The menthol and cold sensation receptor TRPM8 in normal human nasal mucosa and rhinitis.

BACKGROUND: Menthol and cold sensation trigger symptoms and reflex responses in the upper airway, but the underlying molecular mechanisms are unknown. We have therefore studied nerve fibres expressing the menthol and cold receptor TRPM8 in normal human mucosa, and in rhinitis. TRPM8 nerve fibres were compared with those expressing other TRP receptors including TRPV1 (capsaicin and heat receptor), and TRPA1 (mechano-cold receptor). METHODS: Immunohistology and image-analysis were used to study TRP receptors in biopsies of nasal turbinate from control subjects, patients with allergic rhinitis, and non-allergic rhinitis. RESULTS: TRPM8-immunoreactive nerve fibres were observed in the sub-epithelium, and were profuse around blood vessels in deeper regions, where they were markedly greater in number than TRPV1+ fibers. Image analysis of TRPM8 in sub-epithelial and vascular regions showed no significant differences between control and the rhinitis patient groups. TRPA1-immunoreactivity was weak and seen rarely in nerve fibres. CONCLUSION: We show that TRPM8 nerve fibres are abundant in nasal mucosa particularly around blood vessels, and may mediate neurovascular reflexes. TRPM8 antagonists deserve consideration for therapeutic trial in rhinitis.

Increased capsaicin receptor TRPV1-expressing sensory fibres in irritable bowel syndrome and their correlation with abdominal pain.

OBJECTIVE: The capsaicin receptor TRPV1 (transient receptor potential vanilloid type-1) may play an important role in visceral pain and hypersensitivity states. In irritable bowel syndrome (IBS), abdominal pain is a common and distressing symptom where the pathophysiology is still not clearly defined. TRPV1-immunoreactive nerve fibres were investigated in colonic biopsies from patients with IBS, and this was related to abdominal pain. METHODS: Rectosigmoid biopsies were collected from 23 IBS patients fulfilling Rome II criteria, and from 22 controls. Abdominal pain scores were recorded using a validated questionnaire. TRPV1-, substance P- and neuronal marker protein gene product (PGP) 9.5-expressing nerve fibres, mast cells (c-kit) and lymphocytes (CD3 and CD4) were quantified, following immunohistochemistry with specific antibodies. The biopsy findings were related to the abdominal pain scores. RESULTS: A significant 3.5-fold increase in median numbers of TRPV1-immunoreactive fibres was found in biopsies from IBS patients compared with controls (p<0.0001). Substance P-immunoreactive fibres (p = 0.01), total nerve fibres (PGP9.5) (p = 0.002), mast cells (c-kit) (p = 0.02) and lymphocytes (CD3) (p = 0.03) were also significantly increased in the IBS group. In multivariate regression analysis, only TRPV1-immuno-reactive fibres (p = 0.005) and mast cells (p = 0.008) were significantly related to the abdominal pain score. CONCLUSIONS: Increased TRPV1 nerve fibres are observed in IBS, together with a low-grade inflammatory response. The increased TRPV1 nerve fibres may contribute to visceral hypersensitivity and pain in IBS, and provide a novel therapeutic target.

Normalization of substance P levels in rectal mucosa of patients with faecal incontinence treated successfully by sacral nerve stimulation.

BACKGROUND: Sacral nerve stimulation (SNS) may improve faecal incontinence by modulating rectal sensation. This study measured changes in the peripheral expression of various neural epitopes in response to SNS. METHODS: Rectal mucosal biopsies were taken from 12 patients before and after temporary SNS, and from ten responders at 90 days after permanent stimulation. Sections were immunostained for substance P, transient receptor potential vanilloid (TRPV) 1, vasoactive intestinal peptide (VIP) and calcitonin gene-related peptide (CGRP). Levels were compared with those in nine continent controls. RESULTS: Baseline levels of percentage area immunoreactivities of substance P (median 0.51 (95 per cent confidence interval 0.31 to 0.73) versus 0.13 (0.07 to 0.27) per cent; P < 0.001) and TRPV1 (0.76 (0.41 to 1.11) versus 0.09 (0.04 to 0.14) per cent; P < 0.001), but not of VIP (1.26 (0.37 to 2.15) versus 1.28 (0.39 to 2.17); P = 0.943), were significantly greater than in controls. Successful SNS resulted in a significant decrease in substance P immunostaining after temporary (0.15 (0.06 to 0.51) per cent; P = 0.051) and permanent (0.17 (0 to 0.46) per cent; P = 0.051) stimulation. Immunoreactivity of TRPV1, VIP, CGRP and neural markers showed no qualitative change. CONCLUSION: Patients with faecal incontinence demonstrate normalization of raised rectal mucosal substance P levels following successful SNS.

