TRPV4-mediated Ca2 Research Articles

Increased TRPV4 Channel Expression Enhances and Impairs Blood Vessel Function in Hypertension.

Endothelial cell TRPV4 (transient receptor potential vanilloid 4) channels provide a control point that is pivotal in regulating blood vessel diameter by mediating the Ca2+-dependent release of endothelial-derived vasoactive factors. In hypertension, TRPV4-mediated control of vascular function is disrupted, but the underlying mechanisms and precise physiological consequences remain controversial. Here, using a comprehensive array of methodologies, endothelial TRPV4 channel function was examined in intact mesenteric resistance arteries from normotensive Wistar-Kyoto and spontaneously hypertensive rats. Our results show there is a notable shift in vascular reactivity in hypertension characterized by enhanced endothelium-dependent vasodilation at low levels of TRPV4 channel activation. However, at higher levels of TRPV4 activity, this vasodilatory response is reversed, contributing to the aberrant vascular tone observed in hypertension. The change in response, from dilation to constriction, was accompanied by a shift in intracellular Ca2+ signaling modalities arising from TRPV4 activity. Oscillatory TRPV4-evoked IP3 (inositol triphosphate)-mediated Ca2+ release, which underlies dilation, decreased, while the contraction inducing sustained Ca2+ rise, arising from TRPV4-mediated Ca2+ influx, increased. Our findings also reveal that while the sensitivity of endothelial cell TRPV4 to activation was unchanged, expression of the channel is upregulated and IP3 receptors are downregulated in hypertension. These data highlight the intricate interplay between endothelial TRPV4 channel expression, intracellular Ca2+ signaling dynamics, and vascular reactivity. Moreover, the data support a new unifying hypothesis for the vascular impairment that accompanies hypertension. Specifically, endothelial cell TRPV4 channels play a dual role in modulating blood vessel function in hypertension.

Calcium-activated Potassium Channels as Amplifiers of TRPV4-mediated Pulmonary Edema Formation in Male Mice.

As a mechanosensitive cation channel and key regulator of vascular barrier function, endothelial transient receptor potential vanilloid type 4 (TRPV4) contributes critically to ventilator-induced lung injury and edema formation. Ca2+ influx via TRPV4 can activate Ca2+-activated potassium (KCa) channels, categorized into small (SK1-3), intermediate (IK1), and big (BK) KCa, which may in turn amplify Ca2+ influx by increasing the electrochemical Ca2+ gradient and thus promote lung injury. The authors therefore hypothesized that endothelial KCa channels may contribute to the progression of TRPV4-mediated ventilator-induced lung injury. Male C57Bl/6J mice were ventilated for 2 h with low or high tidal volumes in the presence or absence of the nonselective KCa antagonists apamin and charybdotoxin or the selective IK1 antagonist TRAM34. Lung injury was similarly assessed in overventilated, endothelial-specific TRPV4-deficient mice or TRAM34-treated C57Bl/6J mice challenged with intratracheal acid installation. Changes in intracellular calcium Ca2+ concentration ([Ca2+]i) were monitored by real-time imaging in isolated-perfused lungs in response to airway pressure elevation or in human pulmonary microvascular endothelial cells in response to TRPV4 activation with or without inhibition of KCa channels. Analogously, changes in intracellular potassium concentration ([K+]i) and membrane potential were imaged in vitro. Endothelial TRPV4 deficiency or inhibition of KCa channels, and most prominently inhibition of IK1 by TRAM34, attenuated ventilator-induced lung injury as demonstrated by reduced lung edema, protein leak, and quantitative lung histology. All KCa antagonists reduced the [Ca2+]i response to mechanical stimulation or direct TRPV4 activation in isolated lungs. TRAM34 and charybdotoxin yet not apamin prevented TRPV4-induced potassium efflux and membrane hyperpolarization in human pulmonary microvascular endothelial cells. TRAM34 also attenuated the TRPV4 agonist-induced Ca2+ influx in vitro and reduced acid-induced lung injury in vivo. KCa channels, specifically IK1, act as amplifiers of TRPV4-mediated Ca2+ influx and establish a detrimental feedback that promotes barrier failure and drives the progression of ventilator-induced lung injury.

TMAO Impairs Mouse Aortic Vasodilation by Inhibiting TRPV4 Channels in Endothelial Cells.

Trimethylamine oxide (TMAO) is an intestinal flora metabolite associated with risk of cardiovascular diseases. Transient receptor potential vanilloid 4 (TRPV4) is a Ca2+-permeable ion channel that is essential for vasodilation and endothelial function. Currently, there are few studies on the effect of TMAO on TRPV4 channels. In the present study, Ca2+ imaging of vascular tissue showed that TMAO inhibited TRPV4-mediated Ca2+ influx into aortic endothelial cells in a dose-dependent manner. Furthermore, a whole-cell patch clamp assay showed that TMAO blocked TRPV4-mediated cation currents. Notably, results of aortic vascular tension measurement showed that TMAO impaired endothelium-dependent vasodilation in mouse aortic vessels through the TRPV4-NO pathway. Our results indicated that TMAO inhibited Ca2+ entry in endothelial cells and impaired vasodilation through the TRPV4-NO pathway in mice. These results provide scientific evidence for novel pathogenic mechanisms underlying the role of TMAO in cardiovascular disease.

