Ruthenium red: Blocker or antagonist of TRPV channels?

Ruthenium red (RR) is a widely used inhibitor of Transient Receptor Potential (TRP) cation channels and other types of ion channels. Although RR has been generally accepted to inhibit TRP channels by physically blocking the ion permeation pathway, recent structural evidence suggests that it might also function as an antagonist, inducing conformational changes in the channel upon binding that result in closure of the pore. In a recent manuscript published in EMBO Reports, Ruth A. Pumroy and collaborators solve structures of TRPV2 and TRPV5 channels in the presence and absence of activators and RR. The data sheds light on the mechanism of inhibition by RR, while also opening new questions for further investigation.

Molecular details of ruthenium red pore block in TRPV channels.

Transient receptor potential vanilloid (TRPV) channels play a critical role in calcium homeostasis, pain sensation, immunological response, and cancer progression. TRPV channels are blocked by ruthenium red (RR), a universal pore blocker for a wide array of cation channels. Here we use cryo-electron microscopy to reveal the molecular details of RR block in TRPV2 and TRPV5, members of the two TRPV subfamilies. In TRPV2 activated by 2-aminoethoxydiphenyl borate, RR is tightly coordinated in the open selectivity filter, blocking ion flow and preventing channel inactivation. In TRPV5 activated by phosphatidylinositol 4,5-bisphosphate, RR blocks the selectivity filter and closes the lower gate through an interaction with polar residues in the pore vestibule. Together, our results provide a detailed understanding of TRPV subfamily pore block, the dynamic nature of the selectivity filter and allosteric communication between the selectivity filter and lower gate.

MCU Inhibitor Ruthenium Red Alleviates the Osteoclastogenesis and Ovariectomized Osteoporosis via Suppressing RANKL-Induced ROS Production and NFATc1 Activation through P38 MAPK Signaling Pathway.

Osteoporosis is a disorder of bone metabolism that is extremely common in elderly patients as well as in postmenopausal women. The main manifestation is that the bone resorption capacity is greater than the bone formation capacity, which eventually leads to a decrease in bone mass, increasing the risk of fracture. There is growing evidence that inhibiting osteoclast formation and resorption ability can be effective in treating and preventing the occurrence of osteoporosis. Our study is the first time to explore the role of the mitochondrial calcium uniporter (MCU) and its inhibitor ruthenium red (RR) in bone metabolism, clarifying the specific mechanism by which it inhibits osteoclast formation in vitro and plays a therapeutic role in osteoporosis in vivo. We verified the suppressive effects of RR on the receptor activator of nuclear factor-κB ligand (RANKL-)-induced differentiation and bone resorption function of osteoclasts in vitro. The reactive oxygen species (ROS) production stimulated by RANKL and the expression level of P38 MAPK/NFATc1 were also found to be inhibited by RR. Moreover, the promotion of RR on osteogenesis differentiation was investigated by alkaline phosphatase (ALP) and alizarin red S (ARS) staining and the detection of osteogenesis-specific gene expression levels by quantitative polymerase chain reaction (qPCR) and western blotting. Moreover, in ovariectomy (OVX-)-induced osteoporosis models, RR can downregulate the expression and function of the MCU, relieving bone loss and promoting osteogenesis to present a therapeutic effect on osteoporosis. This new finding will provide an important direction for the study of RR and MCU in the study of bone metabolism therapy targets.

TRPV1 and TRPA1 Channels Are Both Involved Downstream of Histamine-Induced Itch.

Two histamine receptor subtypes (HR), namely H1R and H4R, are involved in the transmission of histamine-induced itch as key components. Although exact downstream signaling mechanisms are still elusive, transient receptor potential (TRP) ion channels play important roles in the sensation of histaminergic and non-histaminergic itch. The aim of this study was to investigate the involvement of TRPV1 and TRPA1 channels in the transmission of histaminergic itch. The potential of TRPV1 and TRPA1 inhibitors to modulate H1R- and H4R-induced signal transmission was tested in a scratching assay in mice in vivo as well as via Ca2+ imaging of murine sensory dorsal root ganglia (DRG) neurons in vitro. TRPV1 inhibition led to a reduction of H1R- and H4R- induced itch, whereas TRPA1 inhibition reduced H4R- but not H1R-induced itch. TRPV1 and TRPA1 inhibition resulted in a reduced Ca2+ influx into sensory neurons in vitro. In conclusion, these results indicate that both channels, TRPV1 and TRPA1, are involved in the transmission of histamine-induced pruritus.

