John H. Winston

Nerve growth factor regulates VR-1 mRNA levels in cultures of adult dorsal root ganglion neurons.

&NA; Nerve growth factor (NGF) regulates the nociceptive properties including sensitivity to capsaicin of a subset of dorsal root ganglion neurons, which express the high‐affinity NGF receptor, trkA. Capsaicin sensitivity co‐localizes with the expression of a cloned capsaicin receptor, vanilloid receptor type 1 (VR‐1), which displays properties similar to the native capsaicin response. To determine whether VR‐1 mRNA levels are regulated by NGF, VR‐1 mRNA levels and the ability to respond to capsaicin by release of the neuropeptide calcitonin gene related peptide (CGRP) were measured as a function of NGF concentration in cultures of adult dorsal root ganglion neurons. NGF treatment increased both VR‐1 mRNA expression and capsaicin evoked release of CGRP. These effects were inhibited by treatment with the trkA inhibitor k252a.

P2X receptor-mediated visceral hyperalgesia in a rat model of chronic visceral hypersensitivity

Background: Irritable bowel syndrome (IBS) is a common gastrointestinal disorder characterised by abdominal pain and bloating in association with altered bowel movements. Its pathogenesis and the underlying molecular mechanisms of visceral hyperalgesia remain elusive. Recent studies of somatic and other visceral pain models suggest a role for purinergic signalling mediated by the P2X receptor (P2XR) family. Aims: To examine the role of P2XR signalling in the pathogenesis in a rat model of IBS-like visceral hyperalgesia. Methods: Visceral hypersensitivity was induced by colonic injection of 0.5% acetic acid (AA) in 10-day-old rats and experiments were conducted at 8 weeks of age. Dorsal root ganglion (DRG) neurons innervating the colon were labelled by injection of DiI (1,1′-dioleyl-3,3,3′,3-tetramethylindocarbocyanine methanesulfonate) fluorescence into the colon wall. Results: Visceral hypersensitivity was reversed by TNP-ATP (2′-(or-3′)-O-(trinitrophenyl) ATP), a potent P2X1, P2X3 and P2X2/3 receptor antagonist. Rapid application of ATP (20 μM) induced a fast inactivating current in colon-specific DRG neurons from both control and AA-treated rats. There was a twofold increase in the peak ATP responses in neurons from AA-treated rats. These currents were sensitive to TNP-ATP (100 nM). Under current-clamped conditions, ATP evoked a larger membrane depolarisation in neurons from neonatal AA-treated rats than in controls. P2X3R protein expression was significantly enhanced in colon-specific DRGs 8 weeks after neonatal AA treatment. Conclusions: These data suggest that the large enhancement of P2XR expression and function may contribute to the maintenance of visceral hypersensitivity, thus identifying a specific neurobiological target for the treatment of chronic visceral hyperalgesia.

Molecular and behavioral changes in nociception in a novel rat model of chronic pancreatitis for the study of pain

&NA; The approach to the management of painful chronic pancreatitis has been empirical, primarily due to the lack of information about biological mechanisms producing pain. To facilitate research into pain mechanisms, our aim was to assess a rat model of chronic pancreatitis induced by pancreatic infusion of trinitrobenzene sulfonic acid as a model of painful pancreatitis. Nociception was assessed by measuring mechanical sensitivity of the abdomen and by recording the number of nocifensive behaviors in response to electrical stimulation of the pancreas. Expression of neuropeptides calcitonin gene‐related peptide (CGRP) and substance P (SP) in the thoracic dorsal root ganglia receiving input from the pancreas and nerve growth factor (NGF) in the pancreas were measured. Rats with pancreatitis exhibited marked increase in sensitivity to mechanical probing of the abdomen and increased sensitivity to noxious electrical stimulation of the pancreas. There were significant increases in NGF protein in the pancreas and in expression of neuropeptides CGRP and SP in the sensory neurons from dorsal root ganglia receiving input from the pancreas. We have established quantitative measures of referred nociception and pancreatic hyperalgesia in a rat model of chronic pancreatitis that bears histological similarities to the human disease. This model has considerable construct, face and predictive validity for the human condition. It is of importance for the study of the pathogenesis of pain in this condition and can facilitate the development of new therapeutic options.

Adrenergic Stimulation Mediates Visceral Hypersensitivity to Colorectal Distension Following Heterotypic Chronic Stress

