Characterization and environmental stress-induced expression profiling of transient receptor potential vanilloid (TRPV) channels in the Pacific oyster (Magallana gigas) following short-heatwave and silver exposure.

Cannabidiol is an ultra potent sensitizer for 2-APB responses in rTRPV2 and mTRPV3 channels but not in rTRPV1 through a mechanism that engages channel regions further from the cannabidiol binding site and the pore.

α-Hydroxyl acids (AHAs) from natural sources act as proton donors and topical compounds that penetrate skin and are well known in the cosmetic industry for their use in chemical peels and improvement of the skin. However, little is known about how AHAs cause exfoliation to expose fresh skin cells. Here we report that the transient receptor potential vanilloid 3 (TRPV3) channel in keratinocytes is potently activated by intracellular acidification induced by glycolic acid. Patch clamp recordings and cell death assay of both human keratinocyte HaCaT cells and TRPV3-expressing HEK-293 cells confirmed that intracellular acidification led to direct activation of TRPV3 and promoted cell death. Site-directed mutagenesis revealed that an N-terminal histidine residue, His-426, known to be involved in 2-aminoethyl diphenylborinate-mediated TRPV3 activation, is critical for sensing intracellular proton levels. Taken together, our findings suggest that intracellular protons can strongly activate TRPV3, and TRPV3-mediated proton sensing and cell death in keratinocytes may serve as a molecular basis for the cosmetic use of AHAs and their therapeutic potential in acidic pH-related skin disorders.

TRPV1 is a nociceptive, Ca2+-selective ion channel involved in the development of several painful conditions. Sensitization of TRPV1 responses by cAMP-dependent PKA crucially contributes to the development of inflammatory hyperalgesia. However, the pathways involved in potentiation of TRPV1 responses by cAMP-dependent PKA remain largely unknown. Using HEK cells stably expressing TRPV1 and the mu opioid receptor, we demonstrated that treatment with the adenylate cyclase activator forskolin significantly increased the multimeric TRPV1 species. Pretreatment with the mu opioid receptor agonist morphine reversed this increased TRPV1 multimerization. FRET analysis revealed that treatment with forskolin did not cause multimerization of pre-existing TRPV1 monomers on the plasma membrane and that intracellular pools of TRPV1 exist mostly as monomers in this model. This suggests that increased TRPV1 multimerization occurred from an intracellular store of inactive TRPV1 monomers. Treatment with forskolin also caused an increase in TRPV1 expression on the plasma membrane not resulting from increased TRPV1 expression, and this rapid TRPV1 translocation was inhibited by treatment with morphine. Thus, potentiation of TRPV1 responses by cAMP-dependent PKA involves plasma membrane insertion of functional TRPV1 multimers formed from an intracellular store of inactive TRPV1 monomers. This potentiation occurs rapidly and can be dynamically modulated by activation of the mu opioid receptor under conditions where cAMP levels are raised, such as with inflammation. Increased translocation and multimerization of TRPV1 channels provide a cellular mechanism for fine-tuning of nociceptive responses that allow for rapid modulation of TRPV1 responses independent of transcriptional changes.

Transient receptor potential channels have recently been implicated in physiological functions in a urogenital system. In this study, we investigated the role of transient receptor potential vanilloid 4 (TRPV4) channels in a stretch sensing mechanism in mouse primary urothelial cell cultures. The selective TRPV4 agonist, 4alpha-phorbol 12,13-didecanoate (4alpha-PDD) evoked Ca(2+) influx in wild-type (WT) urothelial cells, but not in TRPV4-deficient (TRPV4KO) cells. We established a cell-stretch system to investigate stretch-evoked changes in intracellular Ca(2+) concentration and ATP release. Stretch stimulation evoked intracellular Ca(2+) increases in a stretch speed- and distance-dependent manner in WT and TRPV4KO cells. In TRPV4KO urothelial cells, however, the intracellular Ca(2+) increase in response to stretch stimulation was significantly attenuated compared with that in WT cells. Stretch-evoked Ca(2+) increases in WT urothelium were partially reduced in the presence of ruthenium red, a broad TRP channel blocker, whereas that in TRPV4KO cells did not show such reduction. Potent ATP release occurred following stretch stimulation or 4alpha-PDD administration in WT urothelial cells, which was dramatically suppressed in TRPV4KO cells. Stretch-dependent ATP release was almost completely eliminated in the presence of ruthenium red or in the absence of extracellular Ca(2+). These results suggest that TRPV4 senses distension of the bladder urothelium, which is converted to an ATP signal in the micturition reflex pathway during urine storage.

