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Received 23 April 1998; accepted after revision 23 July 1998.
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ABSTRACT |
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-nitro-L-argininel-NA; 100 µM) or N -nitro-L-arginine methyl ester (L-NAME; 100 µM) applied to the middle chamber or stimulation chamber decreased the oral contractile response of the LM and CM (by about 30-40 %), but increased the anal relaxation (> 600 %) and exposed an anal contraction (> 1000 % increase) of both muscles. The addition of L-NA to the recording chamber reduced the anal relaxation of the LM and CM and the anal contraction of the LM, but slightly increased the anal contraction of the CM.
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INTRODUCTION |
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Peristalsis in the small and large intestine consists of activation of ascending excitatory and descending inhibitory reflex pathways which produces a synchronous oral contraction and anal relaxation of both the longitudinal and circular muscle layers, respectively, behind and in front of a bolus (Bayliss & Starling, 1899, 1900; Smith & Robertson, 1998). Such reflexes can be activated by radial distension and mechanical and chemical stimulation of the mucosa (Hirst & McKirdy, 1974; Hirst et al. 1975; Costa & Furness, 1976; Smith & Furness, 1988; Bornstein et al. 1991; Smith et al. 1991, 1992a,b). Following the anal relaxation, a late phase of contraction (descending excitation) of both muscle layers provides a further propulsive stimulus to a bolus that has entered the accommodating segment (Hirst et al. 1975; Smith et al. 1992b; Smith & Robertson, 1998).
The longitudinal and circular muscle layers of the intestine are innervated by different populations of motor neurons (Bornstein et al. 1991; Smith et al. 1992a; Costa et al. 1996), which supply both an excitatory and inhibitory innervation to both muscle layers (Costa et al. 1986; Smith & Robertson, 1998). Both oral and anal contractions are, however, due to activation of cholinergic motor neurons which innervate the two muscle layers (Smith & Robertson, 1998). The anal relaxation of the longitudinal and circular muscle is due to the activation of nitrergic motor neurons that release nitric oxide (NO), adenosine triphosphate (ATP) and vasoactive inhibitory polypeptide (VIP) (Sanders & Ward, 1992; He & Goyal, 1993; Foxx-Orenstein & Grider, 1996; Shuttleworth & Sanders, 1996; Smith & Robertson, 1998). The oral contraction also involves activation of ascending cholinergic interneurons, whereas descending inhibition and descending excitation are mediated by both cholinergic and non-cholinergic interneurons (Hirst & McKirdy, 1974; Hirst et al. 1975; Costa & Furness, 1976; Smith & Furness, 1988; Smith et al. 1991, 1992a,b; Smith & Robertson, 1998).
One set of ascending cholinergic interneurons and at least four subpopulations of descending interneurons can be identified immunohistochemically according to their different chemical coding (Messenger & Furness, 1990; McConalogue & Furness, 1993; Pompolo & Furness, 1993; Costa et al. 1996). In the guinea-pig colon, descending interneurons range from 0·5 to 50 mm in length (Messenger & Furness, 1990; McConalogue & Furness, 1993). Some of these descending interneurons are cholinergic (choline acetyltransferase (ChAT)-positive), whereas others are nitrergic (nitric oxide synthase (NOS)-positive), a subset of which may be immunoreactive for both NOS and ChAT, at least in the small intestine (see Pompolo & Furness, 1993). Nitrergic interneurons make synaptic connections with each other and with excitatory and inhibitory motor neurons, suggesting that NO may not only be a neurotransmitter released from inhibitory motor neurons to relax smooth muscle but may also be involved in communication between interneurons (see Pompolo & Furness, 1993; Costa et al. 1996). Nitric oxide could act either as a neurotransmitter or as a neuromodulator of transmitter release as occurs in the CNS (Garthwaite, 1991; Kilbinger, 1996). In fact, transmural stimulation or exogenous NO increases cGMP in a number of myenteric neurons in the colon, suggesting that these neurons are targets for nitrergic nerves (Shuttleworth et al. 1993; Young et al. 1993). Yuan et al. (1995) examined the possibility that NO was involved in neuronal-neuronal transmission in the reflex pathways of the guinea-pig small intestine. They used a three-chambered bath which allowed study of the effects of drugs on various parts of the reflex pathways. They found that NO inhibitors and donors increased and decreased, respectively, the amplitude of evoked inhibitory junction potentials in the circular muscle of the small intestine when they were added to the stimulation chamber but not to the intermediate chamber (see Fig. 1). These drugs did not affect evoked oral excitatory junction potentials. NO was purported to act as a retrograde messenger released from descending, nitrergic interneurons to suppress synaptic activity in intrinsic sensory neurons.
The effects of NO within the myenteric plexus are likely to be complex since inhibitors of NO synthesis and NO donors have both stimulatory and inhibitory effects on acetylcholine release (reviewed in Bartho & Lefebvre, 1995; Kilbinger, 1996).
