Transient Receptor Potential Vanilloid 1 Protein Expression Research Articles

Inhibition of TRPV1 prevented skin irritancy induced by phenoxyethanol. A preliminary in vitro and in vivo study.

Phenoxyethanol is a widely used preservative in personal care products. Transient receptor potential vanilloid 1 (TRPV1) on cell membrane is activated by TRPV1 agonist capsaicin resulting in an opening of the channel for calcium influx, which is linked with neurosensory sensations characterized by itching, burning and stinging of skin. Whether uncomfortable skin sensations caused by phenoxyethanol claimed by people having sensitive skin are also due to activation of TRPV1 has not been reported in the literature. The aim of this study was to evaluate whether the TRPV1 is involved in the induction of itching and burning sensation by phenoxyethanol. The effect of phenoxyethanol on TRPV1 was assessed in vitro on HaCaT cells. The activation of TRPV1 and its inhibition by a TRPV1 antagonist were evaluated by cellular calcium influx. TRPV1 protein expression was also investigated by Western blot. In vivo in a split-face study, phenoxyethanol formulated at 1% was compared to a formulation additionally containing a TRPV1 antagonist. By applying the formulations to the nasolabial fold, the scores of phenoxyethanol-induced sensations were compared to those of the TRPV1 antagonist. In vitro phenoxyethanol induced calcium influx in HaCaT cells in a dose-dependent manner after 20 min. This effect was abolished by a solution containing the TRPV1 antagonist trans-tert-butyl cyclohexanol (ID1609). Phenoxyethanol had no effect on the expression of TRPV1, whereas capsaicin caused a significant downregulation of this receptor in the same experiment. In vivo 1% phenoxyethanol induced a skin burning and itching sensation in a cohort of 60 of 243 Chinese female subjects being sensitive to phenoxyethanol discomfort. The uncomfortable skin sensations were significantly inhibited by ID1609. Different to capsaicin, phenoxyethanol did not downregulate the expression of TRPV1 in HaCaT cells, suggesting that different regulatory mechanisms may exist for capsaicin and phenoxyethanol. Our experiments demonstrated that phenoxyethanol induces skin misperception and uncomfortable skin sensations like itching and burning comparable to capsaicin via activating TRPV1. The stimulation was inhibited by blocking TRPV1 with ID1609. The present data strengthened hitherto studies that TRPV1 plays a critical role in sensitive skin.

Analgesic Effect of Electroacupuncture in a Mouse Fibromyalgia Model: Roles of TRPV1, TRPV4, and pERK.

Fibromyalgia (FM) is among the most common chronic pain syndromes encountered in clinical practice, but there is limited understanding of FM pathogenesis. We examined the contribution of transient receptor potential vanilloid 1 (TRPV1) and TRPV4 channels to chronic pain in the repeated acid injection mouse model of FM and the potential therapeutic efficacy of electroacupuncture. Electroacupuncture (EA) at the bilateral Zusanli (ST36) acupoint reduced the long-lasting mechanical hyperalgesia induced by repeated acid saline (pH 4) injection in mouse hindpaw. Isolated L5 dorsal root ganglion (DRG) neurons from FM model mice (FM group) were hyperexcitable, an effect reversed by EA pretreatment (FM + EA group). The increase in mechanical hyperalgesia was also accompanied by upregulation of TRPV1 expression and phosphoactivation of extracellular signal regulated kinase (pERK) in the DRG, whereas DRG expression levels of TRPV4, p-p38, and p-JNK were unaltered. Blockade of TRPV1, which was achieved using TRPV1 knockout mice or via antagonist injection, and pERK suppressed development of FM-like pain. Both TRPV1 and TRPV4 protein expression levels were increased in the spinal cord (SC) of model mice, and EA at the ST36 acupoint decreased overexpression. This study strongly suggests that DRG TRPV1 overexpression and pERK signaling, as well as SC TRPV1 and TRPV4 overexpression, mediate hyperalgesia in a mouse FM pain model. The therapeutic efficacy of EA may result from the reversal of these changes in pain transmission pathways.

