Transient Receptor Potential Vanilloid 4 Agonist GSK1016790A Research Articles

Activation of Transient Receptor Potential Vanilloid 4 (TRPV4) channels opens Connexin Hemichannels in Lens Epithelial Cells

ATP release from the lens via connexin hemichannels has been hypothesized as a response to TRPV4 activation when the lens is subjected to osmotic swelling. To explore the apparent linkage between TRPV4 activation and hemichannel opening we performed patch-clamp recordings on cultured mouse lens epithelial cells exposed to the TRPV4 agonist GSK1016790A (GSK) in the presence or absence of the TRPV4 antagonist HC067047 (HC). GSK was found to cause a fast, variable and generally large non-selective increase of whole cell membrane conductance evident as a larger membrane current (Im) over a wide voltage range. This response was prevented by HC. The GSK-induced Im increase was proportionally larger at negative voltages and coincided with fast depolarization and the simultaneous disappearance of an outward current, likely a K + current. The initial presence of this outward current in control conditions appeared to be a reliable predictor of a cell’s response to GSK treatment. In some studies, recordings from single cells combining cell-attached and whole-cell patch clamp configurations, were obtained. This novel approach revealed events with a channel conductance 180-270 pS following GSK application through the patch pipette on the cell-attached side. The findings are consistent with TRPV4-dependent opening of connexin hemichannels. Ongoing studies are aimed at confirming the hemichannels identity. NIH: 5R01EY029171-04 and 2R01EY009532-28A1. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

TRPV4 activation prevents lipopolysaccharide-induced painful bladder hypersensitivity in rats by regulating immune pathways.

Chronic inflammation in the urinary bladder is a potential risk factor for bladder dysfunction, including interstitial cystitis/bladder pain syndrome (IC/BPS). Although several studies have reported that activation of transient receptor potential vanilloid 4 (TRPV4) contributes to bladder pain and overactive bladder with a cardinal symptom of acute or chronic cystitis, others have reported its involvement in the protective response mediated by lipopolysaccharides (LPS) to secrete anti-inflammatory/pro-resolution cytokines. Therefore, we investigated the potential benefit of an intravesical TRPV4 agonist for painful bladder hypersensitivity in a rat model of LPS-induced cystitis and determined whether its effects modulate the LPS signal for inflammatory reaction, cytokine release, and macrophage phenotype change. Previously, we showed that repeated intravesical instillations of LPS induce long-lasting bladder inflammation, pain, and overactivity in rats. In the present study, concurrent instillation of the selective TRPV4 agonist GSK1016790A (GSK) with LPS into the rat bladder improved LPS-induced bladder inflammation and reduced the number of mast cells. Furthermore, co-instillation of GSK prevented an increase in bladder pain-related behavior and voiding frequency caused by LPS. Cytokine profiling showed that LPS-stimulated inflammatory events, such as the production and secretion of pro-inflammatory cytokines (CXCL1, CXCL5, CXCL9, CXCL10, CCL3, CCL5, CCL20, and CX3CL1), are suppressed by GSK. Furthermore, TRPV4 activation switched LPS-stimulated pro-inflammatory M1-type macrophages to anti-inflammatory M2-type macrophages. These results suggest that TRPV4 activation in the bladder negatively regulates the pro-inflammatory response induced by LPS and prevents bladder hypersensitivity. These TRPV4 functions may be promising therapeutic targets for refractory IC/BPS.

Mineralocorticoid receptor antagonism improves transient receptor potential vanilloid 4-dependent dilation of cerebral parenchymal arterioles and cognition in a genetic model of hypertension.

In a model of secondary hypertension, mineralocorticoid receptor (MR) antagonism during the development of hypertension prevents the impairment of transient receptor potential vanilloid 4 (TRPV4) activation in parenchymal arterioles (PAs) and cognitive impairment. However, it is unknown whether MR antagonism can improve these impairments when treatment begins after the onset of essential hypertension. We tested the hypothesis that MR activation in stroke-prone spontaneously hypertensive rats (SHRSP) leads to impaired TRPV4-mediated dilation in PAs that is associated with cognitive dysfunction and neuroinflammation. 20-22-week-old male SHRSP ± eplerenone (EPL; 100 mg/kg daily for 4 weeks) were compared to normotensive Sprague-Dawley (SD) rats. Pressure myography was used to assess PA function. Cognition was tested using Y-maze. Neuroinflammation was assessed using immunofluorescence and qRT-PCR. Carbachol-mediated endothelium-dependent dilation was impaired in SHRSP, and MR antagonism improved this without affecting myogenic tone. Dilation to TRPV4 agonist GSK1016790A was impaired in SHRSP, and ELP treatment restored this. Intermediate conductance potassium channel (IKCa)/small conductance potassium channel (SKCa)-mediated dilation was impaired by hypertension and unaffected by EPL treatment. TRPV4 and IKCa/SKCa channel mRNA expression were reduced in PAs from hypertensive rats, and EPL did not improve this. Impairments in PA dilation in SHRSP were associated with cognitive decline, microglial activation, reactive astrogliosis, and neuroinflammation; cognitive and inflammatory changes were improved with MR blockade. These data advance our understanding of the effects of hypertension on cerebral arterioles using a clinically relevant model and treatment paradigm. Our studies suggest TRPV4 and the MR are potential therapeutic targets to improve cerebrovascular function and cognition during hypertension.

