Canine Ileocolonic Junction Research Articles

Propagation of giant migrating contractions between the small intestine, cecum and colon during radiation

Radiation increases the frequency of small intestinal and colonic giant migrating contractions (GMCs). These contractions contribute to the diarrhea and cramping after radiation therapy and are coordinated with one another across the ileocolonic (IC) junction. We investigated the coordination of contractile activity between the small intestine, cecum and colon in five canines following circumferential myotomy on the ileum at the IC junction and compared it to intact animals. Studies were performed before and during a radiation schedule. Myotomy increased the frequency of small intestinal GMCs prior to irradiation. In intact animals, the duration and amplitude of cecal GMCs decreased when multiple contractions occurred in rapid succession. This is in contrast to small intestinal and colonic GMCs and suggests a different mechanism of propagation for GMCs within the cecum. Ileal myotomy dramatically decreased the frequency of propagating radiation-induced colonic GMCs. The total number of colonic GMCs was not altered. Colonic contractile activity was disrupted in intact animals during irradiation. However, after ileal myotomy, irradiation did not affect the pattern of colonic contractile states. Diarrhea in irradiated animals with myotomy started earlier than intact animals. This may be related to the frequency of small intestinal GMCs. Our findings suggest importance of the enteric neural connections at the IC region to contractile disorders of both the small and large intestine. The anatomic relationship between the canine IC junction is similar to the human ileo-appendiceal-colonic region and surgical manipulations of this area may likewise affect human contractile activity.

Pharmacological similarity between nitric oxide and the nitrergic neurotransmitter in the canine ileocolonic junction.

In organ bath experiments, pyrogallol (30-100 microM), and to a lesser extent L-cysteine (3 microM), reduced nitric oxide (NO)-induced relaxation, but not the NO-mediated non-adrenergic non-cholinergic (NANC) relaxation elicited by field stimulation of the canine ileocolonic junction. In contrast, in a superfusion bioassay, pyrogallol (10-30 microM) and L-cysteine (1 microM) inhibited the biological activity of both the transferable nitrergic factor released from the canine ileocolonic junction in response to NANC nerve stimulation and NO, but not that of S-nitroso-L-cysteine, S-nitrosoglutathione or S-nitroso-N-acetyl-D,L-penicillamine. Based on the bioassay experiments, it is concluded that the nitrergic NANC neurotransmitter in the canine ileocolonic junction behaves pharmacologically like NO.

Effect of potassium channel blockade and alpha 2-adrenoceptor activation on the release of nitric oxide from non-adrenergic non-cholinergic nerves.

1. Using a superfusion bioassay cascade, we studied the effect of K+ channel blockers and alpha 2-adrenoceptor agents on the release of a transferable factor, previously characterized as nitric oxide (NO) or a nitric oxide-related substance (NO-R), in response to non-adrenergic non-cholinergic (NANC) nerve stimulation in the canine ileocolonic junction (ICJ). 2. The non-selective K+ channel blockers, 4-aminopyridine (4-AP, 50 microM) and tetraethylammonium (TEA, 1 mM) and the more selective blocker of Ca(2+)-activated K+ channels, charybdotoxin (Leiurus quinquestriatus venom (LQV), 0.4 microgram ml-1), significantly enhanced the release of NO-R induced by low frequency stimulation (2-4 Hz). In the presence of 4-AP and TEA, the release of NO-R was nearly abolished by tetrodotoxin (2 microM), and by L-NG-nitroarginine (L-NOARG, 0.1 mM). Relaxations induced by direct injection of exogenous NO (5-50 pmol) or nitroglycerin (GTN, 10-30 pmol) onto the rabbit aortic detector ring were not affected. 3. The alpha 2-adrenoceptor agonist, UK-14,304 (0.3 microM) inhibited the release of NO-R induced by low (2-4 Hz), but not that induced by high (16 Hz), frequency stimulation. This inhibitory effect was completely reversed by the alpha 2-adrenoceptor antagonist, yohimbine (0.3 microM). Neither UK-14,304 nor yohimbine affected the relaxations induced by exogenous NO (5 pmol) or GTN (10 pmol) on the aortic detector ring.3+

Ca2+ dependency of the release of nitric oxide from non-adrenergic non-cholinergic nerves.