Transient Receptor Potential Vanilloid-4 Has a Major Role in Visceral Hypersensitivity Symptoms

Endothelial dysfunction contributes to the pathogenesis of pulmonary arterial hypertension (PAH) by reducing arterial vasodilation. Endothelium‐dependent vasodilation is an important regulator of pulmonary vascular resistance (PVR) and overall pulmonary arterial pressure (PAP). We recently identified endothelial TRPV4 (transient receptor potential vanilloid 4) channels as important regulators of vasodilation in pulmonary arteries (PAs), and showed that unitary Ca2+ influx events through TRPV4 channels, termed TRPV4 sparklets, activate endothelial nitric oxide synthase (eNOS) to cause vasodilation. Reduced NO bioavailability is a hallmark of endothelial dysfunction in PAH. However, the mechanisms underlying reduced NO bioavailability in PAH are not well understood. We hypothesized that impairment of TRPV4 channel function attenuates NO release in PAH. PAH was induced in mice using three‐week chronic hypoxia (CH) and Sugen 5416 + CH models. Mouse right ventricular pressures (RVP) were measured as an indicator of PAP. RVP was significantly higher in TRPV4−/− mice when compared to the control mice after three weeks of CH. High‐speed Ca2+ imaging was used to analyze TRPV4 sparklets in the native endothelium from resistance‐sized, fourth‐order PAs. Vascular diameter studies were performed on isolated, pressurized (15 mmHg), small PAs. Ca2+ imaging studies were also performed in PAs obtained from control and PAH patients. In CH and Sugen 5416 + CH models, TRPV4 sparklet activity and TRPV4 channel‐dependent vasodilation were significantly lower compared to normoxic control mice. However, TRPV4 mRNA expression and immunofluorescence were not altered in both mouse models of PAH, suggesting an impairment in the regulation of channel function rather than channel expression. TRPV4 sparklet activity was also consistently lower in PAs from PAH patients, when compared to control patients. Elevated levels of superoxide radicals have been shown to contribute to the development of PAH. Superoxides can react with NO to form peroxynitrite (PN). A PN scavenger, uric acid, restored TRPV4 channel activity in CH mice. We, therefore, hypothesized that PN‐induced impairment of TRPV4‐dependent Ca2+ signaling inhibits endothelial function in PAH. Indeed, PN attenuated TRPV4 sparklet activity in a reversible manner. Our results support the concept that PN‐induced regulation of TRPV4‐dependent Ca2+ signaling may contribute to the development of PAH. Moreover, altered TRPV4 channel activity may contribute to the reduced NO bioavailability in PAH.Support or Funding Information1R56HL138496, 5R00HL121484, 17PRE33660762This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

TRPV2 Activation Induces Apoptotic Cell Death in Human T24 Bladder Cancer Cells: A Potential Therapeutic Target for Bladder Cancer

The TRPV4 (transient receptor potential vanilloid 4) ion channel, a member of the vanilloid subfamily of the transient receptor potential channels, is activated by membrane stretch, by non-noxious warm temperatures, and by a range of chemical activators. In the present study we examined the role of phosphorylation in modulating the activation of TRPV4. We expressed TRPV4 in HEK293 cells and activated the channel by cell swelling in a hypotonic solution. TRPV4 channel activation and serine phosphorylation were enhanced by exposure to the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate or by application of bradykinin, which activates PKC via a G-protein-coupled mechanism. The enhancement was inhibited by the PKC inhibitors staurosporine, bisindolylmaleimide I, and rottlerin or by mutation of the serine/threonine residues Ser(162), Thr(175), and Ser(189). The adenylate cyclase activator forskolin also enhanced activation of TRPV4, and the enhancement was antagonized by the selective cyclic AMP-dependent protein kinase (PKA) inhibitor H89 or by mutation of serine residue Ser(824). Sensitization of TRPV4 by both PKC and PKA depended on the scaffolding protein AKAP79, because channel activation and phosphorylation were enhanced by co-transfection of AKAP79 and were antagonized by removal of AKAP79 using small interfering RNA. We conclude that the serine/threonine kinases PKC and PKA enhance activation of the TRPV4 ion channel by phosphorylation at specific sites and that phosphorylation depends on assembly of PKC and PKA by AKAP79 into a signaling complex with TRPV4.

