(-)-Epicatechin acts as a potent agonist of the membrane androgen receptor, ZIP9 (SLC39A9), to promote apoptosis of breast and prostate cancer cells.

(-)-Epicatechin, a flavonoid present in high concentrations in foods such as green tea and cocoa, exerts beneficial and protective effects in numerous disease models, including anti-tumorigenesis and apoptosis in human breast and prostate cancer cells. Potential interactions of (-)-epicatechin and (+)-catechin with the membrane androgen receptor, ZIP9 (SLC39A9), which mediates androgen induction of apoptosis in these cancer cells, were investigated. Both (-)-epicatechin and (+)-catechin were effective competitors of [3H]-testosterone binding to PC-3 prostate cancer cells (nuclear androgen receptor-negative, nAR-null) overexpressing ZIP9 (PC3-ZIP9), with relative binding affinities of 75 % and 28 % that of testosterone, respectively. (-)-Epicatechin (200 nM) mimicked the effects of 100 nM testosterone in inducing apoptosis of PC3-ZIP9 cells, whereas (+)-catechin (concentration range 200 nM-1000 nM) did not significantly increase apoptosis and instead blocked the apoptotic response to testosterone. (-)-Epicatechin also activated androgen-dependent ZIP9 signaling pathways, inducing decreases in cAMP production and elevating intracellular free zinc levels, while (+)-catechin typically lacked these actions. Both (-)-epicatechin and (+)-catechin also bound to cell membranes of MDA-MB-468 breast cancer cells (nAR-null, high ZIP9 expression). MDA-MB-468 cells showed similar apoptotic, cAMP, and free zinc signaling responses to (-)-epicatechin to those observed in PC3-ZIP9 cells, as well as antagonism by (+)-catechin of testosterone-induced apoptosis and modulation of cAMP and caspase-3 levels. Moreover, knockdown of ZIP9 expression in MDA-MB-468 cells with siRNA decreased specific [3H]-testosterone binding of both catechins and blocked the apoptotic and free zinc responses to testosterone and (-)-epicatechin. The results indicate (-)-epicatechin is a potent ZIP9 agonist in breast and prostate cancer cells.

Protective Effect of Aplysin Supplementation on Intestinal Permeability and Microbiota in Rats Treated with Ethanol and Iron.

Aplysin, a kind of phytochemicals or phytonutrients, is purified from red alga Laurencia tristicha. The present study aims to investigate the influence of aplysin on changes of intestinal permeability and microbiota induced by excessive ethanol and iron. Thirty male rats were randomly divided into three groups (10/group): control group (normal saline); ethanol + iron group as EI treated with ethanol (8⁻12 mL/kg/day) and iron (1000 mg/kg) in diet; EI supplemented with aplysin (150 mg/kg/day) group as AEI; the trial lasts for 12 weeks. The result showed that levels of plasma endotoxin, fatty acid-binding protein 2, D-lactic acid, diamine oxidase were increased in rats in the EI group; and significantly decreased by 14%, 17%, 26%, 16%, respectively (p < 0.05) in the AEI group after the 12-week aplysin treatment. Moreover, in the AEI group the amount of Escherichia coli and Bacteroides fragilis were higher, while the amount of Lactobacillus, Bifidobacterium and Clostridium were lower than those in the EI group. The expressions of iron transporters divalent-metal transporter 1(DMT1) and ferroportin 1(FPN1) were significantly upregulated in the EI group compared to those in the control group. In conclusion, aplysin could effectively improve intestinal permeability and intestinal flora disorder induced with excessive ethanol and iron.

Lipid Regulation of Acrosome Exocytosis.

Lipids are critical regulators of mammalian sperm function, first helping prevent premature acrosome exocytosis, then enabling sperm to become competent to fertilize at the right place/time through the process of capacitation, and ultimately triggering acrosome exocytosis. Yet because they do not fit neatly into the "DNA--RNA-protein" synthetic pathway, they are understudied and poorly understood. Here, we focus on three lipids or lipid classes-cholesterol, phospholipids, and the ganglioside G(M1)--in context of the modern paradigm of acrosome exocytosis. We describe how these various- species are precisely segregated into membrane macrodomains and microdomains, simultaneously preventing premature exocytosis while acting as foci for organizing regulatory and effector molecules that will enable exocytosis. Although the mechanisms responsible for these domains are poorly defined, there is substantial evidence for their composition and functions. We present diverse ways that lipids and lipid modifications regulate capacitation and acrosome exocytosis, describing in more detail how removal of cholesterol plays a master regulatory role in enabling exocytosis through at least two complementary pathways. First, cholesterol efflux leads to proteolytic activation of phospholipase B, which cleaves both phospholipid tails. The resultant changes in membrane curvature provide a mechanism for the point fusions now known to occur far before a sperm physically interacts with the zona pellucida. Cholesterol efflux also enables G(M1) to regulate the voltage-dependent cation channel, Ca(V)2.3, triggering focal calcium transients required for acrosome exocytosis in response to subsequent whole-cell calcium rises. We close with a model integrating functions for lipids in regulating acrosome exocytosis.

Vitamin D3 transactivates the zinc and manganese transporter SLC30A10 via the Vitamin D receptor.

