Serotonin exerts a direct modulatory role on bladder afferent firing in mice.

KEY POINTS: Functional disorders (i.e. interstitial cystitis/painful bladder syndrome and irritable bowel syndrome) are associated with hyperexcitability of afferent nerves innervating the urinary tract and the bowel, respectively. Various non-5-HT3 receptor mRNA transcripts are expressed in mouse urothelium and exert functional responses to 5-HT. Whilst 5-HT3 receptors were not detected in mouse urothelium, 5-HT3 receptors expressed on bladder sensory neurons plays a role in bladder afferent excitability both under normal conditions and in a mouse model of chronic visceral hypersensitivity. These data suggest that the role 5-HT3 receptors play in bladder afferent signalling warrants further study as a potential therapeutic target for functional bladder disorders. ABSTRACT: Serotonin (5-HT) is an excitatory mediator that in the gastrointestinal (GI) tract plays a physiological role in gut-brain signalling and is dysregulated in functional GI disorders such as irritable bowel syndrome (IBS). Patients suffering from IBS frequently suffer from urological symptoms characteristic of interstitial cystitis/painful bladder syndrome, which manifests due to cross-sensitization of shared innervation pathways between the bladder and colon. However, a direct modulatory role of 5-HT in bladder afferent signalling and its role in colon-bladder neuronal crosstalk remain elusive. The aim of this study was to investigate the action of 5-HT on bladder afferent signalling in normal mice and mice with chronic visceral hypersensitivity (CVH) following trinitrobenzenesulfonic acid-induced colitis. Bladder afferent activity was recorded directly using ex vivo afferent nerve recordings. Expression of 14 5-HT receptor subtypes, the serotonin transporter (SERT) and 5-HT-producing enzymes was determined in the urothelium using RT-PCR. Retrograde labelling of bladder-projecting dorsal root ganglion neurons was used to investigate expression of 5-HT3 receptors using single cell RT-PCR, while sensory neuronal and urothelial responses to 5-HT were determined by live cell calcium imaging. 5-HT elicited bladder afferent firing predominantly via 5-HT3 receptors expressed on afferent terminals. CVH animals showed a downregulation of SERT mRNA expression in urothelium, suggesting increased 5-HT bioavailability. Granisetron, a 5-HT3 antagonist, reversed bladder afferent hypersensitivity in CVH mice. These data suggest 5-HT exerts a direct effect on bladder afferents to enhance signalling. 5-HT3 antagonists could therefore be a potential therapeutic target to treat functional bladder and bowel disorders.

Interplay between mast cells, enterochromaffin cells, and sensory signaling in the aging human bowel.

BACKGROUND: Advanced age is associated with a reduction in clinical visceral pain perception. However, the underlying mechanisms remain largely unknown. Previous studies have suggested that an abnormal interplay between mast cells, enterochromaffin (EC) cells, and afferent nerves contribute to nociception in gastrointestinal disorders. The aim of this study was to investigate how aging affects afferent sensitivity and neuro-immune association in the human bowel. METHODS: Mechanical and chemical sensitivity of human bowel afferents were examined by ex vivo afferent nerve recordings. Age-related changes in the density of mast cells, EC cells, sensory nerve terminals, and mast cell-nerve micro-anatomical association were investigated by histological and immune staining. KEY RESULTS: Human afferents could be broadly classified into subpopulations displaying mechanical and chemical sensitivity, adaptation, chemo-sensitization, and recruitment. Interestingly human bowel afferent nerve sensitivity was attenuated with age. The density of substance P-immunoreactive (SP-IR) nerve varicosities was also reduced with age. In contrast, the density of ileal and colonic mucosal mast cells was increased with age, as was ileal EC cell number. An increased proportion of mast cells was found in close apposition to SP-IR nerves. CONCLUSIONS & INFERENCES: Afferent sensitivity in human bowel was reduced with advancing age. Augmentation of mast cells and EC cell numbers and the mast cell-nerve association suggest a compensatory mechanism for sensory neurodegeneration.

Translational potential of a mouse in vitro bioassay in predicting gastrointestinal adverse drug reactions in Phase I clinical trials.

