Search

Using neurokinin 1 receptor (NK1R) internalization to measure of substance P release in rat spinal cord slices, we found that it was induced by the adenylyl cyclase (AC) activator forskolin, by the protein kinase A (PKA) activators 6-Bnz-cAMP and 8-Br-cAMP, and by the activator of exchange protein activated by cAMP (Epac) 8-pCPT-2-O-Me-cAMP (CPTOMe-cAMP). Conversely, AC and PKA inhibitors decreased substance P release induced by electrical stimulation of the dorsal root. Therefore, the cAMP signaling pathway mediates substance P release in the dorsal horn. The effects of forskolin and 6-Bnz-cAMP were not additive with NMDA-induced substance P release and were decreased by the NMDA receptor blocker MK-801. In cultured dorsal horn neurons, forskolin increased NMDA-induced Ca 2+ entry and the phosphorylation of the NR1 and NR2B subunits of the NMDA receptor. Therefore, cAMP-induced substance P release is mediated by the activating phosphorylation by PKA of NMDA receptors. Voltage-gated Ca 2+ channels, but not by TRPV1 or TRPA1, also contributed to cAMP-induced substance P release. Activation of PKA was required for the effects of forskolin and the three cAMP analogs. Epac2 contributed to the effects of forskolin and CPTOMe-cAMP, signaling through a Raf - mitogen-activated protein kinase pathway to activate Ca 2+ channels. Epac1 inhibitors induced NK1R internalization independently of substance P release. In rats with latent sensitization to pain, the effect of 6-Bnz-cAMP was unchanged, whereas the effect of forskolin was decreased due to the loss of the stimulatory effect of Epac2. Hence, substance P release induced by cAMP decreases during pain hypersensitivity., (Published by Elsevier Ltd.)

The interaction between NMDA receptors and μ-opioid receptors in primary afferent terminals was studied by using NMDA to induce substance P release, measured as neurokinin 1 receptor internalization. In rat spinal cord slices, the μ-opioid receptor agonists morphine, DAMGO and endomorphin-2 inhibited NMDA-induced substance P release, whereas the antagonist CTAP right-shifted the concentration response of DAMGO. In vivo, substance P release induced by intrathecal NMDA after priming with BDNF was inhibited by DAMGO. ω-Conotoxins MVIIC and GVIA inhibited about half of the NMDA-induced substance P release, showing that it was partially mediated by the opening of voltage-gated calcium (Cav) channels. In contrast, DAMGO or ω-conotoxins did not inhibit capsaicin-induced substance P release. In cultured DRG neurons, DAMGO but not ω-conotoxin inhibited NMDA-induced increases in intracellular calcium, indicating that μ-opioid receptors can inhibit NMDA receptor function by mechanisms other than inactivation of Cav channels. Moreover, DAMGO decreased the ω-conotoxin-insensitive component of the substance P release. Potent inhibition by ifenprodil showed that these NMDA receptors have the NR2B subunit. Activators of adenylyl cyclase and protein kinase A (PKA) induced substance P release and this was decreased by the NMDA receptor blocker MK-801 and by DAMGO. Conversely, inhibitors of adenylyl cyclase and PKA, but not of protein kinase C, decreased NMDA-induced substance P release. Hence, these NMDA receptors are positively modulated by the adenylyl cyclase-PKA pathway, which is inhibited by μ-opioid receptors. In conclusion, μ-opioid receptors inhibit NMDA receptor-induced substance P release through Cav channel inactivation and adenylyl cyclase inhibition., (Published by Elsevier Ltd.)

Patients with functional gastrointestinal disorders (FGIDs) often experience distress, reduced quality of life, a perceived lack of validation, and an unsatisfactory experience with health care providers. A health care provider can provide the patient with a framework in which to understand and legitimize their symptoms, remove self-doubt or blame, and identify factors that contribute to symptoms that the patient can influence or control. This framework is implemented with the consideration of important factors that impact FGIDs, such as gender, age, society, and the patient's perspective. Although the majority of FGIDs, including globus, rumination syndrome, IBS, bloating, constipation, functional abdominal pain, sphincter of Oddi dyskinesia, pelvic floor dysfunction, and extra-intestinal manifestations, are more prevalent in women than men, functional chest pain, dyspepsia, vomiting, and anorectal pain do not appear to vary by gender. Studies suggest sex differences in somatic but not visceral pain perception, motility, and central processing of visceral pain; although further research is required in autonomic nervous system dysfunction, genetics and immunologic/microbiome. Gender differences in response to psychological treatments, antidepressants, fiber, probiotics, and anticholinergics have not been adequately studied. However, a greater clinical response to 5-HT 3 antagonists but not 5-HT 4 agonists has been reported in women compared with men., (Copyright © 2016 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Many chronic pain disorders alternate between bouts of pain and periods of remission. The latent sensitization model reproduces this in rodents by showing that the apparent recovery ("remission") from inflammatory or neuropathic pain can be reversed by opioid antagonists. Therefore, this remission represents an opioid receptor-mediated suppression of a sustained hyperalgesic state. To identify the receptors involved, we induced latent sensitization in mice and rats by injecting complete Freund's adjuvant (CFA) in the hindpaw. In WT mice, responses to mechanical stimulation returned to baseline 3 weeks after CFA. In μ-opioid receptor (MOR) knock-out (KO) mice, responses did not return to baseline but partially recovered from peak hyperalgesia. Antagonists of α2A-adrenergic and δ-opioid receptors reinstated hyperalgesia in WT mice and abolished the partial recovery from hyperalgesia in MOR KO mice. In rats, antagonists of α2A adrenergic and μ-, δ-, and κ-opioid receptors reinstated hyperalgesia during remission from CFA-induced hyperalgesia. Therefore, these four receptors suppress hyperalgesia in latent sensitization. We further demonstrated that suppression of hyperalgesia by MORs was due to their constitutive activity because of the following: (1) CFA-induced hyperalgesia was reinstated by the MOR inverse agonist naltrexone (NTX), but not by its neutral antagonist 6β-naltrexol; (2) pro-enkephalin, pro-opiomelanocortin, and pro-dynorphin KO mice showed recovery from hyperalgesia and reinstatement by NTX; (3) there was no MOR internalization during remission; (4) MORs immunoprecipitated from the spinal cord during remission had increased Ser(375) phosphorylation; and (5) electrophysiology recordings from dorsal root ganglion neurons collected during remission showed constitutive MOR inhibition of calcium channels., Significance Statement: Chronic pain causes extreme suffering to millions of people, but its mechanisms remain to be unraveled. Latent sensitization is a phenomenon studied in rodents that has many key features of chronic pain: it is initiated by a variety of noxious stimuli, has indefinite duration, and pain appears in episodes that can be triggered by stress. Here, we show that, during latent sensitization, there is a sustained state of pain hypersensitivity that is continuously suppressed by the activation of μ-, δ-, and κ-opioid receptors and by adrenergic α2A receptors in the spinal cord. Furthermore, we show that the activation of μ-opioid receptors is not due to the release of endogenous opioids, but rather to its ligand-independent constitutive activity., (Copyright © 2016 the authors 0270-6474/16/360204-18$15.00/0.)

Opiate analgesia in the spinal cord is impaired during neuropathic pain. We hypothesized that this is caused by a decrease in μ-opioid receptor inhibition of neurotransmitter release from primary afferents. To investigate this possibility, we measured substance P release in the spinal dorsal horn as neurokinin 1 receptor (NK1R) internalization in rats with chronic constriction injury (CCI) of the sciatic nerve. Noxious stimulation of the paw with CCI produced inconsistent NK1R internalization, suggesting that transmission of nociceptive signals by the injured nerve was variably impaired after CCI. This idea was supported by the fact that CCI produced only small changes in the ability of exogenous substance P to induce NK1R internalization or in the release of substance P evoked centrally from site of nerve injury. In subsequent experiments, NK1R internalization was induced in spinal cord slices by stimulating the dorsal root ipsilateral to CCI. We observed a complete loss of the inhibition of substance P release by the μ-opioid receptor agonist [D-Ala(2), NMe-Phe(4), Gly-ol(5)]-enkephalin (DAMGO) in CCI rats but not in sham-operated rats. In contrast, DAMGO still inhibited substance P release after inflammation of the hind paw with complete Freund's adjuvant and in naïve rats. This loss of inhibition was not due to μ-opioid receptor downregulation in primary afferents, because their colocalization with substance P was unchanged, both in dorsal root ganglion neurons and primary afferent fibers in the dorsal horn. In conclusion, nerve injury eliminates the inhibition of substance P release by μ-opioid receptors, probably by hindering their signaling mechanisms., (Published by Elsevier Ltd.)

