Transient Receptor Potential Vanilloid Subtype Research Articles

Rutaecarpine prevented dysfunction of endothelial gap junction induced by Ox-LDL via activation of TRPV1

Gap junctions, which is formed by connexins, has been proved to play an important role in the atherogenesis development. Rutaecarpine was reported to inhibited monocyte migration, which indicates its potential for anti-atherosclerosis activity. This study evaluated the effect of rutaecarpine on endothelial dysfunction, and focused on the regulation of connexin expression in endothelial cells by rutaecarpine. Endothelia damage was induced by exposing HUVEC-12 to Ox-LDL (100mg/l) for 24h, which decreased the expression of protective proteins Cx37 and Cx40, but induced atherogenic Cx43 expression, in both mRNA and protein levels, concomitant with the impaired propidium iodide diffusion through the gap junctions. Pretreatment with rutaecarpine effectively recovered the expression of Cx37 and Cx40, but inhibited Cx43 expression, thereby improving gap junction communication and significantly prevented the endothelial dysfunction. Consequently, the cell viability and nitric oxide production were increased, lactate dehydrogenase production was decreased and monocyte adhesion was inhibited. These protective effects of rutaecarpine were remarkably attenuated by pretreatment with capsazepine, a competitive antagonist of transient receptor potential vanilloid subtype 1 (TRPV1). In summary, this study is the first to report that rutaecarpine prevents endothelial injury and gap junction dysfunction induced by Ox-LDL in vitro, which is related to regulation of connexin expression patterns via TRPV1 activation. These results suggest that rutaecarpine has the potential for use as an anti-atherosclerosis agent with a novel mechanism.

Extracellular acidification synergizes with PDGF to stimulate migration of mouse embryo fibroblasts through activation of p38MAPK with a PTX-sensitive manner

The elucidation of the functional mechanisms of extracellular acidification stimulating intracellular signaling pathway is of great importance for developing new targets of treatment for solid tumors, and inflammatory disorders characterized by extracellular acidification. In the present study, we focus on the regulation of extracellular acidification on intracellular signaling pathways in mouse embryo fibroblasts (MEFs). We found extracellular acidification was at least partly involved in stimulating p38MAPK pathway through PTX-sensitive behavior to enhance cell migration in the presence or absence of platelet-derived growth factor (PDGF). Statistical analysis showed that the actions of extracellular acidic pH and PDGF on inducing enhancement of cell migration were not an additive effect. However, we also found extracellular acidic pH did inhibit the viability and proliferation of MEFs, suggesting that extracellular acidification stimulates cell migration probably through proton-sensing mechanisms within MEFs. Using OGR1-, GPR4-, and TDAG8-gene knock out technology, and real-time qPCR, we found known proton-sensing G protein-coupled receptors (GPCRs), transient receptor potential vanilloid subtype 1 (TRPV1), and acid-sensing ion channels (ASICs) were unlikely to be involved in the regulation of acidification on cell migration. In conclusion, our present study validates that extracellular acidification stimulates chemotactic migration of MEFs through activation of p38MAPK with a PTX-sensitive mechanism either by itself, or synergistically with PDGF, which was not regulated by the known proton-sensing GPCRs, TRPV1, or ASICs. Our results suggested that others proton-sensing GPCRs or ion channels might exist in MEFs, which mediates cell migration induced by extracellular acidification in the presence or absence of PDGF.

