Role of alpha(2)-adrenoceptors in the sympathetic inhibition of motility reflexes of guinea-pig ileum

Abstract

1. Sympathetic regulation of the motility of guinea-pig ileum was investigated using mesenteric nerve (MN) stimulation to inhibit motility reflexes, in vitro. 2. Transmural electrical stimulation (5 Hz, 1 s) in intact intestinal segments, or inflation of a balloon against the mucosa in opened segments, evoked contractions of the circular and longitudinal muscles oral to the stimulus. 3. MN stimulation (10 Hz, 5 s) usually abolished contractions of the longitudinal and circular muscles evoked by either electrical or mechanical stimuli. 4. The inhibition was mimicked by UK14,304 (70-100 nM) and abolished by idazoxan (100 nM), revealing an enhancement of circular muscle contractions. There was no evidence for alpha(2)-receptors on the muscle, suggesting sympathetic inhibition was via the myenteric plexus. 5. Possible sites of action of noradrenaline released from sympathetic nerves were investigated using intracellular recordings from the circular muscle in a multichambered organ bath. 6. When in the stimulation chamber, UK14,304 depressed (by 50 %) excitatory junction potentials (EJPs) recorded oral to a distension stimulus, but did not affect inhibitory junction potentials (IJPs) recorded anal to the stimulus. When added to a chamber between the stimulus and recording chambers, UK14,304 depressed EJPs by 40 %, but did not alter IJPs. When in the recording chamber, UK14,304 depressed EJPs by 20 %, but had no effect on IJPs. IJPs were inhibited, however, when UK14,304 was applied to the whole bath. 7. It is concluded that sympathetic activity inhibits intestinal motility mainly via alpha(2)-adrenoceptors on ascending interneurons and intrinsic sensory neurons of the orally directed reflex pathway.

Figure 1. Diagram of the three types of preparation used to study the effects of sympathetic nerves on intestinal motility

A, apparatus used to simultaneously record longitudinal and circular muscle contractions in intact tubes of intestine. A balloon catheter recorded circular muscle activity (as pressure changes, oral end). An isotonic transducer connected to the intestinal wall via a thread and pulley recorded longitudinal muscle length in the same region. Electrodes were used to stimulate both enteric nerves (transmural stimulation) and the perivascular mesenteric nerves. In some experiments an anally placed balloon catheter was used to provide a distension stimulus. B, preparation used to record ascending excitatory reflexes in flat sheets of intestine. A hemispherical balloon set into the base of the bath recorded circular muscle activity (oral end). Distensions were applied via another balloon at the anal end and an electrode used to stimulate the mesenteric nerves. C, apparatus to record ascending EJPs and descending IJPs in the circular muscle. Distensions were applied to the gut wall at two sites in each of two stimulation chambers and reflex responses recorded at the opposite end of the preparation. When the preparation was oriented so that recordings were made at the oral end, ascending EJPs were recorded, and in the opposite orientation the responses seen were IJPs.

Figure 2. Spontaneous and evoked contractions recorded simultaneously in both muscle layers in intact segments of intestine

A, ongoing activity in both the circular (CM, balloon pressure change, mmHg) and longitudinal (LM, transducer output in volts) muscle layers. The circular muscle remained quiescent for long periods, whereas a slowly changing baseline activity was seen in the longitudinal muscle. Occasional larger and faster contractions occurred at random intervals (asterisk), and most often involved both muscle layers. B, contraction seen at the asterisk in A on an expanded time scale. C, contraction evoked in both muscle layers by transmural electrical stimulation (onset at arrow, 1 ms, 100 V, 5 Hz, 1 s train) applied at the anal end of a different segment of intestine. Note differences in the configurations of responses in circular and longitudinal recordings. Pressure and voltage scale bars in C apply to all panels. Time scale in C applies to panels B and C.

Figure 3. Variation in the ascending excitatory reflex evoked by distensions of different volumes

Graphs show the amplitude of the first (^) and tallest (□) components of responses to balloon distension in A, the circular muscle (n = 6 preparations, n = 3 at lowest distension volume) and B, the longitudinal muscle (n = 5, n = 1 at lowest volume). Responses were expressed as a percentage of a standard response to a 0.5 ml distension (% STD). Error bars indicate s.e.m.

