Rapid Fluorescence Lifetime Imaging Reveals That TRPV4 Channels Promote Dysregulation of Neuronal Na+ in Ischemia.

Fluorescence imaging is an indispensable method for analysis of diverse cellular and molecular processes, enabling, for example, detection of ions, second messengers, or metabolites. Intensity-based approaches, however, are prone to artifacts introduced by changes in fluorophore concentrations. This drawback can be overcome by fluorescence lifetime imaging (FLIM) based on time-correlated single-photon counting. FLIM often necessitates long photon collection times, resulting in strong temporal binning of dynamic processes. Recently, rapidFLIM was introduced, exploiting ultra-low dead-time photodetectors together with rapid electronics. Here, we demonstrate the applicability of rapidFLIM, combined with new and improved correction schemes, for spatiotemporal fluorescence lifetime imaging of low-emission fluorophores in a biological system. Using tissue slices of hippocampi of mice of either sex, loaded with the Na+ indicator ING2, we show that improved rapidFLIM enables quantitative, dynamic imaging of neuronal Na+ signals at a full-frame temporal resolution of 0.5 Hz. Induction of transient chemical ischemia resulted in unexpectedly large Na+ influx, accompanied by considerable cell swelling. Both Na+ loading and cell swelling were dampened on inhibition of TRPV4 channels. Together, rapidFLIM enabled the spatiotemporal visualization and quantification of neuronal Na+ transients at unprecedented speed and independent from changes in cell volume. Moreover, our experiments identified TRPV4 channels as hitherto unappreciated contributors to neuronal Na+ loading on metabolic failure, suggesting this pathway as a possible target to ameliorate excitotoxic damage. Finally, rapidFLIM will allow faster and more sensitive detection of a wide range of dynamic signals with other FLIM probes, most notably those with intrinsic low-photon emission.SIGNIFICANCE STATEMENT FLIM is an indispensable method for analysis of cellular processes. FLIM often necessitates long photon collection periods, requiring the sacrifice of temporal resolution at the expense of spatial information. Here, we demonstrate the applicability of the recently introduced rapidFLIM for quantitative, dynamic imaging with low-emission fluorophores in brain slices. RapidFLIM, combined with improved correction schemes, enabled intensity-independent recording of neuronal Na+ transients at unprecedented full-frame rates of 0.5 Hz. It also allowed quantitative imaging independent from changes in cell volume, revealing a surprisingly strong and hitherto uncovered contribution of TRPV4 channels to Na+ loading on energy failure. Collectively, our study thus provides a novel, unexpected insight into the mechanisms that are responsible for Na+ changes on energy depletion.

Identification of TRPV4 as a novel target in invasiveness of colorectal cancer.

BACKGROUND: Emerging evidence has indicated the critical role of TRPV4 in diverse human cancers. However, the underlying molecular mechanism of TRPV4 in colon cancer invasiveness is still unknown. METHODS: Immunohistochemistry staining was used to analyze the expression of TRPV4 and ZEB1 in clinical tissues; Wound healing and transwell assays were applied to determine the cell invasiveness; Western blot was used to explore the relation between TRPV4 and ZEB1. RESULTS: Colon cancer cells were transfected with siRNA against TRPV4 or HC067047 (a selective TRPV4 antagonist), TRPV4 full-length plasmid or siRNA against ZEB1, or both, in order to measure cell migration and invasion. And we found that TRPV4 silencing or inhibition exhibited an inhibitory role in colon cancer cell migration and invasion, coupled with compromised EMT process, and suppressed AKT activity. TRPV4 stimulated expression of ZEB1 and consequently contributed to EMT process and invasiveness. It was also revealed that overexpression of TRPV4 and ZEB1 in clinical patients with local metastasis, and positive correlation between TRPV4 and ZEB1. CONCLUSIONS: Our results uncovered the role of TRPV4 in tumor metastasis and highlighted the potential mechanism of TRPV4-ZEB1 axis in indicating EMT.