Capsaicin Interaction with TRPV1 Channels in a Lipid Bilayer: Molecular Dynamics Simulation

Transient receptor potential vanilloid subtype 1 (TRPV1) is a heat-sensitive ion channel also involved in pain sensation, and is the receptor for capsaicin, the active ingredient of hot chili peppers. The recent structures of TRPV1 revealed putative ligand density within the S1 to S4 voltage-sensor-like domain of the protein. However, questions remain regarding the dynamic role of the lipid bilayer in ligand binding to TRPV1. Molecular dynamics simulations were used to explore behavior of capsaicin in a 1-palmitoyl-2-oleoyl phosphatidylcholine bilayer and with the target S1–S4 transmembrane helices of TRPV1. Equilibrium simulations reveal a preferred interfacial localization for capsaicin. We also observed a capsaicin molecule flipping from the extracellular to the intracellular leaflet, and subsequently able to access the intracellular TRPV1 binding site. Calculation of the potential of mean force (i.e., free energy profile) of capsaicin along the bilayer normal confirms that it prefers an interfacial localization. The free energy profile indicates that there is a nontrivial but surmountable barrier to the flipping of capsaicin between opposing leaflets of the bilayer. Molecular dynamics of the S1–S4 transmembrane helices of the TRPV1 in a lipid bilayer confirm that Y511, known to be crucial to capsaicin binding, has a distribution along the bilayer normal similar to that of the aromatic group of capsaicin. Simulations were conducted of the TRPV1 S1–S4 transmembrane helices in the presence of capsaicin placed in the aqueous phase, in the lipid, or docked to the protein. No stable interaction between ligand and protein was seen for simulations initiated with capsaicin in the bilayer. However, interactions were seen between TRPV1 and capsaicin starting from the cytosolic aqueous phase, and capsaicin remained stable in the majority of simulations from the docked pose. We discuss the significance of capsaicin flipping from the extracellular to the intracellular leaflet and mechanisms of binding site access by capsaicin.

Anti-Inflammatory Effects of Moxibustion on Mice with Adjuvant Arthritis: Role of TRPV1

Aim and Objective: This research was done to observe the anti-inflammatory effect of moxibustion and explore the role of Transient Receptor Potential Vanilloid sub type 1 (TRPV1) on Mice with adjuvant arthritis. Method: Adjuvant arthritis (AA) was established by using the amount of 20 μL of Freund’s Adjuvant Complete (FCA) intradermal injected into right hindfoot plantar. 50 KM mice were randomized into 6 groups by random number table method: control group (n=10), model group (n=8), capsaicin group (n=8), capsazepine group (n=8), moxibustion group (n=8) and Moxibustion+Capsazepine (MC) group (n=8). 16 C57BL/6 wild-type mice are randomized into 2 groups by random number table method: Wild-Type (WT) model group and WT moxibustion group with 8 in each. 14 TRPV1 knockout mice are randomized into 2 groups by random number table method: knockout (KO) model group and KO moxibustion group with 7 in each. Each mouse in the capsaicin group is subcutaneously injected with the amount of 0.1 ml/10 g into the L5, L6 spinal cords. Each mouse in the capsazepine group was intraperitoneally injected with the amount of 0.1 ml/10 g. Similarly, each mouse in the moxibustion group, WT moxibustion group and KO moxibustion group was given a suspended moxibustion with 5 mm × 200 mm specially-made moxa stick for 20 minutes on L5 and L6 spinal cords. Kept the distance between the moxa stick and the skin where L5 and L6 spinal cords located was 10 ± 2 mm that the temperature was stabilized at 46 ± 1°C. Each mouse in MC group was intraperitoneally injected with the amount of 0.1 ml/10 g first, then after 15 minutes was given a suspended moxibustion for 20 minutes on L5 and L6 spinal cords. All the treatments above were performed at the same day when models were successfully establishedand performed once per day for 7 days in total. During the performance, mice in the control group, model group, WT model group and KO model group were of no treatment in any way. The right hindfoot paw volumes of mice in each group were measured and recorded on Day 1, Day 2, Day 4, Day 6 and Day 8 of the experiment. After all treatments, the serum IL-1β and TNF-α level was determined by ELISA. Results: Compared with the model group, the paw volumes in the moxibustion group was significantly decreased (P 0.05). Conclusions: TRPV1 plays a central role in the anti-inflammatory effect of moxibustion, while the effect may be lost if lacked of TRPV1-mediated.