1. The role of Ca2+ in nitrergic neurotransmission was studied in the canine ileocolonic junction. 2. The specific N-type voltage-sensitive Ca2+ channel blocker omega-conotoxin GVIA (CTX, 10-100 nM) significantly reduced the electrically-evoked (2-16 Hz, 1-2 ms pulse width) non-adrenergic non-cholinergic (NANC) relaxations, preferentially affecting those to low frequency stimulation, in circular muscle strips of the ileocolonic junction. In contrast, the nerve-mediated NANC-relaxations in response to acetylcholine (30 microM), gamma-aminobutyric acid (100 microM) and adenosine 5'-triphosphate (100 microM), as well as the relaxations to nitric oxide (NO) (3-10 microM) and nitroglycerin (1 microM), remained unaffected. 3. A NO-related substance (NO-R), released from the ileocolonic junction in response to NANC nerve stimulation (4 and 16 Hz, 2 ms pulse width), was assayed with a superfusion bioassay cascade. CTX (50 nM) reduced the release of NO-R induced by electrical impulses (4 Hz: from 18 +/- 4% to 6 +/- 4%; 16 Hz: from 33 +/- 2% to 14 +/- 4%, n = 5), but not that in response to the nicotinic receptor agonist, 1,1-dimethyl-4-phenylpiperazinium (DMPP, 0.3 mM). In Ca(2+)-free medium, the release of NO-R evoked by electrical impulses or DMPP was inhibited. The L-type Ca2+ channel blockers verapamil (1-3 microM) and nifedipine (1 microM) had no effect. 4. From these results we conclude that the release of NO-R in response to NANC nerve stimulation is Ca(2+)-dependent. The electrically-evoked release of NO-R results from Ca2+ entry through CTX-sensitive N-type voltage-sensitive Ca2+ channels, whereas that induced by nicotinic receptor activation involves CTX-insensitive Ca2+ channels, different from the L- or N-type.

Prejunctional modulation of the nitrergic innervation of the canine ileocolonic junction via potassium channels.

1. The effects of different K+ channel blockers were studied on nitric oxide (NO)-mediated non-adrenergic non-cholinergic (NANC) relaxations in the canine ileocolonic junction. 2. The non-selective blockers of K+ channels, 4-aminopyridine (4-AP) and tetraethylammonium (TEA) and the blocker of large conductance Ca(2+)-activated K+ channels, charybdotoxin, potently enhanced the NANC relaxations induced by low frequency stimulation. The blocker of small conductance Ca(2+)-activated K+ channels, apamin, had no effect on electrically-induced NANC relaxations. 3. NANC nerve-mediated relaxations induced by adenosine 5'-triphosphate (ATP), acetylcholine (ACh) and gamma-aminobutyric acid (GABA) were significantly enhanced by 4-AP and charybdotoxin but not by apamin. TEA significantly enhanced the NANC relaxations in response to GABA and ATP while that in response to ACh was abolished. 4. None of the K+ channel blockers had an effect on the dose-response curve to NO, on the noradrenaline-induced contraction or on the relaxation to nitroglycerine (GTN). 5. From these results we conclude that inhibition of prejunctional K+ channels increases the nitrergic relaxations induced by electrical and chemical receptor stimulation of NANC nerves and thus suggests a regulatory role for these prejunctional K+ channels in the release of NO from NANC nerves in the canine ileocolonic junction.

Alpha 2-adrenoceptor-mediated modulation of the nitrergic innervation of the canine isolated ileocolonic junction.

1. The effects of specific alpha-adrenoceptor agonists and antagonists on electrically-evoked non-adrenergic non-cholinergic (NANC) relaxations, previously demonstrated as nitrergic, were investigated in isolated circular muscle strips of the canine ileocolonic junction. 2. During a substance P-induced contraction and in the presence of atropine and guanethidine, the specific alpha 1-adrenoceptor agonist, phenylephrine and antagonist, prazosin, as well as the specific alpha 2-adrenoceptor antagonist, yohimbine, had no effect on the NANC relaxations evoked by electrical field stimulation. In contrast, clonidine and the more specific alpha 2-adrenoceptor agonist, UK-14,304, significantly reduced the electrically-induced relaxations, preferentially those in response to low frequency stimulation. The inhibitory effect of UK-14,304 on these relaxations was antagonized by yohimbine. 3. During a noradrenaline-induced contraction, clonidine, but not UK-14,304 significantly augmented the relaxations to electrical stimulation. 4. The adrenoceptor agonists and antagonists used had no effect on concentration-response curves to NO or on the relaxation induced by nitroglycerin. 5. These results indicate that stimulation of prejunctional alpha 2-adrenoceptors inhibits the nitrergic NANC relaxations induced by field stimulation and thus suggest prejunctional regulation of nitric oxide release via alpha 2-adrenoceptors in the canine ileocolonic junction.