The TRPV4 channel is a calcium-permeable channel (PCa/PNa ∼ 10). Its expression has been reported in ventricular myocytes, where it is involved in several cardiac pathological mechanisms. In this study, we investigated the implication of TRPV4 in ventricular electrical activity. Left ventricular myocytes were isolated from trpv4+/+ and trpv4-/- mice. TRPV4 membrane expression and its colocalization with L-type calcium channels (Cav1.2) was confirmed using Western blot biotinylation, immunoprecipitation, and immunostaining experiments. Then, electrocardiograms (ECGs) and patch-clamp recordings showed shortened QTc and action potential (AP) duration in trpv4-/- compared with trpv4+/+ mice. Thus, TRPV4 activator GSK1016790A produced a transient and dose-dependent increase in AP duration at 90% of repolarization (APD90) in trpv4+/+ but not in trpv4-/- myocytes or when combined with TRPV4 inhibitor GSK2193874 (100 nM). Hence, GSK1016790A increased calcium transient (CaT) amplitude in trpv4+/+ but not in trpv4-/- myocytes, suggesting that TRPV4 carries an inward Ca2+ current in myocytes. Conversely, TRPV4 inhibitor GSK2193874 (100 nM) alone reduced APD90 in trpv4+/+ but not in trpv4-/- myocytes, suggesting that TRPV4 prolongs AP duration in basal condition. Finally, introducing TRPV4 parameters in a mathematical model predicted the development of an inward TRPV4 current during repolarization that increases AP duration and CaT amplitude, in accord with what was found experimentally. This study shows for the first time that TRPV4 modulates AP and QTc durations. It would be interesting to evaluate whether TRPV4 could be involved in long QT-mediated ventricular arrhythmias.NEW & NOTEWORTHY Transient receptor potential vanilloid 4 (TRPV4) is expressed at the membrane of mouse ventricular myocytes and colocalizes with non-T-tubular L-type calcium channels. Deletion of trpv4 gene in mice results in shortened QT interval on electrocardiogram and reduced action potential duration of ventricular myocytes. Pharmacological activation of TRPV4 channel leads to increased action potential duration and increased calcium transient amplitude in trpv4-/- but not in trpv4-/- ventricular myocytes. To the contrary, TRPV4 channel pharmacological inhibition reduces action potential duration in trpv4+/+ but not in trpv4-/- myocytes. Integration of TRPV4 channel in a computational model of mouse action potential shows that the channel carries an inward current contributing to slowing down action potential repolarization and to increase calcium transient amplitude, similarly to what is observed experimentally. This study highlights for the first time the involvement of TRPV4 channel in ventricular electrical activity.

Homocysteine and cytosolic GSH depletion induce apoptosis and oxidative toxicity through cytosolic calcium overload in the hippocampus of aged mice: Involvement of TRPM2 and TRPV1 channels

Contribution of TRPV channels to osmosensory transduction, thirst, and vasopressin release

Transient receptor potential vanilloid 4 (TRPV4) channel is a physiological sensor for hypo-osmolarity, mechanical deformation, and warm temperature. The channel activation leads to various cellular effects involving Ca(2+) dynamics. We found that TRPV4 interacts with beta-catenin, a crucial component linking adherens junctions and the actin cytoskeleton, thereby enhancing cell-cell junction development and formation of the tight barrier between skin keratinocytes. TRPV4-deficient mice displayed impairment of the intercellular junction-dependent barrier function in the skin. In TRPV4-deficient keratinocytes, extracellular Ca(2+)-induced actin rearrangement and stratification were delayed following significant reduction in cytosolic Ca(2+) increase and small GTPase Rho activation. TRPV4 protein located where the cell-cell junctions are formed, and the channel deficiency caused abnormal cell-cell junction structures, resulting in higher intercellular permeability in vitro. Our results suggest a novel role for TRPV4 in the development and maturation of cell-cell junctions in epithelia of the skin.