Comparative Analysis of Single-Molecule Dynamics of TRPV1 and TRPV4 Channels in Living Cells.

TRPV1 and TRPV4, members of the transient receptor potential vanilloid family, are multimodal ion channels activated by various stimuli, including temperature and chemicals. It has been demonstrated that TRPV channels function as tetramers; however, the dynamics of the diffusion, oligomerization, and endocytosis of these channels in living cells are unclear. Here we undertook single-molecule time-lapse imaging of TRPV1 and TRPV4 in HEK 293 cells. Differences were observed between TRPV1 and TRPV4 before and after agonist stimulation. In the resting state, TRPV4 was more likely to form higher-order oligomers within immobile membrane domains than TRPV1. TRPV1 became immobile after capsaicin stimulation, followed by its gradual endocytosis. In contrast, TRPV4 was rapidly internalized upon stimulation with GSK1016790A. The selective loss of immobile higher-order oligomers from the cell surface through endocytosis increased the proportion of the fast-diffusing state for both subtypes. With the increase in the fast state, the association rate constants of TRPV1 and TRPV4 increased, regenerating the higher-order oligomers. Our results provide a possible mechanism for the different rates of endocytosis of TRPV1 and TRPV4 based on the spatial organization of the higher-order structures of the two TRPV channels.

TRPV4 contributes to ER stress: Relation to apoptosis in the MPP+-induced cell model of Parkinson's disease.

AIMS: Parkinson's disease (PD) is a multifactorial neurodegenerative disorder. Its molecular mechanism is still unclear. Endoplasmic reticulum (ER) stress has been highlighted in PD. Transient receptor potential vanilloid 4 (TRPV4) is a kind of nonselective calcium cation channel. A defined role for TRPV4 in PD has not been reported. The purpose of the present research was to investigate the molecular mechanisms by which TRPV4 regulates ER stress induced by the 1-methyl-4-phenylpyridinium ion (MPP+) in PC12 cells. MAIN METHODS: PC12 cells were pretreated with the TRPV4-specific antagonist HC067047 or transfected with TRPV4 siRNA followed by treatment with MPP+. Cell viability was measured by the CCK-8 Assay. The expression of TRPV4, sarco/endoplasmic reticulum Ca2+-ATPase 2 (SERCA2), glucose-regulated protein 78 (GRP78), glucose-regulated protein 94 (GRP94), C/EBP homologous protein (CHOP), procaspase-12, and tyrosine hydroxylase (TH) was detected by western blot and RT-PCR. KEY FINDINGS: The expression of TRPV4 was upregulated, while cell viability was decreased by MPP+, which was reversed by HC067047. The ER stress common molecular signature SERCA2 was depressed by MPP+. Moreover, MPP+ induced upregulation of GRP78, GRP94, CHOP, and decrease in procaspase-12 and TH. HC067047 and TRPV4 siRNA reversed MPP+-induced ER stress and restored TH production. SIGNIFICANCE: TRPV4 functions upstream of ER stress induced by MPP+ and holds promise as a prospective pharmacotherapy target for PD.

TRPV6 calcium channel directs homeostasis of the mammary epithelial sheets and controls epithelial mesenchymal transition.

Epithelial integrity is lost upon cancer progression as cancer cells detach from the primary tumor site and start to invade to the surrounding tissues. Invasive cancers of epithelial origin often express altered levels of TRP-family cation channels. Upregulation of TRPV6 Ca2+-channel has been associated with a number of human malignancies and its high expression in breast cancer has been linked to both proliferation and invasive disease. The mechanisms behind the potential of TRPV6 to induce invasive progression have, however, not been well elucidated. Here we show that TRPV6 is connected to both E-cadherin-based adherens junctions and intracellular cytoskeletal structures. Loss of TRPV6 from normal mammary epithelial cells led to disruption of epithelial integrity and abnormal 3D-mammo sphere morphology. Furthermore, expression level of TRPV6 was tightly linked to the levels of common EMT markers, suggesting that TRPV6 may have a role in the mesenchymal invasion of breast cancer cells. Thus, either too low or too high TRPV6 levels compromise homeostasis of the mammary epithelial sheets and may promote the progression of pathophysiological conditions.

