Transient Receptor Potential Vanilloid 1 Agonist Research Articles

MP1-03 HERPES SIMPLEX VIRUS (HSV) VECTOR-MEDIATED GENE DELIVERY OF PROTEIN PHOSPHATASE 1α REDUCES BLADDER OVERACTIVITY AND NOCICEPTION IN RATS

You have accessJournal of UrologyUrodynamics/Incontinence/Female Urology: Basic Research I1 Apr 2014MP1-03 HERPES SIMPLEX VIRUS (HSV) VECTOR-MEDIATED GENE DELIVERY OF PROTEIN PHOSPHATASE 1α REDUCES BLADDER OVERACTIVITY AND NOCICEPTION IN RATS Tsuyoshi Majima, Naoki Kawamorita, Hiroki Okada, Yasuhito Funahashi, Momokazu Gotoh, William Goins, Joseph Glorioso, Justus Cohen, Bonnie Hall, Asaff Harel, and Naoki Yoshimura Tsuyoshi MajimaTsuyoshi Majima More articles by this author , Naoki KawamoritaNaoki Kawamorita More articles by this author , Hiroki OkadaHiroki Okada More articles by this author , Yasuhito FunahashiYasuhito Funahashi More articles by this author , Momokazu GotohMomokazu Gotoh More articles by this author , William GoinsWilliam Goins More articles by this author , Joseph GloriosoJoseph Glorioso More articles by this author , Justus CohenJustus Cohen More articles by this author , Bonnie HallBonnie Hall More articles by this author , Asaff HarelAsaff Harel More articles by this author , and Naoki YoshimuraNaoki Yoshimura More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.101AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Increased afferent excitability has been proposed as an important pathophysiological basis of overactive bladder (OAB) and hypersensitive bladder disorders such as interstitial cystitis/bladder pain syndrome (IC/BPS). It has also been reported that transient receptor potential vanilloid-1 (TRPV1) receptors predominantly expressed in C-fiber afferent pathways greatly contribute to afferent sensitization in these disease conditions. We recently found Protein Phosphatase 1α (PP1α) as a modulator of TRPV1 receptor activity using an HSV vector cDNA library screen method to identify genes that inhibit TRPV1 activation, and showed that HSV vector-mediated PP1α expression led to the reduction in capsaicin-induced thermal hyperalgesia. Therefore, this led us to investigate the effect of HSV vectors-mediated gene delivery of PP1α on TRPV1-mediated bladder overactivity and pain-related behavior in rats. METHODS Replication-deficient HSV vectors encoding PP1α or green fluorescent protein (GFP) as control were injected into the bladder wall of adult female Sprague-Dawley rats. Cystometry (CMG) under urethane anesthesia was performed 1 week after viral injection to evaluate bladder overactivity induced by resiniferatoxin (RTX, a TRPV1 agonist). RTX-induced nociceptive behavior such as licking (lower abdominal licking) and freezing (motionless head-turning) was observed 2 weeks after viral injection. Using immunohistochemistry, GFP expression in L6/S1 DRG and the bladder as well as c-Fos positive cells in the L6 spinal cord dorsal horn were evaluated. RESULTS GFP expression was seen in L6/S1 DRG sections. In CMG, the PP1α group showed a significantly (p<0.05) smaller reduction (46.5±1.7%) in intercontraction intervals after RTX infusion than the GFP group (65.7±3.8%). The number of RTX-induced freezing behavior, which is correlated with bladder pain sensation, was significantly (p<0.001) decreased in PP1α vs. GFP groups. The number of c-Fos positive cells in the L6 spinal dorsal horn was significantly (p<0.01) smaller in PP1α vs. GFP rats (24±4 vs. 59±6 cells per section). CONCLUSIONS These results indicate that HSV vectors injected into the bladder wall are transported to lumbosacral DRGs that contain bladder afferent neurons, and that PP1α gene delivery in the bladder is effective in suppressing TRPV1-mediated bladder overactivity and pain behavior. Thus, HSV-mediated PP1α gene therapy could be effective for the treatment of OAB and/or hypersensitive bladder disorders such as IC/BPS. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e1-e2 Peer Review Report Advertisement Copyright & Permissions© 2014MetricsAuthor Information Tsuyoshi Majima More articles by this author Naoki Kawamorita More articles by this author Hiroki Okada More articles by this author Yasuhito Funahashi More articles by this author Momokazu Gotoh More articles by this author William Goins More articles by this author Joseph Glorioso More articles by this author Justus Cohen More articles by this author Bonnie Hall More articles by this author Asaff Harel More articles by this author Naoki Yoshimura More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