Activation of transient receptor potential vanilloid 4 is involved in pressure overload-induced cardiac hypertrophy.

Previous studies, including our own, have demonstrated that transient receptor potential vanilloid 4 (TRPV4) is expressed in hearts and implicated in cardiac remodeling and dysfunction. However, the effects of TRPV4 on pressure overload-induced cardiac hypertrophy remain unclear. In this study, we found that TRPV4 expression was significantly increased in mouse hypertrophic hearts, human failing hearts, and neurohormone-induced hypertrophic cardiomyocytes. Deletion of TRPV4 attenuated transverse aortic constriction (TAC)-induced cardiac hypertrophy, cardiac dysfunction, fibrosis, inflammation, and the activation of NFκB - NOD - like receptor pyrin domain-containing protein 3 (NLRP3) in mice. Furthermore, the TRPV4 antagonist GSK2193874 (GSK3874) inhibited cardiac remodeling and dysfunction induced by TAC. In vitro, pretreatment with GSK3874 reduced the neurohormone-induced cardiomyocyte hypertrophy and intracellular Ca2+ concentration elevation. The specific TRPV4 agonist GSK1016790A (GSK790A) triggered Ca2+ influx and evoked the phosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaMKII). But these effects were abolished by removing extracellular Ca2+ or GSK3874. More importantly, TAC or neurohormone stimulation-induced CaMKII phosphorylation was significantly blocked by TRPV4 inhibition. Finally, we show that CaMKII inhibition significantly prevented the phosphorylation of NFκB induced by GSK790A. Our results suggest that TRPV4 activation contributes to pressure overload-induced cardiac hypertrophy and dysfunction. This effect is associated with upregulated Ca2+/CaMKII mediated activation of NFκB-NLRP3. Thus, TRPV4 may represent a potential therapeutic drug target for cardiac hypertrophy and dysfunction after pressure overload.

Hypertensive Female Mice Are Protected from Impaired Parenchymal Arteriolar TRPV4‐Mediated Dilation and Reactive Astrogliosis

Hypertension is a leading risk factor for cerebral small vessel disease. Our lab has previously demonstrated that cerebral parenchymal arterioles (PAs) are dependent on transient receptor potential vanilloid 4 (TRPV4) function for endothelium‐dependent dilation, and that this dilation is impaired in angiotensin II (AngII)‐hypertensive male mice. This PA dilation impairment is associated with cognitive deficits and increased inflammation. However, sex differences in hypertension‐associated dementia development are not well understood. To better understand the effect of female sex hormones during hypertension, we tested the hypothesis that young, cycling hypertensive female mice will be protected from impaired TRPV4‐mediated PA dilation and reactive astrogliosis observed in male mice. AngII‐filled osmotic minipumps (800ng/kg/min, 4 weeks) were implanted in 16‐18‐week‐old male C56BL/6 mice. Age‐matched female mice received either an AngII dose that matches the dose used in male mice (800ng/kg/min) or a higher dose (1200ng/kg/day) that produces an elevation in blood pressure similar to male mice. Sham mice served as control. Blood pressure was measured by tail‐cuff plethysmography. Pressure myography was used to assess TRPV4‐mediated dilation in PAs. An anti‐glial fibrillary acidic protein (GFAP) antibody was used in paraformaldehyde‐fixed 40μm coronal brain sections to assess the quantity and morphology of astrocytes. Data are presented as means ± SEM (n=5‐10). Male data were compared by Student’s t‐test and female data were compared by one‐way ANOVA unless otherwise stated. Systolic blood pressure was elevated in AngII‐treated male mice (sham: 133 ± 10, AngII: 181 ± 11mmHg; p=0.005) and in 1200ng AngII‐treated female mice (sham: 146 ± 8, 800ng AngII: 161 ± 13, 1200ng AngII: 179 ± 7mmHg; p=0.0482) vs sham. PA dilation in response to the TRPV4 agonist GSK1016790A (10‐9‐10‐5M) was not impaired in AngII‐treated female mice (sham: 85 ± 5, 800ng AngII: 73 ± 7, 1200ng AngII: 77 ± 8% maximum dilation; p=0.4381), contrasting with what we have previously reported in male mice. There was an increase in astrocytes in the corpus callosum of hypertensive male mice (sham: 65 ± 2; AngII: 77 ± 5; p=0.039). Interestingly, sham female mice had more astrocytes than sham male mice, and AngII infusion reduced the quantity (sham: 81 ± 3, 800ng AngII: 71 ± 3, 1200ng AngII: 69 ± 3; p=0.011). Sholl analysis was used to assess astrocyte morphology. Astrocytes from hypertensive male mice had more arborization further from the soma compared to sham (p=0.0348 at 14‐15μm from the soma, by two‐way ANOVA and Bonferroni correction). Female mice receiving the 1200ng dose AngII, however, had fewer arborizations away from the soma compared to sham (p=0.0263 at 13‐15μm from the soma, by two‐way ANOVA and Bonferroni correction). Female mice are resistant to AngII‐induced hypertension and protected from the associated deleterious cerebrovascular effects, supporting our hypothesis.