BACKGROUND & AIMS Chronic stress exacerbates or causes relapse of symptoms such as abdominal pain and cramping in patients with irritable bowel syndrome. We investigated whether chronic stress increases plasma norepinephrine and sensitizes colon-specific dorsal root ganglion (DRG) neurons by increasing expression of nerve growth factor (NGF) in the colon wall. METHODS Heterotypic chronic stress (HeCS) was applied to male Wistar rats and neurologic and molecular responses were analyzed. Tissues were analyzed for NGF expression. RESULTS HeCS significantly increased visceromoter response to colorectal distension; expression of NGF increased in colonic muscularis externa and mucosa/submucosa. Rheobase decreased, resting membrane potential was depolarized, and electrogenesis of action potentials increased in colon-specific thoracolumbar DRG neurons. Luminal administration of resiniferatoxin in distal colon, systemic administration of anti-NGF antibody, or inhibition of the NGF receptor trkA by k252a or antisense oligonucleotides in thoracolumbar DRG blocked the chronic stress-induced visceral hypersensitivity to colorectal distension. Blockade of alpha1/alpha2- and beta1/beta2-adrenergic receptors prevented the stress-induced visceral hypersensitivity and increased expression of NGF in the colon wall. HeCS did not induce any inflammatory response in the colon wall. CONCLUSIONS The peripheral stress mediator norepinephrine induces visceral hypersensitivity to colorectal distension in response to HeCS by increasing the expression of NGF in the colon wall, which sensitizes primary afferents in the absence of an inflammatory response.

Acute pancreatitis results in referred mechanical hypersensitivity and neuropeptide up-regulation that can be suppressed by the protein kinase inhibitor k252a ☆

Although pain is a cardinal feature of pancreatitis, its pathogenesis is poorly understood and treatment remains difficult. Nociceptive sensitization in several somatic pain models has been associated with activation of protein kinases including trkA, protein kinase C, and protein kinase A. We therefore tested the hypothesis that systemic treatment with a kinase inhibitor, k252a, known to inhibit all of these kinases would alleviate pain in an animal model of pancreatitis. Von Frey filament testing of somatic referral regions was evaluated as a method to measure referred pain in a rat model of acute necrotizing pancreatitis induced by L-arginine. Rats with pancreatitis showed increased sensitivity to abdominal stimulation with Von Frey filament. This referred mechanical sensitivity was associated with an 8-fold increase in levels of phosphorylated trkA in the pancreas and with significant up-regulation of both calcitonin gene-related peptide and preprotachykinin mRNA expression in thoracic dorsal root ganglia and with increased calcitonin gene-related peptide and substance P immunoreactivity in spinal cord segment T10. Treatment with the kinase inhibitor k252a suppressed the phosphorylation of trkA in the pancreas as well as reversed both the behavioral changes and the increase in neuropeptide expression associated with pancreatitis.

Diverse Genetic Regulatory Motifs Required for Murine Adenosine Deaminase Gene Expression in the Placenta

Murine adenosine deaminase (ADA) is a ubiquitous purine catabolic enzyme whose expression is subject to developmental and tissue-specific regulation. ADA is enriched in trophoblast cells of the chorioallantoic placenta and is essential for embryonic and fetal development. To begin to understand the genetic pathway controlling Ada gene expression in the placenta, we have identified and characterized a 770-base pair fragment located 5.4 kilobase pairs upstream of the Ada transcription initiation site, which directs reporter gene expression to the placenta of transgenic mice. The expression pattern of the reporter gene reflected that of the endogenous Ada gene in the placenta. Sequence analysis revealed potential binding sites for bHLH and GATA transcription factors. DNase I footprinting defined three protein binding regions, one of which was placenta-specific. Mutations in the potential protein binding sites and footprinting regions resulted in loss of placental expression in transgenic mice. These findings indicate that multiple protein binding motifs are necessary for Ada expression in the placenta.

Differential immune and genetic responses in rat models of Crohn's colitis and ulcerative colitis.

Crohns disease and ulcerative colitis are clinically, immunologically, and morphologically distinct forms of inflammatory bowel disease (IBD). However, smooth muscle function is impaired similarly in both diseases, resulting in diarrhea. We tested the hypothesis that differential cellular, genetic, and immunological mechanisms mediate smooth muscle dysfunction in two animal models believed to represent the two diseases. We used the rat models of trinitrobenzene sulfonic acid (TNBS)- and dextran sodium sulfate (DSS)-induced colonic inflammations, which closely mimic the clinical and morphological features of Crohns disease and ulcerative colitis, respectively. DSS inflammation induced oxidative stress initially in mucosa/submucosa, which then propagated to the muscularis externa to impair smooth muscle function. The muscularis externa showed no increase of cytokines/chemokines. On the other hand, TNBS inflammation almost simultaneously induced oxidative stress, recruited or activated immune cells, and generated cytokines/chemokines in both mucosa/submucosa and muscularis externa. The generation of cytokines/chemokines did not correlate with the recruitment and activation of immune cells. Consequently, the impairment of smooth muscle function in DSS inflammation was primarily due to oxidative stress, whereas that in TNBS inflammation was due to both oxidative stress and proinflammatory cytokines. The impairment of smooth muscle function in DSS inflammation was due to suppression of Gα(q) protein of the excitation-contraction coupling. In TNBS inflammation, it was due to suppression of the α(1C)1b subunit of Ca(v)1.2b channels, CPI-17 and Gα(q). TNBS inflammation increased IGF-1 and TGF-β time dependently in the muscularis externa. IGF-1 induced smooth muscle hyperplasia; both IGF-1 and TGF-β induced hypertrophy. In conclusion, both TNBS and DSS induce transmural inflammation, albeit with different types of inflammatory mediators. The recruitment or activation of immune cells does not correlate directly with the intensity of generation of inflammatory mediators. The inflammatory mediators in TNBS and DSS inflammations target different genes to impair smooth muscle function.

