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 microg/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 microg, intra-arterially). The CRF2 receptor antagonist, astressin2-B (200 microg/kg subcutaneously or 20 microg 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.

Neonatal maternal separation alters stress-induced responses to viscerosomatic nociceptive stimuli in rat.

This study investigated the combined effect of neonatal maternal separation and acute psychological stress on pain responses in adult rats. Long-Evans dams and their male pups were reared under two conditions: 1) 180 min daily maternal separation (MS180) on postnatal days 2-14 or 2) no handling or separation (NH). At 2 mo of age, visceromotor responses to graded intensities of phasic colorectal distension (10-80 mmHg) at baseline as well as following acute 60 min water avoidance stress (WA) were significantly higher in MS180 rats. Both groups showed similar stress-induced visceral hyperalgesia in the presence of naloxone (20 mg/kg ip). MS180 rats had smaller stress-induced cutaneous analgesia in the tail-flick test compared with NH rats, with a residual naloxone-resistant component. MS180 rats showed an enhanced fecal pellet output following WA or exposure to a novel environment. These data suggest that early life events predispose adult Long-Evans rats to develop visceral hyperalgesia, reduced somatic analgesia, and increased colonic motility in response to an acute psychological stressor, mimicking the cardinal features of irritable bowel syndrome.

The role of CNS NMDA receptors and nitric oxide in visceral hyperalgesia.

The studies summarized here document the role of NMDA receptors and nitric oxide in the lumbosacral spinal cord and rostral ventromedial medulla in the maintenance of visceral hyperalgesia. Experiments were conducted in rats in which drugs were administered into either the lumbosacral intrathecal space or directly into the rostral ventromedial medulla. The visceral stimulus was noxious colorectal distension, administered before and 3 h after intracolonic instillation of either saline or 25% zymosan. The visceromotor response to colonic distension was quantified and found to be significantly enhanced in rats in which the colon had previously been treated with zymosan. Enhanced responses to distension were attenuated dose-dependently by intrathecal administration of the NMDA receptor channel blocker MK-801 and by inhibition of the neuronal isoform of nitric oxide synthase (nNOS). In corresponding studies wherein drugs were administered directly into the rostral ventromedial medulla, NMDA receptor antagonism and NOS inhibition dose-dependently attenuated exaggerated responses to colonic distension. Taken together, these data suggest that zymosan-produced visceral hyperalgesia is influenced both at the level of the spinal cord and rostral ventromedial medulla, and that descending facilitatory influences from the rostral ventromedial medulla are important to the maintenance of visceral hyperalgesia.

Involvement of spinal calcitonin gene-related peptide in the development of acute visceral hyperalgesia in the rat.

This study aimed to characterize the role of the neuropeptide calcitonin gene-related peptide (CGRP) in the development of mechanically induced visceral hyperalgesia. Tonic colorectal distension (CRD) was performed in fasted, conscious male Sprague-Dawley rats. The visceromotor reflex associated with noxious CRD was determined as the number of contractions during each of two consecutive tonic distensions (10 min at 60 mmHg), which were separated by a series of phasic distensions (repeated 15-s distensions to 80 mmHg at 30-s intervals). The effect of the CGRP receptor antagonist h-CGRP8-37 given intrathecally (i.t.) (0.03-3 nmol rat-1) or intravenously (i.v.) (20 microg kg-1 bodyweight [bw]) on the visceromotor response was evaluated. The dose for i.v. administration was chosen based on previous results from similar studies. In addition, the effect of a CGRP monoclonal antibody (6 mg kg-1 bw) given intravenously was evaluated. Compared to the baseline response, a significant increase in the number of abdominal contractions was observed during the second tonic distension. The i.t. application of h-CGRP8-37 dose-dependently reduced the numbers of abdominal contractions both during the first and the second tonic distension period, with a maximum effect observed at a peptide concentration of 3 nmol. Intravenous administration of h-CGRP8-37 or of the CGRP antiserum produced a small reduction of the visceromotor response induced by the second tonic distension and had no effect on colonic compliance. The development of mechanically induced colorectal hyperalgesia by repeated tonic distension involves the spinal release of CGRP, while peripheral release of CGRP plays only a minor role.

Role of peripheral N-methyl-D-aspartate (NMDA) receptors in visceral nociception in rats.