BACKGROUND: Motility-related gastrointestinal (GI) adverse drug reactions (GADRs) such as diarrhea and constipation are a common and deleterious feature associated with drug development. Novel biomarkers of GI function are therefore required to aid decision making on the GI liability of compounds in development. METHODS: Fifteen compounds associated with or without clinical GADRs were used to assess the ability of an in vitro colonic motility bioassay to predict motility-related GADRs. Compounds were examined in a blinded fashion for their effects on mouse colonic peristaltic motor complexes in vitro. For each compound concentration-response relationships were determined and the results compared to clinical data. Compounds were also assessed using GI transit measurements obtained using an in vivo rat charcoal meal model. KEY RESULTS: Within a clinically relevant dosing range, the in vitro assay identified five true and three false positives, four true and three false negatives, which gave a predictive capacity of 60%. The in vivo assay detected four true and four false positives, four false and three true negatives, giving rise to a predictive capacity for this model of 47%. CONCLUSIONS & INFERENCES: Overall these results imply that both assays are poor predictors of GADRs. Further analysis would benefit from a larger compound set, but the data show a clear need for improved models for use in safety pharmacology assessment of GI motility.

Curcumin acts via transient receptor potential vanilloid-1 receptors to inhibit gut nociception and reverses visceral hyperalgesia.

BACKGROUND: An antinociceptive effect has been reported for curcumin in animal models and in humans, but the molecular mechanisms of curcumin's effect remain undefined. In this study, we explored the possibility that curcumin inhibit visceral nociception via antagonizing the transient receptor potential vanilloid-1 (TRPV1) receptor. METHODS: The effects of curcumin were explored using two experimental models: viscero-motor response (VMR) to colorectal distension (CRD) in rats and jejunal afferent firing in the ex vivo mouse jejunum preparations [TRPV1 knockout (KO) and wild-type mice, naive and trinitrobenzene sulfonic acid (TNBS)-treated Kunming mice]. In addition, capsaicin-induced calcium transients and whole-cell currents were examined in acutely dissociated dorsal root ganglia (DRG) neurons. KEY RESULTS: In the anesthetized rat, curcumin (4 mg kg(-1)  min(-1) for 3 min) caused a marked and rapidly reversible inhibition of CRD-induced VMRs. In the mouse jejunum, the mesenteric afferent nerve response to ramp distension was attenuated by curcumin (3, 10 µmol L(-1) ), an effect that was significantly reduced in TRPV1 KO mice compared with wild-type (WT) controls. Moreover, in WT mice, curcumin (1-30 µmol L(-1) ) was found to inhibit the afferent responses to capsaicin in a concentration-dependent manner. Trinitrobenzene sulfonic acid-induced hypersensitivity of jejunal afferents was also attenuated by curcumin. Curcumin potently inhibited capsaicin-induced rise in intracellular calcium and inward currents in mouse or rat DRG neurons. CONCLUSIONS & INFERENCES: Our results provide strong evidence that curcumin inhibit visceral nociception via antagonizing TRPV1 and may be a promising lead for the treatment of functional gastrointestinal diseases.

Endocannabinoid modulation of jejunal afferent responses to LPS.

BACKGROUND: Endocannabinoids influence immune function and nociceptive signaling. This study examines cannabinoid modulation of sensory signaling from the GI tract following an acute inflammatory response triggered by systemic administration of bacterial lipopolysaccharide (LPS). METHODS: A segment of proximal jejunum was intubated, to measure intraluminal pressure, in anesthetized rats. Afferent impulse traffic was recorded from a single isolated paravascular nerve bundle supplying the jejunal loop. Drugs and LPS were administered intravenously and changes in afferent firing were determined. KEY RESULTS: The non-selective cannabinoid agonist, WIN55,212-2 (1 mg kg(-1) i.v.) and the anandamide transport inhibitor, VDM11 (1 mg kg(-1) i.v.) but not the fatty acid amide hydrolase (FAAH) inhibitor, URB597 (0.3 mg kg(-1)) caused a significant increase in afferent activity. The WIN55,212-2-induced afferent response was mediated by activation of CB(1) receptors whereas the VDM11 response was mediated by both CB(1) and CB(2) receptor mechanisms. LPS (10 mg kg(-1)) evoked an increase in afferent activity which was significantly reduced in the presence of WIN55,212-2 and VDM11 but not URB597. The inhibitory effect of WIN55,212-2 was prevented by CB(1) but not CB(2) receptor antagonism. In contrast, the inhibitory effect of VDM11 remained unaltered after CB(1) or CB(2) receptor blockade. CONCLUSIONS & INFERENCES: Endocannabinoids play a role in modulating afferent signaling and may represent a target for the treatment of visceral hypersensitivity. In contrast to the effects of blocking endocannabinoid uptake (VDM11), inhibiting breakdown of endocannabinoids (URB597) had no effect on baseline or LPS induced afferent firing. Therefore, uptake of cannabinoids rather than breakdown via FAAH terminates their action in the GI tract.