In this study, we also used a partitioned bath (see Fig. 1) to examine the possibility that NO is involved in neuronal- neuronal transmission within the ascending and descending reflex pathways to both the longitudinal and circular muscle layers of the guinea-pig distal colon that are activated by mechanical stimulation of the mucosa. We present evidence that nitric oxide facilitates cholinergic neurotransmission in ascending reflex pathways and is a powerful depressant of descending cholinergic reflex pathways that regulate both muscle layers.
A preliminary account of these findings has been published (Smith & McCarron, 1998).
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A segment of distal colon was threaded through a greased hole in each of 2 rubber diaphragms which separated 2 partitions, dividing the bath into recording, stimulation and middle chambers (length of middle chamber = 10 mm). The oral or anal dissected end of the colon (7-10 mm in length), which consisted of only longitudinal muscle (LM) and myenteric plexus (MP) (LMMP), the circular muscle having been removed, was pinned to a bead of silicon rubber near the junction with the intact colon in the recording chamber. The other end of the dissected strip was attached to an isometric tension transducer (TLM), which measured movements of the LM. Movements of the circular muscle (CM) were monitored with a frog heart clip attached to the intact segment near the junction with the dissected LMMP strip (within 1-2 mm) and to another isometric tension transducer (TCM). The other end of the segment (15-20 mm in length) was opened and pinned with the mucosa uppermost to the floor of the stimulating chamber. Each chamber contained oxygenated Krebs solution at 37 °C. The fluid in each chamber was kept at different levels so that leaks between chambers could be easily detected. | ||
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METHODS |
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Guinea-pigs (250-350 g) were killed in a specially constructed chamber with an overdose of CO2. The abdomen was then cut open, the distal colon removed and the faecal pellets expelled by gently flushing the lumen with a modified Krebs solution (see below) applied via a syringe to the oral end of the segment. The extrinsic blood vessels and nerves along the mesenteric border of the colon were then carefully trimmed away.
Dissection of the longitudinal muscle
Approximately 5-6 cm of distal colon was pinned lightly to the Sylgard (Dow Corning) floor of a dissection dish. The segment of colon was cut open along the mesenteric border for 7-10 mm at one end and 15-20 mm at the other end. The shorter cut end was pinned flat with the mucosa uppermost and then the mucosa and circular muscle (CM) were removed by sharp dissection, leaving a full-width strip of longitudinal muscle (LM) with attached myenteric plexus from which tension recordings could be made (see Smith & Robertson, 1998). The longer end of the segment was opened and pinned flat with the mucosa uppermost for stimulation (Fig. 1).
Partitioned bath
The segment of colon was then threaded through greased (High Vacuum Grease; Dow Corning) holes in rubber diaphragms in two partitions which divided the organ bath into three chambers: a recording chamber (volume 40 ml) and a stimulation chamber (volume 40 ml) separated by a middle chamber (volume 10 ml) which was 10 mm wide (Fig. 1; Tonini & Costa, 1990; Yuan et al. 1995). Each chamber contained oxygenated Krebs solution at 37°C. The oral or anal end of the segment from which the CM had been removed was mounted over a plastic tube in the recording chamber so that 
5 mm of intact colon, from which recordings of CM activity were made, was also in the recording chamber (Fig. 1). The other end of the segment was then cut open and pinned with the mucosa uppermost to the Sylgard floor of the stimulation chamber.
The methods employed in measuring LM and CM activities were similar to those we have described previously (see Smith & Robertson, 1998).
Longitudinal muscle activity. The full-width strip of LM with attached myenteric plexus was pinned onto a bead of silicon rubber (RTV sealant 732, Dow Corning) close (< 2 mm) to the junction with the intact segment that was mounted on the plastic tube in the recording chamber. The free end of the longitudinal muscle was tied by a silk thread to an isometric tension transducer (Grass FT 03 D).
Circular muscle activity. The mechanical activity of the CM was measured with a frog heart clip attached via the serosal surface to the underlying CM of the intact colon in the recording chamber approximately 1-2 mm from the junction with the dissected strip of LM with intact myenteric plexus (Fig. 1). The clip was attached to the side of the colon (at an angle of 60 deg from the antimesenteric border), which minimized interference with neural pathways running to the dissected LM, and to another isometric tension transducer via a silk thread. Care was taken to ensure that the thread was orthogonal to the long axis of the gut so that only movements of the CM layer were recorded. The tension of the LM and CM was then stretched against the rigid tube in the recording chamber to give an initial tension of 0·75 g. The tensions of the LM and CM layers were recorded on a 4-channel Gilson Medical Electronics 5/6H Recorder.
Oral or anal reflex responses of the LM and CM were evoked by brushing the mucosa in the stimulation chamber with one to ten strokes of an artists' brush (Smith & Furness, 1988; Smith et al. 1991).