Nitric Oxide Inhibits TRPV4‐mediated Hemichannel Opening in Porcine Nonpigmented Ciliary Epithelium

Previous studies showed the presence of connexin 43 (Cx43), connexin 50 (Cx50), and pannexin 1 (Panx1) in the nonpigmented ciliary epithelium (NPE) of the eye, as well as evidence of hemichannel (HC) activity. In the lens, mechanosensitive Transient Receptor Potential Vanilloid 4 (TRPV4)‐mediated calcium entry stimulates hemichannel activity. The aim here is to test for TRPV4 expression in the NPE, determine whether TRPV4 activation causes HC opening, and study the influence of nitric oxide.Immunohistochemistry was used to probe for TRPV4 in porcine ciliary body. Propidium iodide (PI) uptake and ATP release by primary cultured NPE was used to assess HC opening. PI and ATP are large molecules (MW 668 and 507, respectively) that pass through open hemichannels but not the plasma membrane. ATP in the bathing medium was measured by luciferase luminometry and PI uptake by fluorimetry.Abundant expression of TRPV4 was detected in the NPE cell layer of the ciliary body. Cultured NPE cells displayed an increase in PI uptake when exposed to a TRPV4 agonist GSK 1016790A (10 nM) or to hypoosmotic solution (200 mOsm) (Control 4.65 ± 0.24 vs GSK 10.26 ± 0.32 and Hypo 8.26 ± 0.42 fluorescence units per mg protein; n=6, p<0.001). PI uptake also was increased in NPE cells cultured on a flexible membrane and subjected to cyclic stretch for 1‐10 min (10%, 0.5 Hz), the peak response was at at 2 min (Control 1.5 ± 0.03 vs stretch 2.5 ± 0.10; n=6, p<0.001). The increase in PI uptake caused by hypoosmotic solution or by cyclic stretch was abolished by a selective TRPV4 antagonist HC 067047 (10 μM). Hyposmotic solution increased ATP release from 1.12 ± 0.24 to 2.81± 0.27 (n=12, p<0.001) while cyclic stretch increased ATP release from 0.20 ± 0.03 to 0.40 ± 0.03 pmoles/mg protein (n=6, p<0.001). ATP release responses caused by hypoosmotic solution and cyclic stretch were inhibited by HC 067047. Hypoosmotic solution‐induced ATP release was also inhibited by connexin inhibitory peptide, Gap‐27 (200 μM) and nitric oxide (NO) donors, sodium nitroprusside (SNP) and S‐nitroso‐N‐acetylpenicillamine (SNAP).TRPV4 channels are expressed by the NPE. The ability of HC 067047 and Gap‐27 to prevent ATP release and PI uptake points to TRPV4‐dependent opening of connexin HCs in NPE cells. Suppression of ATP release by SNP and SNAP indicates the hemichannel opening mechanism is inhibited by NO.

Blockage of transient receptor potential vanilloid 4 prevents postoperative atrial fibrillation by inhibiting NLRP3-inflammasome in sterile pericarditis mice

The incidence of atrial fibrillation (AF) increases after surgery and is associated with the activation of NLRP3-inflammation. Our previous studies have found that transient receptor potential vanilloid 4 (TRPV4) blockade reduces the susceptibility to AF, but its molecular mechanisms remains unclear. Therefore, we hypothesized that blockage of TRPV4 reduces the incidence of AF by inhibiting NLRP3-inflammasome in sterile pericarditis (SP) mice. In this study, we established SP mice by dusting talcum powder on atrial surfaces. We first confirmed that genetic or pharmacological TRPV4 inhibition reduced the susceptibility to AF in SP mice. We also found that the expression level of NLRP3-inflammasome and inflammatory cytokines significantly increased in the atria of SP mice, which further increased in application the TRPV4 agonist GSK1016790A (GSK101) and decreased in application the TRPV4 antagonist GSK2193874. More importantly, ERK inhibitor (U0126) or NF-κB inhibitor (Bay11–7082) could partially reverse GSK101-induced NLRP3-inflammasome up-regulation. Interestingly, U0126 can reversed GSK101-induced NF-κB phosphorylation, but Bay11–7082 cannot change GSK101-induced ERK phosphorylation. Finally, we shown that the activation of NLRP3-inflammasome and ERK/NF-κB signaling pathway significantly reduced in TRPV4-knockout SP mice. Collectively, our studies indicate that blockage of TRPV4 prevents AF in SP mice by inhibiting NLRP3-inflammasome through the ERK/NF-κB signaling pathway.

Emergent Temporal Signaling in Human Trabecular Meshwork Cells: Role of TRPV4-TRPM4 Interactions.