The role of nitric oxide in serotonin-induced relaxations in the canine terminal ileum and ileocolonic junction.

The role of nitric oxide (NO) in 5-HT-induced non-adrenergic non-cholinergic (NANC) relaxations was studied on circular muscle strips of the canine ileocolonic junction (ICJ) and terminal ileum. During an acetylcholine-induced contraction, NO (10(-5) M) evoked a transient relaxation, whereas 5-HT (10(-4) M) caused an initial NANC relaxation followed by a contraction. This initial relaxation to 5-HT, but not the relaxation to NO, was significantly inhibited by the stereospecific inhibitors of the NO biosynthesis NG-monomethyl-L-arginine (L-NMMA) and NG-nitro-L-arginine (L-NNA). L-arginine, but not D-arginine, prevented the inhibitory effect of L-NMMA and L-NNA. The enantiomer of L-NMMA, D-NMMA, had no effect. Hemoglobin abolished the NO-induced relaxations and significantly inhibited the relaxation to 5-HT. From these experiments it is concluded that the 5-HT-induced NANC relaxation is mediated by NO or a NO releasing substance.

Bioassay of nitric oxide released upon stimulation of non‐adrenergic non‐cholinergic nerves in the canine ileocolonic junction

1. The release and the nature of the inhibitory non-adrenergic non-cholinergic (NANC) neurotransmitter was studied in the canine ileocolonic junction. A circular muscle strip of the canine ileocolonic junction served as donor tissue in a superfusion bioassay in which rings of rabbit aorta with the endothelium removed served as detector tissue. 2. The ileocolonic junction released a labile factor with vasodilator activity upon stimulation of non-adrenergic non-cholinergic (NANC) nerves in response to electrical impulses and the nicotinic receptor agonist 1,1-dimethyl-4-phenylpiperazinium (DMPP). This release was respectively frequency- and concentration-dependent. 3. The release was reduced by the blocker of neuronal conductance, tetrodotoxin, and by the inhibitor of the nitric oxide (NO) biosynthesis NG-nitro-L-arginine. The biological activity was enhanced by superoxide dismutase and eliminated by haemoglobin. Hexamethonium abolished only the release in response to DMPP. 4. Injection of adenosine 5'-triphosphate (ATP) or vasoactive intestinal polypeptide (VIP) onto the cascade induced relaxations of the rabbit aorta but they were different from those induced by NO or the transferable factor. 5. Based on organ bath experiments in which the reactivity of different parts of the circular smooth muscle layer of the ileocolonic junction was investigated, a muscle strip of superficial circular muscle with submucosa was chosen as the detector strip in the bioassay cascade. 6. The ileocolonic junction dose-dependently relaxed in response to nitroglycerin and NO. NO was much more potent in the rabbit aorta than in the canine ileocolonic junction. 7. In conclusion, our results demonstrate the release of a transferable vasorelaxant factor in response to NANC nerve stimulation which behaves pharmacologically like NO but not like ATP or VIP. Therefore, we suggest that NO or a NO releasing substance is the inhibitory NANC neurotransmitter in the canine ileocolonic junction.

Nitric Oxide Release in Response to Stimulation of Nonadrenergic, Noncholinergic Nerves