Figure 4. Effects of hexamethonium and UK14,304 applied to the organ bath on the amplitudes of ascending contractile responses

□, control responses; ▪, responses in the presence of a drug; , responses following washout. As for all subsequent column graphs, column heights represent mean response amplitudes while error bars indicate s.e.m. Effect of hexamethonium (A; 100 μm, n = 4) and UK14,304 (B; 100 nm, n = 7) on ascending responses to transmural stimulation in both the longitudinal and circular muscle layers in intact segments of intestine. Responses in both layers were abolished by both drugs. † Significant difference from control, P < 0.005. C, effect of UK14,304 on ascending contractions of the circular muscle evoked by distension in the opened intestinal preparation. Reflex responses were virtually abolished in the presence of the drug (n = 4, *P < 0.02).

Figure 5. Contractile responses to MN stimulation in both intestinal muscle layers

A, response to stimulation of the MN stimulation (dashed line) in one preparation at 65 V. No contractions occurred during the stimulus, but there was an ‘off’ contraction at the end of the train. MN stimulation at this voltage fully blocked the ascending contraction generated by TM stimulation. B, MN stimulation at 75 V in the same preparation caused an ‘on’ contraction of both muscle layers at a short delay from the onset of the stimulus train.

Figure 6. Effect of MN stimulation on ascending excitatory responses

A, recordings in an intact tube preparation of ascending responses evoked by transmural stimulation (arrows). Upper traces, circular muscle contractions; lower traces, longitudinal muscle activity. One test cycle is shown, consisting of a control response (Pre-control), a test response (Test) during MN stimulation (dashed line) and a recovery response (Post-control), each separated by 5 min intervals. Note the ‘off’ contraction following the MN stimulation train in each case. B, recordings in a flattened preparation. Responses were evoked by distension of an anally placed balloon (0.08 ml). Upper traces, circular muscle contractions; lower traces, distension. a, control response; b, response during MN stimulation (dashed line); c, response during MN stimulation in the presence of 100 nm idazoxan.

Figure 7. Effect of idazoxan (100 nm) on the inhibition by MN stimulation of ascending contractions

Response amplitudes during MN stimulation are expressed as a percentage of their control values in the absence of MN stimulation. □, control responses; ▪, responses during MN stimulation; , responses during MN stimulation in the presence of 100 nm idazoxan; , responses after washout during MN stimulation. A, circular muscle, intact tube preparation; B, longitudinal muscle, intact tube preparation; C, circular muscle, flattened preparation. In all cases, the inhibition of ascending contractions during MN stimulation was abolished by idazoxan. In addition, idazoxan revealed a significant facilitation by MN stimulation of circular muscle responses in both preparations. Symbols indicate significant differences from control, *P < 0.05; †P < 0.01; n = 5 in all cases.

Figure 8. Distension-evoked responses in circular muscle recorded intracellularly

EJPs recorded at the oral end of one preparation (A) and IJPs recorded at the anal end of another preparation (B) when distensions were applied in the chamber adjacent to the recording chamber. Traces between A and B show onset and duration of distension stimuli. a, control responses; b, responses while UK14,304 (100 nm) was present in the stimulus chamber; and c, responses after wash out of the drug. Whereas ascending EJPs evoked in this way were depressed by UK14,304, IJPs recorded at a similar distance, but anally from the stimulation site, were not.

Figure 9. Effects of UK14,304 applied to various chambers on the amplitudes of ascending and descending reflex responses recorded in circular muscle

Responses were recorded in the three-chambered organ bath at one end of the preparation and distensions applied either to the adjacent chamber (Near) or to the more distant one (Far). A, effect of the drug applied to the near chamber on excitatory reflexes (EJPs) evoked in it (near distension) or conducted through it (far distension) (n = 4 in both cases). B, effects of UK14,304 when applied to the whole bath on EJPs elicited by distension in the near chamber (n = 9). C, effects of UK14,304 applied to the recording chamber on EJPs (n = 5 for both near and far chamber distensions). D, effect of UK14,304 when applied to the near chamber on descending reflexes evoked in the near and far chambers (n = 5 in both cases). E, effect of UK14,304, when applied to the recording chamber, on descending reflexes (n = 5 in both cases). F, effect of UK14,304 when applied to the whole bath on IJPs, (near chamber distension, n = 6; far chamber distension, n = 5). Symbols indicate a significant difference from control, *P < 0.03, †P < 0.001.