Trabecular meshwork (TM) cells are phagocytic cells that employ mechanotransduction to actively regulate intraocular pressure. Similar to macrophages, they express scavenger receptors and participate in antigen presentation within the immunosuppressive milieu of the anterior eye. Changes in pressure deform and compress the TM, altering their control of aqueous humor outflow but it is not known whether transducer activation shapes temporal signaling. The present study combines electrophysiology, histochemistry and functional imaging with gene silencing and heterologous expression to gain insight into Ca2+ signaling downstream from TRPV4 (Transient Receptor Potential Vanilloid 4), a stretch-activated polymodal cation channel. Human TM cells respond to the TRPV4 agonist GSK1016790A with fluctuations in intracellular Ca2+ concentration ([Ca2+]i) and an increase in [Na+]i. [Ca2+]i oscillations coincided with monovalent cation current that was suppressed by BAPTA, Ruthenium Red and the TRPM4 (Transient Receptor Potential Melastatin 4) channel inhibitor 9-phenanthrol. TM cells expressed TRPM4 mRNA, protein at the expected 130-150 kDa and showed punctate TRPM4 immunoreactivity at the membrane surface. Genetic silencing of TRPM4 antagonized TRPV4-evoked oscillatory signaling whereas TRPV4 and TRPM4 co-expression in HEK-293 cells reconstituted the oscillations. Membrane potential recordings suggested that TRPM4-dependent oscillations require release of Ca2+ from internal stores. 9-phenanthrol did not affect the outflow facility in mouse eyes and eyes from animals lacking TRPM4 had normal intraocular pressure. Collectively, our results show that TRPV4 activity initiates dynamic calcium signaling in TM cells by stimulating TRPM4 channels and intracellular Ca2+ release. It is possible that TRPV4-TRPM4 interactions downstream from the tensile and compressive impact of intraocular pressure contribute to homeostatic regulation and pathological remodeling within the conventional outflow pathway.

Abstract 12772: Regulation of Blood Pressure by Smooth Muscle TRPV4 Channels Under Normal and Hypertensive Conditions

Introduction: Recent studies suggest that transient receptor potential vanilloid 4 (TRPV4) channel is a functionally important Ca 2+ influx pathway in arterial smooth muscle cells (SMCs). Hypothesis: We hypothesized that SMC TRPV4 (TRPV4 SMC ) channels regulate blood pressure under normal conditions and in hypertension. Results: Radiotelemetric recordings showed lower blood pressures in the SMC-specific TRPV4 -/- (TRPV4 SMC -/- ) mice than the control mice. Phenylephrine (PE, α1 adrenergic receptor or α1AR agonist)-induced constriction was blunted in small mesenteric arteries (MAs) from TRPV4 SMC -/- mice, whereas intraluminal pressure-induced constriction was increased. These data demonstrated that TRPV4 SMC channels contribute to α1AR-induced vasoconstriction but oppose pressure-induced vasoconstriction. Both PE and intraluminal pressure increased the activity of elementary Ca 2+ influx signals through TRPV4 SMC channels (TRPV4 SMC sparklets). TRPV4 agonist GSK1016790A (30 nM) activated the currents through large-conductance, Ca 2+ -sensitive K + (BK) channels in SMCs, whereas phenylephrine had no effect on BK channel currents. These data implied the presence of two functionally distinct pools of TRPV4 SMC channels: 1) α1AR-activated, constrictor TRPV4 SMC channels; and 2) pressure-activated, dilator TRPV4 SMC channels. Next, direct STORM imaging demonstrated that ~30% TRPV4 SMC channels co-localize with α1ARs and ~10% TRPV4 SMC channels co-localize with BK channels. Importantly, only ~3% TRPV4 SMC channels co-localized with both α1ARs and BK channels, supporting the concept of spatially distinct constrictor and dilator TRPV4 SMC channels. Angiotensin II (Ang II, 7-14 days) infusion resulted in a lower increase in blood pressure in TRPV4 SMC -/- mice than the control mice, demonstrating a crucial role of TRPV4 SMC channels in blood pressure elevation in hypertension. Moreover, Ang II infusion in C57BL6 mice increased α1AR-TRPV4 SMC signaling; reduced pressure-activation of TRPV4 SMC channels; and reduced TRPV4 SMC -BK channel signaling. Conclusions: Our results show that increased constrictor TRPV4 SMC channel activity and reduced dilator TRPV4 SMC channel activity combine to elevate blood pressure in hypertension.

Involvement of TRPV4 in changes in rapidly inactivating potassium channels in the early stage of pilocarpine-induced status epilepticus in mice.

