Celine Maillot

A novel water-soluble selective CRF1 receptor antagonist, NBI 35965, blunts stress-induced visceral hyperalgesia and colonic motor function in rats.

The stress response involves the activation of two corticotropin-releasing factor (CRF) receptor subtypes. We investigated the role of CRF1 in stress-related visceral responses. A novel water-soluble tricyclic CRF1 antagonist, NBI 35965 was developed that displayed a high affinity for CRF1 (Ki approximately 4 nM) while having no binding affinity to CRF2. This antagonist also inhibited the stimulation of cAMP induced by sauvagine in CRF1 transfected cells. NBI 35965 administered per orally (p.o.) in rats (1, 3, 10 or 30 mg/kg) inhibited dose-dependently [125I]sauvagine binding selectively at brain sites of CRF1 distribution as shown by ex vivo receptor autoradiography. At the highest doses, NBI 35965 completely prevented [125I]sauvagine labeling in the cortex. NBI 35965 (10 mg/kg) administered p.o. or subcutaneously (s.c.) 1 h before intravenous CRF completely blocked the 81% shortening of distal colonic transit time induced by CRF. NBI 35965 (20 mg/kg s.c.) significantly reduced the defecation in response to water avoidance stress but not that induced by s.c. carbachol. In adult male Long-Evans rats that had undergone maternal separation, acute water avoidance stress significantly increased the visceromotor response to colorectal distention (20-80 mmHg) by 42+/-19% compared with the response before stress. Stress-induced visceral hyperalgesia was abolished by NBI 35965 (20 mg/kg, s.c.). The data show that NBI 35965 is a novel water-soluble selective CRF1 antagonist with bioavailability to the brain upon peripheral administration and that CRF1 receptor signaling pathways are involved in water avoidance stress-induced hyperalgesia to colorectal distention and stimulation of colonic transit.

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.

Peripheral corticotropin-releasing factor induces diarrhea in rats: role of CRF1 receptor in fecal watery excretion

Systemic injection of corticotropin-releasing factor (CRF) stimulates colonic secretory and motor functions, and CRF receptors play a role in stress-related alterations of colonic functions. Stress has also been reported to induce diarrhea and we investigated if peripheral injection of CRF can mimic this response in conscious rats. Intravenous (i.v.) injection of CRF (3, 10 or 30 microg/kg) caused diarrhea in 13%, 63% and 75% of rats, respectively, and dose dependently increased the fecal fluid content by 5.1-, 8.6- and 10.8-fold, while the dried solid weight was increased by 5.2-, 4.9- and 5.8-fold, respectively, compared to the i.v. saline group. CRF actions were rapid in onset and blocked by the CRF1 receptor, antagonist CP-154,526 (butyl-[2,5-dimethyl-7-(2,4,6-trimethylphenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl]ethylamine). These results demonstrate that peripheral CRF induces watery diarrhea, primarily through the activation of CRF1 receptor suggesting a possible role for these pathways in colonic responses to stress.

Peripheral injection of sauvagine prevents repeated colorectal distension-induced visceral pain in female rats.

We investigated the effects of peripheral injection of sauvagine, a CRF2>CRF1 receptor (corticotropin-releasing factor) agonist compared with CRF, on two sets of tonic colorectal distension (CRDs 30, 40, 50 mmHg, 3-min on/off)-induced visceromotor response (VMR) measured as area under the curve (AUC) of abdominal muscle contraction in conscious female rats. Sauvagine (10 or 20 microg/kg, s.c.) abolished the 226.7+/-64.3% and 90.4+/-38.1% increase in AUC to the 2nd CRD compared with the 1st CRD (performed 30 min before) in female Fisher and Sprague-Dawley (SD) rats, respectively. CRF had no effect while the CRF1 antagonist, antalarmin (20 mg/kg, s.c.), alone or with sauvagine, blocked the enhanced response to the 2nd CRD, performed 60 min after the 1st CRD, and reduced further the AUC by 33.5+/-23.3% and 63.5+/-7.2%, respectively in Fisher rats. These data suggest that peripheral CRF2 receptor activation exerts antinociceptive effects on CRD-induced visceral pain, whereas CRF1 contributes to visceral sensitization.

