TRPV1 Immunoreactivity Research Articles

TRPV1, but not P2X3, requires cholesterol for its function and membrane expression in rat nociceptors

We examined the importance of membrane cholesterol for the function and expression of TRPV1 (vanilloid receptor subtype 1) and P2X(3) in adult rat dorsal root ganglion (DRG) neurons. Cholesterol, an essential component of lipid rafts, was depleted using methyl beta-cyclodextrin (MCD). We found that MCD significantly reduced TRPV1-mediated capsaicin- and proton-activated currents. By contrast, inward currents activated by alpha,beta-methylene ATP (alpha,beta-meATP), a non-hydrolysable ATP analogue, were not altered. Immunoreactivity for TRPV1, but not P2X(3), in the plasma membrane was markedly reduced by MCD. A reduction of TRPV1 protein in membrane fractions was found following cholesterol depletion. Our data show that the level of cholesterol determines the activity and the amount of membrane TRPV1, suggesting that TRPV1 might be localized in cholesterol-rich microdomains in nociceptors. The differential dependence on the membrane cholesterol of TRPV1 and P2X(3) may have physiological significance in nociception during inflammation.

Phenotypic characterization of gastric sensory neurons in mice

Recent studies suggest that the capsaicin receptor [transient receptor potential vanilloid (TRPV)1] may play a role in visceral mechanosensation. To address the potential role of TRPV1 in vagal sensory neurons, we developed a new in vitro technique allowing us to determine TRPV1 expression directly in physiologically characterized gastric sensory neurons. Stomach, esophagus, and intact vagus nerve up to the central terminations were carefully dissected and placed in a perfusion chamber. Intracellular recordings were made from the soma of nodose neurons during mechanical stimulation of the stomach. Physiologically characterized neurons were labeled iontophoretically with neurobiotin and processed for immunohistochemical experiments. As shown by action potential responses triggered by stimulation of the upper thoracic vagus with a suction electrode, essentially all abdominal vagal afferents in mice conduct in the C-fiber range. Mechanosensitive gastric afferents encode stimulus intensities over a wide range without apparent saturation when punctate stimuli are used. Nine of 37 mechanosensitive vagal afferents expressed TRPV1 immunoreactivity, with 8 of the TRPV1-positive cells responding to stretch. A small number of mechanosensitive gastric vagal afferents express neurofilament heavy chains and did not respond to stretch. By maintaining the structural and functional integrity of vagal afferents up to the nodose ganglion, physiological and immunohistochemical properties of mechanosensory gastric sensory neurons can be studied in vitro. Using this novel technique, we identified TRPV1 immunoreactivity in only one-fourth of gastric mechanosensitive neurons, arguing against a major role of this ion channel in sensation of mechanical stimuli under physiological conditions.

Peripheral and central distribution of TRPV1, substance P and CGRP of rat corneal neurons

The rat corneal neurons expressing vanilloid receptor TRPV1, substance P (SP) and calcitonin-gene-related peptide (CGRP) were examined. In the cornea, some TRPV1-immunoreactive nerve fibers displayed either SP- or CGRP immunoreactivity also. For observing corneal neuronal elements in the trigeminal ganglion (TG) and in the medulla oblongata, retrograde and anterograde cholera toxin subunit B (CTB) tracing methods combining with triple immunofluorescence technique were performed. The corneal neuronal somata were located in the ophthalmic division of the TG; 37% of them were immunoreactive for TRPV1. One third and three quarters of the corneal TRPV1-immunoreactive neurons co-expressed SP and CGRP, respectively. All of SP-immunoreactive corneal neurons exhibited TRPV1 immunoreactivity. They were predominantly medium-sized (mean ± SE = 638.2 ± 49.5 μm 2) and significantly larger than SP-immunoreactive and TRPV1-immunonegative neurons in the ophthalmic division of the TG. The central projection fibers of corneal neurons co-expressing TRPV1 with SP and CGRP were observed at the subnucleus interpolaris/caudalis transition within trigeminal nucleus. The present study suggests that TRPV1 of the corneal neurons works in close relation to SP and CGRP both in the cornea and CNS for healing and nociceptive transduction.