The rapidly inactivating potassium current (IA ) is important in controlling neuronal action potentials. Altered IA function and K+ channel expression have been found in epilepsy, and activation of the transient receptor potential vanilloid 4 (TRPV4) channel is involved in epilepsy pathogenesis. This study examined whether TRPV4 affects Kv4.2 and K+ channel interacting protein (KCHIP) expression and IA changes following pilocarpine-induced status epilepticus (PISE) in mice. Herein, hippocampal protein levels of Kv4.2 and KCHIP2 increased 3 h-3 d and decreased 7-30 d; that of KCHIP1 increased 3-24 h and decreased 3-30 d post-PISE. The TRPV4 antagonist HC-067047 attenuated the increased protein levels of Kv4.2 and KCHIP2 but not that of KCHIP1 post-PISE. The TRPV4 agonist GSK1016790A increased hippocampal protein levels of Kv4.2 and KCHIP2 but had no effect on KCHIP1 expression. HC-067047 attenuated the increased IA in hippocampal pyramidal neurons 24 h and 3 d post-PISE. GSK1016790A increased IA in hippocampal pyramidal neurons, shifting the voltage-dependent inactivation curve toward depolarization. The GSK1016790A-induced increase of IA was blocked by protein kinase A and calcium/calmodulin-dependent kinase II antagonists but was unaffected by protein kinase C antagonists. We conclude that TRPV4 activation may be responsible for the increases of Kv4.2 and KCHIP2 expression in hippocampi and IA in hippocampal pyramidal neurons in PISE mice, which are likely compensatory measures for hyperexcitability at the early stage of epilepsy.

Role of TRPV4-P2X7 Pathway in Neuropathic Pain in Rats with Chronic Compression of the Dorsal Root Ganglion.

Transient receptor potential vanilloid 4 (TRPV4) is a Ca2+-permeable non-selective cation channel that is involved in the development of neuropathic pain. P2X7 receptor (P2X7) belongs to a class of ATP-gated nonselective cation channels that plays an important role in neuropathic pain. Nevertheless, little is known about the interaction between them for neuropathic pain. In this paper, we investigated role of TRPV4-P2X7 pathway in neuropathic pain. We evaluated the effect of TRPV4-P2X7 pathway on neuropathic pain in a chronic compression of the dorsal root ganglion (DRG) (hereafter termed CCD) model. We analyzed the effect of P2X7 on mechanical and thermal hyperalgesia mediated by TRPV4 in CCD. Furthermore, we assessed the effect of TRPV4 on the expression of P2X7 and the release of IL-1β and IL-6 in DRG after CCD. We found that intraperitoneal injection of TRPV4 agonist GSK-1016790A led to a significant increase of mechanical and thermal hyperalgesia in CCD, which was partially suppressed by P2X7 blockade with antagonist Brilliant Blue G (BBG). Then, we further noticed that GSK-1016790A injection increased the P2X7 expression of CCD, which was decreased by TRPV4 blockade with antagonist RN-1734 and HC-067047. Furthermore, we also discovered that the expressions of IL-1β and IL-6 were upregulated by GSK-1016790A injection but reduced by RN-1734 and HC-067047. Our results provide evidence that P2X7 contributes to development of neuropathic pain mediated by TRPV4 in the CCD model, which may be the basis for treatment of neuropathic pain relief.

TRPV4-Rho signaling drives cytoskeletal and focal adhesion remodeling in trabecular meshwork cells.

Intraocular pressure (IOP) is dynamically regulated by the trabecular meshwork (TM), a mechanosensitive tissue that protects the eye from injury through dynamic regulation of aqueous humor flow. TM compensates for mechanical stress impelled by chronic IOP elevations through increased actin polymerization, tissue stiffness, and contractility. This process has been associated with open angle glaucoma; however, the mechanisms that link mechanical stress to pathological cytoskeletal remodeling downstream from the mechanotransducers remain poorly understood. We used fluorescence imaging and biochemical analyses to investigate cytoskeletal and focal adhesion remodeling in human TM cells stimulated with physiological strains. Mechanical stretch promoted F-actin polymerization, increased the number and size of focal adhesions, and stimulated the activation of the Rho-associated protein kinase (ROCK). Stretch-induced activation of the small GTPase Ras homolog family member A (RhoA), and tyrosine phosphorylations of focal adhesion proteins paxillin, focal adhesion kinase (FAK), vinculin, and zyxin were time dependently inhibited by ROCK inhibitor trans-4-[(1R)-1-aminoethyl]-N-4-pyridinylcyclohexanecarboxamide dihydrochloride (Y-27632), and by HC-067047, an antagonist of transient receptor potential vanilloid 4 (TRPV4) channels. Both TRPV4 and ROCK activation were required for zyxin translocation and increase in the number/size of focal adhesions in stretched cells. Y-27632 blocked actin polymerization without affecting calcium influx induced by membrane stretch and the TRPV4 agonist GSK1016790A. These results reveal that mechanical tuning of TM cells requires parallel activation of TRPV4, integrins, and ROCK, with chronic stress leading to sustained remodeling of the cytoskeleton and focal complexes.

Synthesis and in vitro evaluation of new TRPV4 ligands and biodistribution study of an 11C-labeled radiotracer in rodents

Nine new compounds targeting the transient receptor potential vanilloid-4 (TRPV4) were synthesized and their biological activities toward TRPV4 were determined using freshly isolated mouse skin macrophages through live cell Ca2+ imaging assay. Three compounds 4b, 4c, and 4i exhibited higher percentages of in vitro activation of TRPV4 as 48.1%, 59.3% and 33.5%, which are comparable to 56.4% activation response of the reported TRPV4 agonist GSK1016790A (3). The compound 4i was chosen for 11C-radiosynthesis using its phenol precursor 4g to reacted with [11C]methyl iodide. The radiosynthesis was achieved with good radiochemical yield (16 ± 5%), high chemical and radiochemical purity (>95%), and high molar activity (16–21 GBq/μmol, decay corrected to the end of bombardment, EOB n ≥ 4). Furthermore, the initial ex vivo biodistribution study in rats showed that [11C]4i had higher uptake in kidney, liver and small intestine compared to other tissues with rapid washout.