NMDA receptors in primary afferent terminals can contribute to hyperalgesia by increasing neurotransmitter release. In rats and mice, we found that the ability of intrathecal NMDA to induce neurokinin 1 receptor (NK1R) internalization (a measure of substance P release) required a previous injection of BDNF. Selective knock-down of NMDA receptors in primary afferents decreased NMDA-induced NK1R internalization, confirming the presynaptic location of these receptors. The effect of BDNF was mediated by tropomyosin-related kinase B (trkB) receptors and not p75 neurotrophin receptors (p75(NTR) ), because it was not produced by proBDNF and was inhibited by the trkB antagonist ANA-12 but not by the p75(NTR) inhibitor TAT-Pep5. These effects are probably mediated through the truncated form of the trkB receptor as there is little expression of full-length trkB in dorsal root ganglion (DRG) neurons. Src family kinase inhibitors blocked the effect of BDNF, suggesting that trkB receptors promote the activation of these NMDA receptors by Src family kinase phosphorylation. Western blots of cultured DRG neurons revealed that BDNF increased Tyr(1472) phosphorylation of the NR2B subunit of the NMDA receptor, known to have a potentiating effect. Patch-clamp recordings showed that BDNF, but not proBDNF, increased NMDA receptor currents in cultured DRG neurons. NMDA-induced NK1R internalization was also enabled in a neuropathic pain model or by activating dorsal horn microglia with lipopolysaccharide. These effects were decreased by a BDNF scavenger, a trkB receptor antagonist and a Src family kinase inhibitor, indicating that BDNF released by microglia potentiates NMDA receptors in primary afferents during neuropathic pain., (Published 2014. This article is a U.S. Government work and is in the public domain in the USA.)

Glutamate (Glu) is the primary excitatory neurotransmitter in the central nervous system and plays a critical role in the neuroplasticity of nociceptive networks. We aimed to examine the role of spinal astroglia in the modulation of glutamatergic neurotransmission in a model of chronic psychological stress-induced visceral hyperalgesia in male Wistar rats. We assessed the effect of chronic stress on different glial Glu control mechanisms in the spinal cord including N-methyl-d-aspartate receptors (NMDARs), glial Glu transporters (GLT1 and GLAST), the Glu conversion enzyme glutamine synthetase (GS), and glial fibrillary acidic protein (GFAP). We also tested the effect of pharmacological inhibition of NMDAR activation, of extracellular Glu reuptake, and of astrocyte function on visceral nociceptive response in naive and stressed rats. We observed stress-induced decreased expression of spinal GLT1, GFAP, and GS, whereas GLAST expression was upregulated. Although visceral hyperalgesia was blocked by pharmacological inhibition of spinal NMDARs, we observed no stress effects on NMDAR subunit expression or phosphorylation. The glial modulating agent propentofylline blocked stress-induced visceral hyperalgesia, and blockade of GLT1 function in control rats resulted in enhanced visceral nociceptive response. These findings provide evidence for stress-induced modulation of glia-controlled spinal Glu-ergic neurotransmission and its involvement in chronic stress-induced visceral hyperalgesia. The findings reported in this study demonstrate a unique pattern of stress-induced changes in spinal Glu signaling and metabolism associated with enhanced responses to visceral distension.

Background & Aims: 5-Hydroxytryptamine (5-HT)3 receptor (5-HT3R) antagonists are effective in treating patients with irritable bowel syndrome (IBS) and have anxiolytic effects. Their therapeutic effects are related, in part, to reducing amygdala engagement during expected visceral pain. A single nucleotide polymorphism in HTR3A, c.-42C>T;(C178T; rs1062613), is associated with altered reactivity of the amygdala during emotional face processing in healthy subjects (controls). We evaluated the influence of this single nucleotide polymorphism on amygdala reactivity to emotional faces and nonemotional stimuli in female patients with IBS and controls., Methods: We measured brain responses during an affect-matching paradigm in 54 women (26 with IBS, 29 controls) using functional magnetic resonance imaging. We examined associations between HTR3A c.-42C>T genotype (C/C vs T carrier) and responses in amygdala and other regions of brain that expressed high levels of 5-HT3R., Results: The C/C genotype was associated with greater anxiety symptoms in patients with IBS and controls and increased activation of the amygdala under emotional and nonemotional conditions. Among patients with IBS, C/C genotype was associated with greater symptom ratings. A subset of IBS patients with the C/C genotype had increased amygdala responses to nonemotional stimuli, compared with other subjects with C/C genotype., Conclusions: Regardless of diagnosis, the C/C genotype of the c.-42C>T polymorphism in HTR3A, compared with T carrier status, is associated with increased anxiety and amygdala responsiveness during emotional and nonemotional tasks. This polymorphism was associated with severity of IBS symptoms. Although this genotype is not sufficient for diagnosis of IBS, it is associated with severity of symptoms., (Copyright © 2011 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Intravenous cholecystokinin octapeptide (CCK-8) elicits vago-vagal reflexes that inhibit phasic gastric contractions and reduce gastric tone in urethane-anaesthetized rats. A discrete proximal subdivision of the ventral gastric vagus nerve (pVGV) innervates the proximal stomach, but the fibre populations within it have not been characterized previously.We hypothesized that I.V. CCK-8 injection would excite inhibitory efferent outflow in the pVGV, in contrast to its inhibitory effect on excitatory efferent outflow in the distal subdivision (dVGV), which supplies the distal stomach. In each VGV subdivision, a dual-recording technique was used to record afferent and efferent activity simultaneously, while also monitoring intragastric pressure (IGP). CCK-8 dose dependently (100-1000 pmol kg(-1), I.V.) reduced gastric tone, gastric contractile activity and multi-unit dVGV efferent discharge, but increased pVGV efferent firing. Single-unit analysis revealed a minority of efferent fibres in each branch whose response differed in direction from the bulk response. Unexpectedly, efferent excitation in the pVGV was significantly shorter lived and had a significantly shorter decay half-time than did efferent inhibition in the dVGV, indicating that distinct pathways drive CCK-evoked outflow to the proximal vs. the distal stomach. Efferent inhibition in the dVGV began several seconds before, and persisted significantly longer than, simultaneously recorded dVGV afferent excitation.Thus, dVGV afferent excitation could not account for the pattern of dVGV efferent inhibition. However, the time course of dVGV afferent excitation paralleled that of pVGV efferent excitation. Similarly, the duration of CCK-8-evoked afferent responses recorded in the accessory celiac branch of the vagus (ACV) matched the duration of dVGV efferent responses. The observed temporal relationships suggest that postprandial effects on gastric complicance of CCK released from intestinal endocrine cells may require circulating concentrations to rise to levels capable of exciting distal gastric afferent fibres, in contrast to more immediate effects on distal gastric contractile activity mediated via vago-vagal reflexes initiated by paracrine excitation of intestinal afferents.

The role of NMDA receptors (NMDARs) expressed by primary afferent neurons in nociception remains controversial. The aim of this study was to develop mice with a tissue selective knockdown of NMDARs in these neurons and to evaluate their behavioral responses to different types of painful stimuli. Mice with floxed NMDAR NR1 subunit gene (fNR1) were crossed with mice expressing Cre recombinase under the control of the peripherin promotor (Prph-Cre). Male Prph-Cre+ floxed NR1 mice were compared to Cre- littermates. Both quantitative RT/PCR and Western blotting indicated a ∼75% reduction in NR1 expression in dorsal root ganglia (DRG) extracts with no effect on NR1 expression in spinal cord, brain or the enteric nervous system. Immunocytochemistry with antibodies to NR1 revealed decreased staining in all size classes of DRG neurons. NMDA produced a detectable increase in [Ca2+]i in 60% of DRG neurons cultured from Cre- mice, but only 15% of those from Cre+ mice. Furthermore, the peak [Ca2+]i responses were 64% lower in neurons from Cre+ mice. There was no significant difference between Cre+ and Cre- mice in response latencies to the hotplate or tail withdrawal tests of thermal nociception, nor was there a difference in withdrawal thresholds to mechanical stimuli of the tail or paw. However, compared to Cre- littermates, Cre+ knockdown mice had a 50% decrease in the phase 2 response to formalin injection (P<0.001). There was no effect on phase 1 responses. These results suggest that NMDA receptors expressed by primary afferent nerves play an important role in the development of sensitized pain states., (Copyright © 2011 IBRO. All rights reserved.)