BACKGROUND & AIMS: N-methyl-D-aspartate (NMDA) receptors are ligand-gated ion channels that have an important role in long-term potentiation and memory processing in the central nervous system. The aims in this study were to determine whether NMDA receptors are expressed in the peripheral nervous system and identify their role in mediating behavioral pain responses to colonic distention in the normal gut. METHODS AND RESULTS: Immunohistochemical localization of the NR1 subunit showed that NMDA receptors are expressed on the cell bodies and peripheral terminals of primary afferent nerves innervating the colon. Dorsal root ganglia neurons retrogradely labeled from the colon in short-term culture responded to addition of NMDA with increased intracellular [Ca2+]. Activation of peripheral NMDA receptors in colonic tissue sections caused Ca2+-dependent release of the proinflammatory neuropeptides, calcitonin gene-related peptide and substance P. Behavioral pain responses to noxious mechanical stimulation were inhibited in a reversible, dose-dependent manner by intravenous administration of memantine, a noncompetitive antagonist of the NMDA receptor. Single fiber recordings of decentralized pelvic nerves showed that colorectal distention responsive afferent nerve activity was inhibited by memantine. CONCLUSIONS: Peripheral NMDA receptors are important in normal visceral pain transmission, and may provide a novel mechanism for development of peripheral sensitization and visceral hyperalgesia.

V. Stress and irritable bowel syndrome.

Different types of stress play important roles in the onset and modulation of irritable bowel syndrome (IBS) symptoms. The physiological effects of psychological and physical stressors on gut function and brain-gut interactions are mediated by outputs of the emotional motor system in terms of autonomic, neuroendocrine, attentional, and pain modulatory responses. IBS patients show an enhanced responsiveness of this system manifesting in altered modulation of gastrointestinal motility and secretion and in alterations in the perception of visceral events. Functional brain imaging techniques are beginning to identify brain circuits involved in the perceptual alterations. Animal models have recently been proposed that mimic key features of the human syndrome.

Biphasic modulation of visceral nociception by neurotensin in rat rostral ventromedial medulla.

A potential role for neurotensin in the rostral ventromedial medulla (RVM) in modulation of visceral nociceptive transmission was examined in this study. Microinjection of neurotensin (3-3000 pmol) into the RVM of awake rats produced a dose-dependent inhibition of the visceromotor response (VMR) to noxious colorectal distension (CRD) that lasted 30 to 120 min. Additionally, intra-RVM injection of neurotensin (300 pmol) significantly reduced the slope of the stimulus-response function to graded CRD (20-80 mm Hg), whereas the greatest dose of neurotensin (3000 pmol) completely inhibited the VMR at all intensities of CRD. General motor function was unaffected after intra-RVM injection of neurotensin (3000 pmol). Intra-RVM injection of lesser doses of neurotensin (0.03-0.30 pmol) resulted an enhancement of the VMR to noxious CRD that had a short duration (18-30 min), and produced a leftward shift of the stimulus-response function to graded CRD without a change in the slope of the function. Additionally, intra-RVM injection of the neurotensin-receptor antagonist SR48692 (0.3-300 fmol) in naive animals produced dose-dependent inhibition of VMR to noxious CRD, whereas a lesser dose (0.03 fmol) enhanced the VMR. These data support a role for neurotensin in the RVM in biphasic modulation of visceral nociception. The results obtained with SR48692 suggest that endogenous neurotensin in the RVM modulates VMR to noxious CRD via a prominent interaction with neurotensin receptors that mediate facilitatory influences and a lesser interaction with neurotensin receptors that mediate masked inhibitory influences.

Involvement of excitatory amino acid receptors and nitric oxide in the rostral ventromedial medulla in modulating secondary hyperalgesia produced by mustard oil.