Activation of transient receptor potential vanilloid channel 4 contributes to the development of ethanol-induced gastric injury in mice.

The transient receptor potential vanilloid channel 4 (TRPV4) is associated with the development of several pathologies, particularly gastric disorders. However, there are no studies associating this receptor with the pathophysiology of gastric erosions. The aim of this study was to investigate the role of TRPV4 in the development of ethanol-induced gastric damage in vivo. Gastric lesions were induced by ethanol in Swiss mice pretreated with TRPV4 antagonists, GSK2193874 (0.1; 0.3 and 0.9 mg/kg) or Ruthenium red (0.03; 0.1 or 0.3 mg/kg) or its agonist, GSK1016790A (0.9 mg/kg). Gastric mucosal samples were taken for histopathology, immunohistochemistry, atomic force microscopy and evaluation of antioxidant parameters. The gastric mucus content and TRPV4 mRNA expression were analyzed. Ethanol exposure induced upregulation of gastric mRNA and protein expression of TRPV4. TRPV4 blockade promoted gastroprotection against ethanol-induced injury on macro- and microscopic levels, leading to reduced hemorrhage, cell loss and edema and enhanced gastric mucosal integrity. Moreover, an increase in superoxide dismutase (SOD) and glutathione (GSH) activity was observed, followed by a decrease in malondialdehyde (MDA) levels. TRPV4 blockade during alcohol challenge reestablished gastric mucus content. The combination of TRPV4 agonist and ethanol revealed macroscopic exacerbation of gastric damage area. Our results confirmed the association of TRPV4 with the development of gastric injury, showing the importance of this receptor for further investigations in the field of gastrointestinal pathophysiology and pharmacology.

Mechanotransduction channel Piezo is widely expressed in the spider, Cupiennius salei, mechanosensory neurons and central nervous system.

Mechanosensory neurons use mechanotransduction (MET) ion channels to detect mechanical forces and displacements. Proteins that function as MET channels have appeared multiple times during evolution and occur in at least four different families: the DEG/ENaC and TRP channels, as well as the TMC and Piezo proteins. We found twelve putative members of MET channel families in two spider transcriptomes, but detected only one, the Piezo protein, by in situ hybridization in their mechanosensory neurons. In contrast, probes for orthologs of TRP, ENaC or TMC genes that code MET channels in other species did not produce any signals in these cells. An antibody against C. salei Piezo detected the protein in all parts of their mechanosensory cells and in many neurons of the CNS. Unspecific blockers of MET channels, Ruthenium Red and GsMTx4, had no effect on the mechanically activated currents of the mechanosensory VS-3 neurons, but the latter toxin reduced action potential firing when these cells were stimulated electrically. The Piezo protein is expressed throughout the spider nervous system including the mechanosensory neurons. It is possible that it contributes to mechanosensory transduction in spider mechanosensilla, but it must have other functions in peripheral and central neurons.

Differential sensitivity of cinnamaldehyde-evoked calcium fluxes to ruthenium red in guinea pig and mouse trigeminal sensory neurons.

OBJECTIVE: Transient receptor potential ankyrin 1 (TRPA1) is an excitatory ion channel expressed on a subset of sensory neurons. TRPA1 is activated by a host of noxious stimuli including pollutants, irritants, oxidative stress and inflammation, and is thought to play an important role in nociception and pain perception. TRPA1 is therefore a therapeutic target for diseases with nociceptive sensory signaling components. TRPA1 orthologs have been shown to have differential sensitivity to certain ligands. Cinnamaldehyde has previously been shown to activate sensory neurons via the selective gating of TRPA1. Here, we tested the sensitivity of cinnamaldehyde-evoked responses in mouse and guinea pig sensory neurons to the pore blocker ruthenium red (RuR). RESULTS: Cinnamaldehyde, the canonical TRPA1-selective agonist, caused robust calcium fluxes in trigeminal neurons dissociated from both mice and guinea pigs. RuR effectively inhibited cinnamaldehyde-evoked responses in mouse neurons at 30 nM, with complete block seen with 3 µM. In contrast, responses in guinea pig neurons were only partially inhibited by 3 µM RuR. We conclude that RuR has a decreased affinity for guinea pig TRPA1 compared to mouse TRPA1. This study provides further evidence of differences in ligand affinity for TRPA1 in animal models relevant for drug development.