TRPA1 receptor localisation in the human peripheral nervous system and functional studies in cultured human and rat sensory neurons.

TRPA1 is a receptor expressed by sensory neurons, that is activated by low temperature (<17 degrees C) and plant derivatives such as cinnamaldehyde and isoeugenol, to elicit sensations including pain. Using immunohistochemistry, we have, for the first time, localised TRPA1 in human DRG neurons, spinal cord motoneurones and nerve roots, peripheral nerves, intestinal myenteric plexus neurones, and skin basal keratinocytes. TRPA1 co-localised with a subset of hDRG neurons positive for TRPV1, the heat and capsaicin receptor. The number of small/medium TRPA1 positive neurons (< or =50 microm) was increased after hDRG avulsion injury [percentage of cells, median (range): controls 16.5 (7-23); injured 46 (34-55); P<0.005], but the number of large TRPA1 neurons was unchanged [control 19.5 (13-31); injured 21 (11-35)]. Similar TRPA1 changes were observed in cultured hDRG neurons, after exposure to a combination of key neurotrophic factors NGF, GDNF and NT-3 (NTFs) in vitro. We used calcium imaging to examine responses of HEK cells transfected with hTRPA1 cDNA, and of human and rat DRG neurons cultured with or without added NTFs, to cinnamaldehyde (CA) and isoeugenol (IE). Exposure to NTFs in vitro sensitized cultured human sensory neuronal responses to CA; repeated CA exposure produced desensitisation. In rDRG neurons, low (225 microM) CA preincubation enhanced capsaicin responses, while high (450 microM and 2mM) CA caused inhibition which was partially reversed in the presence of 8 bromo cAMP, indicating receptor dephosphorylation. While TRPA1 localisation is more widespread than TRPV1, it represents a promising novel drug target for the treatment of chronic pain and hypersensitivity.

The potential role of nerve growth factor (NGF) in painful neuromas and the mechanism of pain relief by their relocation to muscle.

Painful neuromas have been successfully treated by surgical procedures including relocation to muscle, but the underlying molecular mechanism remains unclear. Nerve growth factor (NGF) is secreted by tissues and promotes the expression of ion channels and neuropeptides in sensory neurons involved in pain transmission. We hypothesised that excess of NGF may lead to pain in neuromas and that the efficacy of surgical relocation results from deprivation of NGF, i.e. translocation from NGF-rich regions, particularly sub-cutaneous structures associated with injury or inflammation, to NGF-poor structures such as muscle or bone. Using immunohistological methods with primary antibodies to rhNGF, we report that NGF levels were elevated in 13 painful neuromas in comparison with six control nerves. However, in four painful neuromata re-located into muscle with pain relief, the NGF level was similar to that of controls. NGF levels suggest an explanation for the development of painful neuromas and the efficacy of relocation.

Identification and characterization of a novel human vanilloid receptor-like protein, VRL-2.