Activation of KCa channels downstream of TRPV4 amplify pulmonary edema formation

Study objective: In patients with acute respiratory failure (ARDS), mechanical ventilation is the only life-saving treatment. However, mechanical ventilation also can cause ventilator-induced lung injury (VILI) due to the excessive biomechanical forces acting on the distal lung and especially the microvascular endothelium, which in turn further exacerbate endothelial barrier failure thus initiating a vicious cycle leading to irreversible lung damage. The endothelial mechanosensitive transient receptor potential vanilloid 4 (TRPV4) is directly activated by mechanical force and leads to Ca2+ influx, which can activate Ca2+-activated K+ (KCa) channels, categorized into small (SK1-3), intermediate (IK1), and big (BK) KCa. We hypothesized that KCa channels acting downstream of TRPV4 may in turn amplify Ca2+ influx by increasing the electrochemical Ca2+ gradient and thus, promote stretch-induced barrier failure and aggravate lung injury in a positive feedback loop. Methods: Male C57Bl/6J wild-type mice were ventilated for 2 h with low or high tidal volumes in the presence or absence of the non-selective KCa antagonists apamin, charybdotoxin, or the selective IK1 antagonist TRAM34. In a second model, male wild-type mice were challenged with intratracheal HCl instillation (pH 1.5) and ventilated with a low tidal volume for 2h with or without TRAM34. Changes in endothelial Ca2+ concentration ([Ca2+]i) were monitored by real-time imaging of Fura-2 AM in isolated-perfused lungs in response to airway pressure elevation or in human pulmonary microvascular endothelial cells (HPMECs) in response to TRPV4 activation with or without inhibition of KCa channels. Analogously, changes in intracellular potassium concentration ([K+]i) and membrane potential ( Vm) were imaged in vitro. Results: In our established experimental lung injury models, pharmacological inhibition of KCa channels, in particular IK1 attenuated characteristic hallmarks of lung injury in response to high tidal volume ventilation or HCl instillation. Consistent with these findings, inhibition of IK1 channels in the ex vivo perfused lung reduced the sustained [Ca2+]i response to airway pressure elevation over time. In HPMECs, TRPV4-mediated Ca2+ influx induced a pronounced K+ efflux, which in turn caused membrane hyperpolarization. These effects could be prevented by different KCa inhibitors, with IK1 inhibition by TRAM34 showing the most pronounced protective effect. Antagonizing KCa channels as downstream mediators of TRPV4 also reduced endothelial [Ca2+]i transients in response to TRPV4 activation in vitro, indicating that KCa channels, and specifically IK1 serve as amplifiers by increasing the electrochemical gradient for TRPV4-mediated Ca2+ influx. Conclusion: KCa channels, specifically IK1 amplify TRPV4-mediated Ca2+ influx in lung endothelial cells and as such, establish a detrimental feedback that promotes barrier failure and drives the progression of VILI in overventilated lungs. WMK is supported by the German Research Foundation (DFG) (SFB-TR84: subprojects A02 & C09, SFB-1449 subproject B01, SFB 1470 subproject A04, KU1218/9-1, KU1218/11-1, and KU1218/12-1), the Berlin Institute of Health (BIH), the German Federal Ministry of Education and Research (BMBF) in the framework of the projects PROVID (01KI20160A) and SYMPATH (01ZX1906A), and the German Centre for Cardiovascular Research (DZHK). LM is supported by the German Centre for Cardiovascular Research (DZHK) (81X3100216). This is the full abstract presented at the American Physiology Summit 2024 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-dependent Ca2+ influx determines cholesterol dynamics at the plasma membrane