Equilibration.The segment of colon in the bath was equilibrated for 1·5-2 h. During this time the three chambers slowly heated up to a stable working temperature of 37°C. The oxygenated Krebs solution in each chamber was regularly replaced every 30 min throughout each experiment.
Drugs and solutions
Each drug that was applied to a particular chamber was allowed to equilibrate for 20 min before further reflex responses were elicited.
The drugs used in these studies were: atropine sulphate, hexamethonium bromide, N -nitro-L-arginine (L-NA), N -nitro-L-arginine methyl ester (L-NAME) and tetrodotoxin (TTX) (all from Sigma); S-nitroso-N-acetylpenicillamine (SNAP; RBI); and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; Tocris Cookson). Stock solutions (1-10 mM) were prepared in saline or distilled H2O. Drugs were added to the bath in volumes less than 1 % of bath volume. Corresponding volumes of solvents had no effect.
The composition of the modified Krebs solution was (mM): NaCl, 120·35; KCl, 5·9; NaHCO3, 15·5; NaH2PO4, 1·2; MgSO4, 1·2; CaCl2, 2·5; and glucose, 11·5. The solution was gassed continuously with a mixture containing 3 % CO2-97 % O2 (v/v), pH 7·3-7·4.
Analysis of data and statistics
The effects of drugs on the LM and CM layers being stimulated via the mucosa were assessed by comparing the mean of six responses measured before and after equilibration with the drug applied in a particular chamber. The amplitude, duration and area under a stimulated response were measured using a Jandel opaque digitizing tablet and SigmaScan software (Scientific Measuring System, Jandel Scientific, San Rafael, CA, USA) and results were stored on computer. The effect of drugs on the area under a response was commonly used throughout this study (Smith & Robertson, 1998). It reflects the force of the tissue over time and is a sensitive parameter that includes changes in both amplitude and duration. All data are presented as means ±
Responses to mucosal stimulation
Mechanical stimulation of the mucosa evoked a contraction of both the LM and CM oral to the stimulus and a brief transient relaxation of both muscle layers anal to the stimulus (Fig. 2). The oral and anal reflex responses started at the same time in each muscle layer and had a similar duration (duration (measured as 90 % of recovery of response) of oral contraction in LM = 8·3 ± 1·4 s and CM = 11·0 ± 1·8 s, P > 0·05; duration of anal relaxation (measured as 90 % of recovery of response) in LM = 6·5 ± 1·2 s, CM = 7·5 ± 1·3 s; P > 0·5). The oral contraction and anal relaxation of the LM, however, peaked before that of the CM (time to peak of oral contraction (measured from onset to peak of response): LM = 2·9 ± 0·4 s, CM = 6·1 ± 1·1 s, P < 0·01; time to peak of anal relaxation: LM = 4·5 ± 0·3 s, CM = 7·2 ± 0·5 s, P < 0·01) (Fig. 2). The size (area under a stimulated response) of the oral and anal responses depended somewhat on the number of brush strokes applied to the mucosa in quick (2-3 Hz) succession (Smith & Furness, 1988; Smith et al. 1991); maximal responses were obtained with five to ten brush strokes (Fig. 2). On occasions (7 out of 83 responses; n = 30), the anal relaxation was followed by a small 'off' contraction and a slight increase in spontaneous contractile activity; these responses were usually more apparent in the LM than the CM. All responses to mucosal stimulation were neural in origin since they were abolished by TTX (1 µM) applied to any chamber.
The data presented below relating to the reflex responses of the LM and CM were obtained when three to five brush strokes were applied.
Stroking the mucosa with a brush (5 strokes, see arrow) elicited a simultaneous contraction and relaxation of both the LM and CM of the distal colon oral (A) and anal (B) to the stimulus. The size of these responses was somewhat dependent upon the number of strokes (1, 3, 5 and 10) applied for oral (C) and anal (D) responses of LM and CM. Although the duration of the oral and anal responses of the LM and CM were similar, the LM contraction and relaxation peaked before that of the CM (C and D).
Effect of cholinergic antagonists
Hexamethonium (300 µM), an antagonist of nicotinic receptors, substantially reduced the oral contractile responses of both the LM and CM to mucosal stimulation when added to any chamber (stimulation chamber: LM to 7·1 ± 3·4 % and CM to 8·3 ± 4·2 %; middle chamber: LM to 9·6 ± 1·9 % and CM to 6·6 ± 3·7 %; recording chamber: LM to 5·6 ± 3·0 % and CM to 5·2 ± 1·7 % of control; all P < 0·001; n = 5 per chamber), suggesting that the ascending excitatory nervous pathways are cholinergic and consist of chains of interneurons that generally are no longer than the middle chamber. Hexamethonium had no detectable effect on the anal relaxation of either the LM or CM when added to any chamber (P > 0·05). The muscarinic antagonist atropine (1 µM) when added to the recording chamber reduced the oral responses of the LM and CM (LM to 10·0 ± 0·7 % and CM to 19·8 ± 0·6 % of control; n = 6; P < 0·01) suggesting that the oral responses of both muscles are largely generated by the release of acetylcholine from excitatory motor neurons. The oral and anal responses of the LM and CM triggered by a peristaltic wave were similarly affected by cholinergic antagonists (Smith & Robertson, 1998).