Neurogenic adaptation contributes to the afferent response to mechanical stimulation.

This study aimed to characterize the effect of mechanical stimuli on mesenteric afferent nerve signaling in the isolated rat jejunum in vitro. This was done to determine the effect of mechanical stresses and strains relative to nonmechanical parameters (neurogenic adaptation). Mechanical stimulations were applied to a segment of jejunum from 15 rats using ramp distension with water at three rates of distension, a relaxation test (volume maintained constant from initial pressure of 20 or 40 mmHg), and a creep test (pressure maintained constant). Circumferential stress and strain and the spike rate increase ratio were calculated for evaluation of afferent nerve activity during the mechanical stimulations. Ramp distension evoked two distinct phases of afferent nerve signaling as a function of circumferential stress or strain. Changing the volume distension rate did not change the stress-strain relationship, but faster distension rate increased the afferent firing rate (P < 0.05). In the stress relaxation test, the spike rate declined faster and to a greater extent than the stress. In the creep test, the spike rate declined, despite a small increase in the strain. Three classes of mechanosensitive single-afferent units (low, wide dynamic range, and high threshold units) showed different response profiles against stress and strain. Low-threshold units exhibited a near linear relationship against the strain (R(2) = 0.8095), whereas high-threshold units exhibited a linear profile against the stress (R(2) = 0.9642). The afferent response is sensitive to the distension speed and to the stress and strain level during distension. However, the afferent nerve response is not a simple function of either stress or strain. Nonmechanical time-dependent adaptive responses other than those related to viscoelasticity also play a role.

The differential effect of CB1 receptors on the discharge of afferent and efferent fibres supplying the rat jejunum.

BACKGROUND: The cannabinoid receptor (CB(1) ) is expressed on GI sensory neurons and is suggested to play a role in food intake, inflammation and nociception. Expression of CB(1) in the nodose is influenced by nutritional status. Our aim was to determine the functional response of afferent and efferent fibres supplying the proximal jejunum to the CB(1) agonist docosatetraenylethanolamide (DEA) in fed and fasted animals. METHODS: Experiments were performed on anesthetized rats, either fed ad libitum or fasted for 24 h. Blood pressure was recorded via the carotid artery and the proximal jejunum intubated to measure intraluminal pressure. A single paravascular nerve bundle was isolated and attached to an electrode for recording either afferent or efferent impulse traffic. KEY RESULTS: Docosatetraenylethanolamide (1 mg kg(-1) , i.v.) had a depressor effect on blood pressure but surprisingly had little effect on afferent nerve activity in fed animals. In fasted animals the afferent response to DEA was augmented, however, the blood pressure effect was attenuated. In contrast, DEA caused a significant and prolonged increase in efferent firing, which was diminished in fasted animals. Bilateral cervical vagotomy had no effect on the DEA-mediated efferent response, however, hexamethonium (10 mg kg(-1) ) abolished excitation and unmasked an inhibitory effect of DEA. CONCLUSIONS & INFERENCES: Docosatetraenylethanolamide has only a modest effect on intestinal afferent firing but a profound effect on efferent function, which is modulated by changes in nutritional status. The persistent response after vagotomy and block by hexamethonium suggests DEA is acting centrally, although there may be an inhibitory effect at the level of the postganglionic sympathetic neuron.

Signaling mechanisms involved in the intestinal pro-secretory actions of hydrogen sulfide.