Participation of TRPV1 and TRPV2 in the rat laryngeal sensory innervation

Laryngeal sensory innervation is essential to the laryngeal defense system. We investigated the participation of TRPV1 and its homologue TRPV2 in the rat laryngeal sensory innervation using immunohistochemistry and the neuronal tracer, fluoro-gold (FG). After injection of FG into the internal branch of the superior laryngeal nerve, FG-labeled neurons were seen in the rostral part of the nodose ganglion (NG). Neurons immunoreactive for TRPV1 or TRPV2 were distributed throughout the NG. TRPV1 immunoreactivity was seen in 49.0 ± 4.5% of the FG-labeled neurons, while TRPV2 immunoreactivity was seen in 12.5 ± 4.1% of the FG-labeled neurons. These findings suggest that both TRPV1 and TRPV2 participate in laryngeal nociception, but that TRPV1 may have a particularly important role.

Capsaicin receptor (TRPV1) and non-erosive reflux disease

Non-erosive reflux disease (NERD) is a common and heterogeneous disorder. We hypothesized that changes in peripheral innervation may lead to hyperalgesia and contribute to the development of the disorder. Patients referred for evaluation of reflux symptoms with wireless pH monitoring were asked to provide demographic and clinical data and complete a survey related to severity of reflux symptoms. Endoscopies were performed to rule out macroscopic abnormalities of the esophageal mucosa. Biopsies obtained 2 cm above the gastroesophageal junction were stained for protein gene product 9.5 (PGP 9.5; general neuronal marker) and TRPV1 (capsaicin receptor) immunoreactivity. The density of immunoreactive fibers in the esophageal mucosa was determined morphometrically. A total of 39 patients without evidence of Barrett's metaplasia, erosive or ulcerative esophagitis were enrolled. Most patients had daily symptoms. The total esophageal acid exposure time was 5.6+/-0.6%, with 16 patients (41%) having increased acid reflux. Immunoreactivity for PGP 9.5 or TRPV1 was detected in papillary structures as well as within the epithelium (free intra-epithelial endings). Total acid-exposure time, but not symptom score or duration correlated significantly with density of PGP 9.5 immunoreactivity and TRPV1 positive fibers. Even in the absence of macroscopic injury, esophageal acid exposure is associated with changes in mucosal innervation of the esophagus, thus potentially further enhancing symptoms in patients with gastroesophageal reflux.

Comparison of P2X and TRPV1 Receptors in Ganglia or Primary Culture of Trigeminal Neurons and their Modulation by NGF or Serotonin

BackgroundCultured sensory neurons are a common experimental model to elucidate the molecular mechanisms of pain transduction typically involving activation of ATP-sensitive P2X or capsaicin-sensitive TRPV1 receptors. This applies also to trigeminal ganglion neurons that convey pain inputs from head tissues. Little is, however, known about the plasticity of these receptors on trigeminal neurons in culture, grown without adding the neurotrophin NGF which per se is a powerful algogen. The characteristics of such receptors after short-term culture were compared with those of ganglia. Furthermore, their modulation by chronically-applied serotonin or NGF was investigated.ResultsRat or mouse neurons in culture mainly belonged to small and medium diameter neurons as observed in sections of trigeminal ganglia. Real time RT-PCR, Western blot analysis and immunocytochemistry showed upregulation of P2X3 and TRPV1 receptors after 1–4 days in culture (together with their more frequent co-localization), while P2X2 ones were unchanged. TRPV1 immunoreactivity was, however, lower in mouse ganglia and cultures. Intracellular Ca2+ imaging and whole-cell patch clamping showed functional P2X and TRPV1 receptors. Neurons exhibited a range of responses to the P2X agonist α, β-methylene-adenosine-5'-triphosphate indicating the presence of homomeric P2X3 receptors (selectively antagonized by A-317491) and heteromeric P2X2/3 receptors. The latter were observed in 16 % mouse neurons only. Despite upregulation of receptors in culture, neurons retained the potential for further enhancement of P2X3 receptors by 24 h NGF treatment. At this time point TRPV1 receptors had lost the facilitation observed after acute NGF application. Conversely, chronically-applied serotonin selectively upregulated TRPV1 receptors rather than P2X3 receptors.ConclusionComparing ganglia and cultures offered the advantage of understanding early adaptive changes of nociception-transducing receptors of trigeminal neurons. Culturing did not prevent differential receptor upregulation by algogenic substances like NGF or serotonin, indicating that chronic application led to distinct plastic changes in the molecular mechanisms mediating pain on trigeminal nociceptors.