The activities of the transient receptor potential vanilloid 4 (TRPV4), a Ca2+-permeable nonselective cation channel, are controlled by its surrounding membrane lipids (e.g., cholesterol, phosphoinositides). The transmembrane region of TRPV4 contains a cholesterol recognition amino acid consensus (CRAC) motif and its inverted (CARC) motif located in the plasmalemmal cytosolic leaflet. TRPV4 localizes in caveolae, a bulb-shaped cholesterol-rich domain at the plasma membrane. Here, we visualized the spatiotemporal interactions between TRPV4 and cholesterol at the plasma membrane in living cells by dual-color single-molecule imaging using total internal reflection fluorescence microscopy. To this aim, we labeled cholesterol at the cytosolic leaflets of the plasma membrane using a cholesterol biosensor, D4H. Our single-molecule tracking analysis showed that the TRPV4 molecules colocalize with D4H-accessible cholesterol molecules mainly in the low fluidity membrane domains in which both molecules are highly clustered. Colocalization of TRPV4 and D4H-accessible cholesterol was observed both inside and outside of caveolae. Agonist-evoked TRPV4 activation remarkably decreased colocalization probability and association rate between TRPV4 and D4H-accessible cholesterol molecules. Interestingly, upon TRPV4 activation, the particle density of D4H-accessible cholesterol molecules was decreased and the D4H-accessible cholesterol molecules in the fast-diffusing state were increased at the plasma membrane. The introduction of skeletal dysplasia-associated R616Q mutation into the CRAC/CARC motif of TRPV4, which reduced the interaction with cholesterol clusters, could not alter the D4H-accessible cholesterol dynamics. Mechanistically, TRPV4-mediated Ca2+ influx and the C-terminal calmodulin-binding site of TRPV4 are essential for modulating the plasmalemmal D4H-accessible cholesterol dynamics. We propose that TRPV4 remodels its surrounding plasmalemmal environment by manipulating cholesterol dynamics through Ca2+ influx.

Identification of natural antimicrobial peptides mimetic to inhibit Ca2+ influx DDX3X activity for blocking dengue viral infectivity.

Viruses are microscopic biological entities that can quickly invade and multiply in a living organism. Each year, over 36,000 people die and nearly 400 million are infected with the dengue virus (DENV). Despite dengue being an endemic disease, no targeted and effective antiviral peptide resource is available against the dengue species. Antiviral peptides (AVPs) have shown tremendous ability to fight against different viruses. Accelerating antiviral drug discovery is crucial, particularly for RNA viruses. DDX3X, a vital cell component, supports viral translation and interacts with TRPV4, regulating viral RNA metabolism and infectivity. Its diverse signaling pathway makes it a potential therapeutic target. Our study focuses on inhibiting viral RNA translation by blocking the activity of the target gene and the TRPV4-mediated Ca2+ cation channel. Six major proteins from camel milk were first extracted and split with the enzyme pepsin. The antiviral properties were then analyzed using online bioinformatics programs, including AVPpred, Meta-iAVP, AMPfun, and ENNAVIA. The stability of the complex was assessed using MD simulation, MM/GBSA, and principal component analysis. Cytotoxicity evaluations were conducted using COPid and ToxinPred. The top ten AVPs, determined by optimal scores, were selected and saved for docking studies with the GalaxyPepDock tools. Bioinformatics analyses revealed that the peptides had very short hydrogen bond distances (1.8 to 3.6Å) near the active site of the target protein. Approximately 76% of the peptide residues were 5-11 amino acids long. Additionally, the identified peptide candidates exhibited desirable properties for potential therapeutic agents, including a net positive charge, moderate toxicity, hydrophilicity, and selectivity. In conclusion, this computational study provides promising insights for discovering peptide-based therapeutic agents against DENV.

Angiotensin II induces endothelial dysfunction and vascular remodeling by downregulating TRPV4 channels

Angiotensin II (Ang II) is a potent vasoconstrictor of vascular smooth muscle cells (VSMC) and is implicated in hypertension, but it's role in the regulation of endothelial function is not well known. We and others have previously shown that mechanically activated ion channel, Transient Receptor Potential Vanilloid 4 (TRPV4) mediates flow- and/or receptor-dependent vasodilation via nitric oxide (NO) production in endothelial cells. Ang II was demonstrated to crosstalk with TRPV4 via angiotensin 1 receptor (AT1R) and β-arrestin signaling in epithelial and immortalized cells, however, the role of this crosstalk in endothelial cell function is not fully explored. Ang II treatment significantly downregulated TRPV4 protein expression and TRPV4-mediated Ca2+ influx in human EC without altering TRPV4 mRNA levels. Further, TRPV4-induced eNOS phosphorylation and NO production were significantly reduced in Ang II-treated human EC. Importantly, Ang II infusion in mice revealed that, TRPV4/p-eNOS expression and colocalization was reduced in endothelium in vivo. Finally, Ang II infusion induced vascular remodeling as evidenced by decreased lumen to wall ratio in resistant mesenteric arteries. These findings suggest that Ang II induces endothelial dysfunction and vascular remodeling via downregulation of TRPV4/eNOS pathway and may contribute to hypertension, independent of or in addition to its effect on vascular smooth muscle contraction.

TRPV4-mediated Ca2+ deregulation causes mitochondrial dysfunction via the AKT/α-synuclein pathway in dopaminergic neurons.