Ruthenium red attenuates brown adipose tissue thermogenesis in rats.

Ruthenium red (RR) is a non-selective antagonist of the temperature-sensitive Transient Receptor Potential (TRP) channels and it is an important pharmacological tool in thermoregulatory research. However, the effect of RR on thermoeffector activity is not well established. Here we evaluated the effect of RR on cold-defense thermoeffectors induced by menthol, an agonist of the cold-sensitive TRPM8 channel. Adult male Wistar rats were used. Epidermal treatment with menthol raised deep body temperature due to an increase in oxygen consumption (an index of thermogenesis), a reduction in heat loss index (an index of cutaneous vasoconstriction), and an induction in warmth-seeking behavior in a two-temperature choice apparatus. Pretreatment with RR attenuated the menthol-induced increase in deep body temperature and oxygen consumption, but it did not affect heat loss index and warmth-seeking behavior. To stimulate brown adipose tissue thermogenesis, rats were treated with CL 316,243, a potent and selective ß3-adrenoceptor agonist. CL 316,243 increased deep body temperature, which was attenuated by RR pretreatment. We conclude that RR reduces brown adipose tissue thermogenesis induced by menthol and CL 316,243, independent of effects at the thermal sensor level (i.e., TRPM8).

Effects of mitochondria-associated Ca2+ transporters suppression on oocyte activation.

Oocyte activation deficiency leads to female infertility. [Ca2+ ]i oscillations are required for mitochondrial energy supplement transition from the resting to the excited state, but the underlying mechanisms are still very little known. Three mitochondrial Ca2+ channels, Mitochondria Calcium Uniporter (MCU), Na+ /Ca2+ Exchanger (NCLX) and Voltage-dependent Ca2+ Channel (VDAC), were deactivated by inhibitors RU360, CGP37157 and Erastin, respectively. Both Erastin and CGP37157 inhibited mitochondrial activity significantly while attenuating [Ca2+ ]i and [Ca2+ ]m oscillations, which caused developmental block of pronuclear formation. Thus, NCLX and VDAC are two mitochondria-associated Ca2+ transporter proteins regulating oocyte activation, which may be used as potential targets to treat female infertility. SIGNIFICANCE OF THE STUDY: NCLX and VDAC are two mitochondria-associated Ca2+ transporter proteins regulating oocyte activation.

Different Sensitivity of Control and MICU1- and MICU2-Ablated Trypanosoma cruzi Mitochondrial Calcium Uniporter Complex to Ruthenium-Based Inhibitors.

The mitochondrial Ca2+ uptake in trypanosomatids shares biochemical characteristics with that of animals. However, the composition of the mitochondrial Ca2+ uniporter complex (MCUC) in these parasites is quite peculiar, suggesting lineage-specific adaptations. In this work, we compared the inhibitory activity of ruthenium red (RuRed) and Ru360, the most commonly used MCUC inhibitors, with that of the recently described inhibitor Ru265, on Trypanosoma cruzi, the agent of Chagas disease. Ru265 was more potent than Ru360 and RuRed in inhibiting mitochondrial Ca2+ transport in permeabilized cells. When dose-response effects were investigated, an increase in sensitivity for Ru360 and Ru265 was observed in TcMICU1-KO and TcMICU2-KO cells as compared with control cells. In the presence of RuRed, a significant increase in sensitivity was observed only in TcMICU2-KO cells. However, application of Ru265 to intact cells did not affect growth and respiration of epimastigotes, mitochondrial Ca2+ uptake in Rhod-2-labeled intact cells, or attachment to host cells and infection by trypomastigotes, suggesting a low permeability for this compound in trypanosomes.

Antinociceptive activity of 3ß-6ß-16ß-trihydroxylup-20 (29)-ene triterpene isolated from Combretum leprosum leaves in adult zebrafish (Danio rerio).