Remarkable progress has been made recently in identifying a new gene family related to the capsaicin (vanilloid) receptor, VR1. Using a combination of in silico analysis of expressed sequence tag (EST) databases and conventional molecular cloning, we have isolated a novel vanilloid-like receptor, which we call VRL-2, from human kidney. The translated gene shares 46% and 43% identity with VR1 and VRL-1, respectively, and maps to chromosome 12q23-24.1, a locus associated with bipolar affective disorder. VRL-2 mRNA was most strongly expressed in the trachea, kidney, and salivary gland. An affinity-purified antibody against a peptide incorporating the COOH terminal of the receptor localized VRL-2 immunolabel in the distal tubules of the kidney, the epithelial linings of both trachea and lung airways, serous cells of submucosal glands, and mononuclear cells. Unlike VR1 and VRL-1, VRL-2 was not detected in cell bodies of dorsal root ganglia (DRG) or sensory nerve fibers. However, VRL-2 was found on sympathetic and parasympathetic nerve fibers, such as those innervating the arrector pili smooth muscle in skin, sweat glands, intestine, and blood vessels. At least four vanilloid receptor-like genes exist, the newest member, VRL-2 is found in airway and kidney epithelia and in the autonomic nervous system.

Production of pituitary protein 7B2 immunoreactivity by endocrine tumors and its possible diagnostic value.

7B2 is a protein originally isolated from pituitary, which has been shown to be present in the central nervous system and in certain peripheral tissues, with very high concentrations in pancreatic islets. Endocrine and nonendocrine tumors from 185 patients were investigated by RIA for the presence of immunoreactive pituitary protein 7B2. The highest mean concentration of 7B2 immunoreactivity was found in insulinomas [452 +/- 174 (+/- SEM) pmol/g wet wt tissue; n = 16], which was significantly higher than the concentration in normal adult pancreatic tissue (28.3 +/- 4.4 pmol/g; n = 7). High concentrations of 7B2 immunoreactivity also were found in other endocrine tumors. The cellular localization of 7B2 was studied in normal pancreas, pancreas with hyperplastic islets, and endocrine tumors. 7B2 immunoreactivity was localized to B-cells in the normal pancreas and to variable proportions of cells in islet cell hyperplasias, B-cell tumors, and pheochromocytomas. Plasma concentrations of 7B2 immunoreactivity also were determined in 255 patients with established diagnoses of endocrine or nonendocrine tumors. The proportion of patients with elevated plasma concentrations (arbitrarily set at more than 4 SD above the mean) were 42 of 72 with pancreatic islet cell tumors, 7 of 11 with midgut carcinoid tumors, and 5 of 13 with medullary carcinomas of the thyroid. Especially high values were found in patients with glucagonomas (14 of 20), vipomas (12 of 13), and pancreatic polypeptide-producing tumors (5 of 6). Thus, 7B2 immunoreactivity is produced by a variety of different tumors and may serve as a tumor marker, especially in patients with certain pancreatic islet tumors.

Immunocytochemical localisation of katacalcin, a calcium-lowering hormone cleaved from the human calcitonin precursor.

Katacalcin is a newly discovered calcium-lowering hormone predicted from the nucleotide sequence of a cloned cDNA derived from human calcitonin mRNA. The aim of the study was to localise katacalcin by immunocytochemistry at both light and electron microscope levels. Antisera to synthetic katacalcin and calcitonin were used to investigate 8 cases of medullary thyroid carcinoma and 6 normal human thyroids (3 adult and 3 fetal). We have been able to demonstrate the co-localisation of these peptides in normal and neoplastic C-cells in all cases studied. Our results suggest that peptide sequences predicted by recombinant DNA technology can be localised using immunocytochemistry and that the combination of these techniques may have applications in diagnostic pathology.

Ontogeny of peptide- and amine-containing neurones in motor, sensory, and autonomic regions of rat and human spinal cord, dorsal root ganglia, and rat skin.