Visceral hypersensitivity has been implicated as an important pathophysiological mechanism in functional gastrointestinal disorders. In this study, we investigated whether the sustained visceral hyperalgesia induced by repeated psychological stress in rats involves the activation of CRF(1) signaling system using two different antagonists. Male Wistar rats were exposed to 10 consecutive days of water avoidance stress (WAS) or sham stress for 1 h/day, and the visceromotor response to phasic colorectal distension (CRD) was assessed before and after the stress period. Animals were injected subcutaneously with the brain penetrant CRF(1) antagonist, CP-154,526, acutely (30 min before the final CRD) or chronically (via osmotic minipump implanted subcutaneously, during stress) or with the peripherally restricted, nonselective CRF(1) and CRF(2) antagonist, astressin, chronically (15 min before each stress session). Repeated WAS induced visceral hypersensitivity to CRD at 40 and 60 mmHg. CP-154,526 injected acutely significantly reduced stress-induced visceral hyperalgesia at 40 mmHg but not at 60 mmHg. Chronic subcutaneous delivery of astressin reduced the stress-induced visceral hyperalgesia to baseline at all distension pressures. Interestingly, chronically administered CP-154,526 eliminated hyperalgesia and produced responses below baseline at 40 mmHg and 60 mmHg, indicating a hypoalgesic effect of the compound. These data support a major role for CRF(1) in both the development and maintenance of visceral hyperalgesia induced by repeated stress and indicate a possible role of peripheral CRF receptors in such mechanisms.

Separate breeding colonies of C57BL/6 ("B6") mice maintained at the Jackson Laboratories ("J") and NIH ("N") have led to the emergence of two distinct substrains of C57BL/6 mice: C57BL/6J and C57BL/6N. Molecular genetic studies indicate simple sequence-length polymorphisms, single-nucleotide polymorphisms, and copy-number variants among B6 substrains that may contribute to phenotypic differences. We examined differences in motor coordination, pain sensitivity, and conditional fear in the C57BL/6J strain and three N strains: C57BL/6NCrl (Charles River), C57BL/6NTac (Taconic), and C57BL/6NHsd (Harlan Sprague Dawley). Male C57BL/6J mice demonstrated enhanced motor coordination, as measured by the rotarod assay, markedly enhanced pain sensitivity in two assays of acute thermal nociception (e.g., tail withdrawal and hot plate), and a reduced level of conditional fear. The tail withdrawal result was confirmed in a separate laboratory. We also provide a table reviewing previously reported behavioral differences among various B6 substrains and discuss the significance of environmental differences due to obtaining mice form different vendors. These data may be seen as a potential problem and as a potential opportunity. Great care must be taken when working with mice engineered by using B6 embryonic stem cell lines because control groups, backcrosses, and intercrosses could inadvertently introduce behaviorally significant polymorphic alleles or environmental confounds. On the other hand, deliberate crosses between B6 substrains may provide an opportunity to map polymorphic loci that contribute to variability in a trait on largely homogenous backgrounds, which has the potential to improve mapping resolution and aid in the selection of candidate genes.

Women have greater temporal summation of experimental pain stimuli and also have a higher propensity for developing chronic visceral pain conditions. Sex hormone-mediated regulation of N-methyl-d-aspartic acid receptors (NMDARs) in nociceptive pathways is a plausible mechanism that may underlie these phenomena. The aim of this study was to compare the effect of 17-beta-estradiol (E2) in modulation of NMDAR activity in adult male and female rat dorsal root ganglia (DRG) neurons. DRG neurons were collected from adult male or female rats and grown in short-term culture in steroid-free media. NMDAR currents were recorded on small to medium size neurons by whole cell patch clamp using rapid perfusion with saturating concentrations of N-methyl-d-aspartic acid and glycine in the absence of extracellular Mg(2+). We found that the average density of NMDAR currents was 2.8-fold larger in DRG neurons from female rats compared with male rats (P<0.0001). Addition of 100 nM E2 increased NMDAR currents 55+/-15% in female neurons, but only 19+/-7% in male neurons. Potentiation was maximal after 20-40 min and dose dependent with an apparent 50% excitatory concentration of 17-23 nM. This effect was mimicked by E2 conjugated to BSA and attenuated by pretreatment with the protein tyrosine kinase inhibitor lavendustin A (1 microM) or the estrogen receptor (ER) antagonist, ICI 182,780 (1 microM), strongly suggesting activation of a cell surface ER acting through a non-genomic mechanism involving protein tyrosine kinases to increase NMDAR currents. These results identify sex-based differences in both the basal expression and the regulation of the NMDARs in DRG neurons.

N-methyl-D-aspartate (NMDA) receptors (NMDARs) on spinal afferent neurons regulate the peripheral and central release of neuropeptides involved in the development of hyperalgesia. We examined the effect of experimental colitis on the molecular and functional properties of NMDARs on these neurons. Lumbosacral dorsal root ganglia (DRG) were collected from adult rats 5 days after the induction of colitis for whole cell patch-clamp recording, Western blot analysis, and quantitative RT-PCR. Compared with neurons from control rats, those taken from animals with colitis had a threefold higher density of NMDA currents in both retrograde-labeled, colon-specific, and unlabeled DRG neurons. Increased current densities were not observed in DRG neurons taken from thoracic spinal levels. There was no significant change in NMDA or glycine affinity or in voltage-dependent Mg2+ inhibition; however, there was a 10-fold decrease in sensitivity to the NR2B subunit-selective antagonist ifenprodil. Quantitative RT-PCR and Western blot analysis indicated a 28% increase in the expression of NR2B with little or no change in the other three NR2 subunits. The addition of the Src family tyrosine kinase inhibitor PP2 (10 microM) decreased NMDAR currents in neurons from colitis but not control rats. Conversely, pretreatment of DRG neurons from control animals with 100 microM sodium orthovanadate increased NMDAR currents and decreased ifenprodil sensitivity to levels similar to those observed in neurons from animals with colitis. In conclusion, colonic inflammation upregulates the activity of NMDARs in all DRG neurons within ganglia innervating this tissue through mechanisms involving increased expression and persistent tyrosine phosphorylation.

Background & Aims: The neurokinin 1 receptors (NK(1)Rs) and substance P (SP) have been implicated in the stress and/or pain pathways involved in chronic pain conditions. Here we examined the participation of NK(1)Rs in sustained visceral hyperalgesia observed in rats exposed to chronic psychological stress., Methods: Male Wistar rats were exposed to daily 1-hour water avoidance stress (WA) or sham WA for 10 consecutive days. We tested intraperitoneal or intrathecal injection of the NK(1)R antagonist SR140333 on the visceromotor reflex to colorectal distention in both groups at day 11. Real-time reverse-transcription polymerase chain reaction, Western blot, and immunohistochemistry were used to assess the expression of NK(1)Rs and/or SP in samples of colon, spinal cord, and dorsal root ganglia., Results: Both intraperitoneal and intrathecal SR140333 injection diminished the enhanced visceromotor reflex to colorectal distention at day 11 in stressed rats but did not affect the response in control animals. Real-time polymerase chain reaction and Western blotting demonstrated stress-induced up-regulation of spinal NK(1)Rs. Immunohistochemistry showed an increased number of NK(1)R-expressing neurons in the laminae I of the dorsal horn in stressed rats. The expression of NK(1)Rs was decreased in colon from stressed rats compared with control. The expression of SP gene precursor in dorsal root ganglia was unchanged in stressed rats compared with controls., Conclusions: Stress-induced increased NK(1)R expression on spinal neurons and the inhibitory effect of intrathecal NK(1)R antagonist on visceral hyperalgesia support the key contribution of spinal NK(1)Rs in the molecular pathways involved in the maintenance of visceral hyperalgesia observed after chronic WA.

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.

In rodents, maternal pup interactions play an important role in programming the stress responsiveness of the adult organism. The aims of this study were 1) to determine the effect of different neonatal rearing conditions on acute and delayed stress-induced visceral sensitivity as well as on other measures of stress sensitivity of the adult animal; and 2) to determine the role of corticotropin-releasing factor receptor (CRF-R) subtype 1 (CRF(1)R) in mediating visceral hypersensitivity. Three groups of male Long-Evans rat pups were used: separation from their dam for 180 min daily from postnatal days 2-14 (MS180), daily separation (handling) for 15 min (H), or no handling. The visceromotor responses (VMR) to colorectal distension, stress-induced colonic motility, and anxiety-like behavior were assessed in the adult rats. The VMR was assessed at baseline, immediately after a 1-h water avoidance (WA) stress, and 24 h poststress. Astressin B, a nonselective CRF-R antagonist, or CP-154,526, a selective CRF(1)R antagonist, was administered before the stressor and/or before the 24-h measurement. MS rats developed acute and delayed stress-induced visceral hyperalgesia. In contrast, H rats showed hypoalgesia immediately after WA and no change in VMR on day 2. MS rats with visceral hyperalgesia also exhibited enhanced stress-induced colonic motility and increased anxiety-like behavior. In MS rats, both CRF-R antagonists abolished acute and delayed increases in VMR. Rearing conditions have a significant effect on adult stress responsiveness including immediate and delayed visceral pain responses to an acute stressor. Both acute and delayed stress-induced visceral hypersensitivity in MS rats are mediated by the CRF/CRF(1)R system.