The effect of intra-ventral hippocampus administration of TRPV1 agonist and antagonist on spatial learning and memory in male rats

BackgroundThe role of transient receptor potential vanilloid 1 (TRPV1) in peripheral nervous system has been studied well but its role in the central nervous system remains to be studied in detail. The expression of TRPV1 receptors in hippocampus is suggesting that they may play an important role in higher cognitive functions such as learning and memory. MethodsIn the present study, the role of TRPV1 receptors in acquisition and retrieval of spatial memory of male Wistar rats was evaluated by intra-ventral hippocampus administration of TRPV1 selective agonist (OLDA) and antagonist (AMG9810) using Morris water maze. ResultsThe results demonstrated that administration of either OLDA (0.001, 0.01 and 0.1μg/rat) or AMG9810 (0.003, 0.03 and 0.3μg/rat) did not influence memory acquisition or retrieval. ConclusionsThese data suggest that ventral-hippocampal TRPV1 receptors possibly are not involved in spatial learning and memory.

Transient receptor potential vanilloid 1 expression and functionality in mcf-7 cells: a preliminary investigation.

PurposeTransient receptor potential vanilloid 1 (TRPV1) is a nonselective cation channel belonging to the transient receptor potential family, and it is expressed in different neoplastic tissues. Its activation is associated with regulation of cancer growth and progression. The aim of this research was to study the expression and pharmacological characteristics of TRPV1 in cells derived from human breast cancer MCF-7 cells.MethodsTRPV1 presence was assessed by binding studies and Western blotting. Receptor binding characteristics were evaluated through competition assays, while 3-(4,5-dimethylthiazol-2-yl)-2,5,-dipheyltetrazolium bromide reduction assays were performed to confirm an early hypothesis regarding the modulation of cancer cell proliferation. The functionality of TRPV1 was evaluated by measuring Ca2+ uptake in the presence of increasing concentrations of TRPV1 agonists and antagonists.ResultsBinding studies identified a single class of TRPV1 (Bmax 1,492±192 fmol/mg protein), and Western blot showed a signal at 100 kDa corresponding to the molecular weight of human TRPV1. Among the different tested agonists and antagonists, anandamide (Ki: 2.8×10-11 M) and 5-iodoresiniferatoxin (5-I-RTX) (Ki: 5.6×10-11 M) showed the highest degrees of affinity for TRPV1, respectively. All tested TRPV1 agonists and antagonists caused a significant (p<0.05) decrease in cell growth rate in MCF-7 cells. For agonists and antagonists, the efficacy of tested compounds displayed the following rank order: resiniferatoxin>anandamide>capsaicin and 5-I-RTX=capsazepine, respectively.ConclusionThese data indicate that both TRPV1 agonists and antagonists induce significant inhibition of MCF-7 cell growth. Even though the mechanisms involved in the antiproliferative effects of TRPV1 agonists and antagonists should be further investigated, it has been suggested that agonists cause desensitization of the receptor, leading to alteration in Ca2+-influx regulation. By contrast, antagonists cause a functional block of the receptor with consequent fatal dysregulation of cell homeostasis.

Evidence for the Role of Camkinase II and Synapsin I in the Restoration of Neurotransmission in Botulinum Neurotoxin a Intoxicated Nerve Terminals

At the active zones in the nerve terminals, presynaptic Ca2+ is recognized as an important second messenger that regulates neurotransmission. However, the precise role of Ca2+ in the regulation of exocytosis still remains to be elusive. Synapsin I (SYN1) is a member of the synapsin family (SYN1/2/3) of synaptic vesicle phosphoproteins that is phosphorylated by Ca2+/calmodulin dependent protein kinase II (CaMKII) at the Serine 603. At the motor nerve terminals (MNT), SYN I regulates synaptic transmission and the depolarization signal mediated rise in Ca2+ influx stimulates CaMKII dependent phosphorylation of SYN1. Botulinum neurotoxin A (BoNT/A) selectively proteolyzes SNAP-25 to inhibit acetylcholine (ACh) release and causes life-threatening neuroparalysis. In our efforts to develop therapeutic strategies to counteract BoNT/A at its primary site of action, the MNT, we discovered that mouse MNT express transient receptor potential vanilloid 1 (TRPV1) channel protein and that capsaicin (TRPV1 agonist) antagonized the neuroparalytic effects of BoNT/A. Capsaicin, when injected post BoNT/A exposure, significantly accelerated recovery from neuroparalysis by restoring ACh release and muscle functions. Capsaicin treatment also decreased the total duration of paralysis by 50% and synergistically increased the effects of 3,4 diaminopyridine (blocks K+ channels and prolongs action potential) on ACh release measured by stimulus evoked twitch tension in BoNT/A poisoned nerve muscle preparations. Further, BoNT/A treatment, in vitro, decreased the expression of CaMKII and SYN1 and the phosphorylation of SYN1 in cholinergic Neuro 2a cells. Capsaicin treatment, post BoNT/A, reversed this. We hypothesize that at the MNT, capsaicin stimulated Ca2+ influx via TRPV1 triggers exocytosis by facilitating synaptic transmission via CaMKII mediated phosphorylation of SYN1.