A Hot New Twist to Hair Biology: Involvement of Vanilloid Receptor-1 (VR1/TRPV1) Signaling in Human Hair Growth Control

The vanilloid receptor-1 (VR1, or transient receptor potential vanilloid-1 receptor, TRPV1) is activated by capsaicin, the key ingredient of hot peppers. TRPV1 was originally described on sensory neurons as a central integrator of various nociceptive stimuli. However, several human skin cell populations are also now recognized to express TRPV1, but with unknown function. Exploiting the human hair follicle (HF) as a prototypic epithelial-mesenchymal interaction system, we have characterized the HF expression of TRPV1 in situ and have examined TRPV1 signaling in organ-cultured human scalp HF and outer root sheath (ORS) keratinocytes in vitro. TRPV1 immunoreactivity was confined to distinct epithelial compartments of the human HF, mainly to the ORS and hair matrix. In organ culture, TRPV1 activation by capsaicin resulted in a dose-dependent and TRPV1-specific inhibition of hair shaft elongation, suppression of proliferation, induction of apoptosis, premature HF regression (catagen), and up-regulation of intrafollicular transforming growth factor-beta(2). Cultured human ORS keratinocytes also expressed functional TRPV1, whose stimulation inhibited proliferation, induced apoptosis, elevated intracellular calcium concentration, up-regulated known endogenous hair growth inhibitors (interleukin-1beta, transforming growth factor-beta(2)), and down-regulated known hair growth promoters (hepatocyte growth factor, insulin-like growth factor-I, stem cell factor). These findings strongly support TRPV1 as a significant novel player in human hair growth control, underscore the physiological importance of TRPV1 in human skin beyond nociception, and identify TRPV1 as a promising, novel target for pharmacological manipulations of epithelial growth disorders.

Capsaicin receptor TRPV1 in urothelium of neurogenic human bladders and effect of intravesical resiniferatoxin

To study TRPV1 immunoreactivity in the urothelium of patients with neurogenic detrusor overactivity (NDO) before and after treatment with resiniferatoxin (RTX) and controls. Functional capsaicin TRPV1 receptors have been demonstrated in urothelial cells of rodent urinary bladder, and TRPV1-knockout mice exhibit diminished nitric oxide and stretch-evoked adenosine triphosphate release from urothelial cells. In patients with NDO, TRPV1 suburothelial nerve density is increased, which is reversed by successful treatment with intravesical RTX. However, the role of urothelial TRPV1 in human bladder disorders is unknown. Flexible cystoscopic bladder biopsies were obtained from 14 patients with NDO before and after treatment with RTX and from 8 control patients. Using a specific antibody for immunostaining, TRPV1 immunoreactivity in the urothelium was quantified by image analysis. TRPV1 immunoreactivity was observed in basal and apical urothelial cells. Basal cell layer TRPV1 immunoreactivity was significantly increased in NDO compared with control bladders (P = 0.003). In 5 patients who responded clinically to RTX, basal cell layer and total urothelial TRPV1 immunoreactivity decreased significantly after treatment (P = 0.032 and P = 0.016, respectively). The decreases in the basal cell layer TRPV1 immunoreactivity after RTX were comparable to the decreases in suburothelial TRPV1 nerve fibers in the biopsies previously studied from the same patients. Increased urothelial TRPV1 in patients with NDO may play a role in the pathophysiology, in concert with increased TRPV1 nerve fibers. Although it is not known whether similar pathogenic mechanisms are involved in the increase of urothelial and neuronal TRPV1, both may be targeted by successful RTX therapy.