Mutations in the gene encoding the transient receptor potential vanilloid member 4 (TRPV4), a Ca2+ permeable nonselective cation channel, cause TRPV4-related disorders. TRPV4 is widely expressed in the brain; however, the pathogenesis underlying TRPV4-mediated Ca2+ deregulation in neurodevelopment remains unresolved and an effective therapeutic strategy remains to be established. These issues were addressed by isolating mutant dental pulp stem cells from a tooth donated by a child diagnosed with metatropic dysplasia with neurodevelopmental comorbidities caused by a gain-of-function TRPV4 mutation, c.1855C > T (p.L619F). The mutation was repaired using CRISPR/Cas9 to generate corrected isogenic stem cells. These stem cells were differentiated into dopaminergic neurons and the pharmacological effects of folic acid were examined. In mutant neurons, constitutively elevated cytosolic Ca2+ augmented AKT-mediated α-synuclein (α-syn) induction, resulting in mitochondrial Ca2+ accumulation and dysfunction. The TRPV4 antagonist, AKT inhibitor, or α-syn knockdown, normalizes the mitochondrial Ca2+ levels in mutant neurons, suggesting the importance of mutant TRPV4/Ca2+/AKT-induced α-syn in mitochondrial Ca2+ accumulation. Folic acid was effective in normalizing mitochondrial Ca2+ levels via the transcriptional repression of α-syn and improving mitochondrial reactive oxygen species levels, adenosine triphosphate synthesis, and neurite outgrowth of mutant neurons. This study provides new insights into the neuropathological mechanisms underlying TRPV4-related disorders and related therapeutic strategies.

Vasodilators mobilize SK3 channels in endothelial cells to produce arterial relaxation

Endothelial cells (ECs) line the lumen of all blood vessels and regulate functions, including contractility. Physiological stimuli, such as acetylcholine (ACh) and intravascular flow, activate transient receptor potential vanilloid 4 (TRPV4) channels, which stimulate small (SK3)- and intermediate (IK)-conductance Ca2+-activated potassium channels in ECs to produce vasodilation. Whether physiological vasodilators also modulate the surface abundance of these ion channels in ECs to elicit functional responses is unclear. Here, we show that ACh and intravascular flow stimulate rapid anterograde trafficking of an intracellular pool of SK3 channels in ECs of resistance-size arteries, which increases surface SK3 protein more than two-fold. In contrast, ACh and flow do not alter the surface abundance of IK or TRPV4 channels. ACh triggers SK3 channel trafficking by activating TRPV4-mediated Ca2+ influx, which stimulates Rab11A, a Rab GTPase associated with recycling endosomes. Superresolution microscopy data demonstrate that SK3 trafficking specifically increases the size of surface SK3 clusters which overlap with TRPV4 clusters. We also show that Rab11A-dependent trafficking of SK3 channels is an essential contributor to vasodilator-induced SK current activation in ECs and vasorelaxation. In summary, our data demonstrate that vasodilators activate Rab11A, which rapidly delivers an intracellular pool of SK3 channels to the vicinity of surface TRPV4 channels in ECs. This trafficking mechanism increases surface SK3 cluster size, elevates SK3 current density, and produces vasodilation. These data also demonstrate that SK3 and IK channels are differentially regulated by trafficking-dependent and -independent signaling mechanisms in endothelial cells.

Progesterone interacts with the mutational hot-spot of TRPV4 and acts as a ligand relevant for fast Ca2+-signalling

Steroids are also known to induce immediate physiological and cellular response which occurs within minutes to seconds, or even faster. Such non-genomic actions of steroids are rapid and are proposed to be mediated by different ion channels. Transient receptor potential vanilloid sub-type 4 (TRPV4), is a non-specific polymodal ion channel which is involved in several physiological and cellular processes. In this work, we explored the possibilities of Progesterone (P4) as an endogenous ligand for TRPV4. We demonstrate that P4 docks as well as physically interacts with the TM4-loop-TM5 region of TRPV4, a region which is a mutational hotspot for different diseases. Live cell imaging experiments with a genetically encoded Ca2+-sensor suggests that P4 causes quick influx of Ca2+ specifically in the TRPV4 expressing cells, which can be partially blocked by TRPV4-specific inhibitor, suggesting that P4 can act as a ligand for TRPV4. Such P4-mediated Ca2+-influx is altered in cells expressing disease causing TRPV4 mutants, namely in L596P, R616Q, and also in embryonic lethal mutant L618P. P4 dampens, both in terms of “extent” as well as the “pattern” of the Ca2+-influx by other stimulus too in cells expressing TRPV4-Wt, suggesting that P4 crosstalk with the TRPV4-mediated Ca2+-signalling, both in quick and long-term manner. We propose that P4 crosstalk with TRPV4 might be relevant for both acute and chronic pain as well as for other health-related functions.