BACKGROUND: H(2) S actions in the gut involve neural activation. This study aimed to reveal the signaling mechanisms responsible for the pro-secretory effect of H(2) S by using TRPV1 and unselective TRP blockers and inhibitors of other signaling cascades hitherto described to be targeted by H(2) S elsewhere. METHODS: Ussing chamber voltage clamp technique was used to study actions of the H(2) S donor NaHS on secretion in guinea-pig and human colon. NaHS effects on guinea-pig primary afferents were also evaluated. KEY RESULTS: NaHS evoked secretion was significantly reduced in guinea-pig and human tissue by the selective TRPV1 blockers capsazepine, AMG9801, SB705498, BCTC; LY294002 (Phosphatidylinositol-3 kinase (PI3K) inhibitor), SKF96365 (store operated calcium channel blocker), 2-APB (inositol triphosphate blocker), and atropine but not by HC030031 (TRPA1 blocker) or L- and T-type calcium channel antagonists. Actions of TRPV1 antagonists suggested non-competitive inhibition at multiple sites. In guinea-pig colon, Gd(3+) and La(3+) (unselective TRP blockers) had no effects while ruthenium red reduced NaHS effects; in human colon Gd(3+) attenuated NaHS response. NaHS response was inhibited by neurokinin-1 and -3 receptor blockers in guinea-pig and neurokinin-1 and -2 receptor blockade in human tissue. There was cross-desensitization between NaHS and capsaicin responses. NaHS induced capsazepine and LY294002 sensitive afferent discharge. CONCLUSIONS & INFERENCES: H(2) S evokes mucosal secretion by targeting TRPV1 expressing afferent nerves which activate cholinergic secretomotor neurons via release of substance P acting in a species dependent manner on neurokinin-1, -2 or -3 receptors. Besides TRPV1 signaling H(2) S may target intracellular calcium dependent pathways and PI3K.

Differential afferent sensitivity to mucosal lipopolysaccharide from Salmonella typhimurium and Escherichia coli in the rat jejunum.

Postinfectious irritable bowel syndrome may develop subsequent to acute bacterial enteritis. We therefore hypothesized that intestinal afferents may develop hypersensitivity upon exposure to luminal lipopolysaccharide (LPS) from pathogens but not from commensal bacteria and that this may be prostaglandin mediated. Extracellular recordings of jejunal afferents were obtained in vivo from male Wistar rats (n = 5 per group; 300-400 g). Lipopolysaccharide from Escherichia coli (E-LPS), Salmonella typhimurium (S-LPS) or vehicle were infused into the intestinal lumen at 5 mg mL(-1). The selective 5-HT(3)-receptor agonist 2-methyl-5-HT (2m5-HT, 15 microgkg(-1), i.v.) was administered at 15-min intervals before and up to 2 h after S-LPS administration. Intraluminal E-LPS had no effect on mesenteric afferent nerve discharge at baseline. By contrast, afferent discharge increased from 21.7 +/- 0.3 impsec(-1) to 28.8 +/- 3.4 impsec(-1) 40 min after S-LPS administration (mean +/- SEM; P < 0.05) and reached 38.8 +/- 4.1 impsec(-1) after 2 h (P < 0.05). The afferent response to 2m5-HT was enhanced 30 min following S-LPS by 30.9 +/- 3.9% (P < 0.05) and remained elevated thereafter. The increase in baseline discharge and sensitivity to 2m5-HT following S-LPS was prevented by pretreatment with naproxen (COX inhibitor, 10 mgkg(-1) i.v.) or AH-6809 (EP1/EP2 receptor antagonist, 1 mg kg(-1)). Intestinal afferents do not alter their discharge rate to LPS from E. coli but to LPS from the pathogenic bacterium S. typhimurium. The latter response entails afferent sensitisation to 2m5-HT that depends on prostanoid release. This acute sensitisation may prime the intestinal afferent innervation for a later development of persistent hypersensitivity.

Neuronal activation in the nucleus of the solitary tract following jejunal lipopolysaccharide in the rat.

