Vanilloid Receptor 1 Immunoreactivity Research Articles

Cell-type specific expression of vanilloid receptor 1 in the taste cells of rat circumvallate papillae

The present study demonstrates the first-ever characterization of cell types that express the vanilloid receptor 1 (VR1) in the taste buds of rat circumvallate papillae. We performed electron microscopy to identify the subcellular location. The VR1 immunoreactivity was associated with the endoplasmic reticulum, cytoplasmic vesicles, and plasma membrane of taste cells. These results demonstrate the localization of the VR1 in membranous structures of the taste cells. We used double immunofluorescence histochemistry with taste cell type-specific markers to identify the cell types that express the VR1. The VR1 was detected in all functional taste cell types (Type I, Type II, and Type III cells). Together, our data suggest that the VR1 might play different roles according to the cell types within a taste bud.

Cutaneous innervation before and after one treatment period of acupuncture

The effect of acupuncture on nociceptive pain is well documented, but effects on nociceptive itch have been contradictory. To evaluate possible effects of acupuncture on the occurrence, distribution and function of sensory nerve fibres in human skin. Ten subjects were treated by inserting 10 acupuncture needles subcutaneously at the upper lateral aspect of one buttock. The subjects were recruited from an acupuncture clinic and were undergoing specific acupuncture treatment for their disorders. The needles were stimulated (rotated to and fro) twice during the twice-weekly 25-min sessions over 5 weeks. Skin biopsies, diameter 3 mm, were taken before and 3-6 days after local acupuncture. Antibodies to the pan-neuronal marker protein gene product 9.5 (PGP 9.5), calcitonin gene-related peptide (CGRP), vanilloid receptor 1 (VR1) and mu- and delta-opioid receptors were employed to study sensory unmyelinated nerve fibres that transmit nociceptive pain and itch. A histamine prick test using planimetry was used to record experimental itch after acupuncture on the treated area and on the corresponding control skin, and a visual analogue scale was used to evaluate itch. The mean +/- SEM number of CGRP-immunoreactive nerve fibres per biopsy section was reduced from 36.0 +/- 3.3 to 21.3 +/- 4.0 (P = 0.05) after the treatment. PGP 9.5-immunoreactive nerve fibres were found both in the epidermis and in the subpapillary dermis. The mean +/- SEM total number of PGP 9.5-immunoreactive nerve fibres decreased from 249.8 +/- 16.7 to 211.8 +/- 12.0 (P = 0.03). The PGP 9.5-immunoreactive nerve fibres occurring in the dermis appeared more fragmented after the acupuncture compared with pretreatment. VR1 immunoreactivity was found both in the free nerve fibres and in kite-like formations, possibly mast cells, throughout the dermis, sometimes occurring around hair follicles. The mean +/- SEM number of VR1-immunoreactive elements was not significantly influenced by acupuncture, at 33.5 +/- 4.6 vs. 43.0 +/- 4.4 (P = 0.09). No immunoreactivity was found in the skin against mu- and delta-opioid receptors with the antibodies used in this study. Neither histamine-induced itch nor cutaneous responses were influenced by acupuncture. The present data indicate an effect of acupuncture on neuropathic itch but not histamine-mediated itch. Our findings support the opinion that the pain-relieving effects of acupuncture partly depend on its effect on the peripheral innervation.