Role of Transient Receptor Potential Vanilloid 4 Channel in Skin Physiology and Pathology.

Transient receptor potential vanilloid 4 (TRPV4) channel responds to temperature, as well as various mechanical and chemical stimuli. This non-selective cation channel is expressed in several organs, including the blood vessels, kidneys, oesophagus and skin. In the skin, TRPV4 channel is present in various cell types such as keratinocytes, melanocytes and sensory neurons, as well as immune and inflammatory cells, and engages in several physiological actions, from skin homeostasis to sensation. In addition, there is substantial evidence implicating dysfunctional TRPV4 channel-in the form of either deficient or excessive channel activity-in pathological cutaneous conditions such as skin barrier compromise, pruritus, pain, skin inflammation and carcinogenesis. These varied functions, combined with the fact that TRPV4 channel owns pharmacologically-accessible sites, make this channel an attractive therapeutic target for skin disorders. In this review, we summarize the different physiological and pathophysiological effects of TRPV4 in the skin.

Post-mortem histology in transient receptor potential cation channel subfamily V member 6 (TRPV6) under-mineralising skeletal dysplasia suggests postnatal skeletal recovery: a case report.

BACKGROUND: The calcium-selective channel TRPV6 (transient receptor potential cation channel subfamily V member 6) is crucial for maternal-fetal calcium transport across the placenta. TRPV6 mutations have recently been associated with an antenatally severe under-mineralising skeletal dysplasia accompanied by postnatal biochemical abnormalities. This is the first post-mortem report in a patient with TRPV6 skeletal dysplasia. CASE PRESENTATION: The female infant had severe antenatal and postnatal skeletal abnormalities by 20 weeks gestation and was ventilator-dependent from birth. These skeletal abnormalities were apparent at an earlier gestational age than in previous reported cases and a more severe clinical course ensued. Biochemical and skeletal abnormalities, including bone density, improved postnatally but cardiac arrest at 4 months of age led to withdrawal of intensive care. Compound heterozygous TRPV6 variants (c.1978G > C p.(Gly660Arg) and c.1528C > T p.(Arg510Ter)) were identified on exome sequencing. Post-mortem identified skeletal abnormalities but no specific abnormalities in other organ systems. No placental pathology was found, multi-organ histological features reflected prolonged intensive care only. Post-mortem macroscopic examination indicated reduced thoracic size and short, pale and pliable ribs. Histological examination identified reduced number of trabeculae in the diaphyses (away from the growth plates), whereas metaphyses showed adequate mineralisation and normal number of trabeculae, but with slightly enlarged reactive chondrocytes, indicating post-natal skeletal growth recovery. Post-mortem radiological findings demonstrated improved bone density, improved rib width, healed fractures, although ribs were still shorter than normal. Long bones (especially humerus and femur) had improved from initial poorly defined metaphyses and reduced bone density to sharply defined metaphyses, prominent growth restart lines in distal diaphyses and bone-in-bone appearance along diaphyses. CONCLUSIONS: This case provide bone histological confirmation that human skeletal development is compromised in the presence of TRPV6 pathogenic variants. Post-mortem findings were consistent with abnormal in utero skeletal mineralisation due to severe calcium deficit from compromised placental calcium transfer, followed by subsequent phenotypic improvement with adequate postnatal calcium availability. Significant skeletal recovery occurs in the early weeks of postnatal life in TRPV6 skeletal dysplasia.

α1 -Blocker inhibits non-voiding contractions and decreases the level of intravesical prostaglandin E2 in rats with partial bladder outlet obstruction.

