David W. Adelson

Lack of interaction between peripheral injection of CCK and obestatin in the regulation of gastric satiety signaling in rodents.

Obestatin is a new peptide for which anorexigenic effects were recently reported in mice. We investigate whether peripheral injection of obestatin or co-injection with cholecystokinin (CCK) can modulate food intake, gastric motor function (intragastric pressure and emptying) and gastric vagal afferent activity in rodents. Obestatin (30, 100 and 300 microg/kg, i.p.) did not influence cumulative food intake for the 2h post-injection in rats or mice nor gastric emptying in rats. In rats, obestatin (300 microg/kg) did not modify CCK (1 microg/kg, i.p.)-induced significant decrease in food intake (36.6%) and gastric emptying (31.0%). Furthermore, while rats injected with CCK (0.3 microg/kg, i.v.) displayed gastric relaxation, no change in gastric intraluminal pressure was elicited by obestatin (300 microg/kg, i.v.) pre- or post-CCK administration. In in vitro rat gastric vagal afferent preparations, 20 units that had non-significant changes in basal activity after obestatin at 30 microg responded to CCK at 10 ng by a 182% increase. These data show that obestatin neither influences cumulative food intake, gastric motility or vagal afferent activity nor CCK-induced satiety signaling.

CRF2 receptor activation prevents colorectal distension induced visceral pain and spinal ERK1/2 phosphorylation in rats

Background and aims: Activation of corticotropin releasing factor 1 (CRF1) receptors is involved in stress related responses and visceral pain, while activation of CRF2 receptors dampens the endocrine and some behavioural stress responses. We hypothesised that CRF2 receptor activation may influence visceral pain induced by colorectal distension (CRD) in conscious rats, and assessed the possible sites and mechanisms of action. Methods: Male Sprague-Dawley rats were exposed to CRDs (60 mm Hg, 10 minutes twice, with a 10 minute rest interval). Visceromotor responses (VMR) were measured by electromyography or visual observation. Spinal (L6–S1) extracellular signal regulated kinase 1/2 (ERK 1/2) activation following in vivo CRD and CRF2 receptor gene expression in the T13–S1 dorsal root ganglia (DRG) and spinal cord were determined. Inferior splanchnic afferent (ISA) activity to CRD (0.4 ml, 20 seconds) was assessed by electrophysiological recording in an in vitro ISA nerve-inferior mesenteric artery (intra-arterial)-colorectal preparation. Results: In controls, VMR to the second CRD was mean 31 (SEM 4)% higher than that of the first (p<0.05). The selective CRF2 agonist, human urocortin 2 (hUcn 2, at 10 and 20 μg/kg), injected intravenous after the first distension, prevented sensitisation and reduced the second response by 8 (1)% and 30 (5)% (p<0.05) compared with the first response, respectively. RT-PCR detected CRF2 receptor gene expression in the DRG and spinal cord. CRD (60 mm Hg for 10 minutes) induced phosphorylation of ERK 1/2 in neurones of lumbosacral laminae I and IIo and the response was dampened by intravenous hUcn 2. CRD, in vitro, induced robust ISA spike activity that was dose dependently blunted by hUcn 2 (1–3 μg, intra-arterially). The CRF2 receptor antagonist, astressin2-B (200 μg/kg subcutaneously or 20 μg intra-arterially) blocked the hUcn 2 inhibitory effects in vivo and in vitro. Conclusions: Peripheral injection of hUcn 2 blunts CRD induced visceral pain, colonic afferent, and spinal L6-S1 ERK 1/2 activity through CRF2 receptor activation in rats.

Cortagine, a CRF1 agonist, induces stresslike alterations of colonic function and visceral hypersensitivity in rodents primarily through peripheral pathways.