The developmental patterns of neurofilament triplet proteins, peptide and amine immunoreactivities were compared in motor (ventral spinal cord), sensory (dorsal spinal cord, dorsal root ganglia, epidermis), and autonomic (intermediolateral cell columns, dermis) regions in the rat and human. In the rat, neurofilament triplet proteins first appeared in motoneurones (embryonic day 13). In the youngest human fetuses studied (6 weeks), immunoreactivity was present throughout the spinal cord. Peptides and amines occurred later. Calcitonin gene-related peptide, galanin, somatostatin, neuropeptide Y and its C-flanking peptide (CPON) were the first to appear localized to motoneurones (embryonic days 15-17 rat; fetal weeks 6-14 human). Numbers of immunoreactive motoneurones decreased toward birth, but immunoreactive fibers increased in the ventral horn with enkephalin, thyrotrophin-releasing hormone, and the monoaminergic markers 5-hydroxytryptamine and tyrosine hydroxylase (all presumably of supraspinal origin) the last to appear perinatally. In the dorsal horn, particularly in the rat, a transient expression of substance P-, somatostatin-, and neuropeptide Y/CPON-immunoreactive cells was detected (embryonic days 15-17). A pronounced increase of calcitonin gene-related peptide-, galanin-, somatostatin- and substance P- immunoreactive fibers was found perinatally in both species. This coincided with an increased detection of cells in the dorsal root ganglia containing these peptides and the earliest appearance of calcitonin gene-related peptide-, somatostatin-, and substance P-immunoreactive fibers in the rat epidermis. Few antigens were localized to the intermediolateral cell columns before embryonic day 20 (rat), fetal week 20 (human), with thyrotrophin-releasing hormone-, 5-hydroxytryptamine-, tyrosine hydroxylase-, and vasoactive intestinal polypeptide-immunoreactive nerves appearing perinatally. In the rat dermis, tyrosine hydroxylase-immunoreactive fibers (sympathetic fibers) and fibers immunoreactive for neuropeptide Y/CPON and vasoactive intestinal polypeptide were detected from postnatal day 1. In conclusion, 1) peptide and amine immunoreactivity develops in motor before sensory or autonomic regions, 2) many peptide-containing cells are transient in fetal life, and 3) central terminals of dorsal root ganglion cells express peptides before terminals in the skin.

Immunolocalization of SNS/PN3 and NaN/SNS2 sodium channels in human pain states.

The tetrodotoxin-resistant (TTX-R) voltage-gated sodium channel SNS/PN3 and the newly discovered NaN/SNS2 are expressed in sensory neurones, particularly in nociceptors. Using specific antibodies, we have studied, for the first time in humans, the presence of SNS/PN3 and NaN/SNS2 in peripheral nerves, including tissues from patients with chronic neurogenic pain. In brachial plexus injury patients, there was an acute decrease of SNS/PN3- and NaN/SNS2-like immunoreactivity in sensory cell bodies of cervical dorsal root ganglia (DRG) whose central axons had been avulsed from spinal cord, with gradual return of the immunoreactivity to control levels over months. In contrast, there was increased intensity of immunoreactivity to both channels in some peripheral nerve fibers just proximal to the site of injury in brachial plexus trunks, and in neuromas. These findings suggest that the expression of these sodium channels in neuronal cell bodies is reduced after spinal cord root avulsion injury in man, but that pre-synthesized channel proteins may undergo translocation with accumulation at sites of nerve injury, as in animal models of peripheral axotomy. The latter may contribute to positive symptoms, as our patients all showed a positive Tinel's sign. Nerve terminals in distal limb neuromas and skin from patients with chronic local hyperalgesia and allodynia all showed marked increases of SNS/PN3-immunoreactive fibers, but little or no NaN/SNS2-immunoreactivity, suggesting that the former may be related to the persistent hypersensitive state. Axonal immunoreactivity to both channels was similar to control nerves in sural nerve biopsies in a selection of neuropathies, irrespective of nerve inflammation, demyelination or spontaneous pain, including a patient with congenital insensitivity to pain. Our studies suggest that the best target for SNS/PN3 blocking agents is likely to be chronic local hypersensitivity.

Do nerve growth factor-related mechanisms contribute to loss of cutaneous nociception in leprosy?