Effect of paclitaxel on transient receptor potential vanilloid 1 in rat dorsal root ganglion

Peripheral neuropathy is a common adverse effect of paclitaxel treatment. To analyze the contribution of transient receptor potential vanilloid 1 (TRPV1) in the development of paclitaxel-induced thermal hyperalgesia, TRPV1 expression in the rat dorsal root ganglion (DRG) was analyzed after paclitaxel treatment. Behavioral assessment using the tail-flick test showed that intraperitoneal administration of 2 and 4mg/kg paclitaxel induced thermal hyperalgesia after days 7, 14, and 21. Paclitaxel-induced thermal hyperalgesia after day 14 was significantly inhibited by the TRP antagonist ruthenium red (3mg/kg, s.c.) and the TRPV1 antagonist capsazepine (30mg/kg, s.c.). Paclitaxel (2 and 4mg/kg) treatment increased the expression of TRPV1 mRNA and protein in DRG neurons. Immunohistochemistry showed that paclitaxel (4mg/kg) treatment increased TRPV1 protein expression in small and medium DRG neurons 14days after treatment. Antibody double labeling revealed that isolectin B4-positive small DRG neurons co-expressed TRPV1. TRPV1 immunostaining was up-regulated in paw skin day 14 after paclitaxel treatment. Moreover, in situ hybridization histochemistry revealed that most of the TRPV1 mRNA-labeled neurons in the DRG were small or medium in size. These results suggest that paclitaxel treatment increases TRPV1 expression in DRG neurons and may contribute to functional peripheral neuropathic pain.

The Expression and Functionality of Transient Receptor Potential Vanilloid 1 in Ovarian Endometriomas

Pains of various kinds--dysmenorrhea, chronic pelvic pain, and dyspareunia--are the major complaints from women with endometriosis, representing the most debilitating nature of the disease. Despite extensive research, our understanding as how endometriosis causes pain is still fragmentary. In this study, we examined transient receptor potential vanilloid 1 (TRPV1)-positive nerve fibers in ectopic endometrium from women with ovarian endometriomas and in endometrium from women without endometriosis and correlated the density with the severity of dysmenorrhea in cases. We also performed an immunohistochemistry analysis of TRPV1 in ectopic and control endometrium. After finding TRPV1 immunoreactivity in ectopic endometrial cells, we further examined whether TRPV1 is functional in ectopic endometrial stromal cells (EESCs). We found that the density of TRPV1-positive nerve fibers in ectopic endometrial implants is higher than that in control endometrium and correlates positively with the severity of dysmenorrhea in women with endometriosis. In addition, TRPV1 expression was also found to be elevated significantly in EESCs when stimulated with inflammatory mediators such as prostaglandin E2 (PGE(2) ) and tumor necrosis factor-α (TNF-α). Finally, we found that TRPV1 activation can induce the release of nitric oxide (NO) and interleukin 1β (IL-1β) in EESCs. The latter finding appears to be consistent with the reports of increased TRPV1 protein expression following peripheral inflammation. Our results suggest that the increased TRPV1-positive nerve fibers may integrate various stimuli on peripheral terminals or primary sensory neurons and generate hyperalgesia in endometriosis. The expression and functionality of TRPV1 in EESCs also suggest that TRPV1 may have neurosecretory functions that are yet to be elucidated.

Increased neuronal activity in the OVLT of Cyp1a1-Ren2 transgenic rats with inducible Ang II-dependent malignant hypertension

The contribution of angiotensin II (Ang II) to the pathophysiology of hypertension is established based on facts that high levels of circulating Ang II increase vasoconstriction of peripheral arteries causing a rise in blood pressure (BP). In addition, circulating Ang II has various effects on the central nervous system, including the osmosensitive neurons in the organum vasculosum of the lamina terminalis (OVLT). Osmosensitive neurons in the OVLT transduce hypertonicity via the activation of the nonselective cation channel known as transient receptor potential vanilloid 1 (TRPV1), causing membrane depolarization, followed by increased action potential discharge. This effect is absent in mice lacking expression of the TRPV1 gene. Most observations related to the importance of the OVLT in cardiovascular control are mainly based on models of lesion of the entire preoptic periventricular tissue. However, it remains unclear whether neuronal activity and TRPV1 protein expression levels alter in the OVLT of Cyp1a1-Ren2 transgenic rats with inducible Ang II-dependent malignant hypertension. C-fos was used as a marker of neuronal activity. Immunostaining was used to demonstrate distribution of c-fos positive neurons in the OVLT of Cyp1a1Ren2 transgenic rats. Western blot analysis showed increased c-fos and TRPV1 total protein expression levels in the OVLT of hypertensive rats. The present findings demonstrate increased c-fos and TRPV1 expression levels in the OVLT of Cyp1a1-Ren2 transgenic rats with Ang II-dependent malignant hypertension.

