Guy E. Boeckxstaens

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, NG-monomethyl-L-arginine (L-NMMA) and NG-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.

Involvement of nitric oxide in the inhibitory innervation of the human isolated colon

BACKGROUNDnThe exact nature of the inhibitory nonadrenergic noncholinergic (NANC) neurotransmitter in the human colon is still unknown.nnnMETHODSnThe present study was designed to investigate the role of nitric oxide (NO) and adenosine 5-triphosphate (ATP) in circular muscle strips of the human isolated colon.nnnRESULTSnNO and ATP induced tetrodoloxin-resistant relaxations that mimicked those evoked by nerve stimulation. Apamin inhibited the response to ATP, had a variable effect on the relaxations to transmural stimulation, and had no effect on those to NO or nitroglycerin. NG-nitro-L-arginine (L-NNA) concentration dependently reduced the NANC nerve-mediated relaxations, but had no effect on those to ATP, NO, or nitroglycerin; the L-NNA resistant part of the NANC relaxation to nerve stimulation was further reduced by apamin. The inhibitory effect of L-NNA or the combination of L-NNA and apamin was prevented by L-arginine but not by D-arginine.nnnCONCLUSIONSnThese results suggest that NO and another substance, perhaps ATP, are involved in the inhibitory NANC neurotransmission in the circular muscle of the human colon.

Evidence for nitric oxide as mediator of non-adrenergic non-cholinergic relaxations induced by ATP and GABA in the canine gut.

1 The effects of haemoglobin, and the nitric oxide (NO) biosynthesis‐inhibitors NG‐monomethyl‐l‐arginine (l‐NMMA), its enantiomer d‐NMMA, and NG‐nitro‐l‐arginine (l‐NNA) were investigated on non‐adrenergic non‐cholinergic (NANC)‐mediated relaxation of circular muscle strips of the canine terminal ileum and ileocolonic junction induced by electrical stimulation, adenosine 5′‐triphosphate (ATP), γ‐aminobutyric acid (GABA) and NO. 2 Tetrodotoxin, l‐NMMA and l‐NNA, but not d‐NMMA, inhibited the relaxations induced by electrical stimulation, ATP and GABA, but not those in response to NO. 3 The inhibitory effect of l‐NMMA and l‐NNA was prevented by l‐arginine, but not by d‐arginine. l‐Arginine did not potentiate any of the NANC relaxations. 4 Haemoglobin reduced the relaxation induced by electrical stimulation, ATP and GABA, and abolished those in response to NO. 5 Our results demonstrate that the ATP‐ and GABA‐induced relaxations resulting from stimulaton of intramural NANC neurones, in addition to those induced by electrical impulses, are mediated by NO or a NO releasing substance and thus provide further evidence in support of the proposal that NO is the final inhibitory NANC neurotransmitter in the canine terminal ileum and ileocolonic junction.

The role of nitric oxide in inhibitory non-adrenergic non-cholinergic neurotransmission in the canine lower oesophageal sphincter.

1 The role of nitric oxide (NO) in non‐adrenergic non‐cholinergic (NANC) neurotransmission was studied on circular muscle strips of the canine lower oesophageal sphincter (LOS). Electrical field stimulation evoked frequency‐dependent relaxations, which were resistant to adrenergic and cholinergic blockade and abolished by tetrodotoxin. 2 Exogenous administration of NO induced concentration‐dependent and tetrodotoxin‐resistant relaxations which mimicked those in response to electrical stimulation. 3 NG‐nitro‐l‐arginine (l‐NNA), a stereospecific inhibitor of NO‐biosynthesis, inhibited the relaxations induced by electrical stimulation but not those by exogenous NO or vasoactive intestinal polypeptide (VIP). 4 The effect of l‐NNA was prevented by l‐arginine, the precursor of the NO biosynthesis but not by its enantiomer d‐arginine. 5 Haemoglobin abolished the NO‐induced responses and reduced those evoked by electrical stimulation. 6 Cumulative administration of VIP induced concentration‐dependent relaxations, which were slow in onset and sustained. A complete relaxation to VIP was not achieved and the relaxations were not affected by l‐NNA. 7 In conclusion, our results provide evidence that NANC relaxations are mediated by NO, suggesting NO or a NO releasing substance as the final inhibitory NANC neurotransmitter in the canine LOS.

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. Equivalent amounts of the transferable factor, endothelium‐derived relaxing factor and NO, as assessed by the relaxation of the rabbit aorta, failed to affect the 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.

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 ω‐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 μm), γ‐aminobutyric acid (100 μm) and adenosine 5′‐triphosphate (100 μm), as well as the relaxations to nitric oxide (NO) (3–10 μm) and nitroglycerin (1 μm), 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 Ca2+‐free medium, the release of NO‐R evoked by electrical impulses or DMPP was inhibited. The L‐type Ca2+ channel blockers verapamil (1–3 μm) and nifedipine (1 μm) had no effect. 4 From these results we conclude that the release of NO‐R in response to NANC nerve stimulation is Ca2+‐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.

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 (greater than 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.