Role of Calcitonin Gene-related Peptide in Gastric Motility Function: Animal and Human Studies

Diabetes is one of the leading causes of gastroparesis. Gastroparesis is a state of partial gastric paralysis where gastric emptying is delayed due to slower or faster gastric motility. Over the years, many other distinct causes of gastroparesis have emerged, including collagen-related vascular diseases and Parkinson’s disease. Calcitonin gene-related peptide, or CGRP, a potent smooth muscle relaxant, is a 37-amino acid neuropeptide found in the gastrointestinal system that has been shown to slow gastric emptying in healthy rodents. Regulation of its synthesis and release from neurons is achieved in part by activation of the transient receptor potential vanilloid subtype 1 (TRPV1). Regulation of its function is achieved by binding to its own receptor, a complex of the Calcitonin Receptorlike Receptor (CRLR) and Receptor Activity Modifying Protein 1 (RAMP1). In the gastrointestinal system, CGRP regulates the release of somatostatin, also known as Growth Hormone Inhibiting Hormone, or GHIH. Somatostatin functions to inhibit the release of gastric acid thus, demonstrating a possible indirect role of CGRP in the pathogenesis of gastroparesis. Cells present in the gastrointestinal system include mucosal cells, submucosal cells, myenteric neuronal cells, and others. CGRP is localized in the gastric mucosa and the myenteric plexus. The neurons present in the enteric or myenteric nervous system play a vital role in gastric motility functions. CGRP is released from the inhibitory neurons in the enteric or myenteric nervous system. Both serum and gastric CGRP have been found to be decreased in diabetes, but it is not known whether these decreases play a role in the pathogenesis of gastroparesis in diabetic/idiopathic patients. The present review provides information about CGRP, its functions, and its possible roles in gastroparesis. The findings presented can be a great asset to the medical community in directing future research for possible causes and developing treatments for gastroparesis.

The effect of clonidine pretreatment on epidural resiniferatoxin in a neuropathic pain rat model.

Resiniferatoxin (RTX) is an ultrapotent synthetic TRPV1 (transient receptor potential vanilloid subtype 1) agonist with significant initial transient hyperalgesia followed by a prolonged analgesic effect in response to thermal stimulus. Using a rat model of neuropathic pain, we evaluated the effect of pretreatment with clonidine-which has been shown to relieve intradermal capsaicin-induced hyperalgesia-on the initial hyperalgesic response and the thermal analgesic property of RTX. Thirty-six male rats were divided into 6 treatment groups (n=6 each):RTX 500 ng, RTX 1 μg, clonidine 20 μg (Cl), Cl＋RTX 500 ng, Cl＋RTX 1 μg, or normal saline 20 μL (control). We evaluated the short-term (180 min) and long-term (20 days) analgesic effects of RTX after thermal stimulation and mechanical stimulation. RTX had significant initial transient hyperalgesia followed by a prolonged analgesic effect in response to the thermal stimulus, but the RTX 500 ng and RTX 1 μg groups showed no initial short-term thermal hyperalgesic responses when pretreated with clonidine. The Cl＋RTX 1 μg rats' behavior scores indicated that they were more calm and comfortable compared to the RTX 1 μg rats. Even though we cannot precisely confirm that pretreatment with clonidine potentiates or adds to the analgesic effect of RTX, clonidine pretreatment with epidural RTX eliminated the initial RTX-associated hyperalgesic response and systemic toxicity in this neuropathic pain rat model.

Segmental transport of Ca²⁺ and Mg²⁺ along the gastrointestinal tract.