Drugs used to treat pain are associated with adverse effects, increasing the search for new drugs as an alternative treatment for pain. Therefore, we evaluated the antinociceptive behavior and possible neuromodulation mechanisms of triterpene 3ß, 6ß, 16ß-trihydroxylup-20(29)-ene (CLF-1) isolated from Combretum leprosum leaves in zebrafish. Zebrafish (n = 6/group) were pretreated with CLF-1 (0.1 or 0.3 or 1.0 mg/mL; i.p.) and underwent nociception behavior tests. The antinociceptive effect of CFL-1 was tested for modulation by opioid (naloxone), nitrergic (L-NAME), nitric oxide and guanylate cyclase synthesis inhibitor (methylene blue), NMDA (Ketamine), TRPV1 (ruthenium red), TRPA1 (camphor), or ASIC (amiloride) antagonists. The corneal antinociceptive effect of CFL-1 was tested for modulation by TRPV1 (capsazepine). The effect of CFL-1 on zebrafish locomotor behavior was evaluated with the open field test. The acute toxicity study was conducted. CLF-1 reduced nociceptive behavior and corneal in zebrafish without mortalities and without altering the animals' locomotion. Thus, CFL-1 presenting pharmacological potential for the treatment of acute pain and corneal pain, and this effect is modulated by the opioids, nitrergic system, NMDA receptors and TRP and ASIC channels.

Ruthenium red, mitochondrial calcium uniporter inhibitor, attenuates cognitive deficits in STZ-ICV challenged experimental animals.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder with cardinal features of cognitive dysfunction in an individual. Recently, the blockade of mitochondrial calcium uniporter (MCU) exhibits neuroprotective activity in experimental animals. However, the therapeutic potential of MCU has not yet been established in the management of AD. Therefore, the present study explored the therapeutic potential of either Ruthenium red (RR), a MCU blocker, or Spermine, a MCU opener, on the extent of mitochondrial calcium accumulation, function, integrity and bioenergetics in hippocampus, pre-frontal cortex and amygdale of ICV-STZ challenged rats. Experimental AD was induced in male rats by intracerebroventricular injection of streptozotocin (ICV-STZ) on day-1 (D-1) of the experimental protocol at a sub-diabetogenic dose (3 mg/kg) twice at an interval of 48 h into both rat lateral ventricles. RR attenuated ICV-STZ-induced memory-related behavioral abnormalities in Morris water maze and Y-maze tests. RR also attenuated ICV-STZ-induced decrease in the level of acetylcholine and activity of choline acetyltransferase and, increase in the activity of acetylcholinestarase in memory-sensitive rat brain regions. Further, RR attenuated mitochondrial toxicity in terms of reducing mitochondrial calcium accumulation and improving the mitochondrial function, integrity and bioenergetics in memory-sensitive brain regions of ICV-STZ challenged rats. Furthermore, RR attenuated the percentage of apoptotic cells in ICV-STZ challenged rat brain regions. However, Spermine did not alter ICV-STZ-induced behavioral, biochemical and molecular observations in any of the brain regions. These observations indicate the fact that the MCU blockage could be a potential therapeutic option in the management of sporadic type of AD.

Genetic and Chemical Screening in Human Blood Serum Reveals Unique Antibacterial Targets and Compounds against Klebsiella pneumoniae.

Antibiotics halt the growth of bacteria by targeting core, essential physiology that is required for life on standard microbiological media. Many more biochemical and virulence processes, however, are required for bacteria to cause infection in a host. Indeed, chemical inhibitors of the latter processes are overlooked using conventional antibiotic drug discovery approaches. Here, we use human blood serum as an alternative growth medium to explore new targets and compounds. High-throughput screening of genetic and chemical libraries identified compounds targeting biological activities required by Klebsiella pneumoniae to grow in serum, such as nucleobase biosynthesis and iron acquisition, and showed that serum can chemically transform compounds to reveal cryptic antibacterial activity. One of these compounds, ruthenium red, was effective in a rat bloodstream infection model. Our data demonstrate that human serum is an effective tool to find new chemical matter to address the current antibiotic resistance crisis.

TRPV4 channels' dominant role in the temperature modulation of intrinsic contractility and lymph flow of rat diaphragmatic lymphatics.

The lymphatic system drains and propels lymph by extrinsic and intrinsic mechanisms. Intrinsic propulsion depends upon spontaneous rhythmic contractions of lymphatic muscles in the vessel walls and is critically affected by changes in the surrounding tissue like osmolarity and temperature. Lymphatics of the diaphragm display a steep change in contraction frequency in response to changes in temperature, and this, in turn, affects lymph flow. In the present work, we demonstrated in an ex vivo diaphragmatic tissue rat model that diaphragmatic lymphatics express transient receptor potential channels of the vanilloid 4 subfamily (TRPV4) and that their blockade by both the nonselective antagonist Ruthenium Red and the selective antagonist HC-067047 abolished the response of lymphatics to temperature changes. Moreover, the selective activation of TRPV4 channels by means of GSK1016790A mirrored the behavior of vessels exposed to increasing temperatures, pointing out the critical role played by these channels in sensing the temperature of the lymphatic vessels' environment and thus inducing a change in contraction frequency and lymph flow.NEW & NOTEWORTHY The present work addresses the putative receptor system that enables diaphragmatic lymphatics to change intrinsic contraction frequency and thus lymph flow according to the changes in temperature of the surrounding environment, showing that this role can be sustained by TRPV4 channels alone.