Intraperitoneal corticotropin-releasing factor and urocortin induce Fos expression in brain and spinal autonomic nuclei and long lasting stimulation of colonic motility in rats

CRF injected intraperitoneally (i.p.) stimulates colonic motor function and induces Fos expression in colonic myenteric neurons. We investigated central and spinal Fos expression and changes in colonic motility in response to i.p. injection of CRF and urocortin. Ovine CRF(9-33) that is devoid of intrinsic activity at the CRF receptors, was used as control peptide. Myoelectrical activity was monitored for 1 h before and after peptide injection (10 microg/kg, i.p.) in conscious non fasted rats with chronically implanted intraparietal electrodes in the cecum and proximal colon. Brain and lumbosacral spinal cord were processed for Fos immunohistochemistry at 1 h postinjection. CRF and urocortin elicited defecation and a new pattern of ceco-colonic clustered spike bursts that peaked within 15 min and lasted for the 1 h experimental period while CRF(9-33) did not modify baseline myoelectrical activity and defecation. CRF increased significantly Fos expression in the central nucleus of the amygdala (lateral part), parabrachial nucleus (external lateral subnucleus), area postrema, nucleus tractus solitarius, locus coeruleus, paraventricular nucleus of the hypothalamus, the intermediolateral column and area I-VII, X at the L6-S1 level of the spinal cord by 11-, 6.5-, 5.3-, 5.0-, 4.7-, 2.7- and 1.4-fold, respectively compared with i.p. CRF(9-33) injected rats that had little Fos expression. Urocortin induced a similar pattern of Fos response in the brain and the spinal cord. These results indicate that i.p. CRF and urocortin induce a peptide specific activation of brain nuclei receiving viscerosensory inputs and involved in autonomic circuitries whose effector limbs may impact on visceral function.

An impaired accommodation of the proximal stomach to a meal is associated with symptoms after distal gastrectomy.

OBJECTIVE:The aim of this study was to assess gastric emptying and postprandial proximal gastric tone variations depending on the presence of postoperative symptoms after Billroth II gastrectomy (BII).METHODS:A total of 16 consecutive patients were prospectively studied 10 ± 3 months after distal gastrectomy for antral cancer. No evidence of cancer recurrence was detected at the time of the study. Ten patients were asymptomatic after surgery, whereas six patients were considered as symptomatic because of at least one weekly epigastric pain, postprandial fullness, nausea, or vomiting. The fasting fundus volume and tone, the onset of a gastric relaxation after a standard 200-kcal liquid meal, and the characteristics of this relaxation (delay of occurrence, amplitude) were determined with a barostat. Patient results were compared to normal values obtained in 12 healthy volunteers. Gastric emptying studies were performed in all patients using the C13 acid octanoic breath test after a 250-kcal meal.RESULTS:In the patients, both fasting fundus tone (BII 7.0 ± 0.5, controls 8.4 ± 2.4 mm Hg) and volume (BII 108 ± 11, controls 119 ± 29 ml) were not different from controls. Fasting fundus tone was lower in asymptomatic patients than in symptomatic patients (6.5 ± 0.4 vs 8.1 ± 0.5 mm Hg, p < 0.04). Gastric relaxation was observed immediately after the meal in all asymptomatic patients as well as in controls. In contrast, gastric relaxation occurred in only two of six symptomatic patients (p < 0.01); when it occurred it was delayed by the meal and was observed only 23 ± 1 min after food intake. When a relaxation occurred, its amplitude was higher than that observed in controls both in asymptomatic and symptomatic patients (294 ± 21 vs 179 ± 53 ml, p < 0.02).CONCLUSIONS:After distal gastrectomy, gastric accommodation is impaired (i.e., absent or delayed) in symptomatic patients. When relaxation exists in these patients, its amplitude is higher than in control subjects.

Sexual and Asexual Development of Cryptosporidium parvum in Five Oocyst– or Sporozoite-Infected Human Enterocytic Cell Lines

ABSTRACT. The human enterocytic cell lines Caco‐2, HT29, HCT8 and the Caco‐2 clones TC7 and PF11 were studied for their ability to support Cryptosporidium parvum development. Following the addition in cultures of either oocysts or excysted sporozoites, immunofluorescent and transmission electron microscopy revealed the presence of all stages of the parasite life cycle by both procedures, and no difference in the ration of infected cells was found among cell lines. More oocysts were seen in cell monolayers infected with oocysts than with sporozoites (p < 0.0001). The number of meronts observed was the same after either oocysts or sporozoites inoculation. Data suggest that the two methods yield a same cell infection rate.