TRPV4 regulates mitochondrial Ca2+-status and physiology in primary murine T cells based on their immunological state

T cell activation process is critically affected by temperature and intracellular Ca2+-signalling. Yet, the nature and the key molecules involved in such complex Ca2+-signalling is poorly understood. It is mostly assumed that ion channels present in the plasma membrane primarily regulate the cytosolic Ca2+-levels exclusively. TRPV4 is a non-selective Ca2+ channel which can be activated at physiological temperature. TRPV4 is involved in several physiological, pathophysiological process as well as different forms of pain. Here we demonstrate that TRPV4 is endogenously expressed in T cell and is present in the mitochondria of T cells. TRPV4 activation increases mitochondrial Ca2+-levels, and alters mitochondrial temperature as well as specific metabolisms. The TRPV4-dependent increment in the mitochondrial Ca2+ is context-dependent and not just passively due to the increment in the cytosolic Ca2+. Our work also indicates that mitochondrial Ca2+-level correlates positively with a series of essential factors, such as mitochondrial membrane potential, mitochondrial ATP production and negatively correlates with certain factors such as mitochondrial temperature. We propose that TRPV4-mediated mitochondrial Ca2+-signalling and other metabolisms has implications in the immune activation process including immune synapse formation. Our data also endorse the re-evaluation of Ca2+-signalling in T cell, especially in the light of mitochondrial Ca2+-buffering and in higher body temperature, such as in case of fever. Presence of TRPV4 in the mitochondria of T cell is relevant for proper and optimum immune response and may provide evolutionary adaptive benefit. These findings may also have broad implications in different pathophysiological process, neuro-immune cross-talks, and channelopathies involving TRPV4.

Cell Confluence Modulates TRPV4 Channel Activity in Response to Hypoxia.

Transient receptor potential vanilloid 4 (TRPV4) is a polymodal Ca2+-permeable channel involved in various hypoxia-sensitive pathophysiological phenomena. Different tools are available to study channel activity, requiring cells to be cultured at specific optimal densities. In the present study, we examined if cell density may influence the effect of hypoxia on TRPV4 activity. Transiently TRPV4-transfected HEK293T cells were seeded at low or high densities corresponding to non-confluent or confluent cells, respectively, on the day of experiments, and cultured under in vitro normoxia or hypoxia. TRPV4-mediated cytosolic Ca2+ responses, single-channel currents, and Ca2+ influx through the channel were measured using Ca2+ imaging/microspectrofluorimetric assay, patch-clamp, and Bioluminescence Resonance Energy Transfer (BRET), respectively. TRPV4 plasma membrane translocation was studied using confocal microscopy, biotinylation of cell surface proteins, and BRET. Our results show that hypoxia exposure has a differential effect on TRPV4 activation depending on cell confluence. At low confluence levels, TRPV4 response is increased in hypoxia, whereas at high confluence levels, TRPV4 response is strongly inhibited, due to channel internalization. Thus, cell density appears to be a crucial parameter for TRPV4 channel activity.

NADPH oxidase 4 contributes to TRPV4-mediated endothelium-dependent vasodilation in human arterioles by regulating protein phosphorylation of TRPV4 channels.

Impaired endothelium-dependent vasodilation has been suggested to be a key component of coronary microvascular dysfunction (CMD). A better understanding of endothelial pathways involved in vasodilation in human arterioles may provide new insight into the mechanisms of CMD. The goal of this study is to investigate the role of TRPV4, NOX4, and their interaction in human arterioles and examine the underlying mechanisms. Arterioles were freshly isolated from adipose and heart tissues obtained from 71 patients without coronary artery disease, and vascular reactivity was studied by videomicroscopy. In human adipose arterioles (HAA), ACh-induced dilation was significantly reduced by TRPV4 inhibitor HC067047 and by NOX 1/4 inhibitor GKT137831, but GKT137831 did not further affect the dilation in the presence of TRPV4 inhibitors. GKT137831 also inhibited TRPV4 agonist GSK1016790A-induced dilation in HAA and human coronary arterioles (HCA). NOX4 transcripts and proteins were detected in endothelial cells of HAA and HCA. Using fura-2 imaging, GKT137831 significantly reduced GSK1016790A-induced Ca2+ influx in the primary culture of endothelial cells and TRPV4-WT-overexpressing human coronary artery endothelial cells (HCAEC). However, GKT137831 did not affect TRPV4-mediated Ca2+ influx in non-phosphorylatable TRPV4-S823A/S824A-overexpressing HCAEC. In addition, treatment of HCAEC with GKT137831 decreased the phosphorylation level of Ser824 in TRPV4. Finally, proximity ligation assay (PLA) revealed co-localization of NOX4 and TRPV4 proteins. In conclusion, both TRPV4 and NOX4 contribute to ACh-induced dilation in human arterioles from patients without coronary artery disease. NOX4 increases TRPV4 phosphorylation in endothelial cells, which in turn enhances TRPV4-mediated Ca2+ entry and subsequent endothelium-dependent dilation in human arterioles.