Calcium (Ca(2+)) and magnesium (Mg(2+)) ions are involved in many vital physiological functions. Since dietary intake is the only source of minerals for the body, intestinal absorption is essential for normal homeostatic levels. The aim of this study was to characterize the absorption of Ca(2+) as well as Mg(2+) along the gastrointestinal tract at a molecular and functional level. In both humans and mice the Ca(2+) channel transient receptor potential vanilloid subtype 6 (TRPV6) is expressed in the proximal intestinal segments, whereas Mg(2+) channel transient receptor potential melastatin subtype 6 (TRPM6) is expressed in the distal parts of the intestine. A method was established to measure the rate of Mg(2+) absorption from the intestine in a time-dependent manner by use of (25)Mg(2+). In addition, local absorption of Ca(2+) and Mg(2+) in different segments of the intestine of mice was determined by using surgically implanted intestinal cannulas. By these methods, it was demonstrated that intestinal absorption of Mg(2+) is regulated by dietary needs in a vitamin D-independent manner. Also, it was shown that at low luminal concentrations, favoring transcellular absorption, Ca(2+) transport mainly takes place in the proximal segments of the intestine, whereas Mg(2+) absorption predominantly occurs in the distal part of the gastrointestinal tract. Vitamin D treatment of mice increased serum Mg(2+) levels and 24-h urinary Mg(2+) excretion, but not intestinal absorption of (25)Mg(2+). Segmental cannulation of the intestine and time-dependent absorption studies using (25)Mg(2+) provide new ways to study intestinal Mg(2+) absorption.

Distinct Reactivity of Transient Receptor Potential Vanilloid Subtype 1 in a Murine Model of Atopic Dermatitis with Serious Scratching

Background: Abnormality in skin sensitivity may be responsible for unbearable itch in patients with atopic dermatitis (AD). Objectives: We evaluated reactivity of NC/Tnd mice, a model for human AD, against various experimental stimulations. Methods: Several behavioral tests were performed after external stimuli were applied to NC/Tnd mice. Transient receptor potential vanilloid subtype 1 (TRPV1) reactivity of neuronal cells collected from the dorsal root ganglions (DRG) was analyzed with a Ca++ influx test. Finally, we evaluated suppressive effect of capsaicin on atopic itch of NC/Tnd mice. Results: Pain responses to heat, acidic stimulation, and capsaicin injection, which are transduced through TRPV1, were decreased in NC/Tnd mice, when compared to two standard strains BALB/c and C57BL/6 mice. The reactivity of the primary neurons isolated from DRG to capsaicin was markedly reduced in NC/Tnd mice. Topical application of histamine evoked scratching in NC/Tnd mice as well as other two strains; however, the scratching intensities induced by nonhistamine pruritogens were significantly lower in NC/Tnd mice comparing to the two strains. In conventional NC/Tnd mice with AD, topical application of capsaicin reduced the scratching behavior. Conclusion: TRPV1 is associated with both pain and itch sensation; however, abnormalities in TRPV1 reactivity may involve in severe itch in NC/Tnd mice.

Modulation of Transient Receptor Potential Vanilloid Subtype 1 (TRPV<sub>1</sub>) and Norepinephrine Transporters (NET) Protect Against Oxidative Stress, Cellular Injury, and Vascular Dementia

Chronic cerebral hypoperfusion (CCH) is a risk factor for the development of vascular dementia (VaD). CCH participates in a negative role in cognitive impairments. Transient receptor potential vanilloid subtype 1 (TRPV1) participates in cognition, ischemic damage and neuroprotection. Selective norepinephrine transporter (NET) inhibitors have a role in cognitive dysfunction and oxidative stress. The role of TRPV1 and NET in CCH induced VaD is still unknown. The present study has been structured to investigate the role of vanillin; a selective agonist of TRPV1 as well as atomoxetine; a selective NET inhibitor in CCH induced VaD in mice. Permanent bilateral common carotid arteries ligation or two vessel occlusion (2VO) technique was used to induce a stage of chronic cerebral hypoperfusion in mice. 2VO animals have shown significant impairment of locomotion (Actophotometer), motor coordination (Rota rod), learning and memory (Morris water maze). 2VO animals have shown significant reduction in brain catalase, glutathione, and superoxide dismutase, with significant increase in brain infarct size (TTC staining), malondialdehyde and acetyl cholinesterase-AChE activity. Whereas, administration of vanillin as well as atomoxetine has significantly attenuated 2VO induced impaired locomotion, motor coordination, learning and memory, brain damage, brain oxidative stress and higher AChE activity. It may be concluded that 2VO induced CCH has elicited VaD, which was attenuated by vanillin and atomoxetine. Thus, modulators of vanilloid receptors and norepinephrine transporter may be explored further for their benefits in CCH induced VaD.