Role of TRPV1/TRPV3 channels in olanzapine-induced metabolic alteration: Possible involvement in hypothalamic energy-sensing, appetite regulation, inflammation and mesolimbic pathway.

Atypical antipsychotics (AAPs) have the tendency of inducing severe metabolic alterations like obesity, diabetes mellitus, insulin resistance, dyslipidemia and cardiovascular complications. These alterations have been attributed to altered hypothalamic appetite regulation, energy sensing, insulin/leptin signaling, inflammatory reactions and active reward anticipation. Line of evidence suggests that transient receptor potential vanilloid type 1 and 3 (TRPV1 and TRPV3) channels are emerging targets in treatment of obesity, diabetes mellitus and could modulate feed intake. The present study was aimed to investigate the putative role TRPV1/TRPV3 in olanzapine-induced metabolic alterations in mice. Female BALB/c mice were treated with olanzapine for six weeks to induce metabolic alterations. Non-selective TRPV1/TRPV3 antagonist (ruthenium red) and selective TRPV1 (capsazepine) and TRPV3 antagonists (2,2-diphenyltetrahydrofuran or DPTHF) were used to investigate the involvement of TRPV1/TRPV3 in chronic olanzapine-induced metabolic alterations. These metabolic alterations were differentially reversed by ruthenium red and capsazepine, while DPTHF didn't show any significant effect. Olanzapine treatment also altered the mRNA expression of hypothalamic appetite-regulating and nutrient-sensing factors, inflammatory genes and TRPV1/TRPV3, which were reversed with ruthenium red and capsazepine treatment. Furthermore, olanzapine treatment also increased expression of TRPV1/TRPV3 in nucleus accumbens (NAc), TRPV3 expression in ventral tegmental area (VTA), which were reversed by the respective antagonists. However, DPTHF treatment showed reduced feed intake in olanzapine treated mice, which might be due to TRPV3 specific antagonism and reduced hedonic feed intake. In conclusion, our results suggested the putative role TRPV1 in hypothalamic dysregulations and TRPV3 in the mesolimbic pathway; both regulate feeding in olanzapine treated mice.

Ruthenium red improves blastocyst developmental competence by regulating mitochondrial Ca2+ and mitochondrial functions in fertilized porcine oocytes in vitro.

Ruthenium red (RR) inhibits calcium (Ca2+) entry from the cytoplasm to the mitochondria, and is involved in maintenance of Ca2+ homeostasis in mammalian cells. Ca2+ homeostasis is very important for further embryonic development of fertilized oocytes. However, the effect of RR on mitochondria-Ca2+ (mito-Ca2+) levels during in vitro fertilization (IVF) on subsequent blastocyst developmental capacity in porcine is unclear. The present study explored the regulation of mito-Ca2+ levels using RR and/or histamine in fertilized oocytes and their influence on blastocyst developmental capacity in pigs. Red fluorescence intensity by the mito-Ca2+ detection dye Rhod-2 was significantly increased (P < 0.05) in zygotes 6 h after IVF compared to mature oocytes. Based on these results, we investigated the changes in mito-Ca2+ by RR (10 and 20 µM) in presumptive zygotes using Rhod-2 staining and mito-Ca2+ uptake 1 (MICU1) protein levels as an indicator of mito-Ca2+ uptake using western blot analysis. As expected, RR-treated zygotes displayed decreased protein levels of MICU1 and Rhod-2 red fluorescence intensity compared to non-treated zygotes 6 h after IVF. Blastocyst development rate of 20 µM RR-treated zygotes was significantly increased 6 h after IVF (P < 0.05) due to improved mitochondrial functions. Conversely, the blastocyst development rate was significantly decreased in histamine (mito-Ca2+ inhibitor, 100 nM) treated zygotes (P < 0.05). The collective results demonstrate that RR improves blastocyst development in porcine embryos by regulating mito-Ca2+ and MICU1 expression following IVF.