Endothelial Ion Channels and Cell-Cell Communication in the Microcirculation

Endothelial cells in resistance arteries, arterioles, and capillaries express a diverse array of ion channels that contribute to Cell-Cell communication in the microcirculation. Endothelial cells are tightly electrically coupled to their neighboring endothelial cells by gap junctions allowing ion channel-induced changes in membrane potential to be conducted for considerable distances along the endothelial cell tube that lines arterioles and forms capillaries. In addition, endothelial cells may be electrically coupled to overlying smooth muscle cells in arterioles and to pericytes in capillaries via heterocellular gap junctions allowing electrical signals generated by endothelial cell ion channels to be transmitted to overlying mural cells to affect smooth muscle or pericyte contractile activity. Arteriolar endothelial cells express inositol 1,4,5 trisphosphate receptors (IP3Rs) and transient receptor vanilloid family member 4 (TRPV4) channels that contribute to agonist-induced endothelial Ca2+ signals. These Ca2+ signals then activate intermediate and small conductance Ca2+-activated K+ (IKCa and SKCa) channels causing vasodilator-induced endothelial hyperpolarization. This hyperpolarization can be conducted along the endothelium via homocellular gap junctions and transmitted to overlying smooth muscle cells through heterocellular gap junctions to control the activity of voltage-gated Ca2+ channels and smooth muscle or pericyte contraction. The IKCa- and SKCa-induced hyperpolarization may be amplified by activation of inward rectifier K+ (KIR) channels. Endothelial cell IP3R- and TRPV4-mediated Ca2+ signals also control the production of endothelial cell vasodilator autacoids, such as NO, PGI2, and epoxides of arachidonic acid contributing to control of overlying vascular smooth muscle contractile activity. Cerebral capillary endothelial cells lack IKCa and SKCa but express KIR channels, IP3R, TRPV4, and other Ca2+ permeable channels allowing capillary-to-arteriole signaling via hyperpolarization and Ca2+. This allows parenchymal cell signals to be detected in capillaries and signaled to upstream arterioles to control blood flow to capillaries by active parenchymal cells. Thus, endothelial cell ion channels importantly participate in several forms of Cell-Cell communication in the microcirculation that contribute to microcirculatory function and homeostasis.

A novel antagonist of TRPM2 and TRPV4 channels: Carvacrol.

The overload cytosolic free Ca2+ (cCa2+) influx-mediated excessive generation of oxidative stress in the pathophysiological conditions induces neuronal and cellular injury via the activation of cation channels. TRPM2 and TRPV4 channels are activated by oxidative stress, and their specific antagonists have not been discovered yet. The antioxidant and anti-Covid-19 properties of carvacrol (CARV) were recently reported. Hence, I suspected possible antagonist properties of CARV against oxidative stress (OS)/ADP-ribose (ADPR)-induced TRPM2 and GSK1016790A (GSK)-mediated TRPV4 activations in neuronal and kidney cells. I investigated the antagonist role of CARV on the activations of TRPM2 and TRPV4 in SH-SY5Y neuronal, BV-2 microglial, and HEK293 cells. The OS/ADPR and GSK in the cells caused to increase of TRPM2/TRPV4 current densities and overload cytosolic free Ca2+ (cCa2+) influx with an increase of mitochondrial membrane potential, cytosolic (cROS), and mitochondrial (mROS) ROS. The changes were not observed in the absence of TRPM2 and TRPV4 or the presence of Ca2+ free extracellular buffer and PARP-1 inhibitors (PJ34 and DPQ). When OS-induced TRPM2 and GSK-induced TRPV4 activations were inhibited by the treatment of CARV, the increase of cROS, mROS, lipid peroxidation, apoptosis, cell death, cCa2+ concentration, caspase -3, and caspase -9 levels were restored via upregulation of glutathione and glutathione peroxidase. In conclusion, the treatment of CARV modulated the TRPM2 and TRPV4-mediated overload Ca2+ influx and may provide an avenue for protecting TRPM2 and TRPV4-mediated neurodegenerative diseases associated with the increase of mROS and cCa2+.Graphical abstractThe possible TRPM2 and TRPV4 blocker action of carvacrol (CARV) via the modulation oxidative stress and apoptosis in the SH-SY5Y neuronal cells. TRPM2 is activated by DNA damage-induced (via PARP-1 activation) ADP-ribose (ADPR) and reactive oxygen species (ROS) (H2O2), although it is inhibited by nonspecific inhibitors (ACA and 2-APB). TRPV4 is activated by the treatments of GSK1016790A (GSK), although it is inhibited by a nonspecific inhibitor (ruthenium red, RuRe). The treatment of GSK induces excessive generation of ROS. The accumulation of free cytosolic Ca2+ (cCa2+) via the activations of TRPM2 and TRPV4 in the mitochondria causes the increase of mitochondrial membrane depolarization (ΔΨm). In turn, the increase of ΔΨm causes the excessive generation of ROS. The TRPM2 and TRPV4-induced the excessive generations of ROS result in the increase of apoptosis and cell death via the activations of caspase -3 (Casp-3) and caspase -9 (Casp-9) in the neuronal cells, although their oxidant actions decrease the glutathione (GSH) and glutathione peroxidase (GSHPx) levels. The oxidant and apoptotic adverse actions of TRPM2 and TRPV4 are modulated by the treatment of CARV Supplementary InformationThe online version contains supplementary material available at 10.1007/s11011-021-00887-1.