Effect of Surgical and Chemical Sensory Denervation on Non-neural Expression of the Transient Receptor Potential Vanilloid 1 (TRPV1) Receptors in the Rat

Pretreatment with the ultrapotent capsaicin analog resiniferatoxin (RTX) has been applied as a selective pharmacological tool in inflammation and pain studies to desensitize transient receptor potential vanilloid 1 (TRPV1) receptor-expressing sensory nerve endings. The discovery of TRPV1 receptor on non-neural cells challenges systemic RTX desensitization as a method acting exclusively on a population of sensory neurons, but not on non-neural cells. Systemic RTX desensitization was used for chemical denervation and transection of the sciatic and saphenous nerves for surgical denervation in rats. Quantitative real-time PCR and immunohistochemistry were applied to investigate the presence and alterations of the TRPV1 receptor mRNA and protein following chemical and surgical denervation. We provided the first evidence for non-neural TRPV1 immunopositivity and mRNA expression in the rat dorsal paw and plantar skin as well as the oral mucosa. Neither chemical nor surgical denervation influenced the level of TRPV1 receptor mRNA and protein expression in non-neural cells of either skin regions or mucosa. Therefore, RTX and consequently capsaicin remain to be considered as selective neurotoxins for a population of primary afferent neurons.

Evidence for neurotrophic factors associating with TRPV1 gene expression in the inflamed human esophagus

Transient receptor potential vanilloid-1 (TRPV1) receptor has been implicated in the mechanism of acid induced inflammation in gastro-esophageal reflux disease (GERD). It has been demonstrated that the increase in nerve growth factor (NGF) and glial derived neurotrophic factor (GDNF) was associated with the increased expression of TRPV1. We aimed to determine whether expression of TRPV1 was increased in severe inflamed human esophagus, and to test the hypothesis whether the expression of TRPV1 was mediated by neurotrophic factors such as NGF and GDNF. We compared biopsies taken from the distal esophagus of 15 patients with erosive GERD, 16 asymptomatic patients (AP), and 10 healthy controls. We assessed the biopsies with reverse transcription polymerase chain reaction (RT-PCR) and real-time quantitative polymerase chain reaction (qPCR) for TRPV1, NGF, and GDNF. Immunohistochemical analysis of TRPV1 protein expression was also determined. Transient receptor potential vanilloid-1 mRNA level and its protein expression were significantly greater in patients with erosive esophagitis than AP (P < 0.001) and healthy controls (P < 0.001). Nerve growth factor and GDNF gene levels in the esophageal mucosa were also significantly increased in patients with erosive esophagitis compared with AP and healthy controls (all P < 0.001). Transient receptor potential vanilloid-1 mRNA correlated well with NGF (r = 0.61, P < 0.001) and GDNF (r = 0.58, P < 0.001). These results support the association of NGF and GDNF in the up-regulation of TRPV1 receptors in patients with erosive esophagitis.

Inhibition of the expression and function of TRPV4 by RNA interference in dorsal root ganglion

Background: Transient receptor potential vanilloid 4 (TRPV4) calcium channel is an essential mediator of spinal nociceptive transmission in dorsal root ganglion (DRG), but its mechanism in pain transmission is poorly understood. The present study intended to evaluate the effect of small interfering RNA (siRNA) on the expression and function of TRPV4 in DRG neurons and its possible usage in pain relief treatment.Methods: Two pairs of siRNA targeting TRPV4 (siRNAa and siRNAb) were designed and synthesized. A mismatched double-strand RNA was chosen as negative control. siRNAs were transfected into primary culture DRG neurons with cationic lipid-based CodeBreaker reagent. Real-time reverse transcription polymerase chain reaction and Western blot analysis were performed to assess the change of TRPV4 messenger RNA (mRNA) and protein expression in DRG. Calcium imaging and ELISA of substance P were carried out to evaluate the function change of TRPV4. The relationship between TRPV4 and PKC was also observed by immunocytochemistry.Results: TRPV4 mRNA levels in DRG of mismatch and control groups were significantly higher (3·88- and 3·75-fold) than normal. However, siRNAa and siRNAb increased only 1·26- and 1·87-fold, respectively. TRPV4 protein expression was also decreased significantly, as indicated by Western blot. In addition, the function of TRPV4 as ion channel was evaluated by calcium imaging and P substance release before and after siRNA treatment. Inhibition of TRPV4 activity was observed after transfection with both siRNA sequences. Protein kinase C expression, which was thought to be related with TRPV4 function, was inhibited as well.Conclusion: TRPV4 is a key component in hypotonic signal transduction, and designed siRNA inhibits mRNA level, protein expression and function of TRPV4 in DRG neurons. Therefore, synthesized siRNA is suggested to be a potential treatment for TRPV4-related pathological conditions, specifically pain.