Chronic stress plays an important role in the development and exacerbation of symptoms in functional gastrointestinal disorders. To better understand the mechanisms underlying this relationship, we aimed to characterize changes in visceral and somatic nociception, colonic motility, anxiety-related behavior, and mucosal immune activation in rats exposed to 10 days of chronic psychological stress. Male Wistar rats were submitted daily to either 1-h water avoidance (WA) stress or sham WA for 10 consecutive days. The visceromotor response to colorectal distension, thermal somatic nociception, and behavioral responses to an open field test were measured at baseline and after chronic WA. Fecal pellets were counted after each WA stress or sham WA session as a measure of stress-induced colonic motility. Colonic samples were collected from both groups and evaluated for structural changes and neutrophil infiltration, mast cell number by immunohistochemistry, and cytokine expression by quantitative RT-PCR. Rats exposed to chronic WA (but not sham stress) developed persistent visceral hyperalgesia, whereas only transient changes in somatic nociception were observed. Chronically stressed rats also exhibited anxiety-like behaviors, enhanced fecal pellet excretion, and small but significant increases in the mast cell numbers and the expression of IL-1beta and IFN-gamma. Visceral hyperalgesia following chronic stress persisted for at least a month. Chronic psychological stress in rats results in a robust and long-lasting alteration of visceral, but not somatic nociception. Visceral hyperalgesia is associated with other behavioral manifestations of stress sensitization but was only associated with minor colonic immune activation arguing against a primary role of mucosal immune activation in the maintenance of this phenomenon.

In the peripheral nervous system, N-methyl-D-aspartate receptors (NMDAR) expressed on the central and peripheral terminals of primary afferent neurons are involved in nociception. We used single cell imaging of intracellular calcium concentration ([Ca2+]i) and patch clamp techniques to characterize the functional properties of NMDARs on adult rat dorsal root ganglia (DRG) neurons in primary culture and selectively on those innervating the distal colon. In Mg2+-free extracellular solution, rapid perfusion of DRG neurons with 250 microM NMDA and 10 microM glycine caused a significant increase in [Ca2+]i, and elicited inward currents in whole cell patch clamp recordings when the holding potential was -60 mV. Both effects were reversibly inhibited by 200 microM ketamine in a use-dependent manner. The EC50 values for NMDA and glycine were 64 and 1.9 microM with Hill slope coefficients of 1.4 and 1.3, respectively. At negative potentials, extracellular Mg2+ blocked currents in a concentration- and voltage-dependent manner. The IC50 for Mg2+ at a holding potential of -100 mV was 2.0 microM. The NMDAR subtype-selective antagonist, ifenprodil, inhibited 94% of the NMDA and glycine-induced current with an IC50 of 2.6 microM. There was no evidence of multiple binding sites for ifenprodil. There was no significant difference in the NMDAR current density on DRG neurons that had innervated the colon, nor was there a difference in the EC50 for ifenprodil. These results demonstrate that functional NMDARs expressed by DRG neurons innervating both somatic and visceral tissues of adult rats are composed predominantly of NR2B subunits.

Inflammatory proteases (mast cell tryptase and trypsins) cleave protease-activated receptor 2 (PAR2) on spinal afferent neurons and cause persistent inflammation and hyperalgesia by unknown mechanisms. We determined whether transient receptor potential vanilloid receptor 1 (TRPV1), a cation channel activated by capsaicin, protons, and noxious heat, mediates PAR2-induced hyperalgesia. PAR2 was coexpressed with TRPV1 in small- to medium-diameter neurons of the dorsal root ganglia (DRG), as determined by immunofluorescence. PAR2 agonists increased intracellular [Ca2+] ([Ca2+]i) in these neurons in culture, and PAR2-responsive neurons also responded to the TRPV1 agonist capsaicin, confirming coexpression of PAR2 and TRPV1. PAR2 agonists potentiated capsaicin-induced increases in [Ca2+]i in TRPV1-transfected human embryonic kidney (HEK) cells and DRG neurons and potentiated capsaicin-induced currents in DRG neurons. Inhibitors of phospholipase C and protein kinase C (PKC) suppressed PAR2-induced sensitization of TRPV1-mediated changes in [Ca2+]i and TRPV1 currents. Activation of PAR2 or PKC induced phosphorylation of TRPV1 in HEK cells, suggesting a direct regulation of the channel. Intraplantar injection of a PAR2 agonist caused persistent thermal hyperalgesia that was prevented by antagonism or deletion of TRPV1. Coinjection of nonhyperalgesic doses of PAR2 agonist and capsaicin induced hyperalgesia that was inhibited by deletion of TRPV1 or antagonism of PKC. PAR2 activation also potentiated capsaicin-induced release of substance P and calcitonin gene-related peptide from superfused segments of the dorsal horn of the spinal cord, where they mediate hyperalgesia. We have identified a novel mechanism by which proteases that activate PAR2 sensitize TRPV1 through PKC. Antagonism of PAR2, TRPV1, or PKC may abrogate protease-induced thermal hyperalgesia.

The mechanism(s) underlying stress-induced colonic hypersensitivity (SICH) are incompletely understood. Our aims were to assess the acute and delayed (24 h) effect of water avoidance (WA) stress on visceral nociception in awake male Wistar rats and to evaluate the role of two stress-related modulation systems: the substance P/neurokinin-1 receptor (SP/NK(1)R) and the corticotropin-releasing factor (CRF)/CRF(1) receptor (CRF/CRF(1)R) systems, as well as the possible involvement of the sympathetic nervous system. Visceral pain responses were measured as the visceromotor response to colorectal distension (CRD) at baseline, immediately after WA and again 24 h later. The NK(1)R antagonists RP-67580 and SR-140333 and the CRF(1)R antagonist CP-154526 were injected 15 min before WA or 1 h before the CRD on day 2. Chemical sympathectomy was performed by repeated injection of 6-hydroxydopamine. WA stress resulted in a significant increase in the visceromotor response on day 2, but no change immediately after WA. Injection of CP-154526 abolished delayed SICH when applied either before WA stress or before the CRD on day 2. Both NK(1)R antagonists only decreased SICH when injected before the CRD on day 2. Chemical sympathectomy did not affect delayed SICH. Our results indicate that in male Wistar rats, both NK(1)R and CRF(1)R activation, but not sympathetic nervous system activation, play a role in the development of SICH.

N-methyl-D-aspartate (NMDA)receptors (NMDARs) located on peripheral terminals of primary afferents are involved in the transduction of noxious mechanical stimuli. Exploiting the fact that both NMDARs and stretch-activated channels are retained in short-term culture and expressed on the soma of dorsal root ganglia (DRG) neurons, we examined the effect of NMDA on mechanically mediated changes in intracellular calcium concentration ([Ca2+]i). Our aims were to determine whether NMDARs modulate the mechanosensitivity of DRG neurons. Primary cultures of adult rat lumbosacral DRG cells were cultured for 1-3 days. [Ca2+]i responses were determined by Fura-2 ratio fluorescence. Somas were mechanically stimulated with fire-polished glass pipettes that depressed the cell membrane for 0.5 s. Voltage-activated inward Ca2+ currents were measured by the whole cell patch clamp. Stimulation of neurons with 100 microM NMDA in the presence, but not the absence, of co-agonist (10 microM D-serine) caused transient [Ca2+]i responses (101+/-9 nM) and potentiated [Ca2+]i peak responses to subsequent mechanical stimulation more than two-fold (P < 0.001). NMDA-mediated potentiation of mechanically induced [Ca2+]i responses was inhibited by the selective protein kinase C (PKC) inhibitor GF109203X (GFX; 10 microM), which had no independent effects on NMDA- or mechanically induced responses. Short-term treatment with the PKC activator phorbol dibutyrate (1 microM PDBu for 1-2 min) also potentiated mechanically induced [Ca2+]i responses nearly two-fold (P < 0.001), while longer exposure (>10 min) inhibited the [Ca2+]i transients by 44% (P < 0.001). Both effects of PDBu were prevented by prior treatment with GFX. Inhibition of voltage-dependent Ca2+ channels with 25 microM La3+ had no effect on mechanically induced [Ca2+]i transients prior to NMDA, but prevented enhancement of the transients by NMDA and PDBu. NMDA pretreatment transiently enhanced nifedipine-sensitive, voltage-activated Ca2+ currents by a process that was sensitive to GFX. In conclusion, activation of NMDARs on cultured DRG neurons sensitize voltage-dependent L-type Ca2+ channels which contribute to mechanically induced [Ca2+]i transients through a PKC-mediated process.