Propofol Induces Cardioprotection Against Ischemia-Reperfusion Injury via Suppression of Transient Receptor Potential Vanilloid 4 Channel.

Ca2+ entry via the transient receptor potential vanilloid 4 (TRPV4) channel contributes to Ca2+ overload and triggers many pathophysiological conditions, including myocardial ischemia/reperfusion (I/R) injury. Propofol, a widely used intravenous anesthetic, attenuates myocardial I/R injury. However, the mechanism of propofol remains to be examined. The present study aims to test the hypothesis that propofol attenuates myocardial I/R injury through the suppression of TRPV4. We used a murine ex vivo model of myocardial I/R and in vitro cultured myocytes subjected to hypoxia/reoxygenation (H/R). Propofol or TRPV4 antagonist, HC-067047, attenuates myocardial I/R injury in isolated hearts. In addition, propofol, HC-067047, or TRPV4-siRNA attenuates H/R-induced intracellular Ca2+ concentration ([Ca2+]i) increase and cell viability reduction. On the contrary, TRPV4 agonist GSK1016790A exacerbates both ex vivo and in vitro myocardial injury. Pretreatment with propofol reverses the myocardial injury and intracellular Ca2+ overload induced by GSK1016790A at least in vitro. However, neither the combination of propofol and HC-067047 nor applying propofol to cells transfected with TRPV4-siRNA creates additional protective effects. In addition, propofol dose-dependently inhibits TRPV4-mediated Ca2+ entry induced by GSK1016790A and 4α-PDD. Propofol attenuates myocardial I/R injury partially through the suppression of TRPV4 channel and the subsequent inhibition of intracellular Ca2+ overload.

Transient receptor potential vanilloid 4 is involved in the upregulation of connexin expression following pilocarpine-induced status epilepticus in mice

Epilepsy is characterized by spontaneous seizures. Changes in the expression of the connexins (Cxs) have been reported to be involved in epileptogenesis. It has previously been shown that the transient receptor potential vanilloid 4 (TRPV4) plays an important role in the modulation of neuronal excitability, and that application of a TRPV4 antagonist blocks hyperthermia-induced seizures. Accordingly, in the present study, we sought to explore whether TRPV4 is involved in the regulation of Cx expression following pilocarpine-induced status epilepticus (PISE) in mice. We observed that TRPV4 protein levels in hippocampi increased 3 h to 30 d following PISE, peaking 1–3 d after induction, and that pre-application of the TRPV4 antagonist HC-067047 increased the latency to develop SE induced by pilocarpine and reduced the success rate of PISE preparation. We demonstrated that Cx43 protein levels followed a time profile similar to that of TRPV4, and further showed that the increase in Cx43 protein levels on 3 d post-PISE was markedly attenuated by HC-067047. In contrast, the corresponding increase in Cx32 protein levels lagged substantially behind, and these levels were unaffected by HC-067047. Similarly, the TRPV4 agonist GSK1016790A increased the mRNA and protein levels of Cx43, but not those of Cx32. We thus conclude that the upregulation of Cx43 expression by TRPV4 may be involved in the pathophysiology of epilepsy.

TRPV4-induced inflammatory response is involved in neuronal death in pilocarpine model of temporal lobe epilepsy in mice

Activation of transient receptor potential vanilloid 4 (TRPV4) induces neuronal injury. TRPV4 activation enhances inflammatory response and promotes the proinflammatory cytokine release in various types of tissue and cells. Hyperneuroinflammation contributes to neuronal damage in epilepsy. Herein, we examined the contribution of neuroinflammation to TRPV4-induced neurotoxicity and its involvement in the inflammation and neuronal damage in pilocarpine model of temporal lobe epilepsy in mice. Icv. injection of TRPV4 agonist GSK1016790A (GSK1016790A-injected mice) increased ionized calcium binding adapter molecule-1 (Iba-1) and glial fibrillary acidic protein (GFAP) protein levels and Iba-1-positive (Iba-1+) and GFAP-positive (GFAP+) cells in hippocampi, which indicated TRPV4-induced microglial cell and astrocyte activation. The protein levels of nucleotide-binding oligomerization domain-like receptor pyrin domain containing 3 (NLRP3) inflammasome components NLRP3, apoptosis-related spotted protein (ASC) and cysteinyl aspartate-specific protease-1 (caspase-1) were increased in GSK1016790A-injected mice, which indicated NLRP3 inflammasome activation. GSK1016790A also increased proinflammatory cytokine IL-1β, TNF-α and IL-6 protein levels, which were blocked by caspase-1 inhibitor Ac-YVAD-cmk. GSK1016790A-induced neuronal death was attenuated by Ac-YVAD-cmk. Icv. injection of TRPV4-specific antagonist HC-067047 markedly increased the number of surviving cells 3 d post status epilepticus in pilocarpine model of temporal lobe epilepsy in mice (pilocarpine-induced status epilepticus, PISE). HC-067047 also markedly blocked the increase in Iba-1 and GFAP protein levels, as well as Iba-1+ and GFAP+ cells 3 d post-PISE. Finally, the increased protein levels of NLRP3, ASC and caspase-1 as well as IL-1β, TNF-α and IL-6 were markedly blocked by HC-067047. We conclude that TRPV4-induced neuronal death is mediated at least partially by enhancing the neuroinflammatory response, and this action is involved in neuronal injury following status epilepticus.