INTRODUCTION: Inflammation during systemic lipopolysaccharide (LPS) seems to be modulated by the CNS via afferent and efferent vagal pathways. We hypothesized that similar to systemic inflammation, local LPS in the gut lumen may also activate central neurons and aimed to identify potential molecular mechanisms. METHODS: Male Wistar rats were equipped with an exteriorized canula in the proximal jejunum. LPS or vehicle were administered into the jejunum (10 mg ml(-1)). For further study of molecular mechanisms, LPS or vehicle were administered systemically (1 mg kg(-1)). Brain stem activation was quantified by Fos-immunohistochemistry in the vagal nucleus of the solitary tract (NTS) and the Area postrema which is exposed to systemic circulation. Serum LPS concentrations were also determined. RESULTS: Jejunal LPS exposure entailed 91+/-12 (n=7) Fos-positive neurons in the NTS compared to 39+/-9 in controls (n=6; p<0.01), while serum LPS concentrations and Fos-positive neurons in the Area postrema were not different. Systemic LPS triggered 150+/-25 (n=6) and vehicle 52+/-6 Fos-positive neurons (n=7; p<0.01). The Fos count after systemic LPS was reduced to 99+/-30 following pretreatment with the cyclooxygenase inhibitor Naproxen (10 mg kg(-1); p>0.05 versus vehicle controls) and increased to 242+/-66 following the iNOS-inhibitor Aminoguanidine (15 mg kg(-1); p<0.01). In the Area postrema, 97+/-17 (n=6) neurons were counted in animals pretreated with systemic LPS compared to 14+/-4 in controls (n=7, p<0.001). CONCLUSIONS: Central neuronal activation following inflammation after systemic LPS is modulated by cyclooxygenase and NO pathways. Local exposure to bacterial LPS in the gut lumen activates the NTS which may set the stage for efferent vagal modulation of intestinal inflammation.

Purinergic contribution to small intestinal afferent hypersensitivity in a murine model of postinfectious bowel disease.

Increased sensitivity of the afferent innervation of the gastrointestinal tract reportedly underlies symptoms of discomfort and pain in functional bowel disorders. The present investigation aimed to examine whether the purinergic P2X(2) and P2X(3) receptor subunits contribute to the mechanosensitivity of small intestinal afferents in normal mice and in a murine model of postinfectious gut dysfunction. Mesenteric afferent nerve activity was recorded in a mouse jejunum preparation maintained in vitro. As has been shown previously, ramp distension of the jejunal segment evoked biphasic afferent discharge, reflecting activation of low and high threshold fibres. The average pressure-afferent response curve in mice deficient in both P2X(2) and P2X(3) subunits (n = 14) was not significantly different from that of the wild-type control preparations (n = 13). Application of pyridoxal 5-phosphate 6-azophenyl-2 ,4-disulphonic acid (PPADS) (30 micromol L(-1)), a P2X and P2Y antagonist, or 2,4,6-trinitrophenol-adenosine 5'-triphosphate (10 micromol L(-1)), an antagonist selective for homomeric P2X(3) and heteromeric P2X(2/3) receptors, had no effect on the averaged pressure-afferent response curve in wild-type animals. In Trichinella spiralis-infected mice, the magnitude of mesenteric afferent responses to jejunal distension was greater at day 21 and day 56 postinfection compared with the sham control preparations demonstrating the development of afferent hypersensitivity. PPADS had no significant effect upon mechanically evoked afferent discharge rates in sham treated preparations (n = 5), but significantly inhibited afferent sensitivity to jejunal distension in preparations from mice at day 21 (n = 6) and day 56 (n = 7) postinfection. These results suggest that purinergic mechanisms play no role in mechanosensory transduction in the normal small intestine but contribute significantly to postinfectious mechano-hypersensitivity.

Impairment of rectal afferent mechanosensitivity in experimental diabetes in the rat.

Diabetes mellitus results in neuropathy of both somatic and visceral nerves. In diabetic patients with faecal incontinence, impaired rectal sensory function, manifested by a decreased sensitivity to balloon distention is common. This may contribute to unawareness of rectal filling and incontinence. There has been little study to date of visceral mechanosensation in experimental diabetes however. We hypothesized that experimental diabetes would impair mechanosensitivity in rectal afferent nerves. Diabetes was induced in rats by i.p. injection of streptozotocin. Controls were injected with citrate. In vitro recordings were performed from rectal afferent nerves innervating isolated segments of rectum. In control animals, three distinct populations of mechanosensitive fibres were identified. Low threshold fibres responded at low intensity stretch and reached a maximal firing rate at less than 10 g of stretch (11/24 units). Wide dynamic sensitivity units responded at low intensity stretch (<2 g) but encoded stimulus intensity in a linear fashion up to 20 g (12/24 units). High threshold units responded at greater than 5 g. In diabetic animals there was a near complete loss of LT units (1/19) and most (16/29) had properties similar to WD units. However, their response threshold was significantly increased. Firing rates in response to maximal distention did not change in diabetic animals. We conclude that experimental diabetes selectively affects the detection of low threshold 'physiologic' rectal distention, such as that which might occur during rectal filling, prior to defaecation.