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.

Both irritable bowel syndrome and inflammatory bowel diseases share symptoms of altered bowel habits associated with abdominal pain or discomfort. Irritable bowel syndrome has been referred to as a functional bowel disorder, which is diagnosed by a characteristic cluster of symptoms in the absence of detectable structural abnormalities. Inflammatory bowel disease is a heterogeneous group of disorders characterized by various forms of chronic mucosal and/or transmural inflammation of the intestine. In this review, the authors discuss recent evidence suggesting several potential mechanisms that might play a pathophysiologic role in both syndromes. Possible shared pathophysiologic mechanisms include altered mucosal permeability, an altered interaction of luminal flora with the mucosal immune system, persistent mucosal immune activation, alterations in gut motility, and a role of severe, sustained life stressors in symptom modulation. It is proposed that similarities and differences between the two syndromes can best be addressed within the framework of interactions between the central nervous system and the gut immune system. Based on recent reports of low-grade mucosal inflammation in subpopulations of patients meeting current diagnostic criteria for irritable bowel syndrome, therapeutic approaches shown to be effective in inflammatory bowel disease, such as probiotics, antibiotics, and antiinflammatory agents, have been suggested as possible therapies for certain patients with irritable bowel syndrome.

N-methyl-D-aspartate (NMDA) receptors in sensory afferents participate in chronic pain by mediating peripheral and central sensitization. We studied the presence of NMDA receptor subunits in different types of primary afferents. Western blots indicated that rat dorsal root ganglia (DRG) contain NR1, NR2B, NR2C, and NR2D but not NR2A. Real-time RT-PCR showed that NR2B and NR2D were expressed at higher levels than NR2A and NR2C in DRG. Immunofluorescence with an antibody that recognized NR1 and another that recognized NR2A and NR2B showed that NR1 and NR2B colocalized in 90% of DRG neurons, including most A-fibers (identified by the presence of neurofilament 200 kDa). In contrast, an antibody recognizing NR2C and NR2D labeled only neurofilament-negative DRG profiles. This antibody stained practically all DRG cells that contained calcitonin gene-related peptide and neurokinins and those that bound isolectin B4. The percentage of cells immunoreactive for NR1, NR2A/NR2B, and NR2C/NR2D were the same in the T9, T12, L4, and L6 DRG. The intracellular distribution of the NR2 subunits was strikingly different: Whereas NR2A/NR2B immunoreactivity was found in the Golgi apparatus and occasionally at the plasma membrane, NR2C/NR2D immunoreactivity was found in the cytoplasm but not in the Golgi. The NR1 subunit was present throughout the cytoplasm and was more intense in the Golgi. These findings indicate that DRG neurons have two different NMDA receptors, one containing the NR1, NR2D, and possibly the NR2C subunits, found only in C-fibers, and the diheteromer NR1/NR2B, present in the Golgi apparatus of both A- and C-fibers., (Copyright 2002 Wiley-Liss, Inc.)

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.

Nitric oxide (NO) can have opposite effects on peripheral sensory neuron sensitivity depending on the concentration and source of NO, and the experimental setting. The aim of this study was to determine the role of endogenous NO production in the regulation of mechanosensitive Ca(2+) influx of dorsal root ganglion (DRG) neurons. Adult mouse DRG neurons were grown in primary culture for 2-5 days, loaded with Fura-2, and tested for mechanically mediated changes in [Ca(2+)](i) by fluorescent ratio imaging. In the presence of the NOS inhibitors L-NAME, TRIM, or 7-NI, but not the inactive analogue D-NAME, peak [Ca(2+)](i) transients to mechanical stimulation were increased more than 2-fold. Neither La(3+) (25 microM), an inhibitor of voltage activated Ca(2+) channels, or tetrodotoxin (TTX, 1 microM), a selective inhibitor of voltage-gated Na(+) channels, had an effect on mechanically activated [Ca(2+)](i) transients under control conditions. However, in the presence of L-NAME, both La(3+) and TTX partially blocked the [Ca(2+)](i) response. Addition of Gd(3+), a blocker of mechanosensitive cation channels and L-type Ca(2+) channels, at a concentration (100 microM) that markedly inhibited the mechanical response under control conditions, only partially inhibited the response in the presence of L-NAME. The combination of either La(3+) or TTX with Gd(3+) caused near complete inhibition of mechanically stimulated [Ca(2+)](i) transients in the presence of L-NAME. We conclude that focal mechanical stimulation of DRG neurons causes Ca(2+) influx occurs primarily through mechanosensitive cation channels under control conditions. In the presence of NOS inhibitors, additional Ca(2+) influx occurs through voltage-sensitive Ca(2+) channels. These results suggest that endogenously produced NO in cultured DRG neurons decreases mechanosensitivity by inhibiting voltage-gated Na(+) and Ca(2+) channels.

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.

Background & Aims: Intestinal epithelial cell apoptosis occurs continually without apparent permeability defects and is increased in response to intestinal inflammation. We hypothesized that increased, immune-mediated apoptosis during inflammation might result in barrier dysfunction of the epithelium., Methods: T84 cells were cultured as a polarized monolayer and exposed to agonist antibody to Fas. Barrier function was assessed by transepithelial resistance and permeability measurements. Immunofluorescent staining was used to examine junctional protein expression., Results: Fas expression is predominantly basolateral in polarized T84 monolayers. Basolateral cross-linking of the Fas receptor resulted in T84 cell apoptosis and a loss of 50% of the cells within 24 hours. Apoptosis was coincident with a decrease in transepithelial electrical resistance and increased flux of small but not large molecules. Preservation of barrier function was associated with dramatic rearrangement of tight junctions and desmosomal junctions in apoptotic monolayers. E-cadherin-mediated cell contact was maintained between intact cells in the monolayer, thus sealing gaps created by apoptotic cells. Apoptosis and barrier dysfunction could be prevented by caspase inhibition., Conclusions: Immune-mediated apoptosis of intestinal epithelial cells may contribute to the permeability defects associated with inflammatory conditions of the bowel, but the intestinal epithelium is remarkably resilient in the face of apoptosis.

Regulation of cytosolic Ca(2+) is important for a variety of cell functions. The ryanodine receptor (RyR) is a Ca(2+) channel that conducts Ca(2+) from internal pools to the cytoplasm. To demonstrate the presence of the RyR in the pancreatic acinar cell, we performed reverse transcriptase (RT)-PCR, Western blot, immunocytochemistry and microscopic Ca(2+)-release measurements on these cells. RT-PCR showed the presence of mRNA for RyR isoforms 1, 2 and 3 in both rat pancreas and dispersed pancreatic acini. Furthermore, mRNA expression for RyR isoforms 1 and 2 was demonstrated by RT-PCR in individual pancreatic acinar cells selected under the microscope. Western-blot analysis of acinar cell immunoprecipitates, using antibodies against RyR1 and RyR2, showed a high-molecular-mass (>250 kDa) protein band that was much less intense when immunoprecipitated in the presence of RyR peptide. Functionally, permeablized acinar cells stimulated with the RyR activator, palmitoyl-CoA, released Ca(2+) from both basolateral and apical regions. These data show that pancreatic acinar cells express multiple isoforms of the RyR and that there are functional receptors throughout the cell.