Intranasal capsaicin in management of nonallergic (vasomotor) rhinitis.

Capsaicin is a selective transient receptor potential vanilloid 1 (TRPV1) ion channel agonist and has been demonstrated to reduce nerve conduction of nociceptive C fibers in the trigeminal nerve without affecting conduction in Adelta fibers. This chapter reviews the classification of chronic rhinitis subtypes, the prevalence and epidemiology of nonallergic rhinitis (NAR), postulated pathophysiology and mechanisms of NAR including the role of transient receptor potential (TRP) ion channels and discusses the potential therapeutic benefits of capsaicin in the treatment of chronic rhinitis subtypes, specifically NAR. Evidence supports that hypersensitivity of TRP ion channels on sensory afferent neurons innervating nasal mucosa is responsible for inducing NAR symptoms. These symptoms, characterized as excessive nasal glandular secretion, nasal congestion, and headache, are mediated through neuropeptide release during axonal and parasympathetic reflexes which are initiated by a spectrum of nonspecific irritants that activate TRP channels. Rational approaches to treat the pathophysiology of NAR would be to develop therapies with selective TRPV1 agonist activity like capsaicin that target desensitization of TRP ion channels on sensory afferent nerves.

Pharmacologically induced hypothermia via TRPV1 channel agonism provides neuroprotection following ischemic stroke when initiated 90 min after reperfusion

Traditional methods of therapeutic hypothermia show promise for neuroprotection against cerebral ischemia-reperfusion (I/R), however, with limitations. We examined effectiveness and specificity of pharmacological hypothermia (PH) by transient receptor potential vanilloid 1 (TRPV1) channel agonism in the treatment of focal cerebral I/R. Core temperature (T(core)) was measured after subcutaneous infusion of TRPV1 agonist dihydrocapsaicin (DHC) in conscious C57BL/6 WT and TRPV1 knockout (KO) mice. Acute measurements of heart rate (HR), mean arterial pressure (MAP), and cerebral perfusion were measured before and after DHC treatment. Focal cerebral I/R (1 h ischemia + 24 h reperfusion) was induced by distal middle cerebral artery occlusion. Hypothermia (>8 h) was initiated 90 min after start of reperfusion by DHC infusion (osmotic pump). Neurofunction (behavioral testing) and infarct volume (TTC staining) were measured at 24 h. DHC (1.25 mg/kg) produced a stable drop in T(core) (33°C) in naive and I/R mouse models but not in TRPV1 KO mice. DHC (1.25 mg/kg) had no measurable effect on HR and cerebral perfusion but produced a slight transient drop in MAP (<6 mmHg). In stroke mice, DHC infusion produced hypothermia, decreased infarct volume by 87%, and improved neurofunctional score. The hypothermic and neuroprotective effects of DHC were absent in TRPV1 KO mice or mice maintained normothermic with heat support. PH via TRPV1 agonist appears to be a well-tolerated and effective method for promoting mild hypothermia in the conscious mouse. Furthermore, TRPV1 agonism produces effective hypothermia in I/R mice and significantly improves outcome when initiated 90 min after start of reperfusion.

Myocardial Ischemia/Reperfusion Injury: Potential of TRPV1 Agonists as Cardioprotective Agents

Myocardial Ischemia/Reperfusion (I/R) induced injury has widespread detrimental effects partially negating the benefits obtained from early revascularization in Acute Myocardial Infarction. Various complex mechanisms contribute to I/R injury and different agents targeting those specific mechanisms are being studied. Despite continued research and widespread interest, none of them have become incorporated into everyday practice. The TRPV1 (transient receptor potential vanilloid 1) channel is a non selective cation channel predominantly expressed in sensory neurons with the nerve fibers innervating the heart and blood vessels. Multiple studies have demonstrated the importance of the activation of TRPV1 and subsequent release of sensory neurotransmitters in cardioprotection. This review focuses on the role of TRPV1 in prevention of cardiac I/R injury, the work that has been done so far and future implications for TRPV1 agonists as cardioprotective agents.