Impairment of corneal epithelial wound healing in a TRPV1-deficient mouse.

To examine whether the absence or blockage of an ion channel receptor, transient receptor potential vanilloid subtype 1 (TRPV1), affects the healing of an epithelial injury using an experimental model of an epithelial defect in animal cornea. The expression of TRPV1 in the corneal epithelium was examined using immunohistochemistry in mice and rats. The migration of the corneal epithelium was examined in epithelium-debrided rat cornea in organ culture in the presence or absence of a TRPV1 agonist or its antagonist. Epithelial migration and cell proliferation following the debridement were examined in the cornea of a TRPV1-null mouse. Real-time RT-PCR was performed in samples of healing corneas to analyze the expression pattern of epithelial migration-related components (i.e., IL-6, substance P, and TGF-β1). TRPV1 was detected mainly in the basal layer of mouse or rat corneal epithelium. Adding a TRPV1 receptor agonist to the culture medium enhanced epithelial healing in the rat cornea, and a TRPV1 antagonist retarded it in organ culture. The loss of TRPV1 did not affect the histology of the mouse cornea. In vivo analysis showed the loss of TRPV1-impaired re-epithelialization of the debrided area of the corneal epithelium by the suppression of both cell migration and proliferation. The lack of TRPV1 suppressed the expression of IL-6 and substance P but not of TGF-β1 in response to epithelial debridement in mice. TRPV1 signal is required for the upregulation of IL-6 and substance P and the healing of debrided corneal epithelium in mice.

Increased Severity of Inflammation Correlates with Elevated Expression of TRPV1 Nerve Fibers and Nerve Growth Factor on Interstitial Cystitis/Bladder Pain Syndrome

Objectives: Although evidence supports a role for inflammation in interstitial cystitis/bladder pain syndrome (IC/BPS), the mechanism remains unknown. We determined whether inflammation causes an elevated expression of nerve growth factor (NGF) and transient receptor potential vanilloid receptor subtype 1 (TRPV1) and correlated them with the symptoms. Methods: Bladder biopsies were obtained from 53 IC/BPS patients and 27 controls, and hematoxylin and eosin staining, immunostaining and Western blotting were performed to detect inflammation, TRPV1-immunoreactive and PGP9.5-immunoreactive nerve fibers, and NGF, respectively. Symptoms were assessed using the Pelvic Pain/Urgency/Frequency (PUF) questionnaire and pain visual analogue scale scores. Suburothelial nerve fiber density was quantified and correlated with PUF scores. Results: Increased severity of inflammation was correlated with a higher TRPV1-immunoreactive nerve fiber density (r = 0.4113, p = 0.0024) and higher NGF levels (r = 0.3775, p = 0.0052). Suburothelial TRPV1-immunoreactive nerve fiber density was significantly correlated with pain scores and urgency scores (r = 0.3320, p = 0.0145 and r = 0.3823, p = 0.0039, respectively). PGP9.5-immunoreactive nerve fibers were significantly increased in IC/BPS (p = 0.0193) and had a positive relationship with inflammation severity (r = 0.6138, p < 0.0001). Conclusions: Our study revealed increased severity of inflammation correlated with a higher expression of TRPV1-immunoreactive nerve fibers and NGF in IC/BPS and correlated with clinical symptoms.

TRPV1 in Salivary Gland Epithelial Cells Is Not Involved in Salivary Secretion via Transcellular Pathway.