The aim of this study was to characterize plasma membrane pathways involved in the intracellular calcium ([Ca(2+)](i)) response of small DRG neurons to mechanical stimulation and the modulation of these pathways by kappa-opioids. [Ca(2+)](i) responses were measured by fluorescence video microscopy of Fura-2 labeled lumbosacral DRG neurons obtained from adult rats in short-term primary culture. Transient focal mechanical stimulation of the soma, or brief superfusion with 300 nM capsaicin, resulted to [Ca(2+)](i) increases which were abolished in Ca(2+)-free solution, but unaffected by lanthanum (25 microM) or tetrodotoxin (10(-6) M). 156 out of 465 neurons tested (34%) showed mechanosensitivity while 55 out of 118 neurons (47%) were capsaicin-sensitive. Ninty percent of capsaicin-sensitive neurons were mechanosensitive. Gadolinium (Gd(3+); 250 microM) and amiloride (100 microM) abolished the [Ca(2+)](i) transient in response to mechanical stimulation, but had no effect on capsaicin-induced [Ca(2+)](i) transients. The kappa-opioid agonists U50,488 and fedotozine showed a dose-dependent inhibition of mechanically stimulated [Ca(2+)](i) transients but had little effect on capsaicin-induced [Ca(2+)](i) transients. The inhibitory effect of U50,488 was abolished by the kappa-opioid antagonist nor-Binaltorphimine dihydrochloride (nor-BNI; 100 nM), and by high concentrations of naloxone (30-100 nM), but not by low concentrations of naloxone (3 nM). We conclude that mechanically induced [Ca(2+)](i) transients in small diameter DRG somas are mediated by influx of Ca(2+) through a Gd(3+)- and amiloride-sensitive plasma membrane pathway that is co-expressed with capsaicin-sensitive channels. Mechanical-, but not capsaicin-mediated, Ca(2+) transients are sensitive to kappa-opioid agonists.

The mechanisms underlying intracellular Ca2+ waves induced by either mechanical or receptor-mediated stimulation of myocytes isolated from the longitudinal muscle layer of the rabbit distal colon were compared using fura 2 and fluorescence videomicroscopy. Light focal mechanical deformation of the plasma membrane or focal application of substance P resulted in localized intracellular Ca2+ concentration ([Ca2+]i) transients that propagated throughout the cell. In both cases, the Ca2+ response consisted of a transient peak response followed by a delayed-phase response. Substance P-mediated [Ca2+]i responses involved generation of inositol 1,4, 5-trisphosphate and release of Ca2+ from thapsigargin-sensitive stores, whereas mechanically induced responses were partially (29%) dependent on La3+-sensitive influx of extracellular Ca2+ and partially on release of intracellular Ca2+ from thapsigargin-insensitive stores gated by ryanodine receptors. The delayed-phase response in both cases was dependent on extracellular Ca2+. However, although the response to substance P was sensitive to La3+, that after mechanical stimulation was not. In the later case, the underlying mechanism may involve capacitative Ca2+ entry channels that are activated after mechanical stimulation but not by substance P.

In gastrointestinal smooth muscle, intracellular Cl- is maintained at levels higher than its electrochemical equilibrium. Therefore, Cl- efflux through receptor-mediated opening of Cl- channels should result in membrane depolarization and may be sufficient to activate voltage-sensitive calcium channels (VSCCs). To determine the contribution of Cl- channels to receptor-mediated contraction of the longitudinal muscle layer of the rabbit distal colon, we studied the mechanical response of muscle strips to substance P, carbachol and potassium depolarization following the depletion of Cl- i, and in the presence of the Cl- channel blocker 5-nitro-2-(3-phenylpropyl-amino)-benzoate (NPPB). A 60-min incubation of tissues in a HEPES-buffered solution in which NaCl had been replaced by Na isethionate (or Na gluconate) in equimolar amounts resulted in disappearance of phasic contractions, and in a partially reversible reduction of the tonic response to substance P and carbachol, but not to KCl depolarization. When the agonist was applied to tissues in control solution, or to Cl-(-)depleted tissues in a solution in which Na+ was acutely replaced in equimolar amounts by N-methyl-D-glucosamine, the mechanical response to substance P and carbachol was almost abolished. Acute Na+ replacement alone without prior Cl- depletion did not abolish phasic contractions, but reduced the tonic response to substance P and carbachol. Similar to the effect of Cl- depletion, incubation of tissues in NPPB (6.6 x 10(-5) M) reduced the tonic response to substance P and carbachol, and abolished phasic contractions. These findings are consistent with a contribution of a Cl- channel to the receptor-mediated activation of colonic smooth muscle. In addition, the data suggest that transient Cl- channel mediated depolarizations may play a role in the generation of phasic contractions.

Recent studies indicate increased insulin-like growth factor I (IGF-I) expression and altered expression of IGF binding proteins (IGFBP) in the bowel during experimental colitis. This study analyzes the cellular sites of altered IGF-I and IGFBP-expression in large bowel of rats with experimental colitis. Colitis was induced by colonic instillation of 2, 4, 6-trinitrobenzenesulfonic (TNB) acid in ethanol. Animals were sacrificed at 7 days after induction of colitis. Cryostat sections of colon from TNB-treated and control rats were hybridized with 35S-labeled antisense probes for IGF-I, IGFBP-3, IGFBP-4 and IGFBP-5. IGF-I mRNA was up-regulated in lamina propria cells, submucosa and smooth muscle of inflamed colon. IGFBP-3 mRNA was localized to lamina propria and was down-regulated in inflamed colon. IGFBP-4 and IGFBP-5 mRNAs were both up-regulated in inflamed colon. IGFBP-4 mRNA was increased in lamina propria, submucosa and smooth muscle, whereas IGFBP-5 mRNA was increased in smooth muscle. Increased IGF-I expression in mesenchymal layers of colon during experimental colitis supports the hypothesis that IGF-I contributes to hyperplasia and fibrosis in response to inflammation. Altered expression of IGFBP-3, IGFBP-4 and IGFBP-5 in specific bowel layers during colitis suggests that they play a role in modulating IGF-I action.

The regulation of cytosolic Ca2+ is important for a variety of cell functions. One non-inositol 1,4,5-trisphosphate (IP3) compound that may regulate Ca2+ is palmitoyl-coenzyme A (CoA), a fatty acid-CoA that is reported to cause Ca2+ release from intracellular stores of oocytes, myocytes, and hepatocytes. To study the role of palmitoyl-CoA in the pancreatic acinar cell, rat pancreatic acini were isolated by collagenase digestion, permeablized with streptolysin O, and the release of Ca2+ from internal stores was measured with fura-2. Palmitoyl-CoA released Ca2+ from internal stores (EC50 = 14 microM). The palmitoyl-CoA-sensitive pool was distinct from, and overlapping with the IP3-sensitive Ca2+ pool. The effects of submaximal doses of IP3 or cyclic ADP-ribose plus palmitoyl-CoA were additive. Fatty acid-CoA derivatives with carbon chain lengths of 16-18 were the most potent and efficacious. Ryanodine and caffeine or elevated resting [Ca2+] sensitized the Ca2+ pool to the actions of palmitoyl-CoA. Fatty acid-CoA levels in pancreatic acini were measured by extraction with 2-propanol/acetonitrile, followed by separation and quantification using reverse phase high performance liquid chromatography, and were found to be 10.17 +/- 0.93 nmol/mg protein. These data suggest the presence of an IP3-insensitive palmitoyl-CoA-sensitive Ca2+ store in pancreatic acinar cells and suggest that palmitoyl-CoA may be needed for Ca2+-induced Ca2+ release.

Background and Aim: Epidermal growth factor (EGF) and transforming growth factor alpha (TGF-alpha), members of the EGF family of growth factors, protect rat gastric and colonic mucosa against injury. Having shown previously that exogenously applied EGF protects rat colonic mucosa against injury, the aim of the present study was to evaluate the endogenously expressed ligand mediating the protective effect of EGF/TGF-alpha in vivo., Methods: In an experimental model of trinitrobenzene sulphonic acid (TNBS)/ethanol induced colitis in rats EGF and TGF-alpha expression was evaluated using a ribonuclease protection assay, northern blot analysis, western blot analysis, and immunohistochemistry., Results: TGF-alpha mRNA increased 3-4 times at 4-8 hours after induction of colitis and returned to control levels within 24 hours. TGF-alpha immunoreactive protein with a molecular size of about 28 kDa representing TGF-alpha precursors increased markedly after induction of colitis with a peak at 8-12 hours. No fully processed 5.6 kDa TGF-alpha protein was detected in normal or inflamed colon tissue. Only a weak signal for EGF mRNA expression was detected in the rat colon and no EGF protein was observed by immunohistochemistry or western blot analysis., Conclusions: TGF-alpha precursors are the main ligands for the EGF receptor in acute colitis. It is hypothesised that TGF-alpha precursors convey the biological activity of endogenous TGF-alpha peptides during mucosal defence and repair.