Increased expression of bronchial epithelial transient receptor potential vanilloid 1 channels in patients with severe asthma

The airway epithelium is exposed to a range of physical and chemical irritants in the environment that are known to trigger asthma. Transient receptor potential (TRP) cation channels play a central role in sensory responses to noxious physical and chemical stimuli. Recent genetic evidence suggests an involvement of transient receptor potential vanilloid 1 (TRPV1), one member of the vanilloid subfamily of TRP channels, in the pathophysiology of asthma. The functional expression of TRPV1 on airway epithelium has yet to be elucidated. In this study we examined the molecular, functional, and immunohistochemical expression of TRPV1 in asthmatic and healthy airways. Bronchial biopsy specimens and bronchial brushings were obtained from healthy volunteers (n = 18), patients with mild-to-moderate asthma (n = 24), and patients with refractory asthma (n = 22). Cultured primary bronchial epithelial cells from patients with mild asthma (n = 4), nonasthmatic coughers (n = 4), and healthy subjects (n = 4) were studied to investigate the functional role of TRPV1. Quantitative immunohistochemistry revealed significantly more TRPV1 expression in asthmatic patients compared with healthy subjects, with the greatest expression in patients with refractory asthma (P = .001). PCR and Western blotting analysis confirmed gene and protein expression of TRPV1 in cultured primary bronchial epithelial cells. Patch-clamp electrophysiology directly confirmed functional TRPV1 expression in all 3 groups. In functional assays the TRPV1 agonist capsaicin induced dose-dependent IL-8 release, which could be blocked by the antagonist capsazepine. Reduction of external pH from 7.4 to 6.4 activated a capsazepine-sensitive outwardly rectifying membrane current. Functional TRPV1 channels are present in the human airway epithelium and overexpressed in the airways of patients with refractory asthma. These channels might represent a novel therapeutic target for the treatment of uncontrolled asthma.

Analysis of responses to the TRPV4 agonist GSK1016790A in the pulmonary vascular bed of the intact-chest rat

The transient receptor potential vanilloid 4 (TRPV4) channel is a nonselective cation channel expressed on many cell types, including the vascular endothelium and smooth muscle cells. TRPV4 channels play a role in regulating vasomotor tone and capillary permeability. The present study was undertaken to investigate responses to the TRPV4 agonist GSK101790A on the pulmonary and systemic vascular beds in the rat. Intravenous injection of GSK1016790A at doses of 2-10 μg/kg produced dose-dependent decreases in systemic arterial pressure, small decreases in pulmonary arterial pressure, and small increases in cardiac output, and responses were not altered by the cyclooxygenase inhibitor meclofenamate or the cytochrome P-450 inhibitor miconazole. Injection of GSK1016790A at a dose of 12 μg/kg iv produced cardiovascular collapse that was reversible in some animals. GSK1016790A produced dose-related decreases in pulmonary and systemic arterial pressure when baseline tone in the pulmonary vascular bed was increased with U-46619. After treatment with the nitric oxide synthase (NOS) inhibitor N-nitro-l-arginine methyl ester, GSK1016790A produced larger decreases in systemic arterial pressure and dose-dependent increases in pulmonary arterial pressure followed by a small decrease. These results demonstrate that GSK1016790A has vasodilator activity in pulmonary and systemic vascular beds and that when NOS is inhibited, GSK1016790A produced pulmonary vasoconstrictor responses that were attenuated by the L-type Ca(2+) channel antagonist isradipine. The presence of TRPV4 immunoreactivity was observed in small pulmonary arteries and airways. The present data indicate that responses to TRPV4 are modulated differently by NOS in pulmonary and systemic vascular beds and are attenuated by the TRPV4 antagonist GSK2193874.

TRPV4 calcium entry and surface expression attenuated by inhibition of myosin light chain kinase in rat pulmonary microvascular endothelial cells

In previous studies, blockade or gene deletion of either myosin light chain kinase (MLCK) or the mechanogated transient receptor potential vanilloid 4 (TRPV4) channel attenuated mechanical lung injury. To determine their effects on calcium entry, rat pulmonary microvascular endothelial cells (RPMVEC) were labeled with fluo-4 and calcium entry initiated with the TRPV4 agonist, 4α-phorbol 12, 13-didecanoate (4αPDD). Mean calcium transients peaked at ∼25 sec and persisted ∼500 sec. The 4αPDD response was essentially abolished in calcium-free media, or after pretreatment with the MLCK inhibitor, ML-7. ML-7 also attenuated the 4αPDD-induced inward calcium current measured directly using whole-cell patch clamp. Pretreatment with dynasore, an inhibitor of dynamin produced an initial calcium transient followed by a 4αPDD transient of unchanged peak intensity. Automated averaging of areas under the curve (AUC) of calcium transients in individual cells indicated total calcium activity with a relationship between treatment groups of ML-7 + 4αPDD < 4αPDD only < dynasore + 4αPDD. Measurement of biotinylated surface TRPV4 protein indicated a significant reduction after ML-7 pretreatment, but no significant change with dynasore treatment. RPMVEC monolayer electrical resistances were decreased by only 3% with 10 μmol/L 4αPDD and the response was dose-related. Dynasore alone produced a 29% decrease in resistance, but neither ML-7 nor dynasore affected the subsequent 4αPDD resistance response. These studies suggest that MLCK may inhibit mechanogated calcium responses through reduced surface expression of stretch activated TRPV4 channels in the plasma membrane.