Substance P expression in TRPV1 and trkA-positive dorsal root ganglion neurons innervating the mouse lung

In the present study, the co-localisation of substance P (SP) with the vanilloid receptor TRPV1 and the neurotrophin receptor tyrosine kinase trkA was analysed in airway-specific murine dorsal root ganglion (DRG) neurons. DRG neurons labelled with Fast Blue were predominantly found at the segmental levels T2-T5. Immunoreactivity for the receptor TRPV1 was localized to 12% of Fast Blue labelled DRG neurons. Double-labelling immunohistochemistry revealed that a substantial number of them also co-express SP (7.6 +/- 1.1% (mean +/- S.E.M.)), whereas neurons with immunoreactivity for TRPV1 only were found in 4.4 +/- 1.3% of the retrogradely labelled neuronal population. Further analysis of retrogradely labelled neurons showed that their majority expressed trkA (62.8 +/- 1.4%), neurofilament protein 68-kDa (64.8 +/- 1.5%) or glutamate alone (19.5 +/- 1.9%). SP was always expressed in trkA-positive neurons. Based on the extent of co-localization of SP with the receptors TRPV1 and trkA in DRG airway neurons, the present study indicates that the DRG pathway may have effects on the magnitude of neurogenic inflammation in airway diseases such as asthma.

Regulation of TRPV2 by axotomy in sympathetic, but not sensory neurons

Neuropathic pain results from traumatic or disease-related insults to the nervous system. Mechanisms that have been postulated to underlie peripheral neuropathy commonly implicate afferent neurons that have been damaged but still project centrally to the spinal cord, and/or intact neurons that interact with degenerating distal portions of the injured neurons. One pain state that is observed following peripheral nerve injury in the rat is thermal hyperalgesia. The noxious heat-gated ion channel TRPV1 may be responsible for this increased sensitivity, as it is up-regulated in L4 dorsal root ganglion (DRG) neurons following L5 spinal nerve lesion (SpNL). The TRPV1 homologue TRPV2 (or VRL-1) is another member of the TRPV subfamily of TRP ion channels. TRPV2 is a nonselective cation channel activated by high noxious temperatures (>52 °C) and is present in a subset of medium- to large-diameter DRG neurons. To establish whether TRPV2 is endogenous to the spinal cord, we examined its expression in the dorsal horn following rhizotomy. We found no significant decrease in TRPV2 immunoreactivity, suggesting that TRPV2 is endogenous to the spinal cord. In order to determine whether TRPV2, like TRPV1, is regulated by peripheral axotomy, we performed L5 SpNL and characterized TRPV2 distribution in the DRG, spinal cord, brainstem, and sympathetic ganglia. Our results show that peripheral axotomy did not regulate TRPV2 in the DRG, spinal cord, or brainstem; however, TRPV2 was up-regulated in sympathetic postganglionic neurons following injury, suggesting a potential role for TRPV2 in sympathetically mediated neuropathic pain.

Nicotinic acetylcholine receptor subtypes in nociceptive dorsal root ganglion neurons of the adult rat

Stimulation of nicotinic acetylcholine receptors (nAChR) excites peripheral sensory nerve fibres, but also exert antinociceptive effects. The differences in these nAChR-mediated effects could be related to the expression of different nAChR subtypes located on nociceptive neurons. In the present study, we focused on the recently described α10-nAChR subunit, and on α4 and α7 subunits, which are the most abundant subunits in the central nervous system. In nociceptive neurons from thoracic and lumbar dorsal root ganglia (DRG), nAChR subunits were found at transcriptional (RT-PCR), translational (immunohistochemistry) and functional levels. Cultured DRG neurons express mRNA for the subunits α2–7 and α10. The α-subunit proteins 4, 7 and 10 were colocalised in virtually all nociceptive neurons that were identified by immunoreactivity for the vanilloid receptor TRPV-1. These findings were corroborated by current recordings and calcium measurements, which revealed excitatory inward currents and calcium responses in capsaicin sensitive neurons.

Discrete expression of TRPV2 within the hypothalamo‐neurohypophysial system: Implications for regulatory activity within the hypothalamic‐pituitary‐adrenal axis