Cholecystokinin (CCK)-A and CCK-B receptors are highly homologous members of the seven transmembrane domain G-protein-coupled receptor superfamily. Genes of both receptors contain five exons and share a similar exon-intron organization. To determine the structural basis of CCK-A receptor (CCK-AR) functionally coupled to Gs, a series of chimeric mutants were constructed by replacing exons of human CCK-B receptor (CCK-BR), from the second to the fifth (last) exon, with human CCK-AR counterparts. Binding and signal transduction properties of wild-type and chimeric receptors were examined in stably transfected HEK-293 cells. Chimeric receptors that maintained high affinity binding to CCK exhibited dose-dependent increases in intracellular calcium mobilization similar to both wild-type receptors. However, only the wild-type CCK-AR and chimeric mutants containing the second exon of CCK-AR were able to mediate significantly greater increases in intracellular cAMP content and adenylyl cyclase activity compared with wild-type CCK-BR. A CCK-BR mutant was further constructed by replacing five amino acids, Gly-Leu-Ser-Arg-(Arg)-Leu, in the first intracellular loop with the corresponding five CCK-AR specific amino acids, Ile-Arg-Asn-Lys-(Arg)-Met. The resultant receptor maintained high affinity binding to both CCK and gastrin and dose-dependent calcium responses similar to wild-type CCK-BR. However, this first intracellular loop mutant also gained positive cAMP responses to both sulfated CCK-8 and gastrin-17 with EC50 values of 8.5 +/- 1 nM and 23 +/- 7 nM, respectively. These data suggest that the first intracellular loop of CCK-AR is essential for coupling to Gs and activation of adenylyl cyclase signal transduction cascade.

We recently demonstrated upregulation of insulin-like growth factor I (IGF-I) binding sites in the smooth muscle layer of inflamed rat colon. The increase in binding sites was due to increased expression of IGF binding proteins (IGFBPs), which modulate the effects of IGF. To further study the role of IGF in the colon, we investigated whether cultured colonic smooth muscle cells (SMC) express IGF-I receptors and IGFBPs. SMC were isolated by collagenase digestion from rat colonic smooth muscle and grown in primary culture. Equilibrium binding experiments using (125)I-labeled IGF-I showed the presence of an IGF-I receptor with a dissociation constant of 1.96 nM and a maximal binding constant of 53,000 receptors/cell. Competition binding studies with IGF-II and insulin, together with chemical cross-linking experiments, corroborated this conclusion. Western ligand blotting of conditioned medium and Northern analysis of total RNA demonstrated that the cells expressed and secreted IGFBP-4, -5, and -3 with molecular masses of 25, 31, and 45 kDa, respectively. These results, together with our in vivo studies in the rat, support a role for IGF in tissue fibrosis and stricture formation during chronic intestinal inflammation.

Background/aims: The gastrointestinal tract is a major target of insulinlike growth factor (IGF) I. IGF-I binds to two different receptors and to binding proteins (IGFBPs), which act as carriers and mediators. This study investigated the regulation of IGF-I binding sites in rat colitis., Methods: Colitis was induced by colonic instillation of 2,4,6-trinitrobenzenesulfonic acid in ethanol. IGF-I binding sites in colon sections were localized by incubation with 125I-IGF-I. The contribution of binding to the IGF-I receptor was estimated by competition with unlabeled IGF-I, IGF-II, and insulin. Colonic RNA was screened for IGFBPs by Northern hybridization., Results: IGF-I binding sites were increased more than two-fold in the muscularis propria of inflamed colon as soon as 12 hours and up to 1 week after injury. Insulin could not displace this elevated level of binding, even though it could displace IGF-I from the mucosa and muscularis mucosa. Northern hybridization showed a 2-3-fold increase in IGFBP-4 and IGFBP-5 messenger RNA from inflamed colon., Conclusions: Experimental colitis in rats causes an increase in IGF-I binding to the muscularis propria, which represents increased levels of IGFBP-4 and IGFBP-5. These data suggest an important role for IGFBPs in modulating IGF effects during inflammation and tissue repair.

We previously showed that insulin and insulin-like growth factor (IGF)-I and IGF-II caused a dose-dependent increase in permeability through the paracellular pathway of T84 cell monolayers over 3-4 days. Here we have determined which cell surface receptors were involved in this response. Using radioligand binding studies and receptor cross-linking studies, we found that T84 cells possess insulin and IGF-I receptors. There were approximately 20 x 10(3) insulin receptors/cell with a dissociation constant (KD) of 0.5 nM and 29 x 10(3) IGF-I receptors with a KD of 0.6 nM for IGF-I. Cross-linking studies identified the alpha-subunit of insulin and IGF-I receptors with deduced molecular weights of 126 x 10(3) and 128 x 10(3), respectively. IGF-II bound to T84 cells with an apparent KD of approximately 2.0 nM. Radioreceptor cross-linking indicated that IGF-II interacted principally with the IGF-I receptor, although low levels of the IGF-II/mannose 6-phosphate receptor were also expressed on the cell surface. We then correlated the biological effect with the radioligand binding studies. It was first demonstrated that insulin and IGF-I were degraded in medium in the presence of cells. In addition, we showed that continuous exposure for 2-3 days to insulin or IGF-I was required to produce their biological effect on permeability. Taking into account the rate of degradation and the requirement for continuous exposure, we found a close correlation between radioligand binding and the half-maximal effective concentration for the hormonal effects on transepithelial permeability.(ABSTRACT TRUNCATED AT 250 WORDS)

Previous studies have shown that insulin, insulin-like growth factors, and interferon-gamma cause an increase the paracellular permeability across T84 human colonic epithelial cell monolayers. The striking similarity in the time course and the structural changes in the actin cytoskeleton prompted us to test whether these factors influenced the paracellular permeability by a common mechanism. T84 cell monolayers were grown in variously modified media, and the effect of all three factors was tested by measuring the conductance of monolayers mounted in Ussing chambers. Media additions or deletions that modify cellular carbohydrate metabolism markedly influenced the response to all these peptides. In particular, the increase in conductance produced by each of the three peptides was 1) inhibited by addition of short-chain fatty acids, principally n-butyrate and propionate; 2) attenuated by the replacement of media glucose with slowly metabolized and nonmetabolized sugars; 3) potentiated by decreasing the media iron concentration; and 4) inhibited by increasing the media iron concentration. The effects of short-chain fatty acids, glucose, and iron were all shown to be dose dependent. In addition, the ability of high media iron to prevent the increase in permeability caused by all three factors was shown to require ambient oxygen. Together, these results strongly suggest that insulin, insulin-like growth factors, and interferon-gamma increase the permeability across T84 cell monolayers through a common mechanism that is modulated by glucose-derived metabolites and oxidative metabolism.

Background/aims: The role of epidermal growth factor (EGF) in the maintenance of mucosal integrity in the lower gastrointestinal tract is unknown. The aim of this study was to determine the effect of EGF in experimental colitis., Methods: Colitis was induced with 2,4,6-trinitrobenzenesulfonic acid/ethanol enemas. Rats were pretreated with intraperitoneal administration of recombinant human EGF (600 micrograms/kg) or vehicle 1 hour before induction of colitis and daily thereafter until killed at 8 hours, 48 hours, and 1 week. A separate group received an identical dosage and administration of EGF or vehicle for 1 week with treatment initiated 24 hours after the induction of colitis. Colonic tissue was evaluated macroscopically, histologically, and for myeloperoxidase activity., Results: Pretreatment with EGF reduced microscopic erosions at 8 and 48 hours by 74% and 54%, respectively (P < 0.05). At 1 week, microscopic ulcerations and myeloperoxidase activity were reduced by 65% in the EGF-pretreated group (P < 0.05). No significant difference in macroscopic injury, histological damage, or myeloperoxidase activity was noted when EGF treatment was initiated after the induction of colitis., Conclusions: Systemic EGF administration reduces mucosal damage and inflammation in a trinitrobenzenesulfonic acid/ethanol model of colitis in rats through a mechanism involving mucosal protection.