OBJECTIVES: To elucidate the mechanism of action of the α1 -blocker, naftopidil, in partial bladder outlet obstruction animals, by studying non-voiding contractions, and the levels of mediators were measured with resiniferatoxin treatment. METHODS: A total of 35 female Wistar rats were randomly divided into a sham or bladder outlet obstruction group, and rats in each group were given vehicle or resiniferatoxin. Incomplete urethral ligation was applied to the bladder outlet obstruction group. After cystometry, the intravesical level of prostaglandin E2 and adenosine 5'-triphosphate was measured in the instilled perfusate collected. Naftopidil was given at the time of cystometry. RESULTS: In bladder outlet obstruction rats, non-voiding contractions, bladder capacity, and the intravesical levels of prostaglandin E2 and adenosine 5'-triphosphate were markedly increased compared with sham rats. Naftopidil decreased non-voiding contractions, enlarged the bladder capacity, and decreased the intravesical levels of prostaglandin E2 and adenosine 5'-triphosphate. Resiniferatoxin enhanced non-voiding contractions. The effects of naftopidil on non-voiding contractions and the intravesical level of prostaglandin E2 , but not adenosine 5'-triphosphate, were tolerant to resiniferatoxin. CONCLUSIONS: In bladder outlet obstruction rats, one cause of generation of non-voiding contractions might be bladder wall distension, but not transient receptor potential cation channel V1. The increase in intravesical prostaglandin E2 might also be associated with the generation of non-voiding contractions. Naftopidil inhibits the increase in non-voiding contractions and decreases the intravesical level of prostaglandin E2 , which are independent of transient receptor potential cation channel V1.

Transient receptor potential vanilloid (TRPV) channel expression in meningiomas: prognostic and predictive significance.

The TRPV1-4 members of TRPV cation channel subfamily are mainly regarded as polymodal receptors that may be activated by diverse changes in cellular microenvironment and endogenous and exogenous agents. Abnormal expression of these channels has been reported in various tumors but not in meningiomas. Meningioma cells are thought to originate from arachnoid cap cells due to cytological and functional similarities between the two types of cells. To investigate the expression profile of TRPV1-4 channels in meningiomas and compare with TRPV1-4 channel expression in leptomeninges, we used immunohistochemistry in formalin-fixed, paraffin-embedded semi-serial tissue sections from 175 meningiomas with different grades and histological subtypes, and normal brain or meningioma specimens that contained leptomeninges. The labeling index (LI), defined as the percentage of positive (labeled) cells out of the total number of tumor cells counted, was determined. Leptomeninges were TRPV1-4 immunonegative. A significant percentage of tumors exhibited TRPV1-4 channel expression which was independent of the proliferation index of the tumors but was significantly associated with histopathological subtypes. The TRPV1 and TRPV3 immunoexpression was decreased whereas TRPV4 immunoexpression was significantly greater in high-grade (WHO, grade II and III) as compared with low-grade (WHO, grade I) meningiomas. Additionally, TRPV4 emerged as an independent predictor for the degree of malignancy using the binary logistic regression model [dependent variable: grade I versus higher grades (II and III)]. Kaplan-Meier analysis for 102 patients showed no significant association of TRPV1-4 expression with overall survival. The above data support that TRPV1-4 channels are implicated in meningioma pathogenesis, and TRPV4 has predictive significance in the disease.

Excessive mechanical stress induces chondrocyte apoptosis through TRPV4 in an anterior cruciate ligament-transected rat osteoarthritis model.