Expression of vanilloid receptor-1 in epithelial cells of human antral gastric mucosa

ObjectiveCapsaicin, which acts by binding to the vanilloid receptor-1 (VR1), has been shown to give protection against gastric mucosal injury and to enhance healing of gastric ulcers. Although VR1 has recently been reported to be present in non-neural tissues, it is primarily considered to be expressed in nociceptor sensory neurons of small diameter. The aim of the present study was to evaluate the distribution of VR1 immunoreactivity in the normal human gastric mucosa.Material and methodsTen volunteers underwent gastroscopy and biopsies were obtained from the corpus and the antrum. The specimens were labelled immunohistochemically using polyclonal goat anti-VR1 and evaluated at the light- and electronmicroscopic level. Moreover, post-embedding immunogold labelling was performed and subsequently analysed at the electronmicroscopic level.ResultsIn the antrum, VR1 immunoreactivity was located in epithelial cells that fulfilled the criteria of endocrine cells of the “open type”. These cells were located primarily in the neck region of the antral glands and the labelling was concentrated on the microvilli of these cells. At the ultrastructural level, round granulae with differences in electron density were identified in the basal compartment of the labelled cells. VR1 immunoreactivity was also identified in axon-like structures that were located in the lamina propria, often in close vicinity of vessels, in the corpus as well as in the antrum.ConclusionsVR1-immunoreactivity was evident in antral epithelial cells exhibiting characteristics of endocrine-like cells. This may indicate that the gastroprotective effects of capsaicin, which hitherto have been attributed to primary afferent neurons, at least partly may be explained by an action on specific epithelial cells in the antrum.

Cardiac vanilloid receptor 1-expressing afferent nerves and their role in the cardiogenic sympathetic reflex in rats.

Myocardial ischaemia causes the release of metabolites such as bradykinin, which stimulates cardiac sensory receptors to evoke a sympathoexcitatory reflex. However, the molecular identity of the afferent neurons and fibres mediating this reflex response is not clear. In this study, we tested the hypothesis that the cardiogenic sympathoexcitatory reflex is mediated by capsaicin-sensitive afferent fibres. Enhanced immunofluorescence labelling revealed that vanilloid receptor 1 (VR1)-containing afferent nerve fibres were present on the epicardial surface of the rat heart. Resiniferatoxin (RTX), a potent analogue of capsaicin, was used to deplete capsaicin-sensitive afferent fibres in rats. Depletion of these fibres was confirmed by a substantial reduction of VR1 immunoreactivity in the epicardium and dorsal root ganglia. The thermal sensitivity was also diminished in RTX-treated rats. Renal sympathetic nerve activity (RSNA) and blood pressure were recorded in anaesthetized rats during epicardial application of bradykinin or capsaicin. In vehicle-treated rats, epicardial bradykinin (10 microg ml-1) or capsaicin (10 microg ml-1) application produced a significant increase in RSNA and arterial blood pressure. The RSNA and blood pressure responses caused by bradykinin and capsaicin were completely abolished in RTX-treated rats. Furthermore, epicardial application of iodo-RTX, a highly specific antagonist of VR1 receptors, blocked capsaicin- but not bradykinin-induced sympathoexcitatory responses. Thus, these data provide important histological and functional evidence that the heart is innervated by VR1-expressing afferent nerves and these afferent nerves are essential for the cardiogenic sympathoexcitatory reflex during myocardial ischaemia.

Capsaicin-sensitive and -insensitive vagal bronchopulmonary C-fibres in the mouse.

We developed an isolated tracheally perfused (35-37 degrees C) nerve-lung preparation for the study of bronchopulmonary afferent nerve activity in the mouse. Extracellular recordings were made from the vagal sensory neurons located in the jugular-nodose ganglia complex (JNC) with identified receptive fields in the lungs. Analysis of the vagal compound action potential revealed that the mouse vagal C-fibre conduction velocities range from 0.3 to 1.5 m s(-1). A total of 83 bronchopulmonary C-fibres were studied. The sensitivity of the bronchopulmonary C-fibres to the vanilloid receptor 1 (VR1) agonist capsaicin was dependent on conduction velocity. Thus C-fibres with conduction velocities between 0.3 and 0.7 m s(-1) responded to capsaicin (1 microM) while C-fibres with conduction velocities between 0.7 and 1.5 m s(-1) were capsaicin insensitive. Similarly, bradykinin (1 microM) excited only those C-fibres with conduction velocities < 0.7 m s(-1). The response to bradykinin was not mimicked by the B1 receptor agonist [des-Arg9]bradykinin (1 microM) and was abolished by the bradykinin B2 receptor antagonist HOE 140 (1 microM). Adenosine 5'-triphosphate (ATP, 30 microM) activated the C-fibres irrespective of the conduction velocities. This response was mimicked by the selective P2X agonist alpha,beta-methylene-adenosine 5'-triphosphate (30 microM). Consistent with the electrophysiology, morphological analysis revealed that only approximately 40% of the lung-specific small diameter (< 20 microm) JNC neurons consistent with the C-fibre cell bodies display VR1 immunoreactivity. This study describes a convenient in vitro method for the study of mouse bronchopulmonary C-fibres. The results indicate that C-fibres in the mouse lungs are not homogeneous, but can be subclassified into capsaicin-sensitive and capsaicin-insensitive phenotypes.