Background/aims: The lack of pure, proliferative, but not transformed intestinal epithelial cells has impeded progress in understanding their role in chronic intestinal inflammation. To clarify that role, the present study characterized the epithelial cell line VNCC, derived from normal adult dog distal colon., Methods: Cells were cultured on plastic and permeable supports for analysis of eicosanoid production (by radioimmunoassay and high-performance liquid chromatography) and transport characteristics (by Ussing chamber short-circuit determinations)., Results: In culture, VNCC formed confluent monolayers and domes, suggesting formation of tight junctions and active solute absorption. When cultured on permeable supports, VNCC developed modest, but variable, transepithelial resistances (563 +/- 94 omega/cm2) with a spontaneous short-circuit current of 5.0 +/- 0.4 microA/cm2. Forskolin caused a prolonged increase in the short-circuit current, inhibited by amiloride but not bumetanide, suggesting that VNCC display 5'-cyclic adenosine monophosphate-stimulated sodium absorption. VNCC incubated with arachidonic acid released a variety of eicosanoids including 6-keto-prostaglandin (PG)F1 alpha, PGE2, thromboxane B2, and PGF2 alpha, but no hydroxyarachidonate metabolites. Bradykinin stimulated VNCC eicosanoid release., Conclusions: The ability of VNCC to divide and differentiate in culture, to form polarized monolayers capable of active sodium absorption, and to respond to inflammatory mediators with eicosanoid release makes them a unique tool for the study of the interactions of inflammation on colonocyte function.

Epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) are two in a family of growth-promoting peptides for many gastrointestinal epithelia. This study was designed to assess their mitogenic effect on cultured gastric myocytes and to characterize specific EGF receptors on these cells. Single myocytes were isolated from newborn rabbit gastric fundus and placed into tissue culture. The composition of the culture at confluence as assessed by immunostaining with smooth muscle actin-specific monoclonal antibody (CGA7) was > 95% myocytes. To assess the effect of putative growth factors, freshly isolated myocytes were incubated in Dulbecco's modified Eagle's (DME) medium containing 1% fetal bovine serum in the presence or absence of growth factors. After 6 days, cells were incubated in serum-free medium with [3H]thymidine (1 microCi/ml) in the continued presence or absence of growth factors. After 24 h, EGF and TGF-alpha but not insulin-like growth factor I (IGF-I) induced a dose-dependent increase in [3H]thymidine incorporation. Ten nanomolar EGF or TGF-alpha increased [3H]thymidine incorporation more than sixfold over control. EGF was more potent than was TGF-alpha, with apparent median effective dose (ED50) values of 64 +/- 14 pM and 166 +/- 62 pM (p < 0.05), respectively. EGF bound to cultured myocytes with Kd = 7.6 +/- 1.8 nM and Bmax = 27 +/- 11 pmol/mg DNA or 440,000 receptors/cell. TGF-alpha competed for binding at these receptors. Although IGF-I did not stimulate thymidine incorporation, specific high-affinity receptors for IGF-I were detected on gastric myocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

The receptor-binding properties of isolated rabbit colonic circular smooth muscle cells in primary culture have been investigated. In intact smooth muscle, acetylcholine, acting through M2 muscarinic receptors, and vasoactive intestinal polypeptide (VIP), acting through VIP receptors, are two of the principal neurotransmitters mediating contraction and relaxation, respectively. The muscarinic receptor was present in very high levels (600,000 receptors/cell) on freshly isolated colonic smooth muscle cells as shown by binding of the muscarinic receptor antagonist N-methylscopolamine (NMS). However, NMS binding sites decreased rapidly when the cells were placed in primary culture. After 21 h in culture, specific binding of [3H]NMS decreased to 20%, and after 48 h to less than 10% that of preculture values. This loss was not associated with a change in receptor affinity, since Kd was unchanged for the receptors still present. In contrast, high-affinity VIP receptors were expressed on cultured smooth muscle cells but could not be detected on freshly isolated cells. Cultured cells responded to VIP with an increase in intracellular adenosine 3',5'-cyclic monophosphate (cAMP), indicating that the VIP receptors were functionally coupled to adenylate cyclase. Cultured cells also responded to calcitonin gene-related peptide (CGRP) and forskolin with increased production of intracellular cAMP. In contrast, neither VIP nor CGRP elicited an increase in intracellular cAMP when added to freshly isolated cells. Furthermore, freshly isolated cells had a greatly diminished response to forskolin, suggesting that the isolation procedure not only destroyed cell surface receptors for VIP and CGRP, but also damaged the cells sufficiently to decrease cellular adenylate cyclase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Sugar absorption by the biliary ductular epithelium under steady-state conditions was examined using isolated perfused rat liver. The test sugar and mannitol (as a putative marker of paracellular entry) were added to the glucose-free recirculating perfusate each at a concentration of 5 mmol/L, and apparent active biliary ductular absorption equated with the change in concentration of the test sugar relative to that of mannitol. A metabolizable hexose (D-glucose), pentose (D-xylose), and three nonmetabolizable hexoses (alpha-methyl-glucoside, 3-o-methyl-glucose, and L-glucose) were used. All five monosaccharides were well absorbed at constant rates for 2 hours with apparent rates of absorption (mumol.kg body weight-1.min-1, mean +/- SE) of D-glucose, 0.24 +/- 0.01; L-glucose, 0.20 +/- 0.02; 3-o-methyl-glucose, 0.19 +/- 0.02; alpha-methyl-glucoside, 0.16 +/- 0.03; and D-xylose, 0.10 +/- 0.04. The addition of phloridzin to the perfusate inhibited D-glucose absorption in part but did not inhibit L-glucose absorption. When perfusate Na+ was replaced by N-methylglucamine, the bile-plasma ratio of mannitol remained unchanged, as did the apparent absorption rate of D-glucose and 3-o-methyl-glucose. In contrast, absorption of L-glucose and alpha-methyl-D-glucoside gradually ceased. The addition of 15 mmol/L glucose to the perfusate caused decreased bile flow and increased taurocholate concentration in bile, suggesting that glucose absorption by the biliary ductules induced water reabsorption. It is concluded that sugars are absorbed by the biliary ductular system by Na(+)-dependent and Na(+)-independent transport systems, the substrate affinities of which differ from those reported for apical membrane hexose transport systems in renal tubular and intestinal epithelia. Ductular absorption of solutes such as glucose that enter bile passively may have biological use, because ductular absorption decreases the concentration of substrates for bacterial growth in gallbladder bile. On the other hand, ductular absorption of solutes induces reabsorption of biliary water, resulting in decreased bile flow; this might contribute to cholestasis during prolonged hyperalimentation with solutions containing glucose.

We used radioligand binding to tissue homogenates and isometric contraction of muscle strips to characterize the substance P (SP) receptor on gastric smooth muscle from 1- (newborn) and 7-day-old and 4- and 11-wk-old (weanling) rabbits. Scatchard analysis for newborns was curvilinear, suggesting the presence of multiple binding sites. In newborns the dissociation constant (Kd) of high-affinity binding site was 2.2 +/- 0.3 nM, and the maximum binding (Bmax) was 0.57 +/- 0.06 pmol/mg DNA. The number of high-affinity binding sites decreased with age, disappearing by 11 wk. The Kd for the low-affinity site was more than two orders of magnitude greater than that of the high-affinity site. In competitive binding studies with [3H]SP, the order of potency for the neurokinins was SP much greater than neurokinin A (NKA) greater than neurokinin B (NKB), suggesting that the high-affinity binding sites were NK-1 receptors. [125I]NKA is also bound to newborn tissue homogenate with high affinity. With [125I]NKA the order was NKA greater than SP greater than NKB, suggesting that NK-2 receptors were also present. In contraction studies, atropine and tetrodotoxin had no effect on tachykinin-stimulated contraction, suggesting solely myogenic tachykinin effects on this tissue. In newborn rabbits, the potency and efficacy of SP and NKA were similar. The half-maximal effective dose (ED50) of SP was nearly two orders of magnitude less in newborn rabbits than in weanlings; the potency of NKA did not change.(ABSTRACT TRUNCATED AT 250 WORDS)

When grown on permeable supports, the T84 human colonic epithelial cell line forms polarized monolayer cultures with high-resistance tight junctions between adjacent cells. Addition of either insulin-like growth factor (IGF) I or II to the basolateral but not the apical membrane side of established monolayers caused a dose-dependent decrease in transepithelial resistance over a 4-day period. IGF-I was more potent than IGF-II, with half-maximally effective concentrations of 0.7 and 2.2 nM, respectively. Both IGF-I and -II caused a parallel increase in the transepithelial flux rates for Na+ and the extracellular space marker, mannitol, demonstrating that the decrease in electrical resistance was due to increased permeability through the tight junction-regulated paracellular pathway. Simultaneous addition of cycloheximide prevented the decline in electrical resistance, implying that protein synthesis is necessary for the effect of IGF on paracellular permeability. Treatment of monolayers with IGF produced a subtle condensation of the perijunctional actin ring as visualized using rhodamine-labeled phalloidin. These results demonstrate that IGF-I and -II regulate the paracellular permeability of T84 cell monolayers through a receptor-mediated process that probably involves changes in protein synthesis and cytoskeletal structure.