Transient receptor potential vanilloid subtype 1 (TRPV1) was originally found in sensory neurons. Recently, it has been reported that TRPV1 is expressed in salivary gland epithelial cells (SGEC). However, the physiological role of TRPV1 in salivary secretion remains to be elucidated. We found that TRPV1 is expressed in mouse and human submandibular glands (SMG) and HSG cells, originated from human submandibular gland ducts at both mRNA and protein levels. However, capsaicin (CAP), TRPV1 agonist, had little effect on intracellular free calcium concentration ([Ca2+]i) in these cells, although carbachol consistently increased [Ca2+]i. Exposure of cells to high temperature (>43℃) or acidic bath solution (pH5.4) did not increase [Ca2+]i, either. We further examined the role of TRPV1 in salivary secretion using TRPV1 knock-out mice. There was no significant difference in the pilocarpine (PILO)-induced salivary flow rate between wild-type and TRPV1 knock-out mice. Saliva flow rate also showed insignificant change in the mice treated with PILO plus CAP compared with that in mice treated with PILO alone. Taken together, our results suggest that although TRPV1 is expressed in SGEC, it appears not to play any direct roles in saliva secretion via transcellular pathway.

Nitric oxide induces apoptosis associated with TRPV1 channel-mediated Ca2+ entry via S-nitrosylation in osteoblasts

The high-level production of nitric oxide (NO) induced by inflammatory cytokines has been shown to play a key role in the pathogenesis of inflammation-mediated osteoporosis. In the present work, we observed that 1mM of the NO donor sodium nitroprusside (SNP) induced an increase of the cytosolic calcium concentration ([Ca2+]c) in osteoblasts, which was completely abolished by applying an extracellular Ca2+-free buffer. Further experiments showed that the SNP-induced [Ca2+]c increase was specifically blocked by potent antagonists of the transient receptor potential vanilloid subtype 1 (TRPV1) channel: capsazepine, ruthenium red, and La3+ in Ca2+-containing buffer. However, nifedipine, an L-type voltage sensitive Ca2+-channel blocker, failed to suppress the [Ca2+]c elevation caused by SNP. Additionally, 1mM SNP induced osteoblast apoptosis, which was largely inhibited by the blockers of TRPV1, capsazepine and ruthenium red. Interestingly, our data showed that the SNP-induced [Ca2+]c increase was significantly inhibited by N-ethylmaleimide, the blocker of S-nitrosylation modification, instead of inhibitors of the NO–cGMP–PKG pathway. Taken together, our data clearly demonstrated that the NO donor SNP resulted in apoptosis associated with TRPV1 channel-mediated Ca2+ entry via S-nitrosylation in osteoblasts.

Disrupting Sensitization of Transient Receptor Potential Vanilloid Subtype 1 Inhibits Inflammatory Hyperalgesia

Transient receptor potential vanilloid subtype 1 (TRPV1) is a heat-sensitive ion channel that plays a key role in enhanced pain sensation after inflammation, but directly blocking TRPV1 causes hyperthermia and decreased sensitivity to painful levels of heat in animals and humans. Here we explore an alternative analgesic strategy in which the modulation of TRPV1 is inhibited by antagonizing the interaction between TRPV1 and A kinase anchoring protein 79 (AKAP79), a scaffolding protein essential for positioning serine-threonine kinases adjacent to target phosphorylation sites. We first defined key residues in the domain in TRPV1 that interacts with AKAP79, and we then used this information to construct short peptides capable of preventing TRPV1-AKAP79 interaction. An effective peptide, when coupled to a TAT sequence conferring cell permeability, was found to be analgesic in three mouse models of inflammatory hyperalgesia. These results demonstrate the potential value of interfering with the interaction between TRPV1 and AKAP79 as a novel analgesic strategy.