We have recently reported a model of secondary hyperalgesia in which facilitation of the thermal nociceptive tail-flick reflex following topical mustard oil is largely dependent on descending influences from the rostral ventromedial medulla (RVM). The current study was designed to examine a potential role for excitatory amino acid receptors and nitric oxide in the RVM in modulating this hyperalgesia. Topical application of mustard oil (100%) to the lateral surface of the hind leg of awake rats produced a short-lived (60 min) facilitation of the tail-flick reflex that was dose-dependently attenuated by microinjection of the selective N-methyl-D-aspartate (NMDA) receptor antagonist APV (1-100 fmol) into the RVM. Microinjection of a greater dose of APV (1000 fmol) into the RVM produced a significant inhibition of the tail-flick reflex in the presence, but not absence, of mustard oil. In contrast, microinjection of the non-NMDA receptor antagonist DNQX (10 nmol) into the RVM further enhanced the magnitude and duration of the hyperalgesic response, and produced a facilitation of the tail-flick reflex following injection into the RVM of naive animals. Similar to APV, microinjection of the nitric oxide synthase inhibitor L-NAME (100-1000 nmol) into the RVM attenuated mustard oil hyperalgesia, while the greatest dose (1000 nmol) produced a significant inhibition of the tail-flick reflex in the presence, but not absence, of mustard oil. A role for nitric oxide synthase in the RVM in mustard oil hyperalgesia was further demonstrated by a significant increase in the number of NADPH-d labeled cells in the RVM at the time of maximal hyperalgesia. Involvement of NMDA receptors and nitric oxide in the RVM in descending nociceptive facilitation was supported by the observation that microinjection of either NMDA or the NO* donor GEA 5024 into the RVM of naive animals dose-dependently facilitated the tail-flick reflex. The hyperalgesia produced by NMDA injection into the RVM was blocked by prior intra-RVM injection of either APV or L-NAME. These results support the notion that secondary hyperalgesia produced by mustard oil involves concurrent activation of dominant descending facilitatory, as well as masked inhibitory systems from the RVM. Additionally, the data suggest that descending facilitation involves activation of NMDA receptors and production NO* in the RVM, whereas inhibition involves activation of non-NMDA receptors in the RVM.

A role for spinal nitric oxide in mediating visceral hyperalgesia in the rat.

BACKGROUND & AIMS: Intracolonic instillation of zymosan in rats produces hyperalgesia (i.e., facilitates the visceromotor response to colorectal distention) mediated by activity at spinal N-methyl-D-aspartate (NMDA) and non-NMDA receptors. Nitric oxide (NO*) production often increases after NMDA receptor activation; NO* may then function as a further messenger. This study was designed to investigate the role of spinal NO* in this model of visceral hyperalgesia. METHODS: Zymosan or saline was given intracolonically, and the visceromotor response to noxious colorectal distention (80 mm Hg, 20 seconds) was measured 3 hours afterward. RESULTS: There was a significant enhancement of the visceromotor response in zymosan-, but not saline-treated, rats. This hyperalgesia was dose-dependently and reversibly attenuated by intrathecal administration of the nonselective NO* synthase (NOS) inhibitor NG-nitro-L-arginine methyl ester (100-800 nmol) as well as by the selective neuronal NOS inhibitor ARL 17477 (30-600 nmol). In support of these observations, there was a significant increase in the number of cells labeled for NADPH diaphorase or neuronal NOS in the lumbosacral spinal cord after intracolonic instillation of zymosan, but not saline. CONCLUSIONS: These data suggest that colonic inflammation induces the expression of neuronal NOS in the spinal cord and that increased production of spinal NO* is necessary for maintenance of zymosan-produced visceral hyperalgesia.

Intracolonic zymosan produces visceral hyperalgesia in the rat that is mediated by spinal NMDA and non-NMDA receptors.

The present study examined the effects of colonic inflammation on reflex responses to colorectal distension (CRD) in awake rats. Visceromotor responses (VMR) to CRD were recorded in rats that received either no treatment or intracolonic saline or zymosan. Three hours following zymosan treatment (25 mg/ml; 1 ml) VMR response magnitudes were significantly increased at all intensities of CRD tested (10-80 mmHg). The enhanced responses to CRD were attenuated in a dose-dependent fashion by intrathecal administration of the non-competitive N-methyl-D-aspartate (NMDA) receptor channel blocker MK-801 to 60% of control and by the non-NMDA receptor antagonist DNQX to less than 20% of control. The metabotropic receptor antagonist AP-3 was without effect. Signs of multi-focal colonic inflammation were clearly present 3 h after zymosan treatment, characterized by an ingress of inflammatory cells and damaged crypts in and around these foci. Taken together these findings suggest that tissue inflammation increases the sensitivity of the colon to mechanical stimuli, leading to enhanced responses to CRD. This enhancement involves the activation of spinal NMDA as well as non-NMDA receptors, but not metabotropic receptors.