TRPV4-Mediated Calcium Influx and Ciliary Activity in Human Native Airway Epithelial Cells

The transient receptor potential vanilloid 4 (TRPV4) is a calcium permeable ion channel expressed in airway epithelial cells. Based on studies of cell lines and animals, TRPV4 has been suggested to play a role in the regulation of ciliary beat frequency (CBF). Whether the same is true for human ciliated epithelial cells is not known. Therefore, the aim was to examine the expression and function of TRPV4 in human native nasal epithelial cells. Expression of TRPV4 mRNA in nasal epithelial cells and in the cell lines BEAS2B and 16HBE was confirmed by quantitative real-time PCR. A marked apical TRPV4 immunoreactivity was observed in nasal epithelial cells using immunocytochemistry. Responses to pharmacological modulation of TRPV4 were assessed with calcium imaging and CBF measurements. The TRPV4 agonist GSK1016790A produced concentration-dependent calcium responses in TRPV4-expressing HEK293, BEAS2B and 16HBE cells, and the TRPV4 antagonist HC067047 caused a rightward shift of the GSK1016790A concentration-response curves. Nasal epithelial cells responded to the TRPV4 agonist GSK1016790A with increased intracellular calcium signals and increased CBF, followed by cessation of ciliary beating and cell death. These effects were prevented or inhibited by the TRPV4 antagonist HC067047, the TRP channel blocker ruthenium red or removal of extracellular calcium. We conclude that TRPV4 is expressed in human primary nasal epithelial cells and modulates epithelial calcium levels and CBF. Thus, TRPV4 may participate in mucociliary clearance and airway protection. However, exaggerated activation of TRPV4 may result in epithelial cell death.

Abstract 537: Segment-specific Intrathecal Injection of Metformin Protects Against High Fat Diet-induced Impairment in Afferent Renal Nerves and Renal Function

Metformin (Met), a drug for treating type 2 diabetes by suppressing glucose production and increasing insulin sensitivity, may possess neuroprotective and neurogenesis effects. We test the hypotheses 1) that high fat diet (HFD) intake impairs afferent renal nerves expressing transient receptor potential vanilloid 1 (TRPV1) channels, leading to increased efferent renal nerve activity (ERNA), decreased renal function, and hypertension; and 2) that Met given intrathecally (i.t.) to segments (T8-L3) supplying the kidney prevents HFD-induced impairment in afferent renal nerves, renal function, and hypertension. Met (1 ng/kg, i.t. daily) or vehicle was given to rats fed a HFD or normal fat diet (Con) for 8 weeks. Glucose or insulin tolerance was impaired to the same degree between rats fed HFD with and without Met treatment. HFD decreased renal TRPV1 expression, plasma levels of calcitonin gene-related peptide, and afferent renal nerve activity (ARNA, Con: 126±11, Met: 136±14, HFD: 77±7, HFD+Met: 121±8, p&lt;0.05) in response to intra-pelvis perfusion of capsaicin (a TRPV1 agonist at 4 mM, 3 min, 20 ml/min), which were prevented by Met. HFD increased renal tyrosine hydroxylase contents, ERNA responses to intrathecal injection of muscimol (a GABA-A receptor agonist at 3 nmol/kg), and urinary norepinephrine levels, which were prevented by Met. HFD decreased creatinine clearance (Con: 0.40±0.04, Met: 0.52±0.07, HFD: 0.12±0.05, HFD+Met: 0.30±0.04 ml/min/100 gbwt, p&lt;0.05) and urinary Na+ excretion, and increased levels of urinary albumin, plasma urea, renal collagen deposition, renal connective tissue growth factor, and systolic blood pressure (Con: 132±1, Met: 130±1, HFD: 145±3, HFD+Met: 132±1 mmHg, p&lt;0.05), which were prevented or attenuated by Met. Thus, HFD impairs TRPV1-positive afferent renal nerves and renal function, and increases renal sympathetic nerve activity and blood pressure. Segment-specific intrathecal injection of Met protects against HFD-induced impairment in afferent renal nerves and renal function, and prevents HFD-induced hypertension.