Role of NADPH oxidases in TRPV4‐mediated vasodilation of human arterioles

NADPH oxidases (NOXs) are a major source of reactive oxygen species (ROS) in the vascular system and play diverse roles in cardiovascular health and disease. We recently reported that H2O2, a diffusible ROS, regulates protein phosphorylation of endothelial transient receptor potential vanilloid 4 (TRPV4) channels and TRPV4‐mediated vasodilation in human coronary arterioles (HCA) from subjects with coronary artery disease (CAD). The endogenous source of H2O2 involved in TRPV4 regulation remains unclear. In this study, we investigated the expression of different NOX isoforms in human arterioles and their potential role in the regulation of TRPV4‐mediated arteriolar dilation.Human adipose arterioles (HAA) and HCA were dissected from fresh adipose and heart tissues obtained as discarded surgical specimens. The mRNA and protein expression of NOXs were detected by RT‐PCR and Western blotting. TRPV4 agonist‐induced vasodilation was examined in cannulated arterioles (100–200 μm internal diameter at 60 mmHg) by measuring diameter changes using video microscopy.RT‐PCR analysis revealed consistent expression of NOX2 and 4 but low expression of other isoforms (NOX1, 3, and 5) in HAA and HCA. Using endothelium‐intact and denuded arterioles (n=3 tissues/each) as well as freshly isolated endothelial cells and smooth muscle cells (n=2 tissues/each), we further found that NOX2 and 4 are more abundantly expressed in endothelial cells and at a significantly higher level than other NOX isoforms. Consistent with mRNA expression, NOX2 and 4 proteins were detected in HAA and HCA using Western blot analysis. In cannulated arterioles, TRPV4 agonist GSK1016790A (10−9–10−7 M) induced potent and concentration‐dependent dilation in HAA (maximal dilation at 10−7 M, 76±6%, logEC50 −8.0±0.1, n=6) and HCA (maximal dilation, 69±8%, logEC50 −7.9±0.1, n=5). This dilation was significantly reduced by the small molecule NOX2 inhibitor GSK2795039 (1 μM) in both HAA (22±8%, n=6, p< 0.05 vs control) and HCA (22±10%, n=5, p<0.05 vs control). Interestingly, NOX4 inhibitor GKT137831 (1 μM) also significantly reduced the dilation in HAA (maximal dilation at 10−7 M, 18±8% vs. 72±8% in control, n=6, p<0.05). Both NOX2 and 4 inhibitors did not affect sodium nitroprusside (10−10–10−4 M, an endothelium‐independent vasodilator)‐induced dilation in HAA (maximal dilation at 10−4 M, 94±2% with GSK2795039 vs. 94±2% of control, and 96±3% with GKT137831 vs. 97±3% of control, n=4 and 3, respectively).These results indicate that NOX2 and NOX4 represent two main isoforms of NOXs expressed in endothelial cells of human arterioles and play a potentially important role in TRPV4‐mediated endothelium‐dependent vasodilation. Future studies will explore whether the mechanisms of TRPV4 regulation involves NOX2/4‐derived and H2O2‐mediated channel protein phosphorylation or other signaling pathways in endothelial cells.Support or Funding InformationNational Heart, Lung and Blood Institute Grant RO1‐HL 096647This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Intravesical Activation of the Cation Channel TRPV4 Improves Bladder Function in a Rat Model for Detrusor Underactivity