Effect of inhibiting NO synthesis on oral responses
These experiments investigated whether endogenous nitric oxide (NO) modified neuronal-neuronal conduction in those ascending excitatory reflex pathways responsible for the oral contraction of both the LM and CM. L-NA (100 µM), which at this concentration is a specific competitive inhibitor of NOS activity (Keef et al. 1997), reduced the oral contractile response of the LM and CM to mucosal stimulation when applied to the middle chamber (Fig. 3B and D), the stimulation chamber (Fig. 3E), or the recording chamber (Fig. 3F). The further addition of SNAP (10 µM) to a particular chamber, after the prior addition of L-NA (100 µM) to the same chamber, significantly restored the reduced oral contractile responses of both muscle layers (Fig. 3C and G).
A, control oral responses of LM and CM. B, oral responses after L-NA (100 µM) in the middle chamber. C, oral responses in the presence of L-NA (100 µM) and SNAP (10 µM) in the middle chamber. D-G, cumulative data for the effect of L-NA (100 µM) on oral responses of LM and CM when added to the middle chamber (D), the stimulation chamber (E) and the recording chamber (F). G, effect of SNAP (10 µM) and L-NA (100 µM added previously) on oral responses of LM and CM when added simultaneously to both the middle and stimulation chambers. Here and in the following figures: Mid ch, middle chamber; Stim ch, stimulation chamber; Rec ch, recording chamber; C, control; * P < 0·05; ** P < 0·01; *** P < 0·001; **** P < 0·0001.
L-NAME (100 µM; 2 experiments per chamber, data not shown) had similar inhibitory effects to L-NA.
Effect of inhibiting guanylate cyclase on oral responses. Nitric oxide usually exerts its actions indirectly in cells by activating guanylate cyclase. This enzyme can be either in a constitutive or in a soluble form, and activation increases cGMP which in turn leads via protein kinase G (PKG) to phosphorylation of a number of proteins including ion channels (Garthwaite, 1991; Sanders & Ward, 1992; Shuttleworth & Sanders, 1996). To test whether NO was acting via similar intracellular pathways within neurons in enteric reflex pathways we investigated the effects of ODQ (10 µM), a specific soluble guanylate cyclase inhibitor (Garthwaite et al. 1995), on the reflex responses of the LM and CM. ODQ at the concentration used in our study blocks long-term potentiation in some CNS neurons (Boulton et al. 1995) and nerve-evoked relaxations of the longitudinal muscle in the mouse caecum (Young et al. 1996). ODQ (10 µM) reduced the evoked oral contractions of both the LM and CM when added to the middle chamber (Fig. 4B and E) and then to the stimulation chamber (Fig. 4C and F).
The subsequent addition of SNAP (10 µM) simultaneously to both the middle and stimulation chambers did not, however, reverse the inhibition of the oral responses of the LM and CM caused by prior addition of ODQ to these chambers (Fig. 4D and G).
A, control oral responses of LM and CM to mucosal stimulation (5 strokes, applied at arrow). B, oral responses after ODQ (10 µM) was initially added to the middle chamber. C, oral responses after ODQ (10 µM) was then added to the stimulation chamber. D, oral responses after the subsequent addition of SNAP (10 µM) applied simultaneously to both the middle and stimulation chambers containing ODQ (10 µM). E and F, cumulative data for the effect of ODQ (10 µM) on oral responses of LM and CM when added to the middle chamber (E) and to both the middle and stimulation chambers (F). G, effect of SNAP (10 µM) after ODQ (10 µM) on the oral responses of LM and CM when added simultaneously to both the middle and stimulation chambers. The control was ODQ alone in the middle and stimulation chambers.
Effect of inhibiting NO synthesis on anal responses
The effects of the NO synthesis inhibitors L-NA and L-NAME on the descending nervous pathways were markedly different and opposite to their effects on ascending nervous pathways.
Effect of NO inhibitors in the middle and stimulation chambers. L-NA (100 µM; n = 25), when added to the middle chamber or to the stimulation chamber, or to the two chambers simultaneously, consistently caused a large increase in the duration of the relaxation response of both the LM and CM (Figs 5B and 6A-C). This increased relaxation response was also followed by a large increase in the 'off' contraction of the LM and CM, which occurred at about the same time in the two muscle layers (Figs 5B and 6E -G). The duration of the relaxation response increased from 6·40 ± 0·93 s (LM) and 7·00 ± 1·00 s (CM) to 26·00 ± 3·70 s (LM) and 19·80 ± 3·12 s (CM) after L-NA (P < 0·0001; 40 responses from n = 20). The duration of the anal contractile response in the presence of L-NA, which was negligible in control tissues, was 21·00 ± 4·6 s (LM) and 31·20 ± 5·68 s (CM). The time from the onset of the relaxation to the peak of the contraction was similar for the LM (36·80 ± 2·35 s) and CM (38·60 ± 2·37 s) (P > 0·05).