Corticotropin-releasing factor (CRF) 1 receptor (CRF(1)) activation in the brain is a core pathway orchestrating the stress response. Anatomical data also support the existence of CRF signaling components within the colon. We investigated the colonic response to intraperitoneal (ip) injection of cortagine, a newly developed selective CRF(1) peptide agonist. Colonic motor function and visceral motor response (VMR) were monitored by using a modified miniaturized pressure transducer catheter in adult conscious male Sprague-Dawley rats and C57Bl/6 mice. Colonic permeability was monitored by the Evans blue method and myenteric neurons activation by Fos immunohistochemistry. Compared with vehicle, cortagine (10 microg/kg ip) significantly decreased the distal colonic transit time by 45% without affecting gastric transit, increased distal and transverse colonic contractility by 35.6 and 66.2%, respectively, and induced a 7.1-fold increase in defecation and watery diarrhea in 50% of rats during the first hour postinjection whereas intracerebroventricular (icv) cortagine (3 microg/rat) had lesser effects. Intraperitoneal (ip) cortagine also increased colonic permeability, activated proximal and distal colonic myenteric neurons, and induced visceral hypersensitivity to a second set of phasic colorectal distention (CRD). The CRF antagonist astressin (10 mug/kg ip) abolished ip cortagine-induced hyperalgesia whereas injected icv it had no effect. In mice, cortagine (30 microg/kg ip) stimulated defecation by 7.8-fold, induced 60% incidence of diarrhea, and increased VMR to CRD. Stresslike colonic alterations induced by ip cortagine in rats and mice through restricted activation of peripheral CRF(1) receptors support a role for peripheral CRF(1) signaling as the local arm of the colonic response to stress.

Inflammation-induced changes in primary afferent-evoked release of substance P within trigeminal ganglia in vivo

Substance P (SP) is synthesized in a subset of nociceptive sensory neurons and is released from their peripheral and central terminals. Here we demonstrate with the use of in vivo microdialysis and radioimmunoassay techniques that SP is also released within trigeminal ganglia following intraganglionic application of KCl, veratridine or capsaicin, and after electrical stimulation of peripheral afferent fibers. Both the basal and KCl-evoked release of SP are shown to be dependent on extracellular calcium. Using the turpentine-induced model of unilateral orofacial inflammation we also show that both the basal and KCl-evoked release of SP within trigeminal ganglia are greatly increased on the inflamed side 48 h after induction of inflammation. Coupled with previous demonstrations of excitatory effects of SP on sensory neurons, these results suggest that SP fulfils the role of a non-synaptically released diffusible chemical messenger that may modulate the somatic excitability of neurons within sensory ganglia in inflammatory pain states.

Chylomicron components activate duodenal vagal afferents via a cholecystokinin A receptor-mediated pathway to inhibit gastric motor function in the rat

Nutrients in the intestine initiate changes in secretory and motor function of the gastrointestinal (GI) tract. The nature of the ‘sensors’ in the intestinal wall is not well characterized. Intestinal lipid stimulates the release of cholecystokinin (CCK) from mucosal entero‐endocrine cells, and it is proposed that CCK activates CCK A receptors on vagal afferent nerve terminals. There is evidence that chylomicron components are involved in this lipid transduction pathway. The aim of the present study was to determine (1) the pathway mediating reflex inhibition of gastric motility and (2) activation of duodenal vagal afferents in response to chylomicrons. Mesenteric lymph was obtained from awake rats fitted with lymph fistulas during intestinal perfusion of lipid (Intralipid, 170 μmol h−1, chylous lymph) or a dextrose and/or electrolyte solution (control lymph). Inhibition of gastric motility was measured manometrically in urethane‐anaesthetized recipient rats in response to intra‐arterial injection of lymph close to the upper GI tract. Chylous lymph was significantly more potent than control lymph in inhibiting gastric motility. Functional vagal deafferentation by perineural capsaicin or CCK A receptor antagonist (devazepide, 1 mg kg−1, I.V.) significantly reduced chylous lymph‐induced inhibition of gastric motility. The discharge of duodenal vagal afferent fibres was recorded from the dorsal abdominal vagus nerve in an in vitro preparation of the duodenum. Duodenal vagal afferent nerve fibre discharge was significantly increased by close‐arterial injection of CCK (1‐100 pmol) in 43 of 83 units tested. The discharge of 88 % of CCK‐responsive fibres was increased by close‐arterial injection of chylous lymph; devazepide (100 μg, I.A.) abolished the afferent response to chylous lymph in 83 % of these units. These data suggest that in the intestinal mucosa, chylomicrons or their products release endogenous CCK which activates CCK A receptors on vagal afferent nerve fibre terminals, which in turn initiate a vago‐vagal reflex inhibition of gastric motor function.