Tannic acid, a vasodilator present in wines and beverages, stimulates Ca2+ influx via TRP channels in bEND.3 endothelial cells.

Tannic acid (TA) is a polyphenol compound present in wines and many beverages. Although previous works have shown that TA could cause vasodilation in an endothelial cell (EC)-dependent manner, there is hitherto no report showing whether TA could raise EC cytosolic Ca2+ concentration. In this work we examined the effects of TA on cytosolic Ca2+ of mouse brain bEND.3 EC. TA (1-30 µM) caused a slow elevation in cytosolic Ca2+ level in a concentration-dependent manner. At 30 µM, TA triggered Ca2+ influx without causing intracellular Ca2+ release. TA-triggered Ca2+ influx was suppressed by Ni2+ (a non-specific Ca2+ channel blocker), ruthenium red and SKF 96365 (non-specific TRP channel blockers), CBA (a selective TRPM4 inhibitor) and M 084 (a selective TRPC4/C5 blocker). However, TA-triggered Ca2+ influx pathway was not permeable to Mn2+. Our results suggest TA activated TRP channels, possibly TRPM4 and TRPC4/C5, to promote influx of Ca2+.

Polynuclear Ruthenium Amines Inhibit K2P Channels via a "Finger in the Dam" Mechanism.

The trinuclear ruthenium amine ruthenium red (RuR) inhibits diverse ion channels, including K2P potassium channels, TRPs, the calcium uniporter, CALHMs, ryanodine receptors, and Piezos. Despite this extraordinary array, there is limited information for how RuR engages targets. Here, using X-ray crystallographic and electrophysiological studies of an RuR-sensitive K2P, K2P2.1 (TREK-1) I110D, we show that RuR acts by binding an acidic residue pair comprising the "Keystone inhibitor site" under the K2P CAP domain archway above the channel pore. We further establish that Ru360, a dinuclear ruthenium amine not known to affect K2Ps, inhibits RuR-sensitive K2Ps using the same mechanism. Structural knowledge enabled a generalizable design strategy for creating K2P RuR "super-responders" having nanomolar sensitivity. Together, the data define a "finger in the dam" inhibition mechanism acting at a novel K2P inhibitor binding site. These findings highlight the polysite nature of K2P pharmacology and provide a new framework for K2P inhibitor development.

Determining the effect of calcium on cell death rate and perforation formation during leaf development in the novel model system, the lace plant (Aponogeton madagascariensis).

Programmed cell death (PCD) is the destruction of unwanted cells through an intracellularly mediated process. Perforation formation in the lace plant (Aponogeton madagascariensis) provides an excellent model for studying developmentally regulated PCD. Ca2+ fluxes have previously been identified as important signals for PCD in plants and mammals. The fundamental goal of this project was to determine the influence of Ca2+ on the rate of cell death and perforation formation during leaf development in the lace plant. This was investigated using the application of various known calcium modulators including lanthanum III chloride (LaCl3 ), ruthenium red and calcium ionophore A23187. Detached lace plant leaves at an early stage of development were treated with these modulators in both short- and long-term exposure assays and analysed using live cell imaging. Results from this study indicate that calcium plays a vital role in developmentally regulated PCD in the lace plant as application of the modulators significantly altered the rate of cell death and perforation formation during leaf development. In conclusion, this study exemplifies the suitability of the lace plant for live cell imaging and detached leaf experiments to study cell death and provides insight into the importance of Ca2+ in developmentally regulated PCD in planta.

Central Transient Receptor Potential Vanilloid 4 Contributes to Systemic Water Homeostasis through Urinary Excretion.

Intracerebroventricular (icv) injection of transient receptor potential vanilloid 4 (TRPV4) agonists 4α-phorbol-12, 13-didecanoate (4α-PDD) and GSK101690A increased urinary excretion under the physiological condition. TRPV4 antagonists ruthenium red and HC-067047 significantly blocked increased urinary volume after intragastric administration of water and 4α-PDD-induced diuresis. Administration of the TRPV4 agonists did not significantly change the plasma concentration of vasopressin or atrial natriuretic factor. Pretreatment with indomethacin inhibited the diuresis induced by 4α-PDD. Moreover, icv injection of prostaglandin (PG) F2α produced diuretic effects. These findings indicate that central TRPV4 regulates urine excretion, which contributes to systemic water homeostasis in vivo. The underlying mechanisms are suggested to involve PG synthesis, but not release of vasopressin or atrial natriuretic factor.