A, control anal responses of LM and CM to 3 and 5 brush strokes of the mucosa. B, effects of L-NA (100 µM) in the middle chamber. C, effects of L-NA (100 µM) and SNAP (10 µM) applied to the middle chamber on the mucosal responses to brush strokes. D, responses after further addition of L-NA (100 µM) to the stimulation chamber. E, responses after further addition of SNAP (10 µM) to the stimulation chamber.
Cumulative data for the effects of L-NA (100 µM) added to particular chambers on anal relaxation (A-D) and anal contraction (E-H) of LM and CM (n = 8 per chamber). Note that in H, the decrease in anal contraction of LM and increase in contraction of CM after L-NA in the recording chamber. In D and H, the control was L-NA alone in the middle and stimulation chambers.
The effects of L-NAME (100 µM) were similar, if not identical, to those produced by L-NA added to the same chambers (data not presented).
Spontaneous descending complexes. In some preparations (26 out of 50), an anal relaxation-contraction complex, which was identical in both amplitude and duration to the evoked events, occurred spontaneously at a frequency of 5·75 ± 0·5 min following the application of L-NA to the middle or stimulation chambers. These spontaneous events were not observed after mucosal stimulation. This suggested that although these events could propagate anally as a complex, the interneurons regulating their spread were normally restrained by the inhibiting effects of NO. These complexes may be similar to the spontaneous descending waves of contraction observed in the rabbit and guinea-pig distal colon (Mackenna & McKirdy, 1972; Costa & Furness, 1976) and migrating myoelectric activity in the mouse colon (Bywater et al. 1989).
Effect of NO donors in the middle and stimulation chambers. After the addition of L-NA (100 µM) to the middle or stimulation chamber, the subsequent addition of an NO donor such as SNAP (10 µM; n = 5 for each chamber) or sodium nitroprusside (SNP; 10 µM; n = 2 for each chamber, data not presented) completely reversed the increases in anal relaxation and contractile responses produced by L-NA (Figs 5C and 7). An example of the effects of SNAP on the evoked anal responses of the LM and CM are shown in Fig. 5. Figure 5B shows the increased evoked anal relaxation and late contractile response following the addition of L-NA (100 µM) to the middle chamber. The subsequent addition of SNAP (10 µM) to this chamber reversed both the evoked relaxation and the evoked contraction (Fig. 5C). Following the further addition of L-NA (100 µM) to the stimulation chamber there was again an increase in the evoked anal responses (Fig. 5D). Subsequent addition of SNAP (10 µM) to the stimulation chamber restored the responses back to control levels (Fig. 5E).
Cumulative data for the effects of SNAP (10 µM) on increased anal relaxation (A and B) and anal contraction (C and D) of LM and CM produced by L-NA (100 µM) added either to the middle chamber (A and C) or to both the middle and stimulation chambers prior to the addition of SNAP to these chambers (n = 5 for each chamber).
Effect of inhibiting guanylate cyclase on anal responses. ODQ (10 µM) added to either the middle or stimulation chamber, or to the two chambers simultaneously, increased both the evoked anal relaxation and anal contraction of the LM and CM (Fig. 8B, E and F (relaxation), H and I (contraction)), effects identical to those of L-NA. The subsequent addition of SNAP (10 µM) to the same chamber had no significant effect on the anal responses of either the LM or CM to mucosal stimulation (Fig. 8D, G (relaxation) and J (contraction)).
A, control anal responses of LM and CM. B, responses after ODQ (10 µM) was initially added to the middle chamber. C, responses after ODQ (10 µM) was then added to the stimulation chamber. D, responses after SNAP (10 µM) was applied simultaneously to both the middle and stimulation chambers containing ODQ. E and F, cumulative data (n = 5 per chamber) for the effect of ODQ (10 µM) on the anal relaxation responses of LM and CM when added to the middle chamber (E) and to both the middle and stimulation chambers (F). G, effect of SNAP (10 µM), in the presence of ODQ, on anal relaxation of LM and CM when added simultaneously to both the middle and stimulation chambers. H and I, cumulative data for the effect of ODQ (10 µM) on the anal contractile responses of LM and CM when added to the middle chamber (H) and to both the middle and stimulation chambers (I). J, effect of SNAP (10 µM), in the presence of ODQ, on anal contraction of LM and CM when added simultaneously to both the middle and stimulation chambers (n = 5). In G and J, the control (C) was ODQ alone.