The gaseous mediator, hydrogen sulphide, inhibits in vitro motor patterns in the human, rat and mouse colon and jejunum.

Hydrogen sulphide (H2S) has been recently proposed as a transmitter in the brain and peripheral tissues. Its role in the gastrointestinal tract is still unknown despite some data which suggest an involvement mediating smooth muscle relaxation. The aim of this study was to investigate the effect of this gas on intestinal segments from mouse jejunum and colon, and muscular strips from the human and rat colon. In isolated segments of mouse colon and jejunum, bath applied sodium hydrogen sulphide (NaHS) (a H2S donor) caused a concentration-dependent inhibition of spontaneous motor complexes (MCs) (IC(50) 121 micromol L(-1) in the colon and 150 micromol L(-1) in the jejunum). This inhibitory effect of NaHS on MCs was (i) unaffected by tetrodotoxin (TTX), capsaicin, pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate and N-nitro-L-arginine suggesting a non-neural effect and (ii) significantly reduced by apamin 3 micromol L(-1). NaHS concentration-dependently inhibited the spontaneous motility in strips from human colon (IC(50) 261 micromol L(-1)) and rat colon (IC(50) 31 micromol L(-1)). The inhibitory effect of NaHS on colonic strips was (i) unaffected by the neural blocker TTX (1 micromol L(-1)) with IC(50) 183 micromol L(-1) for the human colon and of 26 micromol L(-1) for the rat colon and (ii) significantly reduced by glybenclamide (10 micromol L(-1)), apamin (3 micromol L(-1)) and TEA (10 mmol L(-1)) with IC(50) values of 2464, 1307 and 2421 micromol L(-1) for human strips, and 80, 167 and 674 micromol L(-1) for rat strips respectively. We conclude that H2S strongly inhibits in vitro intestinal and colonic motor patterns. This effect appears to be critically dependent on K channels particularly apamin-sensitive SK channels and glybenclamide-sensitive K (ATP) channels.

5-HT system in the gut: roles in the regulation of visceral sensitivity and motor functions.

5-Hydroxytryptamine (5-HT) is a major transmitter molecule within the gastrointestinal tract. It is contained in enterochromaffin (EC) cells, which form part of the epithelial lining of the gut and in enteric neurones in the submucosal and myenteric plexuses. 5-HT is present in murine mucosal mast cells in the lamina propria and some studies have suggested that human mast cells may also contain 5-HT especially in conditions associated with mastocytosis. The strategic positioning of the enteric and extrinsic sensory innervation in close proximity to these sources of 5-HT, in conjunction with their demonstrated sensitivity to this mediator, suggests the involvement of 5-HT in the transduction of visceral stimuli and reflex responses affecting motor and secretory function. Under physiological conditions, the release of 5-HT from these storage sites may result in the orchestration of reflexes responsible for transit of material along the bowel at a rate that is appropriate for digestion and absorption of nutrients. However, in the pathophysiological state, 5-HT acting together with other inflammatory mediators may cause inappropriate intestinal secretomotor activity and/or initiate sensations such as nausea or discomfort/pain. Current evidence suggests that the bioavailability of 5-HT within the gut wall is altered in a number of post-inflammatory models of gut dysfunction with increased numbers of EC cells and mast cells with increased 5-HT content in proximity to sensory nerve endings, and decreased serotonin reuptake mechanisms. Changes may also occur in the sensory innervation or pathways within the central nervous system. These processes may contribute to pain mechanisms in the irritable bowel syndrome, in which visceral hypersensitivity is a predominant feature and may also contribute to motor dysfunction leading to altered bowel habit.

An educational and audit tool to reduce prescribing error in intensive care.

AIM: To reduce prescribing errors in an intensive care unit by providing prescriber education in tutorials, ward-based teaching and feedback in 3-monthly cycles with each new group of trainee medical staff. METHODS: Prescribing audits were conducted three times in each 3-month cycle, once pretraining, once post-training and a final audit after 6 weeks. The audit information was fed back to prescribers with their correct prescribing rates, rates for individual error types and total error rates together with anonymised information about other prescribers' error rates. RESULTS: The percentage of prescriptions with errors decreased over each 3-month cycle (pretraining 25%, 19%, (one missing data point), post-training 23%, 6%, 11%, final audit 7%, 3%, 5% (p<0.0005)). The total number of prescriptions and error rates varied widely between trainees (data collection one; cycle two: range of prescriptions written: 1-61, median 18; error rate: 0-100%; median: 15%). CONCLUSION: Prescriber education and feedback reduce manual prescribing errors in intensive care.