BackgroundImprovement of bladder emptying by modulating afferent nerve activity is an attractive therapeutic strategy for detrusor underactivity. Transient receptor potential vanilloid 4 (TRPV4) is a sensory ion channel in urothelial cells that contribute to the detection of bladder filling. ObjectiveTo investigate the potential benefit of intravesical TRPV4 agonists in a pelvic nerve injury rat model for detrusor underactivity. Design, setting, and participantsFemale wild-type and Trpv4 knockout rats underwent sham surgery or bilateral pelvic nerve injury (bPNI). Four weeks later, rats underwent cystometry with infusion of the TRPV4 agonist GSK1016790A. Bladders were harvested for in vitro pharmacological studies, quantitative reverse polymerase chain reaction and immunohistochemistry. Outcome measurements and statistical analysisData are expressed as median ± interquartile range. Statistical comparisons were made using the Mann-Witney U test and Wilcoxon signed rank test as appropriate. Results and limitationsRats with bPNI showed a phenotype characteristic of detrusor underactivity with lower-amplitude voiding contractions, decreased voiding frequency, and increased postvoid residual. Intravesical application of GSK1016790A increased voiding frequency and reduced postvoid residual in wild-type, but not Trpv4−/−, rats. In isolated bladder strips, GSK1016790A did not induce relevant contractions, indicating that the observed improvements in bladder function are the result of increased afferent signalling through TRPV4 activation, rather than a local effect on the detrusor. The altered urinary phenotype of Trpv4−/− mice was not apparent in the Trpv4−/− rat model, suggesting species-related functional variations. Our results are limited to the preclinical setting in rodents. ConclusionsIntravesical activation of TRPV4 improves bladder dysfunction after bPNI by increasing afferent signalling. Patient summaryWe demonstrate that the sensory protein transient receptor potential vanilloid 4 (TRPV4) can be targeted to improve bladder function in animals that have iatrogenic injury to the nerves innervating the bladder. Further research is required to determine whether these results can be translated to patients with an underactive bladder.

TRPV4 is functionally expressed in oligodendrocyte precursor cells and increases their proliferation.

Oligodendrocytes, which differentiate from oligodendrocyte precursor cells (OPCs), ensheath axons with myelin, play an essential role in rapid conduction of action potentials and metabolically support neurons. Elucidation of the mechanisms underlying the proliferation, migration, differentiation, and survival of OPCs is considered indispensable for determining the causes of central nervous system diseases. However, the relationship between these functions of OPCs and their intracellular Ca2+ signaling has not been fully elucidated. Here, we investigated the function of transient receptor potential vanilloid 4 (TRPV4), a Ca2+-permeable channel that responds to hypo-osmolarity, mild temperature, mechanical stimulation, and endogenous arachidonic acid metabolites, in OPCs. Trpv4 mRNA was detected in OPCs in vivo and in primary cultured rat OPCs. In Ca2+ imaging experiments, treatment with the selective TRPV4 agonist GSK1016790A induced sustained elevation of the intracellular Ca2+ concentration in OPCs in a concentration-dependent manner, which was almost completely suppressed by co-treatment with the selective TRPV4 antagonist HC067047. Stimulation of TRPV4 by GSK1016790A augmented OPC proliferation, which was abolished by co-treatment with HC067047, the intracellular Ca2+ chelator BAPTA-AM, and the protein kinase C inhibitor bisindolylmaleimide II. By contrast, GSK1016790A did not significantly affect the migration or differentiation of OPCs. Taken together, these results suggest that TRPV4 is functionally expressed in OPCs and increases the proliferation of these cells without affecting their ability to differentiate into oligodendrocytes.

Activation of Transient Receptor Potential Vanilloid 4 Impairs the Dendritic Arborization of Newborn Neurons in the Hippocampal Dentate Gyrus through the AMPK and Akt Signaling Pathways.

Neurite growth is an important process for the adult hippocampal neurogenesis which is regulated by a specific range of the intracellular free Ca2+ concentration ([Ca2+]i). Transient receptor potential vanilloid 4 (TRPV4) is a calcium-permeable channel and activation of it causes an increase in [Ca2+]i. We recently reported that TRPV4 activation promotes the proliferation of stem cells in the adult hippocampal dentate gyrus (DG). The present study aimed to examine the effect of TRPV4 activation on the dendrite morphology of newborn neurons in the adult hippocampal DG. Here, we report that intracerebroventricular injection of the TRPV4 agonist GSK1016790A for 5 days (GSK1016790A-injected mice) reduced the number of doublecortin immunopositive (DCX+) cells and DCX+ fibers in the hippocampal DG, showing the impaired dendritic arborization of newborn neurons. The phosphorylated AMP-activated protein kinase (p-AMPK) protein level increased from 30 min to 2 h, and then decreased from 1 to 5 days after GSK1016790A injection. The phosphorylated protein kinase B (p-Akt) protein level decreased from 30 min to 5 days after GSK1016790A injection; this decrease was markedly attenuated by the AMPK antagonist compound C (CC), but not by the AMPK agonist AICAR. Moreover, the phosphorylated mammalian target of rapamycin (mTOR) and p70 ribosomal S6 kinase (p70S6k) protein levels were decreased by GSK1016790A; these changes were sensitive to 740 Y-P and CC. The phosphorylation of glycogen synthase kinase 3β (GSK3β) at Y216 was increased by GSK1016790A, and this change was accompanied by increased phosphorylation of microtubule-associated protein 2 (MAP2) and collapsin response mediator protein-2 (CRMP-2). These changes were markedly blocked by 740 Y-P and CC. Finally, GSK1016790A-induced decrease of DCX+ cells and DCX+ fibers was markedly attenuated by 740 Y-P and CC, but was unaffected by AICAR. We conclude that TRPV4 activation impairs the dendritic arborization of newborn neurons through increasing AMPK and inhibiting Akt to inhibit the mTOR-p70S6k pathway, activate GSK3β and thereby result in the inhibition of MAP2 and CRMP-2 function.