State-dependent phosphorylation of epsilon-isozyme of protein kinase C in adult rat dorsal root ganglia after inflammation and nerve injury.

The epsilon-isozyme of protein kinase C (PKCepsilon) and the vanilloid receptor 1 (VR1) are both expressed in dorsal root ganglion (DRG) neurons and are reported to be predominantly and specifically involved in nociceptive function. Using phosphospecific antibody against the C-terminal hydrophobic site Ser729 of PKCepsilon as a marker of enzyme activation, the state-dependent activation of PKCepsilon, as well as the expression of VR1 in rat DRG neurons, was evaluated in different experimental pain models in vivo. Quantitative analysis showed that phosphorylation of PKCepsilon in DRG neurons was significantly up-regulated after carrageen- and Complete Freund's Adjuvant-induced inflammation, while it was markedly down-regulated after chronic constriction injury. A double-labeling study showed that phosphorylation of PKCepsilon was expressed predominantly in VR1 immunoreactivity positive small diameter DRG neurons mediating the nociceptive information from peripheral tissue to spinal cord. The VR1 protein expression showed no significant changes after either inflammation or chronic constriction injury. These data indicate that functional activation of PKCepsilon has a close relationship with the production of inflammatory hyperalgesia and the sensitization of the nociceptors. Inflammatory mediator-induced activation of PKCepsilon and subsequent sensitization of VR1 to noxious stimuli by PKCepsilon may be involved in nociceptor sensitization.

Selective increase of tumour necrosis factor‐alpha in injured and spared myelinated primary afferents after chronic constrictive injury of rat sciatic nerve

Chronic constriction of the sciatic nerve, leading to a hyperalgesic state, results in a partial lesion wherein some axons are injured and others remain intact. Here we sought to characterize reactive changes which occur in DRG cell bodies of injured and uninjured axons projecting to skin and muscle. Using immunohistochemistry combined with flurorogold and fluororuby retrograde labelling to define DRG cell bodies associated with injured and uninjured axons, we analysed the DRG immunoreactivity (IR) for tumour necrosis factor-alpha (TNF), interleukin-10 (IL-10), the sensory neuron-specific channel vanilloid receptor 1 (VR1), isolectin B4 (IB4) and calcitonin-gene-related peptide (CGRP) 4 days after a unilateral chronic constriction injury (CCI) of the rat sciatic nerve. TNF IR was predominantly localized in neuronal DRG cells. In DRG with an intact nerve, TNF IR was present in 45%, IL-10 IR in 46%, VR1 IR in 44%, IB4 IR in 51% and CGRP IR in 40% of all neuronal profiles. Four days after CCI, TNF IR was increased in medium-sized neurons, whereas IR for IL-10, VR1 and IB4, predominantly present in small neurons, was reduced. Importantly, not only injured but also adjacent spared neurons contributed markedly to increased TNF IR. Neurons projecting to both muscle and skin displayed upregulated TNF IR after CCI. TNF in medium-sized neurons colocalized with neurofilament and trkB, but not with IB4, trkA or RET, suggesting a selective phenotypic switch in presumably low-threshold myelinated primary afferents. Spared myelinated fibres with intact sensory functions but upregulated TNF expression may contribute to behavioural changes observed after nerve injury.