A TRPV4 Channel C-terminal Folding Recognition Domain Critical for Trafficking and Function

The Ca(2+)-permeable transient receptor potential vanilloid subtype 4 (TRPV4) channel mediates crucial physiological functions, such as calcium signaling, temperature sensing, and maintaining cell volume and energy homeostasis. Noticeably, most disease-causing genetic mutations are concentrated in the cytoplasmic domains. In the present study, we focused on the role of the TRPV4 C terminus in modulating protein folding, trafficking, and activity. By examining a series of C-terminal deletions, we identified a 20-amino acid distal region covering residues 838-857 that is critical for channel folding, maturation, and trafficking. Surface biotinylation, confocal imaging, and fluorescence-based calcium influx assay demonstrated that mutant proteins missing this region were trapped in the endoplasmic reticulum and unglycosylated, leading to accelerated degradation and loss of channel activity. Rosetta de novo structural modeling indicated that residues 838-857 assume a defined conformation, with Gly(849) and Pro(851) located at critical positions. Patch clamp recordings confirmed that lowering the temperature from 37 to 30 °C rescued channel activity of folding-defective mutants. Moreover, biochemical tests demonstrated that, in addition to participating in C-C interaction, the C terminus also interacts with the N terminus. Taken together, our findings indicate that the C-terminal region of TRPV4 is critical for channel protein folding and maturation, and the short distal segment plays an essential role in this process. Therefore, selectively disrupting the folding-sensitive region may present therapeutic potential for treating overactive TRPV4-mediated diseases, such as pain and skeletal dysplasias.

Targeting TRPV3 for the Development of Novel Analgesics.

Decades of characterization of the transient receptor potential vanilloid subtype 1 (TRPV1) has led to the realization of its central role in thermosensation and pain perception. A large number of pharmaceutical companies have had interest in developing TPRV1 antagonists for the treatment of pain. The subsequent discovery of multiple other members of this TRPV family has not gone unnoticed. TRPV3 exhibits approximately 40% homology to TRPV1, and has common as well as distinct features from TRPV1 in channel physiology, expression and function. Here we review the current understanding of TRPV3 channel biology, activation, sensitization and the consequences of TRPV3 manipulation for thermosensation and nociception, as well as additional considerations regarding the expression of TRPV3 in the skin. We weigh in on the available evidence in the context of potential development of TRPV3 modulating agents as analgesics.

Paradoxic effects of propofol on visceral pain induced by various TRPV1 agonists

Intraperitoneal injection of propofol inhibits subsequent acetic acid-induced writhing response in mice. Propofol increases the sensitivity of dorsal root ganglion neurons to capsaicin through transient receptor potential ankyrin subtype-1 (TRPA1) and protein kinase Cε (PKCε)-mediated phosphorylation of transient receptor potential vanilloid subtype-1 (TRPV1). Intraperitoneal co-injection of propofol may increase visceral nociception induced by TRPV1 agonists via sensitization of TRPV1. Therefore, we investigated the effects of intraperitoneal co-injection of propofol on nociception induced by acetic acid and capsaicin. The number of writhing movements induced by acetic acid or nociception time by capsaicin with or without propofol were counted. Neonatal capsaicin-treated mice were also used to demonstrate the role of TRPV1 in the effects of propofol on nociception, induced by TRPV1 agonists. Co-injection of propofol resulted in a pronociceptive effect on the writhing response induced by acetic acid, while the same dose of propofol ameliorated the response to capsaicin. The writhing response to intraperitoneal acetic acid was sharply inhibited following neonatal treatment with capsaicin. Co-injection with propofol reduced the number of writhing movements induced by acetic acid in neonatal capsaicin-treated mice. These results suggest that propofol binds to TRPV1 at the capsaicin-binding pocket.