Effect of thiol modulators and Cu/Zn superoxide dismutase inhibition on nitrergic relaxations in the rat gastric fundus

1 The effects of superoxide anion generators before and after treatment with inhibitors of Cu/Zn superoxide dismutase (Cu/Zn SOD) and the effects of thiol‐modulating agents were investigated on nitrergic relaxations to electrical stimulation of non‐adrenergic non‐cholinergic (NANC) nerves of the rat gastric fundus and on relaxations to authentic nitric oxide (NO) and nitroglycerin. 2 The superoxide anion generators, pyrogallol (30 μm) and duroquinone (30–60 μm), significantly inhibited the relaxations to NO (0.03‐3 μm) but not nitrergic relaxations to NANC nerve stimulation (0.5–8 Hz) or those to ATP (10 μm). Treatment of the rat gastric fundus with the inhibitors of Cu/Zn SOD, diethyldithiocarbamate (DETC, 1 mM for 2 h) or triethylenetetramine (TETA, 100 μm for 2 h) had no effect on the relaxations to NANC nerve stimulation (1 − 8 Hz), NO (0.03 − 3 μm) or on those to ATP (10 μm). 3 After treatment of the rat gastric fundus with DETC (1 mM) but not after treatment with TETA (100 μm), pyrogallol (30 μm) and duroquinone (30–60 μm) significantly inhibited the nitrergic relaxations to electrical stimulation (0.5–8 Hz) and those to NO (0.03‐3 μm). This inhibitory effect of pyrogallol and duroquinone was prevented by addition of exogenous SOD (250 units ml−1). Pyrogallol but not duroquinone also inhibited the NO‐independent relaxations to ATP (10 μm). 4 The thiol modulators, buthionine sulphoximine (1 mM for 2 h) and ethacrynic acid (30 γU for 2 h), significantly inhibited the relaxations to nitroglycerin (0.03‐3 μm) but had no effect on the nitrergic relaxations to electrical stimulation (0.5–8 Hz) or on those to NO (0.03–10 γUM) and ATP (10 μm). The thiol modulators, sulphobromophthalein (100 μm for 2 h) and diamide (30–100 μm for 2 h) did not affect the relaxations to nitroglycerin, or those to NANC nerve stimulation and NO. 5 In summary, thiol modulators significantly inhibited the thiol‐dependent relaxations to nitroglycerin but not those to NANC nerve stimulation or NO. Relaxations to nitrergic stimulation were decreased by superoxide anion generators only after inhibition of Cu/Zn SOD. These results suggest that the nitrergic NANC neurotransmitter in the rat gastric fundus is not a nitrosothiol but more likely free NO, which is protected from breakdown by tissue SOD.

The role of increased nitric oxide in the vascular hyporeactivity to noradrenaline in long-term portal vein ligated rats

To test the possible role of nitric oxide production in long-term portal vein ligation in the rat, where the hyperdynamic circulation was reported to be absent, in vivo experiments on isolated thoracic aortic rings from partial portal vein ligated or sham-operated rats were performed, 6 months postoperatively. The concentration-response curves to noradrenaline of both intact and endothelium-denuded rings from portal hypertensive rats were significantly shifted to the right as compared to those from sham-operated animals. In intact rings, addition of NG-nitro-L-arginine, a specific inhibitor of nitric oxide synthase, resulted in a significant shift of the curves to the left in sham-operated and portal vein ligated rats. In endothelium-denuded rings, addition of NG-nitro-L-arginine resulted in a significant shift of the curves to the left in portal vein ligated but not in sham-operated animals. After blockade of the nitric oxide biosynthesis with NG-nitro-L-arginine, the negative logarithm of the concentration of nonadrenaline causing half-maximal response did not significantly differ any more between portal vein ligated and sham-operated rats; in endothelium-denuded rings hyporeactivity to noradrenaline persisted in portal vein ligated rats. Only in the intact rings did NG-nitro-L-arginine significantly increase the maximal contractions. No differences were demonstrated in endothelium-dependent relaxations to acetylcholine between sham-operated and portal hypertensive animals. From these results, it can be concluded that in vitro aortic hyporeactivity to noradrenaline is still present in long-term portal vein ligated rats, and that it results at least partially from activation of the L-arginine: nitric oxide pathway in the aortic vascular wall.

GABAA receptor-mediated stimulation of non-adrenergic non-cholinergic neurones in the dog ileocolonic junction.

1 The inhibitory effects of γ‐aminobutyric acid (GABA), the GABAA receptor agonist homotaurine and the GABAB receptor agonist (±)‐baclofen were investigated on circular muscle strips of the dog terminal ileum and ileocolonic junction. 2 In the presence of atropine, GABA and homotaurine induced concentration‐dependent relaxations, similar to the non‐adrenergic non‐cholinergic (NANC)‐mediated relaxations evoked by electrical stimulation or by acetylcholine. The ileocolonic junction was more sensitive to GABA and homotaurine than the ileum. (±)‐Baclofen had no effect. Cross desensitization only occurred between GABA and homotaurine. 3 The GABAA receptor antagonist bicuculline shifted the concentration‐response curves to GABA and homotaurine to the right. The maximal relaxation to GABA remained unaffected. 4 GABA‐induced relaxations were not inhibited by timolol, guanethidine, domperidone, hexamethonium and desensitization to ATP, but were abolished by tetrodotoxin. 5 Bicuculline, and pretreatment with GABA or (±)‐baclofen had no effect on the NANC‐evoked relaxations to electrical stimulation and acetylcholine. 6 In conclusion, GABA stimulates GABAA receptors located on inhibitory NANC neurones in the dog ileocolonic junction. Our results suggest that it is unlikely that GABA is the final inhibitory NANC neurotransmitter.