Topography of the Vanilloid Receptor in the Human Bladder: More Than Just the Nerve Fibers

We determined the presence and distribution of vanilloid receptor-1 in the human bladder and confirmed or rejected previous findings of other groups that used indirect methods or vanilloid receptor-1 antibodies made by immunizing experimental animals. Also, we tested the reproducibility of results using commercially available antibodies against the N-terminus and C-terminus of the vanilloid receptor.A total of 11 normal bladder tissue samples were obtained from cystectomy specimens and fresh frozen processed. Specimens were studied by immunohistochemistry and confocal laser microscopy using 3 vanilloid receptor-1 antibodies. Immunohistochemical co-localization studies for neurofilament, neuronal nitric oxide synthase and nerve growth factor were performed.Our results confirm the presence of vanilloid receptor-1 on nonmyelinated and myelinated nerve fibers. There was vanilloid receptor-1 immunoreactivity on smooth muscle cells but different sensitivities for the antibodies. There was immunoreactivity on interstitial cells located in the suburothelium and intermuscular septa of the muscularis. There was co-localization of neuronal nitric oxide synthase with interstitial cells but not with neurofilament. No co-localization was found for nerve growth factor and vanilloid receptor-1.Vanilloid receptor-1 is located on small unmyelinated and myelinated nerve fibers. In addition, vanilloid receptor-1 is also present on interstitial cells in the suburothelium. There is smooth muscle cell immunoreactivity but a difference in antibodies raised against the C-terminus and N-terminus. These data suggest that the current hypothesis about the mechanism of action of vanilloids is through blocking the afferent reflex arc must be revised and the function of interstitial cells deserves further attention.

Distribution and characterization of vanilloid receptors in the rat stomach

The cloned vanilloid receptor-1 (VR1) is recognized as a common molecular target for protons, noxious heat, and vanilloids. The presence of VR1 in the dorsal root, trigeminal, and nodose ganglia has been firmly established, but it is unclear in the gut, despite this VR1 may be important for gastric mucosal homeostasis. In this study we used an antibody and a radioligand to show the distribution of vanilloid receptors (VRs) in rat stomach and to characterize it. The deafferentiation of capsaicin-sensitive nerves in rats was induced by consecutive injections of capsaicin. VR1-immunopositive nerve endings were predominantly found in the mucous neck cells of the proliferation zone, and around blood vessels in the submucosa. Radioreceptor assay using [ 3H]-resiniferatoxin (RTX) revealed the existence of high affinity and single-class binding site in the membrane fractions of the mucosa. Capsaicin completely inhibited the specific binding of [ 3H]-RTX. Both the VR1 immunoreactivity and the receptor density of [ 3H]-RTX binding sites significantly reduced by the application of capsaicin for prolonged periods of time in the mucosa of rats. Our results indicate that VRs are expressed in the rat stomach, and suggest that they may be involved in mucosal protection by increasing cell proliferation and blood flow.

Vanilloid receptor 1 immunoreactivity in inflamed human bowel

Vanilloid receptor 1 (VR1) is expressed by sensory neurons. Once activated, these neurons evoke the sensation of burning pain and release neuropeptides that induce neurogenic inflammation. We used immunoblotting and immunostaining to estimate the density of VR1 in colonic tissues of patients with inflammatory bowel disease and of controls. Our study results indicate that VR1 immunoreactivity is greatly increased in colonic nerve fibres of patients with active inflammatory bowel disease. Thus, the discovery of new drugs that can bind the VR1 receptor, or antagonise endogenous inflammatory substances that activate this receptor, could lead to new therapies for pain and dysmotility.