Abstract 509: Role of TRPV1 and TRPV4 Channels in the Renal Vasculature

Transient receptor potential vanilloid 1 (TRPV1) and vanilloid 4 (TRPV4) cation channels have been recently identified to promote endothelium-dependent relaxation of small mesenteric arteries of mice, with calcium sparklets providing elementary calcium influx through single TRPV4 channels. The role of TRPV1 and TRPV4 in the renal vasculature is largely unknown. We hypothesized that TRPV1 and TRPV4 play an important role in endothelium-dependent vasodilation of renal blood vessels. We performed myography experiments on isolated renal arteries and perfused isolated kidneys of C57BL/6J mice. Isolated mesenteric arteries served as control vessels. The TRPV1 agonist capsaicin relaxed mesenteric arteries with an EC 50 of ~9 nM, but not large renal arteries (EC 50 &gt;10 μM). This relaxation was inhibited by the TRPV1 antagonist capsazepine (10 μM). The capsaicin effect was absent in vessels of TRPV1 knock-out mice. Cytochrome P450 (CYP) 4A- and CYP4F-derived hydroxyeicosatetraenoic acid (20-HETE), which is a proinflammatory mediator of endotoxin-induced acute systemic inflammation, activated TRPV1 channels in patch clamp studies. The TRPV4 agonist GSK1016790A relaxed renal and mesenteric arteries with EC 50 of ~12 nM and 70 nM, respectively. These effects were inhibited by a specific TRPV4 antagonist, AB159908 (10 μM). Capsaicin and GSK1016790A produced vascular dilation in isolated kidneys with EC 50 of ~11 nM and ~2 nM, respectively. The capsaicin effects were absent in kidneys of TRPV1 knock-out mice, whereas the effects of GSK1016790A were also observed in TRPV1 knock-out mice. Our results demonstrate that TRPV4 channels are capable to promote endothelial relaxation in large and small renal arteries, whereas TRPV1 channels play a specific vasodilatory role in small arteries of the kidney. We suggest that specific activation of TRPV1 and TRPV4 may be a novel promising strategy for treatment of sepsis-associated renal vasoconstriction.

Effect of TRPV1 channel on the proliferation and apoptosis in asthmatic rat airway smooth muscle cells

ABSTRACTBackground: Hyperplasia of airway smooth muscle cells (ASMC) is a major contributor to airway remodeling in asthma. Transient receptor potential vanilloid 1 (TRPV1) is an important channel to mediate Ca2+ influx. This study explores the expression of TRPV1 channel and its effect on the proliferation and apoptosis in rat ASMC, in order to find a new target to treat airway remodeling in asthma. Methods: Rats were sensitized and challenged with ovalbumin to replicate asthmatic models. Proliferating cell nuclear antigen (PCNA) was detected by immunohistochemistry. Reverse transcriptase-polymerase chain reaction, immunocytochemistry, and Western blot were used to detect the mRNA and protein expression of TRPV1 channel. Intracellular calcium ([Ca2+]i) was detected using confocal fluorescence Ca2+ imaging. [3H] thymidine incorporation and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay were used to observe the DNA synthesis and proliferation. TUNEL assay was used to detect the apoptosis of ASMC. Results: (1) The expression of PCNA was significantly increased in intact asthmatic rat ASMC. (2) The expression of TRPV1 channel was significantly increased in asthmatic rat ASMC. (3) [Ca2+]i in ASMC of the asthmatic group was significantly increased. After treatment with TRPV1 agonist capsaicin (CAP), [Ca2+]i was further increased, whereas [Ca2+]i was decreased after administration of TRPV1 antagonist capsazepine (CPZ) in ASMC of the asthmatic group. (4) The DNA synthesis and absorbance of MTT were significantly increased, while apoptosis was significantly decreased in asthmatic ASMC. CAP further enhanced proliferation and decreased apoptosis. CPZ significantly inhibited the effect of CAP in asthmatic ASMC. Conclusion: TRPV1 channel was involved in the regulation of proliferation and apoptosis in asthmatic ASMC.

