David E. Burleigh

Ng-nitro-l-arginine reduces nonadrenergic, noncholinergic relaxations of human gut

The aim of this study was to determine whether nonadrenergic, noncholinergic inhibitory neurotransmission in human circular sigmoid colonic and internal anal sphincter muscle involves release of a nitric oxide-like substance. Colonic and sphincter muscle respond to electrical field stimulation by giving nonadrenergic, noncholinergic relaxations. After-contractions always occur in colonic muscle but only sometimes in sphincter muscle. Ng-Nitro-L-arginine abolished relaxations of sphincter muscle and partially reduced those of colonic muscle. After-contractions were undiminished as were relaxations of sphincter muscle to sodium nitroprusside. The effects of Ng-nitro-L-arginine were reversed by L-arginine. The results suggest that nitric oxide is possibly the neurotransmitter mediating nonadrenergic, noncholinergic relaxations of the human internal anal sphincter muscle.

Smooth muscle cholinergic denervation hypersensitivity in diverticular disease

BACKGROUND Evidence from clinical and laboratory investigations into the causes of diverticular disease suggests that disturbances in cholinergic activity are important, the effector mechanisms of which have yet to be established. We aimed to investigate the role of smooth muscle and neural cholinergic activity in the pathogenesis of this disease. METHODS Two investigators independently did a blinded immunohistochemical image analysis of localising antibodies to choline acetyltransferase, co-localised with protein gene product (PGP)--a marker of general neural tissue-and smooth muscle muscarinic M3 receptors, on three histological sections of sigmoid colons from ten patients with diverticular disease and ten controls, after resections for rectal tumours. We also did isotonic organ bath experiments to assess muscle strip sensitivities to exogenous acetylcholine. FINDINGS In circular muscle, activity of choline acetyltransferase was lower in patients with diverticular disease than in controls: median percentage surface area of choline acetyltransferase over PGP was 17.5% (range 10.0-37.0) in patients with diverticular disease and 47.0% (29.0-54.0) in controls (p<0.0001). M3 receptors were upregulated in patients with diverticular disease compared with controls: the median surface area was 13.2% (6.0-23.3) in patients with diverticular disease and 2.5% (1.6-3.7) in controls (p<0.0001). The sensitivity to exogenous acetylcholine was increased in patients with diverticular disease (mean -log EC(50) 5.6 [SD 0.3]) compared with controls (4.9 [0.5]; difference 0.7 [95% CI 0.3-1.1], p=0.006). In longitudinal muscle, choline acetyltransferase activity was lower in patients with diverticular disease (median 19.5%, range 12.0-30.0) than in controls (47.0%, 35.0-60.0; p<0.0001), with upregulation of M3 receptors in diverticular disease (diverticular disease 7.8% [1.9-20.4], controls 1.7% [0.8-3.0]; p<0.0001). However, sensitivity to exogenous acetylcholine did not differ between the two groups (diverticular disease mean 5.6% [SD 0.3], controls 5.2% [0.4]; difference 0.4% [95% CI -0.02-0.7], p=0.06). INTERPRETATION Our results suggest that cholinergic denervation hypersensitivity can affect smooth muscle. Upregulation of smooth muscle M3 receptors might account for specific clinical, physiological, and pharmacological abnormalities associated with diverticular disease.

Neural and Pharmacologic Factors Affecting Motility of the Internal Anal Sphincter

Abstract An important factor contributing toward anal continence is the degree of muscle contraction shown by the internal anal sphincter and how this is influenced by the autonomic innervation of the muscle. In this review various investigations, in vivo in patients or volunteers and in vitro using human sphincter muscle strips, are discussed. These investigations have led to a greater understanding of the influence on the internal anal sphincter of extrinsic and intrinsic nerves. The actions of drugs and electrical field stimulation on sphincter muscle strips is discussed and provides additional information about the receptors and nerves that might be involved in the motility of the sphincter in vivo. The innervation of the internal anal sphincter of a number of animal species (cat, dog, rabbit, and monkey) is discussed briefly.

Evidence for the involvement of a 5-HT4 receptor in the secretory response of human small intestine to 5-HT.

5‐Hydroxytryptamine increases transmucosal short‐circuit current across human isolated small intestinal mucosa. The competitive 5‐HT4 antagonist, DAU 6285 evoked a concentration‐dependent, dextral and parallel shift of the concentration‐response curve to 5‐HT, with no alteration of the maximum response. Schild analysis of this antagonism produced a Schild regression with a slope of 1.00 and an apparent pA2 estimate of 6.17. It appears that a 5‐HT4 receptor may mediate the short‐circuit current response of human small intestinal mucosa to 5‐HT.

Longitudinal muscle shows abnormal relaxation responses to nitric oxide and contains altered levels of NOS1 and elastin in uncomplicated diverticular disease

Objective  Recent evidence challenges the ‘low‐fibre/high‐colonic intraluminal pressure’ hypothesis of diverticular disease (DD) and raises the possibility that other mechanisms are involved. Although bowel wall smooth muscle is known to be hypercontractile in DD, the nature of its relaxation is unknown. The present study investigated colonic smooth muscle responses to nitric oxide, as well as the smooth muscle contents of neural nitric oxide and elastin associated with the disease.