TRPV4 and the Regulation of Vascular Tone

Recent studies have introduced the importance of transient receptor potential vanilloid subtype 4 (TRPV4) channels in the regulation of vascular tone. TRPV4 channels are expressed in both endothelium and vascular smooth muscle cells and can be activated by numerous stimuli including mechanical (eg, shear stress, cell swelling, and heat) and chemical (eg, epoxyeicosatrienoic acids, endocannabinoids, and 4α-phorbol esters). In the brain, TRPV4 channels are primarily localized to astrocytic endfeet processes, which wrap around blood vessels. Thus, TRPV4 channels are strategically localized to sense hemodynamic changes and contribute to the regulation of vascular tone. TRPV4 channel activation leads to smooth muscle cell hyperpolarization and vasodilation. Here, we review recent findings on the cellular mechanisms underlying TRPV4-mediated vasodilation; TRPV4 channel interaction with other proteins including transient receptor potential channel 1, small conductance (K(Ca)2.3), and large conductance (K(Ca)1.1) calcium-activated potassium-selective channels; and the importance of caveolin-rich domains for these interactions to take place.

Occludin is required for TRPV1-modulated paracellular permeability in the submandibular gland

Occludin plays an important role in maintaining tight junction barrier function in many types of epithelia. We previously reported that activation of transient receptor potential vanilloid subtype 1 (TRPV1) in rabbit submandibular gland promoted salivary secretion, partly by an increase in paracellular permeability. We have now explored the role of occludin in TRPV1-modulated paracellular permeability in a rat submandibular gland cell line SMG-C6. Both TRPV1 and occludin were expressed in SMG-C6 cells, and capsaicin induced redistribution of occludin, but not claudin-3, claudin-4 or E-cadherin, from the cell membrane into the cytoplasm. Capsaicin also decreased transepithelial electrical resistance (TER) and increased the Trypan Blue and FITC-dextran flux. Capsazepine (CPZ), a TRPV1 antagonist, inhibited the capsaicin-induced occludin redistribution and TER decrease. Moreover, occludin knockdown by shRNA suppressed, whereas occludin re-expression restored, the TER response to capsaicin. Mechanistically, TRPV1 activation increased ERK1/2 and MLC2 phosphorylation. PD98059, an ERK1/2 kinase inhibitor, abolished the capsaicin-induced MLC2 phosphorylation, whereas ML-7, an MLC2 kinase inhibitor, did not affect ERK1/2 phosphorylation, suggesting that ERK1/2 is the upstream signaling molecule of MLC2. Capsaicin also induced F-actin reorganization, which was abolished by CPZ, PD98059 and ML-7, indicating that TRPV1 activation altered F-actin organization in an ERK1/2- and MLC2-dependent manner. Furthermore, either PD98059 or ML-7 could abolish the capsaicin-induced TER response and occludin redistribution, whereas knockdown of ERK1/2 further confirmed that the TRPV1-modulated paracellular permeability was ERK1/2 dependent. Taken together, these results identified a crucial role of occludin in submandibular epithelial cells, and more importantly, demonstrated that occludin was required to mediate TRPV1-modulated paracellular permeability.

Expression-dependent pharmacology of transient receptor potential vanilloid subtype 1 channels in Xenopus laevis oocytes

Transient receptor potential vanilloid subfamily member 1 channels are polymodal sensors of noxious stimuli and integral players in thermosensation, inflammation and pain signaling. It has been shown previously that under prolonged stimulation, these channels show dynamic pore dilation, providing a pathway for large and otherwise relatively impermeant molecules. Further, we have shown recently that these nonselective cation channels, when activated by capsaicin, are potently and reversibly blocked by external application of quaternary ammonium compounds and local anesthetics. Here we describe a novel phenomenon in transient receptor potential channel pharmacology whereby their expression levels in Xenopus laevis oocytes, as assessed by the magnitude of macroscopic currents, are negatively correlated with extracellular blocker affinity: small current densities give rise to nanomolar blockade by quaternary ammoniums and this affinity decreases linearly as current density increases. Possible mechanisms to explain these data are discussed in light of similar observations in other channels and receptors.