The potential role of mechanosensitive ion channels in substrate stiffness-regulated Ca2+ response in chondrocytes

ABSTRACT Purpose The stiffness of the pericellular matrix (PCM) decreases in the most common degenerative joint disease, osteoarthritis (OA). This study was undertaken to explore the potential functional role of transient receptor potential vanilloid 4 (TRPV4), Piezo1, and Piezo2 in transducing different PCM stiffness in chondrocytes. Methods and Results Polydimethylsiloxane (PDMS) substrates with different stiffness (designated 197 kPa, 78 kPa, 54 kPa, or 2 kPa, respectively) were first prepared to simulate the decrease in stiffness of the PCM that chondrocytes encounter in osteoarthritic cartilage. Next, the TRPV4-, Piezo1-, or Piezo2-knockdown primary chondrocytes (designated TRPV4-KD, Piezo1-KD, or Piezo2-KD cells) were seeded onto these different PDMS substrates. Then, using a Ca2+-imaging system, substrate stiffness-regulated intracellular Ca2+ influx ([Ca2+] i ) in chondrocytes was examined to investigate the role of TRPV4, Piezo1, and Piezo2 in Ca2+ signaling in response to different stiffness. Results showed that the characteristics of intracellular [Ca2+] i in chondrocytes regulated by PDMS substrate exhibited stiffness-dependent differences. Additionally, stiffness-evoked [Ca2+] i changes were suppressed in TRPV4-KD, Piezo1-KD, or Piezo2-KD cells compared with control siRNA-treated cells, implying that any channel is fundamental for Ca2+ signaling induced by substrate stiffness. Furthermore, TRPV4-mediated Ca2+ signaling played a central role in the response of chondrocytes to 197 kPa and 78 kPa substrate, while Piezo1/2-mediated Ca2+ signaling played a central role in the response of chondrocytes to 54 kPa and 2 kPa substrate. Conclusions Collectively, these findings indicate that chondrocytes might perceive and distinguish the different PCM stiffness by using different mechanosensitive ion channels.

Mitochondria regulate TRPV4-mediated release of ATP.

Ca2+ influx via TRPV4 channels triggers Ca2+ release from the IP3 -sensitive internal store to generate repetitive oscillations. Although mitochondria are acknowledged regulators of IP3 -mediated Ca2+ release, how TRPV4-mediated Ca2+ signals are regulated by mitochondria is unknown. We show that depolarised mitochondria switch TRPV4 signalling from relying on Ca2+ -induced Ca2+ release at IP3 receptors to being independent of Ca2+ influx and instead mediated by ATP release via pannexins. TRPV4-evoked Ca2+ signals were individually examined in hundreds of cells in the endothelium of rat mesenteric resistance arteries using the indicator Cal520. TRPV4 activation with GSK1016790A (GSK) generated repetitive Ca2+ oscillations that required Ca2+ influx. However, when the mitochondrial membrane potential was depolarised, by the uncoupler CCCP or complex I inhibitor rotenone, TRPV4 activation generated large propagating, multicellular, Ca2+ waves in the absence of external Ca2+ . The ATP synthase inhibitor oligomycin did not potentiate TRPV4-mediated Ca2+ signals. GSK-evoked Ca2+ waves, when mitochondria were depolarised, were blocked by the TRPV4 channel blocker HC067047, the SERCA inhibitor cyclopiazonic acid, the PLC blocker U73122 and the inositol trisphosphate receptor blocker caffeine. The Ca2+ waves were also inhibited by the extracellular ATP blockers suramin and apyrase and the pannexin blocker probenecid. These results highlight a previously unknown role of mitochondria in shaping TRPV4-mediated Ca2+ signalling by facilitating ATP release. When mitochondria are depolarised, TRPV4-mediated release of ATP via pannexin channels activates plasma membrane purinergic receptors to trigger IP3 -evoked Ca2+ release.

The involvement of TRPV4 on the hypoxia-induced oxidative neurotoxicity and apoptosis in a neuronal cell line: Protective role of melatonin

The hypoxia (HYPX)-mediated excessive generation of mitochondrial free reactive oxygen species (mROS) and the overload Ca2+ influx via the inhibition of TRPV4 are controlled by the treatment of antioxidants. However, the molecular mechanisms underlying melatonin (MLT)'s neuroprotection remains elusive. We investigated the role of MLT via modulation of TRPV4 on oxidative neurodegeneration and death in SH-SY5Y neuronal cells. The SH-SY5Y cells were divided into five groups as follows: control, MLT (1 mM for 2 h), HYPX (200 μM CoCl2 for 24 h), HYPX + MLT, and HYPX + TRPV4 blockers (ruthenium red-1 μM for 30 min). The HYPX caused to the increase of TRPV4 current density and overload Ca2+ influx with an increase of mitochondrial membrane potential and mROS generation. The changes were not observed in the absence of TRPV4. When HYPX exposure and TRPV4 agonist (GSK1016790A)-induced TRPV4 activity were inhibited by the treatment of ruthenium red or MLT, the increase of mROS, lipid peroxidation, apoptosis, Zn2+ concentrations, TRPV4, caspase -3, caspase -9, Bax, and Bcl-2 expressions were restored via upregulation of reduced glutathione, glutathione peroxidase, and total antioxidant status. The levels of apoptosis and cell death in the cells were enriched with increases of caspase -3 and -9 activations, although they were decreased by MLT treatment. In conclusion, the treatment of MLT modulates HYPX-mediated mROS, apoptosis, and TRPV4-mediated overload Ca2+ influx and may provide an avenue for protecting HYPX-mediated neurological diseases associated with the increase of mROS, Ca2+, and Zn2+ concentration.