Short-circuit current responses to 5-hydroxytryptamine in human ileal mucosa are mediated by a 5-HT4 receptor

5-Hydroxytryptamine (5-HT) increases short-circuit current when added to the serosal side of human isolated ileal mucosa; mucosally applied 5-HT was ineffective. Tetrodotoxin reduced both basal short-circuit current and increases in short-circuit current due to electrical field stimulation of mucosal nerves. However, neither tetrodotoxin, ondansetron nor methysergide plus ketanserin affected 5-HT induced increases in short-circuit current. Application of SDZ 205-557 (2-diethylaminoethyl-(2-methoxy-4-amino-5-chloro) benzoate) to the tissue caused a significant increase in the concentration ratio between two successive 5-HT response curves. It is concluded that the effect of 5-HT on short-circuit current of human ileal mucosa appears to be due to stimulation of a 5-HT4 receptor.

Antisecretory actions of a novel vasoactive intestinal polypeptide (VIP) antagonist in human and rat small intestine

1 Vasoactive intestinal peptide (VIP) has been demonstrated in intestinal mucosal neurones and elicits chloride secretion from enterocytes. These findings have led to the proposal that VIP is a secretomotor neurotransmitter. Confirmation of such a role may now be possible with the development of PG 97–269, a high‐affinity, selective antagonist of VIP type 1 (VPAC1) receptor, which is expressed by gut epithelial cells. We have evaluated the VIP antagonism and antisecretory potential of this novel compound using in vitro and in vivo models of intestinal secretion. 2 Monolayers of the human colonic cell line (T84) and muscle‐stripped preparations of rat jejunum and human ileum were set up in Ussing chambers for recording of transepithelial resistance and short‐circuit current. Ussing chambers were modified to allow electrical stimulation of mucosal neurones. Effects of PG 97–269 on enterotoxin‐induced secretion were investigated in perfused rat jejunum in vivo. 3 PG 97–269 competitively antagonised VIP in T84 monolayers. In rat jejunum and human ileum, responses to VIP were inhibited as were responses of rat jejunum to electrical stimulation of mucosal neurons. 4 In perfused rat jejunum, PG 97–269 abolished the effects of VIP on fluid and electrolyte transport and attenuated cholera toxin and Escherichia coli heat labile toxin‐induced net fluid and electrolyte secretion. 5 PG 97–269 is a competitive antagonist of enterocyte VIP receptors and effectively inhibits responses of rat and human intestinal mucosa to VIP. Antagonism of secretory responses to electrical stimulation of mucosal neurons and lumenal application of enterotoxins imply a secretory role for VIP in these processes.

Functional evidence for a 5-HT2B receptor mediating contraction of longitudinal muscle in human small intestine.

Application of 5‐hydroxytryptamine induces contraction of longitudinal muscle strips from human terminal ileum. The response was resistant to antagonism by ketanserin, ondansetron or DAU6285, but was non‐surmountably antagonized by methysergide. The selective 5‐HT2B/2C receptor antagonist, SB 200646A evoked a concentration‐dependent, parallel and dextral displacement of the concentration‐response curve to 5‐HT, yielding a pA2 estimate of 7.17. Application of yohimbine, a 5‐HT1 and 5‐HT2B receptor antagonist, also induced a rightward shift of the response curve to 5‐HT, yielding a pA2 estimate of 8.10. In conclusion, it appears that a 5‐HT2B receptor mediates the contractile response of the longitudinal muscle of human small intestine to 5‐HT.

Human colonic mucosa possesses a mixed population of 5-HT receptors.

The aim of this study was to characterise the 5-HT receptor(s) mediating secretory responses of isolated human colonic mucosa to 5-HT. Sheets of muscle-stripped mucosa from proximal (ascending) and distal (sigmoid) human colon were set up in Ussing chambers for measurement of short-circuit current (Isc). Serosal application of 5-HT led to non-neural, concentration-dependent increases in Isc. Desensitisation to 5-HT was observed with ascending and sigmoid colonic mucosa. Using selective 5-HT antagonists we have shown that 5-HT induces secretion in sigmoid colon via a 5-HT2A receptor. In ascending colon a combination of 5-HT2A and 5-HT4 receptors appears to be involved.

Subcutaneous morphine reduces intestinal propulsion in rats partly by a central action

Rats were given intracerebroventricular or intravenous injections of the quarternary opioid receptor antagonist N,N-diallyl-normorphinium (DANM. 100 microgram). Ten min later morphine (7.5 mg/kg) or loperamide (10 mg/kg) was injected subcutaneously. Intestinal propulsion was assessed by measuring the progress of radioactive chromium (Na51(2) CrO4, 0.5 microCi) along the small intestine. The radioactive chromium was instilled into the proximal duodenum 20 min after injection of morphine or loperamide, and the animals sacrificed 35 min after giving radioactive chromium. Morphine and loperamide both inhibit intestinal propulsion. DANM (100 microgram i.c.v.) reduces the effect of morphine but not loperamide. Intravenous injection of DANM does not reduce anti-propulsive action of morphine. By itself DANM neither increases nor decreases intestinal propulsion. These experiments indicate that morphine, when administered by a peripheral route, reduces small intestinal propulsion in the rat partly through an action on brain opioid receptors.