Summary: The nature of the inhibitory nonadrenergic, noncholinergic (NANC) neurotransmitter was studied in the canine ileocolonic junction. In a first series of experiments, circular muscle strips were mounted in organ baths filled with aerated Krebs-Ringer solution for isometric tension recording. During a norepinephrine-induced contraction and in the presence of atropine, nitric oxide (NO) induced tetrodotoxin-resistant relaxations that were similar to the NANC relaxations obtained by electrical stimulation and acetylcholine (ACh). Inhibition of the NO biosynthesis by NG-monomethyl-l-arginine (l-NMMA) and NG-nitro-l-arginine (l-NNA) resulted in an increase in basal tension and inhibited the relaxations to electrical stimulation and ACh, but not those to NO. These effects were partly reversed by l-arginine, but not by d-arginine. Hemoglobin also raised the basal tension and reduced the relaxations to electrical stimulation and ACh and abolished those to NO. In a second series of experiments, we demonstrated the release of a labile factor with vasodilator activity upon stimulation of NANC nerves of the canine ileocolonic junction using a superfusion bioassay cascade. The release of this factor was inhibited by tetrodotoxin and l-NNA, whereas it was increased by l-arginine. The biological activity was enhanced by superoxide dismutase and eliminated by hemoglobin. Our results suggest that NO or an NO-releasing substance represents the NANC neurotransmitter in the canine ileocolonic junction.

Nitric Oxide Release in Response to Stimulation of Nonadrenergic, Noncholinergic Nerves

The nature of the inhibitory nonadrenergic, noncholinergic (NANC) neurotransmitter was studied in the canine ileocolonic junction. In a first series of experiments, circular muscle strips were mounted in organ baths filled with aerated Krebs-Ringer solution for isometric tension recording. During a norepinephrine-induced contraction and in the presence of atropine, nitric oxide (NO) induced tetrodoxin-resistant relaxations that were similar to the NANC relaxations obtained by electrical stimulation and acetylcholine (ACh).

Non-adrenergic non-cholinergic relaxation mediated by nitric oxide in the canine ileocolonic junction

The nature of the inhibitory non-adrenergic non-cholinergic (NANC) neurotransmitter was studied in circular muscle strips of the canine terminal ileum and ileocolonic junction. Nitric oxide (NO) induced tetrodotoxin-resistant NANC relaxation, similar to that induced by electrical stimulation or acetylcholine (ACh). Incubation with the stereospecific inhibitors of NO biosynthesis, N G-monomethyl-L-arginine (L-NMMA) and N G-nitro-L-arginine (L-NNA), resulted in an increase of basal tension in the ileocolonic junction which was partly reversed by L-arginine but not by D-arginine. Moreover, L-NMMA and L-NNA, but not D-NMMA, concentration dependently inhibited the NANC relaxation in response to electrical stimulation and ACh, but not that in response to NO or nitroglycerin. This inhibitory effect was reversed by L-arginine but not by D-arginine. Hemoglobin reduced the NANC relaxation in response to electrical stimulation, ACh and nitroglycerin, and abolished the responses to NO. Our results suggest that NO or a NO releasing substance mediates the NANC relaxation in the canine terminal ileum and ileocolonic junction. Non-adrenergic non-cholinergic (NANC) neurotransmission; Ileum (canine); Ileocolonic junction (canine); Nitric oxide

Nitric oxide as an inhibitory non-adrenergic non-cholinergic neurotransmitter

Inhibitory non-adrenergic non-cholinergic (NANC) nerves are thought to be important in the autonomic innervation of the gastrointestinal tract and other organ systems. The nature of their neurotransmitter is still debated. Speculation that nitric oxide (NO), formed from L-arginine in neuronal structures and other cells, could act as a neurotransmitter, is not yet supported by demonstration of its release upon nerve stimulation. Using a superfusion bioassay, we report the release of a vasorelaxant factor upon stimulation of the NANC nerves in the canine ileocolonic junction. Several pieces of evidence, including the selectivity of the bioassay tissues, chemical instability, inactivation by superoxide anion and haemoglobin, inhibition by NG-nitro-L-arginine (L-NNA) and potentiation by L-arginine all indicated that NO accounted for the biological activity of this transferable NANC factor.

Acetylcholine is an indirect inhibitory transmitter in the canine ileocolonic junction

The effects of cholinergic agents, electrical stimulation and vasoactive intestinal polypeptide (VIP) were studied on transverse muscle strips of the canine ileum, ileocolonic junction and colon. Acetylcholine, methacholine and carbachol caused concentration-dependent contractions in the three gut tissues. Only acetylcholine (> 10 −5 M) evoked transient relaxations in the ileum and the ileocolonic junction before the onset of contractions. During contractions by noradrenaline, acetylcholine induced relaxations, which were enhanced by atropine; electrical stimulation also caused frequency-dependent relaxations. Propranolol or naloxone did not affect the relaxations. Hexamethonium, cocaine or lidocaine inhibited the relaxations induced by acetylcholine but not those evoked by electrical stimuli. Tetrodotoxin inhibited all relaxations. VIP did not evoke relaxation in the ileocolonic junction. These data indicate that acetylcholine stimulates nicotinic receptors on non-adrenergic non-cholinergic neurons, which do not release VIP or opioids. It is thus suggested that there is a nicotinic inhibitory innervation at the canine ileum and ileocolonic junction.