Influence of the pattern of jejunal distension on mesenteric afferent sensitivity in the anaesthetized rat.

Vagal, spinal and intestino-fugal fibres all potentially transmit mechanosensory afferent information from the gastrointestinal tract. We aimed to characterize the relative mechanosensitivity of these three different afferent populations supplying the rat jejunum. Afferent nerve discharge was recorded from pentobarbitone-anaesthetized rats during different distension protocols. Saline ramp distension (1 mL min(-1)) and barostat ramp distension (2 mmHg 4 s(-1)) each evoked biphasic responses but with the latter significantly attenuated especially at low distending pressures. Barostat controlled phasic distensions (10-50 mmHg, 25 s) evoked an afferent response with a peak at the onset of distension adapting to a plateau level that was maintained and comparable to the barostat ramp responses at the corresponding pressures. Chronic subdiaphragmatic vagotomy significantly attenuated the low pressure component of the response to balloon ramp distension and both peak and plateau responses to phasic distension. Single unit analysis showed an absence of low threshold afferent activity after vagotomy while the response to fibres with wide-dynamic range and high threshold sensitivity were preserved hexamethonium had no effect on the responses to either ramp or phasic distension. These findings suggest that the nature of the distension stimulus is critical in determining the pattern of response observed from the various subpopulations of afferents supplying the bowel wall.

Intestinal secretory and absorptive function in Trichinella spiralis mouse model of postinfective gut dysfunction: role of bile acids.

OBJECTIVE: Observations showing that bile acid malabsorption is frequent in irritable bowel syndrome (IBS) suggest that alterations in bile acid-induced secretion and absorption could contribute to IBS-associated diarrhoea. The secretory response to bile acids, fluid transport and bile absorption was examined in intestinal tissues from a Trichinella spiralis mouse model of postinfectious gut dysfunction in vitro. Changes in the protein expression of apical sodium-dependent bile acid transporter (ASBT) were also measured. DESIGN: T. spiralis-infected mice were killed at 18 and 25 days postinfection. Jejunal, ileal, proximal and distal colon segments were exposed to taurodeoxycholic acid (TDCA) or cholic acid. Short circuit current (SCC) increases were determined. Tritiated taurocholic acid (3H-TCA) absorption was determined in everted jejunal and ileal sacs. ASBT protein expression was determined by Western blot analysis and immunohistochemistry. RESULTS: Basal SCC increased in ileum and distal colon at 18 and 25 days postinfection, respectively. Ileal SCC responses to TDCA and cholic acid were enhanced at 18 days postinfection. Distal colon SCC response to TDCA was raised at 18 days postinfection but was significantly reduced by 25 days. Ileal 3H-TCA uptake was significantly reduced at 18 and 25 days postinfection. Surprisingly, increased ASBT expression was observed in infected animals. CONCLUSIONS: In a T. spiralis model of postinfectious gut dysfunction, decreased bile absorption and enhanced secretion in response to bile acids was observed. Decreased absorption was not, however, caused by decreased ASBT as increased expression was observed. If similar events occur postinfection, the combined effects of these disturbances may contribute to some symptoms observed in postinfectious IBS patients.

Bladder afferent sensitivity in wild-type and TRPV1 knockout mice.