Mechanisms involved in abdominal nociception induced by either TRPV1 or TRPA1 stimulation of rat peritoneum

Abdominal pain is a frequent symptom of peritoneal cavity irritation, but little is known about the role of the receptors for irritant substances, transient receptor potential vanilloid 1 (TRPV1) and ankyrin 1 (TRPA1), in this painful condition. Thus, we investigated the abdominal nociception caused by peritoneal stimulation with TRPV1 (capsaicin) and TRPA1 (allyl isothiocyanate, AITC) agonists and their mechanisms in rats. The intraperitoneal (i.p.) injection of either capsaicin or AITC (0.03–10mg/kg) induced short-term (up to 20min) and dose-dependent abdominal nociception, and also produced c-fos expression in spinal afferents of the dorsal horn. TRPV1 antagonism prevented (94±4% inhibition) nociception induced by capsaicin but not by AITC. In contrast, the TRPA1 antagonism almost abolished AITC-induced nociception (95±2% inhibition) without altering the capsaicin response. Moreover, nociception induced by either capsaicin or AITC was reduced by the desensitisation of TRPV1-positive sensory fibres with resiniferatoxin (73±18 and 76±15% inhibitions, respectively) and by the NK1 receptor antagonist aprepitant (56±5 and 53±8% inhibitions, respectively). Likewise, the i.p. injections of capsaicin or AITC increased the content of substance P in the peritoneal fluid. Nevertheless, neither the mast cell membrane stabiliser cromoglycate, nor the H1 antagonist promethazine, nor depletion of peritoneal macrophages affected abdominal nociception induced either by capsaicin or AITC. Accordingly, neither capsaicin nor AITC increased the histamine content in the peritoneal fluid or provoked peritoneal mast cell degranulation in vitro. Collectively, our findings suggest that TRPV1 and TRPA1 stimulation in the peritoneum produces abdominal nociception that is mediated by sensory fibres activation.

Role of endogenous TRPV1 agonists in a postburn pain model of partial-thickness injury

Oxidized linoleic acid metabolites (OLAMs) are a class of endogenous transient receptor potential vanilloid 1 (TRPV1) channel agonists released on exposure of tissue to transient noxious temperatures. These lipid compounds also contribute to inflammatory and heat allodynia. Because persistent pain after a burn injury represents a significant clinical challenge for treatment, we developed an in vivo rat model of partial-thickness cutaneous thermal injury and examined whether TRPV1 and specific OLAM metabolites play a role in mediating postburn pain injury. This peripheral model of burn injury had marked thermal allodynia peaking at 24h after thermal injury, with allodynia being maintained for up to 7d. Immunohistochemical characterization of tissue taken from injury sites revealed an increase in leukocyte/macrophage infiltration that was colocalized with TRPV1-positive fibers. Using this peripheral thermal injury model, we found that pharmacological blockade of peripheral TRPV1 receptors reduced thermal allodynia by about 98%. Moreover, there was a significant increase in OLAM levels compared to naive controls in hind paw skin biopsies. Additional studies of the metabolism of [C14]-linoleic acid in skin biopsies revealed the role of the cytochrome P450 (CYP) system in mediating the metabolism of linoleic acid after thermal injury. Finally, we demonstrated that direct inhibition of OLAMs using OLAM antibodies and indirect inhibition using the CYP inhibitor ketoconazole significantly reduced postburn thermal allodynia. Collectively, these findings point to a novel role of the OLAMs and CYP-related enzymes in generating postburn allodynia via activation of peripheral TRPV1.

A review of the high-concentration capsaicin patch and experience in its use in the management of neuropathic pain

In the European Union, the high-concentration capsaicin patch is licensed for the management of neuropathic pain conditions in nondiabetic patients, including postherpetic neuralgia (PHN) and HIV-associated distal sensory polyneuropathy (HIV-DSP). However, in the USA, the Food and Drug Administration approved its use only in PHN patients. Capsaicin is a transient receptor potential vanilloid-1 agonist, which increases the intracellular calcium ion concentration. This triggers calcium-dependent protease enzymes causing cytoskeletal breakdown and leads to the loss of cellular integrity and 'defunctionalization' of nociceptor fibres. Efficacy and therapeutic effect has been shown in several clinical studies of PHN and HIV-DSP. The high-concentration capsaicin patch and its practical application are different from low-concentration creams; one application can help for up to 3 months. The process of setting up of a service to use the capsaicin 8% patch is also discussed.