The role of mast cells in the pathogenesis of pain in chronic pancreatitis

BackgroundThe biological basis of pain in chronic pancreatitis is poorly understood. Mast cells have been implicated in the pathogenesis of pain in other conditions. We hypothesized that mast cells play a role in the pain of chronic pancreatitis.We examined the association of pain with mast cells in autopsy specimens of patients with painful chronic pancreatitis. We explored our hypothesis further using an experimental model of trinitrobenzene sulfonic acid (TNBS) -induced chronic pancreatitis in both wild type (WT) and mast cell deficient mice (MCDM).MethodsArchival tissues with histological diagnoses of chronic pancreatitis were identified and clinical records reviewed for presence or absence of reported pain in humans. Mast cells were counted.The presence of pain was assessed using von Frey Filaments (VFF) to measure abdominal withdrawal responses in both WT and MCDM mice with and without chronic pancreatitis.ResultsHumans with painful chronic pancreatitis demonstrated a 3.5-fold increase in pancreatic mast cells as compared with those with painless chronic pancreatitis.WT mice with chronic pancreatitis were significantly more sensitive as assessed by VFF pain testing of the abdomen when compared with MCDM.ConclusionHumans with painful chronic pancreatitis have an increased number of pancreatic mast cells as compared with those with painless chronic pancreatitis. MCDM are less sensitive to mechanical stimulation of the abdomen after induction of chronic pancreatitis as compared with WT. Mast cells may play an important role in the pathogenesis of pain in chronic pancreatitis.

A Rat Model of Chronic Gastric Sensorimotor Dysfunction Resulting From Transient Neonatal Gastric Irritation

BACKGROUND & AIMS Although several pathophysiologic abnormalities have been noted in functional dyspepsia (FD), their pathogenesis is poorly understood. We hypothesized that chronic gastric hypersensitivity and gastric motor dysfunction seen in FD patients can be modeled in rats by transient gastric irritation during the neonatal period, a time of known neuronal vulnerability to long-term plasticity. METHODS Ten-day-old male rats received 0.2 mL 0.1% iodoacetamide (IA) in 2% sucrose daily by oral gavages for 6 days; controls received 2% sucrose. Rats in both groups were then followed to adulthood (8-10 weeks) at which point behavioral, visceromotor, and great splanchnic nerve responses to graded gastric balloon distention (GD; 20-80 mm Hg) and gastric motor function were tested. RESULTS IA-treated rats exhibited hypersensitivity to GD in a dose-dependent manner, as compared with the control group. The threshold of afferent nerve activation was lower and nerve responses to GD were significantly increased in IA-treated rats. Although IA-treated rats ingested food at a lower rate, gastric emptying was not significantly different between IA and control groups. However, gastric accommodation was significantly reduced in the IA group. No significant gastric pathology was seen in hypersensitive adult rats compared with controls. CONCLUSIONS These studies demonstrate that gastric irritation in the neonatal period can result in chronic gastric hypersensitivity and gastric motor dysfunction in adults even in the absence of significant detectable gastric pathology. Our results offer insight into the pathogenesis of chronic functional dyspepsia and provide a potential model for further study to this important clinical problem.

The ClC-3 chloride transport protein traffics through the plasma membrane via interaction of an N-terminal dileucine cluster with clathrin

ClC-3 is a ubiquitously expressed chloride transport protein that is present in synaptic vesicles and endosome/lysosome compartments. It is largely intracellular but has been observed at the plasma membrane as well. The aim of this study was to identify the pathways and regulation of ClC-3 trafficking to intracellular sites. At the steady state, ∼94% of transfected ClC-3 was localized intracellularly, and only 6% was at the plasma membrane. Pulse labeling with [35S]methionine and biotinylation demonstrated that about 25% of newly synthesized ClC-3 traffics through the plasma membrane. We used both immunofluorescence microscopy and biotinylation assays to assess the trafficking of ClC-3. Plasma membrane ClC-3 was rapidly endocytosed (t½ ∼ 9 min); a portion entered a recycling pool that returned to the cell surface after internalization, and the remainder trafficked to more distal intracellular compartments. ClC-3 associated with clathrin at the plasma membrane. Coimmunoprecipitation and glutathione S-transferase pulldown assays demonstrated that the N terminus of ClC-3 binds to clathrin. Alanine replacement of a dileucine acidic cluster within the cytosolic N terminus (amino acids 13-19) resulted in a molecule that had decreased endocytosis and increased surface expression. This replacement also abolished interaction with clathrin as assessed both by coimmunoprecipitation and glutathione S-transferase pulldown assays. We conclude that ClC-3 is primarily an intracellular transport protein that is transiently inserted into the plasma membrane where it is rapidly endocytosed. Internalization of ClC-3 depends on the interaction between an N-terminal dileucine cluster and clathrin.