Repeated psychological stress-induced alterations of visceral sensitivity and colonic motor functions in mice: Influence of surgery and postoperative single housing on visceromotor responses

Visceral pain modulation by chronic stress in mice has been little studied. Electromyography (EMG) recording of abdominal muscle contractions, as a proxy to the visceromotor response (VMR), requires electrode implantation and post-surgical single housing (SH) which could affect the VMR to stress. To test this hypothesis, male mice had electrode implantation surgery (S) plus SH, or no surgery and were group housed (NS-GH) or single housed (NS-SH) and exposed to either water avoidance stress (WAS, 1 h/day) or left undisturbed in their home cages for 10 days. The VMR to phasic ascending colorectal distension (CRD) was assessed before (basal) and 24 h after 10 days of WAS or no stress using a surgery-free method of intraluminal colonic pressure (ICP) recording (solid-state manometry). WAS heightened significantly the VMR to CRD at 30, 45, and 60 mmHg in S-SH vs. NS-GH, but not compared to NS-SH conscious mice. Compared to basal CRD, WAS increased VMR at 60 mmHg in the S-SH group and decreased it at 30–60 mmHg in NS-GH mice, while having no effect in NS-SH mice. The average defecation during the hour of repeated WAS over 10 days was 1.9 and 2.4 fold greater in S-SH vs. NS-GH and NS-SH mice, respectively. These data indicate that the combination of S-SH required for VMR monitoring with EMG is an important component of repeated WAS-induced post-stress visceral hypersensitivity and defecation in mice.

Intracisternal urocortin inhibits vagally stimulated gastric motility in rats: role of CRF2

Corticotropin‐releasing factor (CRF) acts in the brain to inhibit thyrotropin‐releasing hormone (TRH) analogue, RX‐77368‐induced vagal stimulation of gastric motility. We investigated CRF receptor‐mediated actions of rat urocortin (rUcn) injected intracisternally (ic) on gastric motor function. Urethane‐anaesthetized rats with strain gauges on the gastric corpus were injected i.c. with rUcn and 20 min later, with i.c. RX‐77368. CRF antagonists were injected i.c. 10 min before rUcn. RX‐77368 (1.5, 3, 10, 30 and 100 ng, i.c.) dose‐dependently increased corpus contractions, expressed as total area under the curve (AUC, mV min−1) to 2.6±2.5, 6.1±5.9, 9.8±2.6, 69.7±21.7 and 74.9±28.7 respectively vs 0.2±0.1 after i.c. saline. Ucn (1, 3 or 10 μg) inhibited RX‐77368 (30 ng)‐induced increase in total AUC by 28, 62 and 93% respectively vs i.c. saline+RX‐77368. The CRF1/CRF2 antagonist, astressin‐B (60 μg, i.c.) completely blocked i.c. rUcn (3 μg, i.c.)‐induced inhibition of gastric motility stimulated by RX‐77368 (30 ng). The selective CRF2 antagonist, astressin2‐B (30, 60 or 100 μg, i.c.) dose‐dependently prevented i.c. rUCn action while the CRF1 antagonist, NBI‐27914 did not. In conscious rats, rUcn (0.6 or 1 μg, i.c.) inhibited gastric emptying of an ingested chow meal by 61 and 92% respectively. rUcn action was antagonized by astressin2‐B. These data show that i.c. rUcn acts through CRF2 receptors to inhibit central vagal gastric contractile response and postoprandial emptying.