AIMS: Chondrocyte apoptosis is the most common pathological feature of cartilage in osteoarthritis (OA). Excessive mechanical stress can induce chondrocyte apoptosis and destroy cartilage tissue. Transient receptor potential channel vanilloid 4 (TRPV4) is a mechanosensitive ion channel that mediates chondrocyte response to mechanical stress. Here, we investigated the potential role of TRPV4 in chondrocyte apoptosis induced by excessive mechanical stress. MAIN METHODS: Using a rat OA anterior cruciate-ligament transection (ALCT) model, we detected immunolocalization of calmodulin protein and mRNA and protein levels of TRPV4, calmodulin, and cleaved caspase-8 in articular cartilage. Primary chondrocytes were isolated and cultured in vitro, and Fluo-4AM staining was used to assess intracellular Ca2+ levels in order to evaluate TRPV4-mediated Ca2+ influx. Flow cytometry and western blot were performed to detect apoptosis and apoptosis-related protein levels in chondrocytes, respectively. KEY FINDINGS: TRPV4 was upregulated in ALCT-induced OA articular cartilage, and we found that administration of a TRPV4 inhibitor attenuated cartilage degeneration. Additionally, TRPV4 specifically mediated extracellular Ca2+ influx, leading to chondrocyte apoptosis in vitro, which was inhibited by transfection of TRPV4 small-interfering RNA or administration of a TRPV4 inhibitor. Moreover, increased Ca2+ influx triggered apoptosis by upregulating FAS-associated protein with death domain and cleaved caspase-3, -6, -7, and -8 levels, with these effects abolished by TRPV4 knockdown or TRPV4 inhibition. SIGNIFICANCE: These results indicated that TRPV4 was upregulated in OA articular cartilage, and that excessive mechanical stress might induce chondrocyte apoptosis via TRPV4-mediated Ca2+ influx, suggesting TRPV4 as a potential drug target in OA.

New Insights of Uterine Leiomyoma Pathogenesis: Endocannabinoid System.

BACKGROUND The aim of this study was to determine if components of the endocannabinoid system are modulated in uterine leiomyomas (fibroids). Components studied included cannabinoid receptors 1 (CB1) and 2 (CB2); the G protein-coupled receptor GPR55; transient potential vanilloid receptor 1 (TRPV1) and the endocannabinoid modulating enzymes N-acylphosphatidylethanolamine-specific phospholipase D (NAPE-PLD) and fatty acid amide hydrolase (FAAH), and their N-acylethanolamine (NAE) ligands: N-arachidonylethanolamine (AEA), N-oleoylethanolamine (OEA), and N-palmityolethanaolamine (PEA). MATERIAL AND METHODS Transcript levels of CB1, CB2, TRPV1, GPR55, NAPE-PLD, and FAAH were measured using RT-PCR and correlated with the tissue levels of the 3 NAEs in myometrial tissues. The tissues studied were: 1) fibroids, 2) myometrium adjacent/juxtaposed to the fibroid lesions, and 3) normal myometrium. Thirty-seven samples were processed for NAE measurements and 28 samples were used for RT-PCR analyses. RESULTS FAAH expression was significantly lower in fibroids, resulting in a NAPE-PLD: FAAH ratio that favors higher AEA levels in pre-menopausal tissues, whilst PEA levels were significantly lower, particularly in post-menopausal women, suggesting PEA protects against fibroid pathogenesis. The CB1: CB2 ratio was lower in fibroids, suggesting that loss of CB1 expression affects the fibroid cell phenotype. Significant correlations between reduced FAAH, CB1, and GPR55 expression and PEA in fibroids indicate that the loss of these endocannabinoid system components are biomarkers of leiomyomata. CONCLUSIONS Loss of expression of CB1, FAAH, GPR55, and PEA production are linked to the pathogenesis of uterine fibroids and further understanding of this might eventually lead to better disease indicators or the development of therapeutic potentials that might eventually be used in the management of uterine fibroids.

Impaired Junctions and Invaded Macrophages in Oral Epithelia With Oral Pain.

Recurrent or chronic oral pain is a great burden for patients. Recently, the links between epithelial barrier loss and disease were extended to include initiation and propagation. To explore the effects of pathohistological changes in oral epithelia on pain, we utilized labial mucosa samples in diagnostic labial gland biopsies from patients with suspected Sjögren's syndrome (SS), because they frequently experience pain and discomfort. In most labial mucosa samples from patients diagnosed with SS, disseminated epithelial cellular edema was prevalent as ballooning degeneration. The disrupted epithelia contained larger numbers of infiltrating macrophages in patients with oral pain than in patients without pain. Immunohistochemistry revealed that edematous areas were distinct from normal areas, with disarranged cell-cell adhesion molecules (filamentous actin, E-cadherin, ß-catenin). Furthermore, edematous areas were devoid of immunostaining for transient receptor potential channel vanilloid 4 (TRPV4), a key molecule in adherens junctions. In an investigation on whether impaired TRPV4 affect cell-cell adhesion, calcium stimulation induced intimate cell-cell contacts among oral epithelial cells from wild-type mice, while intercellular spaces were apparent in cells from TRPV4-knockout mice. The present findings highlight the relationship between macrophages and epithelia in oral pain processing, and identify TRPV4-mediated cell-cell contacts as a possible target for pain treatment.