Transient receptor potential channel proteins (TRPs) constitute a steadily growing family of ion channels with a range of purported functions. It has been demonstrated that TRPV2 is activated by moderate thermal stimuli and, in the rat, is expressed in medium to large diameter dorsal root ganglion neurons. In this study, antisera specific for the human TRPV2 homologue were raised and characterized for immunohistochemical use. Subsequently, thorough investigation was made of the localization of this cation channel in the macaque primate brain. TRPV2-immunoreactive material was highly restrictively localized to hypothalamic paraventricular, suprachiasmatic, and supraoptic nuclei. Confocal double- and triple-labeling studies demonstrated that TRPV2 immunoreactivity is preferentially localized to oxytocinergic and vasopressinergic neurons. Few, if any, cells in these regions expressed TRPV2 immunoreactivity in the absence of oxytocin immunoreactivity or vasopressin immunoreactivity. Expression in the paraventricular and supraoptic nuclei suggests that TRPV2 is likely to play a fundamental role in mediating cation transport in neurohypophysial neurons. TRPV2 has been shown to be translocated upon cell activation and neurons expressing TRPV2 immunoreactivity in vivo are among those known to engage in sporadic, intense activity. Taken together, these data suggest that this channel may play a vital role in mediating physiological activities associated with oxytocin and vasopressin release such as parturition, lactation, and diuresis. These data may also implicate the involvement of TRPV2 in disorders of the hypothalamic-pituitary-adrenal axis, including anxiety, depression, hypertension, and preterm labor.

Painful diabetic neuropathy (pdn): a morphological study of capsaicin receptor distribution

Capsaicin receptor TRPV1 is a non‐selective ligand‐gated channel activated by different noxious stimuli. Previous studies suggested that an increased density of TRPV1 positive axons in the skin could be involved in the pathogenesis of neuropathic pain. To investigate the expression of TRPV1 in PDN patients, skin biopsies from 6 patients with PDN and 10 controls, and sural nerve biopsies from 2 patients with PDN and 2 controls were studied using the polyclonal anti‐human TRPV1 antibody. Skin nerve fibers widely expressed TRPV1 immunoreactivity. The density of TRPV1 positive dermal and intra‐epidermal nerve fibers (IENF) did not differ from that obtained using the PGP 9.5 antibody (the standard marker for IEFN recognition). Confocal analysis demonstrated that TRPV1 and anti unique‐tubulin‐1 antibody (TuJ1) co‐localized in all axons. In PDN patients IENF density was significantly reduced, but no difference in immunostaining was detectable using TRPV1, PGP 9.5 and TuJ1 antibodies. Sural nerve unmyelinated fibers and few small‐myelinated fibers were intensely stained by TRPV1. DPN patients disclosed a severe reduction in unmyelinated fiber density, but residual fibers were recognized by TRPV1. These data suggest that TRPV1 is widely expressed in skin and sural nerve unmyelinated axons, but its expression is not increased in PDN. Other mechanisms, such as changes in proximal axon or neuron excitability, are more likely involved in the pathogenesis of neuropathic pain in diabetic neuropathy.

Differential pH and capsaicin responses of griffonia simplicifolia IB4 (IB4)-positive and IB4-negative small sensory neurons

Protons play a key role in nociception caused by inflammation and ischaemia, but little is known about the relative sensitivities of different dorsal root ganglion (DRG) neurons. We have therefore examined the responses in vitro of rat DRG cells classified according to whether or not they bind Griffonia simplicifolia IB4 (IB4), a lectin which is widely used to distinguish between two major populations of small diameter neurons. Under voltage-clamp conditions, proton-activated inward currents were found in approximately 90% of small DRG neurons and showed one of three waveforms: transient, sustained or mixed. The majority of IB4-positive (IB4+) neurons (63%) gave rise to sustained inward currents that were sensitive to capsazepine. In contrast, the most prevalent waveform in small IB4-negative (IB4−) neurons (69%) was a mixed response containing transient and sustained components. The transient component was inhibited by amiloride whilst the sustained component showed a variable sensitivity to capsazepine. We also found that more IB4+ cells responded to capsaicin and, on average, gave rise to a larger magnitude of response than small IB4− neurons, consistent with their higher prevalence and greater amplitude of vanilloid receptor 1 (TRPV1)-like acid responses. The increase in intracellular Ca 2+ induced by capsaicin was also slightly greater in IB4+ neurons and in these cells its magnitude correlated with the level of TRPV1 immunoreactivity. Our data suggest that acid-sensing ion channels (ASICs) and TRPV1 are the major acid-sensitive receptors in small IB4− neurons, whilst TRPV1 is the predominant one in IB4+ neurons. Because ASIC-like responses were approximately 10-fold more sensitive to changes in H + than TRPV1-like responses, we speculate that small IB4− rather than IB4+ neurons play an essential role in sensing acid. Our results also highlight differences in capsaicin responses between IB4+ and IB4− small neurons and reveal the close link between capsaicin responses and levels of TRPV1 expression.