colon strips is not dependent on the release of acetylcholine from excitatory cholinergic neurons [2,4,24]. However, the motor migrating complexes observed in the isolated mouse colon are dependent on cholinergic neurotransmission [3]. Activation of intrinsic nerves by the nicotinic, ganglionic receptor agonist, DMPP, abolished

the GCs for a short time. When the GCs resumed, the resting tone was reduced by 22%. This transient inhibitory effect of DMPP suggests that nicotinic receptors are present on inhibitory motor neurons. A similar relaxing effect has been reported in the longitudinal muscle of the rat distal colon only when precontracted by carbachol 1 ␮M, and was attributed to ATP and NO transmission [32]. Acetylcholinesterase inhibition with neostigmine contracted the circular smooth muscle in a concentration–dependent manner, converting by this way GCs into phasic contractions. It was suggested that after inhibition of its hydrolysis, acetylcholine could diffuse to postjunctional receptors located on smooth muscle cells [33] and induce contraction of the tissue by activation of muscarinic receptors. Indeed, neostigmine was shown to cause airway smooth muscle contraction and inositol monophosphate accumulation [34]; inositol phosphate is the product of hydrolysis of inositol triphosphate released by activation of muscarinic receptors. An elevated resting tone and a high frequency of phasic contractions are consistent with the prokinetic activity of this drug [15,35].

Fig. 9. Effects of cumulative addition of diazoxide on the giant contractions of the middle colon. (a) Typical recording of the effect of diazoxide. (b) Effect of diazoxide on the resting tone and amplitude of the giant contractions. Values are mean ± S.E.M. (n = 8); *: p ≤ 0.05 with respect to basal control (B); ns: non significant.

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The inhibition of NOS by l-NAME was without effect on the GCs whereas l-arginine, the substrate of this enzyme to produce NO, increased their frequency and duration. The insensitivity of the rabbit colon to l-NAME is contrasting with that of other species. Interestingly, l-NAME and Nω-nitro-l-arginine (l-NNA) failed to alter the spontaneous contractions of rabbit intestine [36]. It was reported that blockade of NOS increased the amplitude and frequency of the spontaneous contractile activity of human and rat colon [2,21,24,27,37–39]. Regional sensitivity of contractions to NOS inhibition by l-NNA is correlated to nNOS positive cells density; proximal and distal colon of the rat are less sensitive to l-NNA [21]. It could be argued that the concentration of l-NAME (100 ␮M) used in this study was insufficient to block NOS activity. The prolonging effect of l-arginine on GCs duration and the marked inhibitory effect of SNP suggest that the tonic release of inhibitory mediators controls both the amplitude and frequency of the spontaneous contractions as it has been reported previously [24,39,6]. To test the involvement of cGMP in the tonic inhibition by NO, we used methylene blue as an inhibitor of soluble guanylate cyclase [19]. Methylene blue has been reported to antagonize the relaxing effect of SNP in various smooth muscle preparations [40–42]. In the present study, methylene blue converted the GCs pattern into high tone phasic contractions. By increasing the tone of the circular smooth muscle, we can postulate that methylene blue blocks the relaxation of the muscle during the falling phase of the GCs by lowering the concentration of cGMP in the tissue. However, it could not be ruled out that methylene blue increased the tone and force generation, preventing in this manner the ability of the muscle to generate GCs. A high tone seems to block the generation of GCs as observed during the tonic contraction of the strips induced by carbachol or neostigmine. There is evidence that NO through cGMP is responsible for the opening of small conductance Ca2+ -dependent K+ channels [8,36,43]. Blockade of K+ channels by TEA and dequalinium, non-selective and selective blockers, respectively, increased the resting tone and abolished the rhythmic GCs. In some strips (8/21) treated with TEA, the amplitude and frequency of GCs were increased, suggesting that GCs could be controlled by more than one type of K+ channels, each presenting a different sensitivity to TEA. Abolition of GCs of the rabbit colon by dequalinium suggests that they are regulated by apaminsensitive Ca2+ -dependent K+ channels. It has been shown in rabbit colon that NO increases KCa current by increasing the open probability of Ca2+ -dependent K+ channels and this effect was mediated in part by cGMP [8]. The complete inhibition of rhythmic mechanical activity by diazoxide is due to the opening of ATP-sensitive K+ channels, the resulting hyperpolarisation of the smooth muscle cells is responsible for the reduction of their excitability and contractility. The lack of effect of diazoxide on the resting tone suggests that the tone of the muscle is independent of ATP-sensitive K+ channels state. In conclusion, the present study provides evidence that all regions of the rabbit colon develop both phasic contractions and GCs. The dominance of one pattern of contractions over the other depends probably on the release of both inhibitory (NO, ATP) and excitatory (acetylcholine, tachykinins) neuro-

Fig. 10. Schematic summary of the present findings depicting the control of GCs of the circular smooth muscle of the colon. KATP : ATP-dependent K+ channels; KCa : Ca2+ -dependent K+ channels; AChE: acetylcholine esterase; MR: muscarinic receptors; nAChR: nicotinic receptors; NOS: intracellular nitric oxide synthase.(→) Activation; ( ) inhibition.

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