While sensory loss in leprosy skin is the consequence of invasion by M. leprae of Schwann cells related to unmyelinated fibres, early loss of cutaneous pain sensation, even in the presence of nerve fibres and inflammation, is a hallmark of leprosy, and requires explanation. In normal skin, nerve growth factor (NGF) is produced by basal keratinocytes, and acts via its high affinity receptor (trk A) on nociceptor nerve fibres to increase their sensitivity, particularly in inflammation. We have therefore studied NGF- and trk A-like immunoreactivity in affected skin and mirror-site clinically-unaffected skin from patients with leprosy, and compared these with non-leprosy, control skin, following quantitative sensory testing at each site. Sensory tests were within normal limits in clinically-unaffected leprosy skin, but markedly abnormal in affected skin. Sub-epidermal PGP 9.5- and trk A- positive nerve fibres were reduced only in affected leprosy skin, with fewer fibres contacting keratinocytes. However, NGF-immunoreactivity in basal keratinocytes, and intra-epidermal PGP 9.5-positive nerve fibres, were reduced in both sites compared to non-leprosy controls, as were nerve fibres positive for the sensory neurone specific sodium channel SNS/PN3, which is regulated by NGF, and may mediate inflammation-induced hypersensitivity. Keratinocyte trk A expression (which mediates an autocrine role for NGF) was increased in clinically affected and unaffected skin, suggesting a compensatory mechanism secondary to reduced NGF secretion at both sites. We conclude that decreased NGF- and SNS/PN3-immunoreactivity, and loss of intra-epidermal innervation, may be found without sensory loss on quantitative testing in clinically-unaffected skin in leprosy; this appears to be a sub-clinical change, and may explain the lack of cutaneous pain with inflammation. Sensory loss occurred with reduced sub-epidermal nerve fibres in affected skin, but these still showed trk A-staining, suggesting NGF treatment may restore pain sensation.

A comparative study of serum total thyroxine estimation on unextracted serum by radioimmunoassay and by competitive protein binding.

A rapid and precise radioimmunoassay (RIA) for serum total thyroxine (T4) on as little as 1-10 mul of unextracted serum is described. Results in hypothyroidism (overt and borderline), in euthyroid subjects in pregnant and oestrogen-medicated subjects, and in hyperthyroidism (overt and borderline) are compared with the results on the same sera by an established competitive protein binding technique (Ames' Tetralute) on unextracted serum from a different laboratory. The correlation between the two methods was excellent (r= 0.94) and no significant difference between overall appeared to measure total T4 Reliaby in sera containing only 1-3 or 2-6 nmol/1. Both methods predicted the clinical outcome in borderline hypothyroidism and borderline hyperthyroidism equally well and both gave normal results in T3-toxicosis. It is concluded that both techniques reliably measure total T4- RIA appears to have advantages of sensitivity and precision (especially in the hypothroid range), of simplicity, and of low cost.

A computer-assisted stereological quantification program: OpenStereo.

At present, the stereological assessment of histological sections is made possible by the use of manual counting techniques which estimate measurement parameters. These methods are tedious, time-consuming, and subject to operator error. This paper describes a UNIX-based computer program, OpenStereo, which was developed to facilitate the quantitative investigation of innervation and vascularity from histological sections. We designed OpenStereo to reduce operator error and increase the efficiency of stereological point counting for volume estimation and intercept counting for surface area analysis. The program was written in the C language for the Sun Workstation and uses the XView graphics user interface. Digital images, obtained by a variety of modalities, may be processed using stereological point counting, interceptions, planimetry, or thresholding techniques. The program displays selected images in a random fashion for analysis or processing and records the number of manually selected points or interceptions. Delineation of the reference space provides the computer with the data necessary to calculate volume or surface densities. The efficiency of OpenStereo was demonstrated by performing a pilot study on the quantification of innervation in the normal human colon and ileum. This stereological package benefits from the features of the X-windowing environment and has proved to be suitable for what has hitherto been a tedious and time-consuming task.

Effect of streptozotocin-diabetes on the level of VIP mRNA in myenteric neurones.