An efficient nonlinear hybridization chain reaction-based sensitive fluorescent assay for in situ estimation of calcium channel protein expression on bone marrow cells.

A sensitive and highly efficient approach to monitor the expression of proteins on live cells was urgently needed to demonstrate its factor and mechanism and most important for clinical diagnostics and molecular biology. Herein, we developed a simple and highly efficient strategy, nonlinear hybridization chain reaction (nonlinear HCR), for the sensitive determination of proteins on live cells with transient receptor potential vanilloid 4 (TRPV4) and RAW264.7 cells as a model. Unlike the normal hybridization chain reaction (HCR) with multiplicative amplification, an exponential amplified fluorescent response could be obtained in theory based on the proposed nonlinear HCR. As a result, the nonlinear HCR generated a significant enhancement about 3 times compared with the normal HCR and 10 times compared with the directly immunofluorescence assay. Based on the proposed nonlinear HCR, the fluorescent signals increased with the concentration of TRPV4 in the range from 10 pg/mL to 100 ng/mL with a detection limit of 2.8 pg/mL, which would be useful for the sensitive detection of proteins in cell lysis or on cell surface. At the same time, the significant improvements via nonlinear HCR were achieved in the fluorescent imaging system compared with traditional immunofluorescence staining and normal HCR, proving the significant value of nonlinear HCR-based amplification strategy. Success in the establishment of the highly efficient nonlinear HCR strategy offered a simple and sensitive approach to demonstrate the concentration of special proteins on cell and other proteins and nucleotide potentially, revealing a simple and efficient technology for research fields of clinical diagnostics and molecular biology.

Selective ablation of TRPV1 by intrathecal injection of resiniferatoxin in rats increases renal sympathoexcitatory responses and salt sensitivity.

This study tested the hypothesis that selective ablation of transient receptor potential vanilloid type 1 (TRPV1)-positive nerve fibers by intrathecal injection of resiniferatoxin (RTX) enhances renal sympathoexcitatory responses and salt sensitivity. Intrathecal injection of RTX (1.8 µg/kg) to the levels of lower thoracic and upper lumbar spinal cord (T8-L3) increased mean arterial pressure (MAP) in rats fed a normal (NS, 1% NaCl) or high-sodium (HS, 8% NaCl) diet for 4 weeks compared to vehicle-treated rats (NS: 121 ± 2 vs. 111 ± 2; HS: 154 ± 2 vs. 134 ± 2 mm Hg, both P < 0.05), with a greater increase in HS compared to NS rats (9 ± 1% vs. 15 ± 1%, P < 0.05). TRPV1 contents were decreased in T8-L3 segments of spinal dorsal horn but not in corresponding dorsal root ganglia and the kidney following RTX treatment (P < 0.05). Selective activation of GABA-A receptors with intrathecal T8-L3 segment-injection of muscimol (3 nmol/kg) decreased renal sympathetic nerve activity and increased urinary excretion in both NS and HS rats, with a greater effect in RTX-treated compared to vehicle-treated rats (P < 0.05). Chronic activation of GABA-A receptors with muscimol (50 mg/kg/day × 2, p.o.) abolished RTX treatment-induced pressor effects in NS and HS rats. GAD65/67, a GABA synthetase, in the spinal cord was downregulated and tyrosine hydroxylase in the kidney upregulated in NS or HS rats treated with RTX (P < 0.05). Thus, selective ablation of TRPV1-positive central terminals of sensory neurons plays a prohypertensive role possibly via inhibition of spinal GABA system especially with HS intake, suggesting that activation of TRPV1 in central terminals of sensory neurons may convey an antihypertensive effect.

