Wendy J. Winchester

Jejunal afferent nerve sensitivity in wild-type and TRPV1 knockout mice

The aim of this study was to investigate the contribution of the TRPV1 receptor to jejunal afferent sensitivity in the murine intestine. Multiunit activity was recorded in vitro from mesenteric afferents supplying segments of mouse jejunum taken from wild‐type (WT) and TRPV1 knockout (TRPV1−/−) animals. In WT preparations, ramp distension of the gut (up to 60 mmHg) produced biphasic changes in afferent activity so the pressure–response curve had an initial rapid increase in afferent discharge followed by a second phase of slower increase in activity. Afferent response to distension was significantly lower in TRPV1−/− than in WT mice. Single‐unit analysis revealed three functional types of afferent fibres: (1) low‐threshold fibres (2) wide dynamic range fibres and (3) high‐threshold fibres. There was a marked downward shift of the pressure–response curve for wide dynamic range fibres in the TRPV1−/− mice as compared to the WT controls. The afferent response to intraluminal hydrochloric acid (20 mm) was also attenuated in the TRPV1−/− mice. In contrast, the response to bath application of bradykinin (1 μm, 3 ml) was not significantly different between the two groups. The TRPV1 antagonist capsazepine (10 μm) significantly attenuated the nerve responses to distension, intraluminal acid and bradykinin, as well as the spontaneous discharge in WT mice. The WT jejunal afferents responded to capsaicin with rapid increases in afferent activity, whereas TRPV1−/− afferents were not at all sensitive to capsaicin. Previous evidence indicates that TRPV1 is not mechanosensitive, so the results of the present study suggest that activation of TRPV1 may sensitize small intestinal afferent neurones.

Regional brain activation in conscious, nonrestrained rats in response to noxious visceral stimulation.

&NA; Preclinical drug development for visceral pain has largely relied on quantifying pseudoaffective responses to colorectal distension (CRD) in restrained rodents. However, the predictive value of changes in simple reflex responses in rodents for the complex human pain experience is not known. Male rats were implanted with venous cannulas and with telemetry transmitters for abdominal electromyographic (EMG) recordings. [14C]‐iodoantipyrine was injected during noxious CRD (60 mmHg) in the awake, nonrestrained animal. Regional cerebral blood flow (rCBF)‐related tissue radioactivity was quantified by autoradiography and analyzed in the three‐dimensionally reconstructed brain by statistical parametric mapping. 60‐mmHg CRD, compared with controls (0 mmHg) evoked significant increases in EMG activity (267 ± 24% vs. 103 ± 8%), as well as in behavioral pain score (77 ± 6% vs. 3 ± 3%). CRD elicited significant increases in rCBF as expected in sensory (insula, somatosensory cortex), and limbic and paralimbic regions (including anterior cingulate cortex and amygdala). Significant decreases in rCBF were seen in the thalamus, parabrachial nucleus, periaqueductal gray, hypothalamus and pons. Correlations of rCBF with EMG and with behavioral pain score were noted in the cingulate, insula, lateral amygdala, dorsal striatum, somatosensory and motor regions. Our findings support the validity of measurements of cerebral perfusion during CRD in the freely moving rat as a model of functional brain changes in human visceral pain. However, not all regions demonstrating significant group differences correlated with EMG or behavioral measures. This suggests that functional brain imaging captures more extensive responses of the central nervous system to noxious visceral distension than those identified by traditional measures.

Dual role of 5-HT3 receptors in a rat model of delayed stress-induced visceral hyperalgesia.