Transient Receptor Potential Vanilloid 4 Mediates Hypotonicity‐Induced Enhancement of Synaptic Transmission in Hippocampal Slices

Changes in cerebrospinal fluid osmotic pressure modulate brain excitability. Transient receptor potential vanilloid 4 (TRPV4), which is sensitive to hypotonic stimulation, is expressed in hippocampus. The present study investigated the effect of hypotonic stimulation on hippocampal synaptic transmission and the role of TRPV4 in hypotonicity-action using electrophysiological recording and pharmacological technique. Accompanied with the decrease in paired pulse facilitation, field excitatory postsynaptic potential (fEPSP) was enhanced by hypotonicity and TRPV4 agonist 4α-PDD in hippocampal slices, which was sensitive to TRPV4 antagonist HC-067047. Hypotonicity-induced increase in fEPSP was blocked by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist, but not N-methyl-d-aspartate receptor or N- or P/Q-type voltage-gated calcium channel antagonist. High voltage-gated calcium current (ICa ) in hippocampal CA3 pyramidal neurons was not affected by hypotonicity. AMPA-activated current (IAMPA ) in hippocampal CA1 pyramidal neurons was increased by hypotonicity and 4α-PDD, which was attenuated by HC-067047. Inhibition of protein kinase C or protein kinase A markedly attenuated hypotonicity-increased IAMPA , whereas antagonism of calcium/calmodulin-dependent protein kinase II had no such effect. TRPV4 is involved in hypotonicity-induced enhancement of hippocampal synaptic transmission, which may be mediated through promoting presynaptic glutamate release and increasing postsynaptic AMPA receptor function.

Abstract LB-33: TRPV1 suppresses intestinal tumorigenesis by negatively regulating EGFR signaling.

Abstract The intestinal epithelium is the most rapidly self-renewing tissue. This dynamic process is mainly driven by Wnt, Notch and EGFR signaling in the crypts of Lieberkühn. These signaling pathways are negatively regulated by multiple control mechanisms to restrain intestinal epithelial cell (IEC) turnover and thereby reduce the risk of oncogenic transformation. Here we report a novel homeostatic feedback loop, intrinsic to IEC, between the EGFR and a member of the transient receptor potential (TRP) ion channel family. We identified that TRP vanilloid 1 (TRPV1), a polymodal pain receptor, is expressed by IEC and that this channel negatively regulates EGFR-induced cell proliferation in vivo. Intestinal crypt cultures (organoids) from Trpv1-/- mice showed hyperproliferation, crypt elongation and a reduced requirement for exogenous EGF compared to WT controls. Importantly, genetic deletion of Trpv1 increased the intestinal tumor load in multiple intestinal neoplasia (Apc+/min) mice, which was reversed by EGFR kinase inhibitor treatment. Continuous oral administration of a dietary TRPV1 agonist, capsaicin, suppressed intestinal tumorigenesis in Apc+/min mice in a TRPV1-dependent manner. In addition we found that capsaicin worked synergistically with a COX2 inhibitor, celecoxib, in the prevention of intestinal neoplasia development in these mice. Mechanistically, we show that TRPV1 is activated by EGFR signaling through phospholipase C (PLC). TRPV1 signaling subsequently restrains EGFR signaling through Ca2+-dependent triggering of protein tyrosine phosphatase (PTP) activity. Together, these findings suggest an intrinsic mechanism that exerts negative feedback on EGFR signaling through Ca2+ channel TRPV1 that thereby acts as a tumor suppressor in the intestinal epithelium. Citation Format: Petrus R. de Jong, Naoki Takahashi, Alexandra R. Harris, Jihyung Lee, Samuel Bertin, Eyal Raz. TRPV1 suppresses intestinal tumorigenesis by negatively regulating EGFR signaling. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-33. doi:10.1158/1538-7445.AM2013-LB-33

TRPV4 Ca2+ sparklets in myoendothelial projections (MEPs) regulate vascular function

Endothelial cells (ECs) lining blood vessels regulate vascular tone. MEPs connect ECs to adjacent smooth muscle cells (SMCs) via gap junctions. We discovered elementary Ca2+ signals (“sparklets”) through single TRPV4 (transient receptor potential vanilloid 4) channels at MEPs (Sonkusare et. al., Science, 2012). TRPV4 sparklets dilate 3rd order mouse mesenteric arteries (MAs) via EC intermediate‐conductance, Ca2+ sensitive K+ (IK) channels. The current study elucidates the mechanism by which muscarinic receptor stimulation dilates MAs via TRPV4 channels. Carbachol (CCh) increased sparklets (5‐fold) ONLY at MEPs, and not elsewhere on the EC membrane. Protein kinase C (PKC) activation similarly increased sparklets only at MEPs. CCh‐activation of sparklets was prevented by PKC inhibitor Go‐6976. Inward rectifier K+ (Kir) currents were present in ECs but not in SMCs. Dilations to CCh, TRPV4 agonist GSK1016790A, and IK/SK opener NS309 were inhibited (70%) by Kir channel blocker Ba2+. The results support a MEP‐localized signaling circuit of PKC‐TRPV4‐IK‐Kir channels which mediates vasodilatory input to the SM.