Association of cough hypersensitivity with tracheal TRPV1 activation and neurogenic inflammation in a novel guinea pig model of citric acid-induced chronic cough.

Objective This study was performed to establish a novel model of citric acid-induced chronic cough in guinea pigs and to investigate the pathogenesis of cough hypersensitivity. Methods Healthy conscious guinea pigs inhaled citric acid (0.4 M) for 3 minutes twice daily for 25 days. Cough reactivity was evaluated, substance P (SP) and calcitonin gene-related peptide (CGRP) in bronchoalveolar lavage fluid were detected, and transient receptor potential cation channel subfamily V member 1 (TRPV1) protein expression in the trachea and bronchus was determined. Tracheal and bronchial tissues were examined for TRPV1. Results Inhalation of 0.4 M citric acid increased coughing in a time-dependent manner: coughing peaked at 15 days and reached the lowest level at 25 days. This was accompanied by similar changes in SP, CGRP, and TRPV1 protein expression. TRPV1 was mainly observed in the mucosal and submucosal layer of the trachea and bronchi. The areas of TRPV1 positivity in the trachea and bronchi of citric acid-treated animals were significantly larger than in the control group. Conclusions Repeated inhalation of citric acid can be employed to establish a chronic cough model in guinea pigs. Cough hypersensitivity in this model is related to tracheal TRPV1 activation and neurogenic inflammation.

H89 dihydrochloride hydrate and calphostin C lower the body temperature through TRPV1.

The transient receptor potential vanilloid (TRPV1) serves as a negative regulator of body temperature, and during fever conditions its expression can lead to a decrease in temperature. TRPV1 is regulated by a variety of enzymes; however, it is currently unclear whether the regulation of TRPV1 phosphorylation may serve a role in the increase in TRPV1 expression during fever. In the present study, using an in vivo experimental method, rat brain ventricles were injected with the protein kinase A (PKA) antagonist, H89, and the protein kinase C (PKC) antagonist, calphostin C, and fever was induced using lipopolysaccharide (LPS) in order to detect the expression of TRPV1 and phosphorylated (p­)TRPV1, the intracellular Ca2+ concentration [(Ca2+)i] of hypothalami and rat body temperature. The results demonstrated that following the generation of fever using LPS, the expressions of TRPV1 and p­TRPV1, and hypothalamic [Ca2+]i markedly increased. In addition, following an injection with the PKA or PKC antagonist, the temperature increased further due to the inhibition of p­TRPV1. Thus, it was hypothesized that PKA and PKC may be involved in TRPV1 phosphorylation, resulting in a temperature reduction during LPS­induced fever conditions.

Role of the endothelial caveolae microdomain in shear stress-mediated coronary vasorelaxation.

In this study, we determined the role of caveolae and the ionic mechanisms that mediate shear stress-mediated vasodilation (SSD). We found that both TRPV4 and SK channels are targeted to caveolae in freshly isolated bovine coronary endothelial cells (BCECs) and that TRPV4 and KCa2.3 (SK3) channels are co-immunoprecipitated by anti-caveolin-1 antibodies. Acute exposure of BCECs seeded in a capillary tube to 10 dynes/cm2 of shear stress (SS) resulted in activation of TRPV4 and SK currents. However, after incubation with HC067047 (TRPV4 inhibitor), SK currents could no longer be activated by SS, suggesting SK channel activation by SS was mediated through TRPV4. SK currents in BCECs were also activated by isoproterenol or by GSK1016790A (TRPV4 activator). In addition, preincubation of isolated coronary arterioles with apamin (SK inhibitor) resulted in a significant diminution of SSD whereas preincubation with HC067047 produced vasoconstriction by SS. Exposure of BCECs to SS (15 dynes/cm2 16 h) enhanced the production of nitric oxide and prostacyclin (PGI2) and facilitated the translocation of TRPV4 to the caveolae. Inhibition of TRPV4 abolished the SS-mediated intracellular Ca2+ ([Ca2+] i ) increase in BCECs. These results indicate a dynamic interaction in the vascular endothelium among caveolae TRPV4 and SK3 channels. This caveolae-TRPV4-SK3 channel complex underlies the molecular and ionic mechanisms that modulate SSD in the coronary circulation.