Effect of NO inhibitors in the recording chamber. Drugs in the recording chamber can affect not only connections between interneurons and motor neurons but also neuromuscular transmission. Nitric oxide appears to be a major inhibitory neurotransmitter to the CM and LM in the large intestine (Sanders & Ward, 1992; Watson et al. 1996; Young et al. 1996; Smith & Robertson, 1998); NO may also cause both a direct relaxation of the LM and a neurally evoked contraction, sensitive to both atropine and TTX, of the LM (Bartho & Lefebvre, 1995). To determine whether the large increase in the relaxation and contractile responses produced by blocking the effects of endogenous NO on neurotransmission (see Fig. 5) was due to the activation of nitrergic inhibitory motor neurons to both muscle layers, the effects of adding L-NA (100 µM) to the recording chamber after the drug was first added to both the stimulation and middle chambers (Fig. 9A) were examined. Under these circumstances, the further addition of L-NA (100 µM; n = 7) or L-NAME (100 µM; n = 3) to the recording chamber reduced the relaxation response of both the LM and CM (Figs 9C and 6D), suggesting that the relaxation of both muscle layers was mediated in part by NO released from inhibitory motor neurons. These inhibitors of NO synthesis also reduced the anal contractile response of the LM but produced a slight but significant (P < 0·05) increase in the anal contractile response of the CM (Figs 9C and 6H).
SNAP (10 µM) in the recording chamber relaxed both the LM (by 6·4 ± 0·3 mN, n = 5, P < 0·01) and CM (by 4·3 ± 0·6 mN, n = 5, P < 0·01). This relaxation was unaffected by TTX (1 µM; n = 4).
A, D and G, control anal mucosal responses of LM and CM. B, E and H, anal relaxation and contractile responses of the LM and CM produced by L-NA (100 µM) added simultaneously to both the middle and stimulation chambers. C, effect of the addition of L-NA (100 µM) to the recording chamber. Note that the relaxation of the LM and CM has been reduced as has the area under the late contraction of the LM. F, effect of hexamethonium (Hex; 300 µM) in the middle chamber. I, effect of addition of atropine (1 µM) to the recording chamber, after the tone of LM and CM had been readjusted back to its original level. The three pairs of traces in each column are from the same experiment.
Effect of cholinergic antagonists on anal responses
In both the myenteric plexus and CNS, NO donors and inhibitors of NOS either inhibit or stimulate acetycholine release (reviewed in Bartho & Lefebvre, 1995; Kilbinger, 1996). As described above, nicotinic blockers substantially reduced the evoked oral contraction of the LM and CM, when added to any chamber, suggesting that endogenous NO was involved in neurotransmission by facilitating acetylcholine release from cholinergic ascending interneurons. We therefore investigated whether the large biphasic inhibitory then excitatory responses of the LM and CM produced by blocking NO release in descending reflex pathways were also mediated by cholinergic interneurons. Hexamethonium (300 µM) added to either the middle or stimulation chambers blocked the increases in the anal relaxation and late contraction of both the LM and CM (Figs 9F, 10A-C (relaxation) and D-F (contraction)). Also, hexamethonium (300 µM) almost abolished the increase in the evoked anal relaxation and anal contraction of the LM and CM when added to the recording chamber (Fig. 10C (relaxation) and F (contraction)).
These results suggest that NO may suppress neurotransmission in chains of descending cholinergic interneurons. Also, many of the inhibitory and excitatory motor neurons activated in the recording chamber by these cholinergic interneurons probably have short projections to the LM and CM (< 5 mm).
The muscarinic antagonist atropine (1 µM), when added to the recording chamber, reduced the rhythmic contractions and tone of both muscle layers as we reported previously (Smith & Robertson, 1998). The tone of the LM was also reduced by 25·3 ± 6·7 % (P < 0·001) and to a lesser extent that of the CM by 9·3 ± 3·2 % (P < 0·02; n = 7). After the tone had been restored by increasing the tension of the muscle back to control levels it was found that atropine in the recording chamber had reduced not only the area under the oral contractile response of both muscles as described above but also blocked the increased anal contractile responses of the LM and CM produced by the prior addition of L-NA (100 µM) in the middle and stimulation chamber (Figs 9H and I and 10G).
In all panels, controls (C) are the anal relaxation and contractile responses of the LM and CM to L-NA (100 µM) added to both the middle and stimulation chambers. A-C (anal relaxation) and D-F (anal contraction): inhibitory effect of hexamethonium (Hex; 300 µM; n = 5 per chamber) on these responses when added to the middle (A and D), stimulation (B and E) or recording chambers (C and F). G, effect of atropine (1 µM) on the anal contraction of LM and CM. Note that hexamethonium blocked any contraction when added to any chamber but left a small residual relaxation.