Abstract Despite its beneficial effect in IBS patients, the mechanism of action of the 5‐HT3 receptor (5‐HT3R) antagonist alosetron is still incompletely understood. We aimed to characterize the effect and site(s) of action in a model of stress‐induced sensitization of visceral nociception in rats. Adult male Wistar rats were equipped for recording of visceromotor response (VMR) to phasic colorectal distension (CRD; 10–60 mm Hg). VMR to CRD was recorded 24 h after an acute session of water avoidance (WA) stress (post‐WA). Baseline and post‐WA responses were measured in rats exposed to WA or sham‐WA, treated with alosetron at 0.3 mg/kg subcutaneously (s.c.) 25 nmol intrathecally (i.t.) or vehicle before post‐WA CRD. Some rats were treated with capsaicin/vehicle on the cervical vagus nerve and received alosetron (0.3 mg/kg, s.c.) 15 min before post‐WA CRD. WA stress led to visceral hyperalgesia 24 h later. Alosetron (0.3 mg/kg, s.c.), failed to inhibit WA‐induced exacerbation of VMR to CRD. Stress‐induced visceral hyperalgesia was abolished when alosetron was injected intrathecally (P < 0.05) in intact rats or subcutaneously (0.3 mg/kg) in capsaicin‐pretreated animals (P < 0.05). Capsaicin‐pretreatment did not affect the exacerbating effect of stress on visceral sensitivity. Alosetron had no inhibitory effect on normal visceral pain responses when administered subcutaneously or intrathecally. We demonstrated that 5‐HT3Rs on central terminals of spinal afferents are engaged in the facilitatory effect of stress on visceral sensory information processing. In addition, we showed that stress‐induced sensitization of visceral nociception is independent of 5‐HT3R activation on vagal afferents.

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

Abstract  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 pleateau 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.

Sex differences in functional brain activation during noxious visceral stimulation in rats.

ABSTRACT Studies in healthy human subjects and patients with irritable bowel syndrome suggest sex differences in cerebral nociceptive processing. Here we examine sex differences in functional brain activation in the rat during colorectal distention (CRD), a preclinical model of acute visceral pain. [14C]‐iodoantipyrine was injected intravenously in awake, non‐restrained female rats during 60‐ or 0‐mmHg CRD while electromyographic abdominal activity (EMG) and pain behavior were recorded. Regional cerebral blood flow‐related tissue radioactivity was analyzed by statistical parametric mapping from autoradiographic images of three‐dimensionally reconstructed brains. Sex differences were addressed by comparing the current data with our previously published data collected from male rats. While sex differences in EMG and pain scores were modest, significant differences were noted in functional brain activation. Females showed widespread changes in limbic (amygdala, hypothalamus) and paralimbic structures (ventral striatum, nucleus accumbens, raphe), while males demonstrated broad cortical changes. Sex differences were apparent in the homeostatic afferent network (parabrachial nucleus, thalamus, insular and dorsal anterior cingulate cortices), in an emotional–arousal network (amygdala, locus coeruleus complex), and in cortical areas modulating these networks (prefrontal cortex). Greater activation of the ventromedial prefrontal cortex and broader limbic/paralimbic changes in females suggest greater engagement of affective mechanisms during visceral pain. Greater cortical activation in males is consistent with the concept of greater cortical inhibitory effects on limbic structures in males, which may relate to differences in attentional and cognitive attribution to visceral stimuli. These findings show remarkable similarities to reported sex differences in brain responses to visceral stimuli in humans.

Amitriptyline modifies the visceral hypersensitivity response to acute stress in the irritable bowel syndrome

Background  Acute physical stress causes alteration in gut autonomic function and visceral hypersensitivity in patients with irritable bowel syndrome (IBS). We have developed a model to measure this stress response.

UDP-glucose modulates gastric function through P2Y14 receptor-dependent and -independent mechanisms