Understanding bladder afferent pathways may reveal novel targets for therapy of lower urinary tract disorders such as overactive bladder syndrome and cystitis. Several potential candidate molecules have been postulated as playing a significant role in bladder function. One such candidate is the transient receptor potential vanilloid 1 (TRPV1) ion channel. Mice lacking the TRPV1 channel have altered micturition thresholds suggesting that TRPV1 channels may play a role in the detection of bladder filling. The aim of this study was therefore to investigate the role of TRPV1 receptors in controlling bladder afferent sensitivity in the mouse using pharmacological receptor blockade and genetic deletion of the channel. Multiunit afferent activity was recorded in vitro from bladder afferents taken from wild-type (TRPV+/+) mice and knockout (TRPV1-/-) mice. In wild-type preparations, ramp distension of the bladder to a maximal pressure of 40 mmHg produced a graded increase in afferent activity. Bath application of the TRPV1 antagonist capsazepine (10 mum) caused a significant attenuation of afferent discharge in TRPV1+/+ mice. Afferent responses to distension were significantly attenuated in TRPV1-/- mice in which sensitivity to intravesical hydrochloric acid (50 mm) and capsaicin (10 microm) were also blunted. Altered mechanosensitivity occurred in the absence of any changes in the pressure-volume relationship during filling indicating that this was not secondary to a change in bladder compliance. Single-unit analysis was used to classify individual afferents into low-threshold and high-threshold fibres. Low threshold afferent responses were attenuated in TRPV1-/- mice compared to the TRPV1+/+ littermates while surprisingly high threshold afferent sensitivity was unchanged. While TRPV1 channels are not considered to be mechanically gated, the present study demonstrates a clear role for TRPV1 in the excitability of particularly low threshold bladder afferents. This suggests that TRPV1 may play an important role in normal bladder function.

Impact of 5-HT3 receptor blockade on colonic haemodynamic responses to ischaemia and reperfusion in the rat.

5-HT(3) receptor antagonists are clinically available for treating patients with irritable bowel syndrome (IBS) but their use is restricted because of a link with some episodes of ischaemic colitis. However, the role of 5-HT3 receptors in regulating colonic blood flow has not been systematically investigated. Thus, we examined acute and chronic treatment with alosetron, a potent and selective antagonist of the 5-HT3 receptor, on baseline colonic blood flow and haemodynamic responses during occlusion and reactive hyperaemia in the pentobarbitone-anaesthetized rat. Colonic haemodynamics were assessed using ultrasonic recordings of superior mesenteric blood flow (MBF) and laser Doppler recordings of colonic vascular perfusion (VP). Blood pressure (BP) was also monitored and in some experiments tissue oxygen was detected polarographically. Alosetron (10, 30, 100 microg kg(-1), i.v.) had no effect on baseline haemodynamics nor responses to nitric oxide synthase inhibition with N(omega)-nitro-l-arginine methyl ester (l-NAME) (16 mg kg(-1)). Arterial occlusion (5 min) reduced MBF (-98.6 +/- 0.6%) and VP (-70.7 +/- 5.4%) followed by a post-occlusion reactive hyperaemia (MBF = +94.5 +/- 19.1%; VP = +60.0 +/- 22.3%) the magnitude of which was unchanged following acute (30 microg kg(-1)) or chronic alosetron administration (0.5 mg kg(-1) twice daily, 5 days). Alosetron did not significantly alter baseline colonic blood flow in the anaesthetized rat; nor did it interfere with vascular control mechanisms activated during occlusion and reactive hyperaemia.

Mechanisms underlying mechanosensitivity of mesenteric afferent fibers to vascular flow.

Spinal afferent neurons, with endings in the intestinal mesenteries, have been shown to respond to changes in vascular perfusion rates. The mechanisms underlying this sensitivity were investigated in an in vitro preparation of the mesenteric fan devoid of connections with the gut wall. Afferent discharge increased when vascular perfusion was stopped ("flow off"), a response localized to the terminal vessels just prior to where they entered the gut wall. The flow-off response was compared following pharmacological manipulations designed to determine direct mechanical activation from indirect mechanisms via the vascular endothelium or muscle. Under Ca(2+)-free conditions, responses to flow off were significantly augmented. In contrast, the myosin light chain kinase inhibitor wortmannin (1 microM, 20 min) did not affect the flow-off response despite blocking the vasoconstriction evoked by 10 microM l-phenylephrine. This ruled out active tension, generated by vascular smooth muscle, in the response to flow off. Passive changes caused by vessel collapse during flow off were speculated to affect sensory nerve terminals directly. The flow-off response was not affected by the N-, P-, and Q-type Ca(2+) channel blocker omega-conotoxin MVIIC (1 muM intra-arterially) or the P2X receptor/ion channel blocker PPADS (50 microM). However, ruthenium red (50 microM), a blocker of nonselective cation channels, greatly reduced the flow-off response and also abolished the vasodilator response to capsaicin. Our data support the concept that mesenteric afferents sense changes in vascular flow during flow off through direct mechanisms, possibly involving nonselective cation channels. Passive distortion in the fan, caused by changes in blood flow, may represent a natural stimulus for these afferents in vivo.