Antinociceptive Effect of Ghrelin in a Rat Model of Irritable Bowel Syndrome Involves TRPV1/Opioid Systems.

BACKGROUND/AIMS: Irritable bowel syndrome (IBS), defined as recurrent abdominal pain and changes in bowel habits, seriously affects quality of life and ability to work. Ghrelin is a brain-gut hormone, which has been reported to show antinociceptive effects in peripheral pain. We investigated the effect of ghrelin on visceral hypersensitivity and pain in a rat model of IBS. METHODS: Maternal deprivation (MD) was used to provide a stress-induced model of IBS in Wistar rats. Colorectal distension (CRD) was used to detect visceral sensitivity, which was evaluated by abdominal withdrawal reflex (AWR) scores. Rats that were confirmed to have visceral hypersensitivity after MD were injected with ghrelin (10 µg/kg) subcutaneously twice a week from weeks 7 to 8. [D-Lys3]-GHRP-6 (100 nmol/L) and naloxone (100 nmol/L) were administered subcutaneously to block growth hormone secretagogue receptor 1α (GHS-R1α) and opioid receptors, respectively. Expression of transient receptor potential vanilloid type 1 (TRPV1) and µ and κ opioid receptors (MOR and KOR) in colon, dorsal root ganglion (DRG) and cerebral cortex tissues were detected by western blotting, quantitative real-time polymerase chain reaction (qRT-PCR), immunohistochemical analyses and immunofluorescence. RESULTS: Ghrelin treatment increased expression of opioid receptors and inhibited expression of TRPV1 in colon, dorsal root ganglion (DRG) and cerebral cortex. The antinociceptive effect of ghrelin in the rat model of IBS was partly blocked by both the ghrelin antagonist [D-Lys3]-GHRP-6 and the opioid receptor antagonist naloxone. CONCLUSION: The results indicate that ghrelin exerted an antinociceptive effect, which was mediated via TRPV1/opioid systems, in IBS-induced visceral hypersensitivity. Ghrelin might potentially be used as a new treatment for IBS.

[Effect of high-fat diet on expression of transient receptor potential vanilloid 1 in respiratory tract and dorsal root ganglion of mice].

OBJECTIVE: To investigate the effect of high-fat diet on the expression of transient receptor potential vanilloid 1 (TRPV1) in the respiratory system and the dorsal root ganglion (DRG) of mice, as well as its effect on the excitability of sensory neurons. METHODS: A total of 20 C57BL/6 mice were randomly divided into normal-diet (ND) group and high-fat diet (HFD) group, with 10 mice in each group. The mice were given corresponding diets and body weights were monitored. After 7 weeks of feeding, lung tissue, bronchial tissue, and DRG at thoracic segments 3-4 were collected and immunohistochemical staining was performed. A patch clamp was used to measure the number of action potentials and TRPV1 current intensity in the DRG. RESULTS: After 7 weeks of feeding, the HFD group had significantly greater mean weight gain than the ND group (6.4±2.6 g vs 2.3±0.5 g; P<0.001). The HFD group had significantly higher expression of TRPV1 in the bronchus, pulmonary alveoli, and DRG than the ND group (P<0.05). Compared with the ND group, the HFD group had significant increases in the TRPV1 current intensity and number of action potentials in the DRG (P<0.05). CONCLUSIONS: High-fat diet induces a significant increase in body weight and leads to high expression of TRPV1 and high excitability in the respiratory system and the peripheral sensory neurons. This suggests that TRPV1 may be an important factor in the physiopathological mechanisms of bronchial hyperresponsiveness.