The transient receptor potential vanilloid 4 (TRPV4) channel in the mesenteric endothelium plays a pivotal role in mediating Ca2+ entry leading to membrane hyperpolarization. We previously showed that endothelial cells in the cerebral circulation express functional TRPV4 channels, which are less sensitive to the TRPV4 agonist GSK1016790A (GSK101) when compared to their peripheral counterparts. This suggests that TRPV4 channels are differentially regulated, depending on the vascular bed and/or cellular environment. Here, we employed patch clamp electrophysiology and endothelial cells freshly isolated from C57BL/6 mouse brain capillaries (cECs) and pial arteries to explore possible mechanisms underlying the disparate regulation of TRPV4 channels. While conventional whole‐cell recordings in cECs revealed a TRPV4 current comparable to that previously recorded in peripheral ECs, perforated patch clamp electrophysiology indicated reduced sensitivity to GSK101; an observation that suggested a role for an intracellular factor. Along this suggestion, inclusion of physiological levels of intracellular ATP, but not GTP or ATP‐γ‐S, rendered cECs less sensitive to TRPV4 agonists (GSK101 or 4αPDD). The latter finding proposed a potential role for ATP hydrolysis/kinase activity in the inhibitory regulation of TRPV4. Consistent with a hypothetical role for kinases, intracellular ATP was insufficient to suppress TRPV4 current in Mg2+‐free pipette solutions. Pharmacological suppression of protein kinases A, C or G failed to reverse the ATP‐mediated effect. Alternatively, inhibitors of the phosphatidylinositol 4‐kinase (PIK93, wortmannin, phenylarsine oxide and LY294002) significantly masked the inhibitory effect of ATP on TRPV4 currents; suggesting that ATP may act as a precursor for phosphoinositides that suppress TRPV4 activity. In line with this hypothesis, intracellular application of exogenous PtdIns(4,5)P2 (diC8‐PIP2), but not PtdIns(4)P, suppressed GSK101‐induced currents. Accumulation of endogenous PtdIns(4,5)P2 (U73122) similarly inhibited TRPV4 current. Furthermore, scavenging PtdIns(4,5)P2 using poly‐L‐lysine reversed ATP‐induced inhibition. This regulatory mechanism is not delimited to capillaries; importantly, a similar inhibitory effect of PtdIns(4,5)P2 or ATP was evident in arterial ECs isolated from pial cerebral arteries. In conclusion, TRPV4 channel activity is regulated in the cerebral circulation due to, at least in part, modulation by the phosphoinositide PtdIns(4,5)P2. The inhibitory role of PtdIns(4,5)P2 represents a mechanism underlying the tighter control over TRPV4 channel activity in cerebral arteries and capillaries. It may further allude to a possible crosstalk between G‐protein coupled receptors, that dynamically alter cellular PtdIns(4,5)P2 levels, and the TRPV4 channel. These regulatory cascades may have broad implications for TRPV4 channel control in diverse cell types beyond the endothelium.Support or Funding InformationSupported by: Totman Medical Research Trust, Fondation Leducq, and NIH (R01 HL121706, PO1 HL‐095488, R37‐DK‐053832).

Transient Receptor Potential Vanilloid 4 channels participate in mouse ventricular action potentia

Sodium salicylate (SA), a cyclooxygenase inhibitor, has been shown to increase insulin sensitivity and to suppress inflammation in obese patients and animal models. Transient receptor potential vanilloid 1 (TRPV1) is a nonselective cation channel expressed in afferent nerve fibers. Cyclooxygenase-derived prostaglandins are involved in the activation and sensitization of TRPV1. This study tested whether the metabolic and renal effects of SA were mediated by the TRPV1 channel. Wild-type (WT) and TRPV1−/− mice were fed a Western diet (WD) for 4 months and received SA infusion (120mg/kg/day) or vehicle for the last 4 weeks of WD feeding. SA treatment significantly increased blood pressure in WD-fed TRPV1−/− mice (p < 0.05) but not in WD-fed WT mice. Similarly, SA impaired renal blood flow in TRPV1−/− mice (p < 0.05) but not in WT mice. SA improved insulin and glucose tolerance in both WT and TRPV1−/− mice on WD (all p < 0.05). In addition, SA reduced renal p65 and urinary prostaglandin E2, prostaglandin F1α, and interleukin-6 in both WT and TRPV1−/− mice (all p < 0.05). SA decreased urine noradrenaline levels, increased afferent renal nerve activity, and improved baroreflex sensitivity in WT mice (all p < 0.05) but not in TRPV1−/− mice. Importantly, SA increased serum creatinine and urine kidney injury molecule-1 levels and decreased the glomerular filtration rate in obese WT mice (all p < 0.05), and these detrimental effects were significantly exacerbated in obese TRPV1−/− mice (all p < 0.05). Lastly, SA treatment increased urine albumin levels in TRPV1−/− mice (p < 0.05) but not in WT mice. Taken together, SA-elicited metabolic benefits and anti-inflammatory effects are independent of TRPV1, while SA-induced sympathetic suppression is dependent on TRPV1 channels. SA-induced renal dysfunction is dependent on intact TRPV1 channels. These findings suggest that SA needs to be cautiously used in patients with obesity or diabetes, as SA-induced renal dysfunction may be exacerbated due to impaired TRPV1 in obese and diabetic patients.