Sensory neurons responsive to mucosal stimulation activate ascending cholinergic interneurons which then activate excitatory cholinergic motor neurons to both the LM and CM. The sensory neurons also activate descending cholinergic interneurons which activate inhibitory motor neurons and cholinergic motor neurons to both the LM and CM. The release of acetycholine (ACh) is facilitated by nitric oxide (NO). This may derive from activation of nitrergic interneurons, nitrergic motor neurons or interstitial cells of Cajal (ICC). On the other hand, ACh release from descending cholinergic interneurons is suppressed by NO. The NO is likely to be derived from a separate population of descending nitrergic neurons or from the same (nitrergic/cholinergic) interneurons. n, nicotinic receptor.
We have demonstrated, for the first time, that endogenous NO facilitates cholinergic neurotransmission in ascending excitatory nervous pathways and suppresses cholinergic neurotransmission in descending inhibitory and excitatory nervous pathways to both LM and CM. These opposite effects of NO within the polarized reflex pathways may help to explain why both stimulation and inhibition of acetylcholine release from the myenteric plexus have been observed with NO donors and inhibitors of NO synthesis (see Bartho & Lefebvre, 1995; Kilbinger, 1996).
Mucosal reflex responses
Mechanical stimulation of the mucosa evoked a contraction and relaxation of both the LM and CM, oral and anal to the stimulus, respectively, confirming our previous hypothesis that neurons in the peristaltic reflex pathways of the distal colon are connected so that both the LM and CM layers contract and relax synchronously (see Smith & Robertson, 1998). However, responses of the LM peak before those in the CM. These faster movements of the LM may have important mechanical consequences for propulsion, and may be why the two muscle layers sometimes appear to exhibit reciprocal activity (see McKirdy, 1972; Smith & Robertson, 1998).
In the present study descending excitation, which is normally triggered by a peristaltic wave (Smith & Robertson, 1998), was only observed when NO synthesis was inhibited. NO synthesis inhibitors also produced a prolonged increase in the duration of the relaxation. Both these events sometimes spontaneously propagated down the colon as a complex, suggesting that the same descending interneurons may activate both inhibitory and excitatory motor neurons to the LM and CM. The complex probably represents a balance between the effects of inhibitory and excitatory neurotransmitters on the muscle, because in some records the contractile responses appeared to break through during the relaxation giving the appearance of an apparent delay in the relaxation, which was not so readily observed in atropine (see Figs 5D, and 9E and I).
Endogenous NO appears, therefore, to regulate the spread, timing and size of events associated with descending peristaltic pathways in the colon, as it appears to in the oesophagus (Knudsen et al. 1994).
Cholinergic and non-cholinergic reflex pathways
The oral contractile (ascending excitation) and anal relaxation (descending inhibition) responses of both the LM and CM to mucosal stimulation were mediated by cholinergic ascending and non-cholinergic descending neural pathways, respectively, since hexamethonium blocked the oral responses but had little effect on anal relaxation (see Smith et al. 1992b; Smith & Robertson, 1998).
Surprisingly, L-NA added to the middle or stimulation chambers increased the duration of the anal relaxation and revealed a large anal excitatory response (descending excitation) in both muscle layers. Both these events were mediated by short descending cholinergic interneurons (length < 10 mm) since they were also blocked by hexamethonium in any chamber (Fig. 10). They were, therefore, also mediated by inhibitory and excitatory motor neurons with short projections to both muscle layers (Smith et al. 1988; Costa et al. 1996; Fig. 11). The excitatory motor neurons to the LM and CM are cholinergic and likely to receive convergent synaptic inputs from both ascending and descending cholinergic interneurons (see Bornstein et al. 1991; Smith et al. 1992a; Pompolo & Furness, 1995; Smith & Sanders, 1995; Smith & Robertson, 1998).
NO in nervous reflex pathways
L-NA (or L-NAME) significantly decreased the oral contraction but increased the anal relaxation and revealed an anal contraction of both the LM and CM when added to the middle or stimulation chambers. These effects were partially reversed by the NO donor SNAP. ODQ, an inhibitor of soluble guanylate cyclase (Garthwaite et al. 1995), had the same effects as L-NA and L-NAME. SNAP, however, had no effect in the presence of ODQ, suggesting that ODQ was acting specifically and that the NO donated by SNAP was acting through soluble guanylate cyclase. These results suggest that NO affects neurotransmission in ascending and descending nervous pathways by acting through soluble guanylate cyclase in cholinergic interneurons.
The NO that modifies the descending cholinergic interneurons in the colon is presumably released from reflexly activated descending nitrergic interneurons (NOS immunoreactive), which can be up to 50 mm in length in the guinea-pig colon (Messenger & Furness, 1990; McConalogue & Furness, 1993). These descending nitrergic neurons could influence descending cholinergic interneurons situated in parallel pathways (Fig. 11; see Pompolo & Furness, 1993). Alternatively, some descending cholinergic interneurons could also be nitrergic (i.e. display both NOS and ChAT immunoreactivity) (see Messenger & Furness, 1990; McConalogue & Furness, 1993; Costa et al. 1996). This seems unlikely, however, since stimulation of cGMP immunoreactivity by transmural stimulation, NO or NO donors in strips of small and large intestine occurs in non-nitrergic myenteric neurons, which are presumably the targets for nitrergic neurons (Shuttleworth et al. 1993; Young et al. 1993). Our results with ODQ suggest that some of these target neurons could include ascending and descending cholinergic interneurons.