Pregabalin decreases visceral pain and prevents spinal neuronal activation in rats

We read the recent article by Houghton et al ( Gut 2007, Apr 19 [Epub ahead of print]), reporting that pregabalin, a new generation of α2δ ligand, increased sensory thresholds to normal levels in 26 patients with irritable bowel syndrome (IBS) and baseline rectal hypersensitivity, in a randomised double blind, placebo controlled, parallel group study. The authors concluded that α2δ ligands are worthy of further physiological and clinical investigations for diseases affecting gut sensory function. Experimental studies to date indicate that pregabalin prevents colorectal allodynia and hyperalgesia in rats exposed to intracolonic trinitrobenzene-sulphonic acid1 or septic shock.2 Visceral hyperalgesia and symptoms in IBS are, however, characterised by the absence of overt colonic damage or mucosal abnormality. In the study we describe here, pregabalin given orally in a rat non-inflammatory model of repeated tonic colorectal-distension-induced hypersensitivity3 prevented visceral hyperalgesia and blunted lumbosacral spinal neurone activation. Adult male Sprague–Dawley rats with or without electrodes implanted in the abdominal muscles were given orally (po) either vehicle (water, 1 ml/rat) or pregabalin (10 or 30 mg/kg; Parke Davis, Fresnes, France) and placed individually in Bollman cages. After a 60 minute stabilisation period, basal abdominal contractions were monitored for 10 minutes, then isobaric colorectal distension …

Cholinergic giant migrating contractions in conscious mouse colon assessed by using a novel noninvasive solid-state manometry method: modulation by stressors

There is a glaring lack of knowledge on mouse colonic motility in vivo, primarily due to unavailability of adequate recording methods. Using a noninvasive miniature catheter pressure transducer inserted into the distal colon, we assessed changes in colonic motility in conscious mice induced by various acute or chronic stressors and determined the neurotransmitters mediating these changes. Mice exposed to restraint stress (RS) for 60 min displayed distal colonic phasic contractions including high-amplitude giant migrating contractions (GMCs), which had peak amplitudes >25 mmHg and occurred at a rate of 15-25 h(-1) of which over 50% were aborally propagative. Responses during the first 20-min of RS were characterized by high-frequency and high-amplitude contractions that were correlated with defecation. RS-induced GMCs and fecal pellet output were blocked by atropine (0.5 mg/kg ip) or the corticotrophin releasing factor (CRF) receptor antagonist astressin-B (100 microg/kg ip). RS activated colonic myenteric neurons as shown by Fos immunoreactivity. In mice previously exposed to repeated RS (60 min/day, 14 days), or in transgenic mice that overexpress CRF, the duration of stimulation of phasic colonic contractions was significantly shorter (10 vs. 20 min). In contrast to RS, abdominal surgery abolished colonic contractions including GMCs. These findings provide the first evidence for the presence of frequent cholinergic-dependent GMCs in the distal colon of conscious mice and their modulation by acute and chronic stressors. Noninvasive colonic manometry opens new venues to investigate colonic motor function in genetically modified mice relevant to diseases that involve colonic motility alterations.

Substance P release and neurokinin 1 receptor activation in the rat spinal cord increase with the firing frequency of C-fibers.

Both the firing frequency of primary afferents and neurokinin 1 receptor (NK1R) internalization in dorsal horn neurons increase with the intensity of noxious stimulus. Accordingly, we studied how the pattern of firing of primary afferent influences NK1R internalization. In rat spinal cord slices, electrical stimulation of the dorsal root evoked NK1R internalization in lamina I neurons by inducing substance P release from primary afferents. The stimulation frequency had pronounced effects on NK1R internalization, which increased up to 100 Hz and then diminished abruptly at 200 Hz. Peptidase inhibitors increased NK1R internalization at frequencies below 30 Hz, indicating that peptidases limit the access of substance P to the receptor at moderate firing rates. NK1R internalization increased with number of pulses at all frequencies, but maximal internalization was substantially lower at 1-10 Hz than at 30 Hz. Pulses organized into bursts produced the same NK1R internalization as sustained 30 Hz stimulation. To determine whether substance P release induced at high stimulation frequencies was from C-fibers, we recorded compound action potentials in the sciatic nerve of anesthetized rats. We observed substantial NK1R internalization when stimulating at intensities evoking a C-elevation, but not at intensities evoking only an Adelta-elevation. Each pulse in trains at frequencies up to 100 Hz evoked a C-elevation, demonstrating that C-fibers can follow these high frequencies. C-elevation amplitudes declined progressively with increasing stimulation frequency, which was likely caused by a combination of factors including temporal dispersion. In conclusion, the instantaneous firing frequency in C-fibers determines the amount of substance P released by noxious stimuli.