Transient receptor potential channel TRPV4 mediates TGF-β1-induced differentiation of human ventricular fibroblasts.

Cardiac fibroblasts (CFs) are principal extracellular matrix-producing cells. In response to injury, CFs transdifferentiate into myofibroblasts. Intracellular calcium (Ca2+) signaling, involved in fibroblast proliferation and differentiation, is activated in fibroblasts through transient receptor potential (TRP) channels, but the function of these channels has not been investigated in human ventricular CFs. Under evaluation in this study, was the role of TRP channels in the differentiation of human ventricular CFs induced by transforming the growth factor beta (TGF-β), a pro-fibrotic cytokine. Human ventricular CFs were used in this study. The differentiation of CFs into myofibroblast was induced with TGF-β and was identified by the expression of smooth muscle actin. Results indicate that Ca2+ signaling was an essential component of ventricular CF dif-ferentiation. CFs treated with TGF-β demonstrated increased expression of a TRP channel, TRPV4, both at the mRNA and protein levels, which corresponded with CF-myofibroblast trans-differentiation, as evidenced by the upregulation of α-smooth muscle actin, a myofibroblast marker, and plasminogen activator inhibitor-1, which are fibrogenesis markers. An agonist of TRPV4 induced the conversion of CFs into myofibroblasts, whereas it's antagonist as well a Ca2+ chelating agent reduced it, indicating that the Ca2+ influx throughTRPV4 is required for CF trans-differentiation. Overall, these results dem-onstrate that TRPV4-mediated Ca2+ influx participates in regulating the differentiation of human ventricular CFs into myofibroblasts through the MAPK/ERK pathway. Overall, these results demonstrate that TRPV4-mediated Ca2+ influx participates in regulating the differentiation of human ventricular CFs into myofibroblasts through the MAPK/ERK pathway.

TRPV4 Complexes With the Na+/Ca2+ Exchanger and IP3 Receptor 1 to Regulate Local Intracellular Calcium and Tracheal Tension in Mice

Intracellular Ca2+ is critical for regulating airway smooth muscle (ASM) tension. A rapid rise in the intracellular Ca2+ concentration ([Ca2+]i) of ASM cells is crucial for modulating the intensity and length of the ASM contraction. Because this rapid increase in [Ca2+]i largely depends on the balance between Ca2+ released from intracellular Ca2+ stores and extracellular Ca2+ entry, exploring the mechanisms mediating Ca2+ transport is critical for understanding ASM contractility and the pathogenesis of bronchial contraction disorders. Transient receptor potential vanilloid 4 (TRPV4) is a highly Ca2+-permeable non-selective cation channel that mediates Ca2+ influx to increase [Ca2+]i, which then directly or indirectly regulates the contraction and relaxation of ASM. The [Ca2+]i returns to basal levels through several uptake and extrusion pumps, such as the sarco(endo)plasmic reticulum Ca2+ ATPase and inositol 1,4,5-trisphosphate receptors (IP3Rs), the plasmalemmal Ca2+ ATPase, and the plasma membrane Na+/Ca2+ exchanger (NCX). Thus, to further understand ASM tension regulation in normal and diseased tissue, the present study examined whether an interaction exists among TRPV4, IP3Rs, and NCX. The TRPV4-specific and potent agonist GSK1016790A increased [Ca2+]i in mouse ASM cells, an effect that was completely blocked by the TRPV4-specific antagonist HC067047. However, GSK1016790A induced relaxation in mouse tracheal rings precontracted with carbachol in vitro. To determine the mechanism underlying this TRPV4-induced relaxation of ASM, we blocked specific downstream molecules. We found that the GSK1016790A-induced relaxation was abolished by the NCX inhibitors KB-R7943 and LiCl but not by specific inhibitors of the Ca2+-activated large-, intermediate-, or small-conductance K+ channels (BKCa, IK, and SK3, respectively). The results of co-immunoprecipitation (co-IP) assays showed an interaction of TRPV4 and IP3R1 with NCXs. Taken together, these findings support a physical and functional interaction of TRPV4 and IP3R1 with NCXs as a novel TRPV4-mediated Ca2+ signaling mechanism and suggest a potential target for regulation of ASM tension and treatment of respiratory diseases, especially tracheal spasm.