The effect of streptozotocin-induced diabetes on the production of vasoactive intestinal polypeptide messenger RNA in the myenteric neurones of 8 weeks diabetic rat intestine was examined using in situ hybridization. Total ganglion cell number per length (mm) of tissue section was measured using NADH diaphorase histochemistry. Although ganglion cells were more numerous in the control preparations compared with diabetic samples, a significantly greater number of vasoactive intestinal polypeptide messenger RNA-containing cells was detected in the diabetic tissues. These observations suggest that there is either a decrease in the breakdown of vasoactive intestinal polypeptide messenger RNA or an increase in its synthesis in myenteric neurones of diabetic rat intestine.

P2X3 receptor in injured human sensory neurons.

The ATP-gated cation channel P2X3 is expressed selectively by rat sensory neurones, and may play a role in nociception by binding ATP released from damaged or inflamed tissues. However, the distribution of this channel in human sensory neurons is not known. Using a specific antibody, we have demonstrated intense P2X3 immunoreactivity within a subset (60%) of small/medium diameter sensory neurones and fine nerve fibres in intact post-mortem human dorsal root ganglia (DRG). Co-localization studies showed < 15% overlap with the trkA immunostaining in DRG, indicating that P2X3 was expressed predominantly in sensory neurons that are also isolectin B4 positive. There was a significant decrease in numbers of P2X3-like immunoreactive neurons in human DRG after central axotomy (to 36%), similar to the decrease in rat DRG after peripheral axotomy. However, Western blotting demonstrated a specific 66 kDa band in human DRG and peripheral organs, including intestine, where histochemistry showed P2X3 immunoreactivity in myenteric plexus neurons. Thus P2X3 antagonists may be analgesic, but are unlikely to have a selective effect on pain in humans.

GDNF and its receptor component Ret in injured human nerves and dorsal root ganglia.

Glial cell line-derived neurotrophic factor (GDNF) is trophic to motor and sensory neurones in animal models. GDNF mRNA is up-regulated in Schwann cells after peripheral nerve injury in rats. We have quantified and localized GDNF and its receptor component Ret, for the first time in any species, in injured human peripheral nerves and dorsal root ganglia (DRG) avulsed from the spinal cord. Significantly higher levels of GDNF were found in nerve distal to the site of the injury than in proximal or intact nerve, and in avulsed DRG than in post-mortem control DRG. GDNF immunostaining was seen in Schwann cells and in DRG neurones, especially of small and medium size, with significantly increased numbers of medium sized sensory neurones immunoreactive for GDNF after avulsion. Ret immunoreactivity was restricted to DRG neurones and axons, with no significant changes in numbers of positive DRG cells after injury. Our findings suggest that GDNF may play a role in injured human nerves and sensory ganglia, particularly in medium sized sensory neurones.

Pancreastatin distribution and plasma levels in the pig.

Pancreastatin is a peptide isolated from porcine pancreas which has insulin-suppressive actions in vitro and sequence homology with chromogranin A. Using radioimmunoassay and immunocytochemistry we investigated whether pancreastatin has a more widespread distribution and a possible endocrine role in the pig. Pancreastatin immunoreactivity was found in plasma, adrenal gland, pancreas, anterior pituitary and throughout the gastrointestinal tract. The immunoreactivity was colocalized with chromogranin immunoreactivity in endocrine cells and ultrastructurally (in the pancreas) to storage granules. Characterization of pancreastatin-like immunoreactivity, using gel permeation and high performance liquid chromatography, separated 3 different pancreastatin-like immunoreactive forms: one molecular form, indistinguishable from synthetic pancreastatin 1-49, was predominant in pancreas and thyroid and released into the circulation postprandially. However, a high dose (greater than 1 nmol/l) infusion of pancreastatin 33-49 (the biologically active moiety in vitro) into conscious pigs had no effect on either basal or glucose-stimulated insulin secretion.

ATP-gated ion channel P2X(3) is increased in human inflammatory bowel disease.