Ascending nitrergic interneurons are absent, so it is unclear how NO could regulate ascending cholinergic interneurons. Both ascending excitatory and inhibitory nervous pathways exist in the distal colon; i.e. inhibitory motor neurons activated by ascending cholineric interneurons (Smith et al. 1992b). NO could possibly be generated in the cell bodies of inhibitory motor neurons by synaptic activity in ascending interneurons and feed back to facilitate nervous transmission in a manner similar to that proposed by Yuan et al. (1995) for interactions between interneurons and sensory neurons. Ascending cholinergic interneurons could also be influenced by descending nitrergic interneurons, local nitrergic interneurons, or even interstitial cells of Cajal (ICC), which contain NOS (Fig. 11; see Shuttleworth & Sanders, 1996).
The actual mechanisms underlying the actions of NO within the nervous pathways were unresolved by our methods. NO could act postsynaptically to regulate ion channels on the soma that would then alter the excitability of cholinergic interneurons or presynaptically to facilitate or depress synaptic transmission from nerve terminals as proposed for long-term potentiation (LTP) and long-term depression (LTD), respectively, in the CNS (see Garthwaite, 1991; Kilbinger, 1996). The most dominant response in myenteric neurons to reflex stimulation of ascending and descending reflex pathways is a burst of fast excitatory postsynaptic potentials (FEPSPs) (Hirst et al. 1975; Bornstein et al. 1991; Smith et al. 1991; Smith & Sanders, 1995). The calcium influx associated with FEPSPs (see Trouslard et al. 1993) could possibly lead to stimulation of NOS activity and NO production in nitrergic neurons, which in turn could lead to either depression or facilitation of acetycholine release in neighbouring neurons as in some cholinergic systems in the CNS (see Kilbinger, 1996).
NO involvement in muscle responses
The increase in the inhibitory response produced by L-NA in the middle or stimulation chambers, was abolished by the further addition of L-NA in the recording chamber, suggesting that L-NA was blocking the release of NO from inhibitory motor neurons innervating both the LM and CM. Nitric oxide is reportedly a major inhibitory neurotransmitter to both muscle layers in the colon (see Sanders & Ward, 1992; Watson et al. 1996; Young et al. 1996; Smith & Robertson, 1998). In support, SNAP in the recording chamber relaxed both muscle layers. L-NA in the recording chamber also reduced anal contraction of the LM but increased that of the CM (see Smith & Robertson, 1998). NO may excite the LM indirectly via stimulation of cholinergic motor neurons or by direct actions on the muscle (see Bartho & Lefebvre, 1995). Presumably, the increase in the CM response after L-NA is due to the removal of underlying nitrergic inhibition of the muscle (see Sanders & Ward, 1992) (Fig. 11).
Conclusions
NO facilitates neurotransmission in ascending cholinergic interneurons but depresses cholinergic neurotransmission in descending cholinergic interneurons; these effects of NO on neurons are mediated through soluble guanylate cyclase.
It is likely that, once activation of low-threshold ascending excitatory pathways initiates peristalsis, NO release is suppressed. This reduction in NO release may be expected to inhibit further activation of ascending cholinergic interneurons but increase activity in descending cholinergic interneurons, thereby allowing the inhibitory-excitatory complex to migrate down the bowel (see Smith & Roberston, 1998).
S. L. McCarron is a visiting research scholar from the University of Ulster, Coleraine, UK. This work was supported by a grant from the National Institutes of Health, no. NIDAA 10793.
Corresponding author
T. K. Smith: Department of Physiology and Cell Biology, Anderson Medical Sciences Building/352, University of Nevada School of Medicine, Reno, NV 89503, USA.
Email: tks{at}physio.unr.edu
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RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References
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Figure 2. Oral and anal mucosal reflex responses of LM and CM
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Figure 3. Effect of L-NA and SNAP on oral responses
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Figure 4. Effect of ODQ and SNAP on oral responses
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Figure 5. Effect of L-NA and SNAP on anal responses
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Figure 6. Summary of effects of L-NA on anal responses
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Figure 7. Summary of effects of SNAP on anal responses
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Figure 8. Effect of ODQ and SNAP on anal responses
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Figure 9. Effect of L-NA, hexamethonium and atropine on anal responses
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Figure 10. Summary of effects of hexamethonium and atropine on anal responses
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Figure 11. Modulation of cholinergic transmission by nitric oxide
DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References
REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References
Acknowledgements
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