P2Y receptors have been reported to modulate gastrointestinal functions. The newest family member is the nucleotide-sugar receptor P2Y14. P2ry14 mRNA was detected throughout the rat gut, with the highest level being in the forestomach. We investigated the role of the receptor in stomach motility using cognate agonists and knockout (KO) mice. In rat isolated forestomach, 100 microM UDP-glucose and 100 muM UDP-galactose both increased the baseline muscle tension (BMT) by 6.2+/-0.6 and 1.6+/-0.6 mN (P<0.05, n=3-4), respectively, and the amplitude of contractions during electrical field stimulation (EFS) by 3.7+/-1.7 and 4.3+/-2.5 mN (P<0.05, n=3-4), respectively. In forestomach from wild-type (WT) mice, 100 microM UDP-glucose increased the BMT by 1.0+/-0.1 mN (P<0.05, n=6) but this effect was lost in the KO mice (change of -0.1+/-0.1 mN, n=6). The 100 microM UDP-glucose also increased the contraction amplitude during EFS in this tissue from the WT animals (0.9+/-0.4 mN, P < 0.05, n=6) but not from the KO mice (0.0+/-0.2 mN, n=6). In vivo, UDP-glucose at 2,000 mg/kg ip reduced gastric emptying in rats by 49.7% (P<0.05, n=4-6) and in WT and KO mice by 56.1 and 66.2%, respectively (P<0.05, n=7-10) vs. saline-treated control animals. There was no significant difference in gastric emptying between WT and KO animals receiving either saline or d-glucose. These results demonstrate a novel function of the P2Y14 receptor associated with contractility in the rodent stomach that does not lead to altered gastric emptying after receptor deletion and an ability of UDP-glucose to delay gastric emptying without involving the P2Y14 receptor.

Spontaneous hypersensitivity in mesenteric afferent nerves of mice deficient in the sst2 subtype of somatostatin receptor

Somatostatin is an inhibitory peptide present in abundance in the gastrointestinal (GI) tract. The effects of somatostatin are mediated through its interaction with a family of G‐protein‐coupled receptors, namely sst1–5. Previous evidence suggested that the sst2 receptor mediates an inhibitory role of somatostatin on GI afferent nerve sensitivity. In the present study we further evaluated mechanical and chemical sensitivity of mesenteric afferents in mice deficient in the sst2 receptor. Multi‐unit recordings were made from mesenteric afferents from mouse jejunal segments perfused in vitro. Ramp distension of the jejunum up to 60 mmHg induced biphasic increases in afferent activity in both wild‐type (WT) and sst2 gene knock‐out (KO) mice. However, the level of afferent activity was significantly higher in the KO (n= 15) compared to the WT (n= 16) mice across the entire pressure range. The mesenteric afferent sensitivity to acid was evaluated by intraluminal infusion of hydrochloric acid (HCl 20 mm) for 2 min. Peak afferent discharge rate following acid infusion was significantly greater in KO (36.76 ± 6.47 impulses s−1, n= 7) than in WT preparations (16.53 ± 3.91 impulses s−1, n= 5, P < 0.01). The response to bath‐applied bradykinin (1 μm, 3 ml) was not significantly different in the KO and the WT preparations. It is interesting that in the WT preparations, octreotide inhibited both low‐ and high‐threshold mechanosensory responses, whereas in the sst2 KO group it appeared to inhibit the low‐threshold responses preferentially and failed to affect the high‐threshold responses. The results of the present investigation demonstrate that sst2 deficiency is associated with exaggerated jejunal afferent sensitivity to both mechanical and chemical stimulations, suggesting that somatostatin plays an important inhibitory role in the control of visceral sensitivity by interacting with the sst2 receptor.

Discovery of GSK345931A: An EP(1) receptor antagonist with efficacy in preclinical models of inflammatory pain.

Herein we describe the medicinal chemistry programme to identify a potential back-up compound to the EP(1) receptor antagonist GW848687X. This work started with the lipophilic 1,2-biaryl benzene derivative 4 which displayed molecular weight of 414.9g/mol and poor in vivo metabolic stability in the rat and resulted in the identification of compound 7i (GSK345931A) which demonstrated good metabolic stability in the rat and lower molecular weight (381.9g/mol). In addition, 7i (GSK345931A) showed measurable CNS penetration in the mouse and rat and potent analgesic efficacy in acute and sub-chronic models of inflammatory pain.

GSK962040: a small molecule motilin receptor agonist which increases gastrointestinal motility in conscious dogs

Background  GSK962040, a small molecule motilin receptor agonist, was identified to address the need for a safe, efficacious gastric prokinetic agent. However, as laboratory rodents lack a functional motilin system, studies in vivo have been limited to a single dose, which increased defecation in rabbits. Motilin agonists do not usually increase human colonic motility, so gastric prokinetic activity needs to be demonstrated.