P2X(3) is a novel ATP-gated cation channel that is selectively expressed by small-diameter sensory neurones in rodents, and may play a role in nociception by binding ATP released from damaged or inflamed tissues. We have studied, for the first time, P2X(3) immunoreactivity in human inflammatory bowel disease, using Western blotting and immunohistochemistry. A major 66-kDa specific protein was found by Western blotting in all colon extracts. In the inflamed group there was a significant two-fold increase in the relative optical density of the 66-kDa band (21.2 +/- 3.1; n=8) compared to controls (11.4 +/- 3.7; n=8; P=0.009). In the control colon, P2X(3)-immunoreactive neurones were scattered throughout the myenteric and submucosal plexuses, with some neurones showing immunopositive axons/dendrites. The pattern of immunostaining was similar to the neuronal marker peripherin. In general, the intensity of the staining was greater in myenteric than submucosal neurones. The number of P2X(3)-immunoreactive neurones was significantly increased in the myenteric plexus of inflamed colon compared to controls (n=13; P=0.01). In humans, unlike rodents, P2X(3) is thus not restricted to sensory neurones. Increased P2X(3) in inflamed intestine suggests a potential role in dysmotility and pain, for which it represents a new therapeutic target.

Increased acid-sensing ion channel ASIC-3 in inflamed human intestine.

OBJECTIVES: Acid-sensing ion channels (ASICs) are expressed by rat sensory neurons and may mediate pain associated with tissue acidosis after inflammation or injury. Our aim was to examine the molecular forms and localization of ASICs in human intestine and dorsal root ganglia using immunochemical techniques, and to measure the effects of inflammation and injury. DESIGN AND METHODS: Inflamed Crohn's disease intestine and injured human dorsal root ganglia, with appropriate controls, were studied by Western blotting and immunohistochemistry, using specific affinity-purified ASIC antibodies. RESULTS: In the Western blot, there was a significant three-fold increase in the mean relative optical density of the ASIC-3 55-kDa band (but not ASIC-1 or ASIC-2) in full-thickness inflamed intestine, as well as in separated muscle and mucosal layers. There was a corresponding trend for an increased immunoreactive density and increased number of ASIC-3-positive neurons in the myenteric and sub-mucous plexus of inflamed intestine. In dorsal root ganglia, immunoreactivity for all ASICs was restricted to a sub-population (about 50%) of small-diameter (nociceptor) sensory neurons, and was generally less intense after injury. CONCLUSIONS: Increased ASIC-3 in inflamed intestine suggests a role in pain or dysmotility, for which ASICs represent new therapeutic targets.

Angiotensin II type 2 receptor (AT2 R) localization and antagonist-mediated inhibition of capsaicin responses and neurite outgrowth in human and rat sensory neurons.

BACKGROUND: The angiotensin II (AngII) receptor subtype 2 (AT2 R) is expressed in sensory neurons and may play a role in nociception and neuronal regeneration. METHODS: We used immunostaining with characterized antibodies to study the localization of AT2 R in cultured human and rat dorsal root ganglion (DRG) neurons and a range of human tissues. The effects of AngII and AT2 R antagonist EMA401 on capsaicin responses in cultured human and rat (DRG) neurons were measured with calcium imaging, on neurite length and density with Gap43 immunostaining, and on cyclic adenosine monophosphate (cAMP) expression using immunofluorescence. RESULTS: AT2 R expression was localized in small-/medium-sized cultured neurons of human and rat DRG. Treatment with the AT2 R antagonist EMA401 resulted in dose-related functional inhibition of capsaicin responses (IC50 = 10 nmol/L), which was reversed by 8-bromo-cAMP, and reduced neurite length and density; AngII treatment significantly enhanced capsaicin responses, cAMP levels and neurite outgrowth. The AT1 R antagonist losartan had no effect on capsaicin responses. AT2 R was localized in sensory neurons of human DRG, and nerve fibres in peripheral nerves, skin, urinary bladder and bowel. A majority sub-population (60%) of small-/medium-diameter neuronal cells were immunopositive in both control post-mortem and avulsion-injured human DRG; some very small neurons appeared to be intensely immunoreactive, with TRPV1 co-localization. While AT2 R levels were reduced in human limb peripheral nerve segments proximal to injury, they were preserved in painful neuromas. CONCLUSIONS: AT2 R antagonists could be particularly useful in the treatment of chronic pain and hypersensitivity associated with abnormal nerve sprouting.