Flow across the canine ileocolonic junction: role of the ileocolonic sphincter.

The role of the canine ileocolonic sphincter (ICS) in influencing transit of fluids and absorption of water in the ileum was assessed in loops with or without the ICS. Two sets of four dogs each were equipped with isolated ileal loops 40 cm long, attached to the intact bowel through a neuromuscular bridge. In one set, the loop included the ICS and it terminated in an end colostomy; in the other, the loop ended immediately proximal to the ICS, as an ileostomy. Electrical and mechanical signals were recorded from serosal electrodes and strain gauges, respectively, during perfusion of loops with an isotonic solution at rates from 0.7 to 10.0 ml/min. Loops without an ICS propelled fluid by steady, continuous flow at all rates of perfusion; loops with the ICS in place moved fluids in this way only at 10 ml/min. At greater rates of perfusion, fasting cycles of motility were inhibited in all loops and a random pattern of motility developed; this presumably facilitated more steady flow of fluids. At the slowest rate of perfusion, transit times were longer when the ICS was present, but at faster rates both loops showed similar transit times. Presence of the ICS was associated with increased volumes of fluid in the loop and increased absorption of water, especially when loops were perfused slowly. The canine ICS influences flow of saline and absorption in the terminal ileum, but only when flow rates are low.

Tone of canine ileocolonic junction: topography and response to phasic contractions.

We wished to define the physiology of the canine ileocolonic sphincter (ICS) and to examine function in relation to the region's anatomy. Prolonged recordings of tone at the ICS were made from seven dogs with isolated ileocolonic loops, and the effects of ileal and colonic distension on sphincteric tone were assessed. In acute experiments, pull-through pressures were measured at the ICS, and the location of the high-pressure zone was related to the region's anatomy. Basal tone at the ICS of approximately 30 cmH2O was confirmed; tone was augmented by colonic and ileal distension. The high-pressure zone was always centered on the anatomic ICS, but it extended into the adjacent ileum and colon for a total length of 2.0 cm (range 1.5-3.0 cm). Phasic contractions contributed to the maintenance of tone at the ICS, and a coordinated pattern of phasic contractions appears to contribute to the sphincteric properties of the region.

Distinctive patterns of interdigestive motility at the canine ileocolonic junction

Studies of interdigestive motor activity in the distal ileum, ileocolonic sphincter, and proximal colon were performed in two groups of dogs: (i) in those with ileocolonic loops that maintained neuromuscular continuity with the proximal intestine, we used an intraluminal side-hole assembly; (ii) in those with intact bowel, we attached extraluminal strain gauge transducers. Both groups of animals were equipped also with serosal electrodes. Positioning of motor sensors could be defined accurately by (a) a clear separation of rhythmic frequencies between those of the distal ileum (mean 10.8 cycles/min) and proximal colon (mean 6.2 cycles/min) and (b) the characteristic motor patterns of the ileocolonic sphincter. Analysis of motor activity in the segment demonstrated four distinct patterns: (i) irregular, apparently random phasic waves, (ii) rhythmic bursts of contractions corresponding to phase III of the migrating motor complex, (iii) other slowly propagated bursts of rhythmic activity (mean duration 3.6 min) recurring on average every 10 min, and (iv) single, prolonged (mean duration 16.7 s), high amplitude (mean 271 cmH2O) waves that propagated rapidly (“prolonged propagated contractions”). The latter two patterns appear unique to, or unusually prominent in, this region. A high degree of motor coordination between the distal ileum, ileocolonic sphincter, and proximal colon was also recognized. Thus, the patterns of motor activity suggest a specialized function for the distal 30 cm of ileum, the ileocolonic sphincter, and the proximal colon. These unique and coordinated patterns of motility deserve further exploration, especially in relation to ileocolonic transit and actions of the sphincter as a barrier to colo-ileal reflux.