Vitamin D3 regulates genes critical for human health and its deficiency is associated with an increased risk for osteoporosis, cancer, diabetes, multiple sclerosis, hypertension, inflammatory and immunological diseases. To study the impact of vitamin D3 on genes relevant for the transport and metabolism of nutrients and drugs, we employed next-generation sequencing (NGS) and analyzed global gene expression of the human-derived Caco-2 cell line treated with 500nM vitamin D3. Genes involved in neuropeptide signaling, inflammation, cell adhesion and morphogenesis were differentially expressed. Notably, genes implicated in zinc, manganese and iron homeostasis were largely increased by vitamin D3 treatment. An ∼10-fold increase in ceruloplasmin and ∼4-fold increase in haptoglobin gene expression suggested a possible association between vitamin D and iron homeostasis. SLC30A10, the gene encoding the zinc and manganese transporter ZnT10, was the chiefly affected transporter, with ∼15-fold increase in expression. SLC30A10 is critical for zinc and manganese homeostasis and mutations in this gene, resulting in impaired ZnT10 function or expression, cause manganese intoxication, with Parkinson-like symptoms. Our NGS results were validated by real-time PCR in Caco-2 cells, as well as in duodenal biopsies taken from healthy human subjects treated with 0.5µg vitamin D3 daily for 10 days. In addition to increasing gene expression of SLC30A10 and the positive control TRPV6, vitamin D3 also increased ZnT10 protein expression, as indicated by Western blot and cytofluorescence. In silico identification of potential vitamin D responsive elements (VDREs) in the 5'-flanking region of the SLC30A10 promoter and dual-luciferase reporter assay showed enhanced promoter activity in the presence of vitamin D receptor (VDR) and retinoid X receptor (RXR) constructs, as well as vitamin D3, but not when one of these factors was absent. Electrophoretic mobility shift assay (EMSA) and competition EMSA revealed binding of select sequences, namely, nt -1623/-1588 and nt -1758/-1723 relative to the transcription start site, to VDR-containing nuclear extracts. In conclusion, we have shown that vitamin D3 transactivates the SLC30A10 gene in a VDR-dependent manner, resulting in increased ZnT10 protein expression. Because SLC30A10 is highly expressed in the small intestine, it is possible that the control of zinc and manganese systemic levels is regulated by vitamin D3 in the intestine. Zinc, manganese and vitamin D are important for bone metabolism and brain health. Future examination of a possible role for supplementation or chelation of zinc and manganese, alongside vitamin D3 administration, will further our understanding of its potential benefit in the treatment of specific illnesses, such as osteoporosis and Parkinson's disease.

A short-term zinc-deficient diet decreases bone formation through down-regulated BMP2 in rat bone.

We investigated the effects of a short-term dietary zinc deficiency on bone metabolism. Zinc deficiency increased the mRNA expression of zinc uptake transporters such as Zip1, Zip13, and Zip14 in bone. However, zinc deficiency might not maintain zinc storage in bone, resulting in a decrease in bone formation through downregulation of the expression levels of osteoblastogenesis-related genes.

Soybean extracts increase cell surface ZIP4 abundance and cellular zinc levels: a potential novel strategy to enhance zinc absorption by ZIP4 targeting.

Dietary zinc deficiency puts human health at risk, so we explored strategies for enhancing zinc absorption. In the small intestine, the zinc transporter ZIP4 functions as an essential component of zinc absorption. Overexpression of ZIP4 protein increases zinc uptake and thereby cellular zinc levels, suggesting that food components with the ability to increase ZIP4 could potentially enhance zinc absorption via the intestine. In the present study, we used mouse Hepa cells, which regulate mouse Zip4 (mZip4) in a manner indistinguishable from that in intestinal enterocytes, to screen for suitable food components that can increase the abundance of ZIP4. Using this ZIP4-targeting strategy, two such soybean extracts were identified that were specifically able to decrease mZip4 endocytosis in response to zinc. These soybean extracts also effectively increased the abundance of apically localized mZip4 in transfected polarized Caco2 and Madin-Darby canine kidney cells and, moreover, two apically localized mZip4 acrodermatitis enteropathica mutants. Soybean components were purified from one extract and soyasaponin Bb was identified as an active component that increased both mZip4 protein abundance and zinc levels in Hepa cells. Finally, we confirmed that soyasaponin Bb is capable of enhancing cell surface endogenous human ZIP4 in human cells. Our results suggest that ZIP4 targeting may represent a new strategy to improve zinc absorption in humans.

3-Hydro-2,2,5,6-tetramethylpyrazine: A novel inducer of zinc transporter-1 in HepG2 human hepatocellular carcinoma cells.

Dihydropyrazine compounds, including 3-hydro-2,2,5,6-tetramethylpyrazine (DHP-3), are low-molecular-weight glycation products spontaneously generated in vivo and also ingested via food. Our preliminary study using microarray analysis demonstrated that DHP-3 induced zinc transporter-1 (ZnT-1) in HepG2 cells. It is well known that the increase of intracellular zinc is a sensitive stimulating factor for ZnT-1 protein induction; however, there is little information about the induction of ZnT-1 by low-molecular-weight chemical compounds. Here, we attempted to clarify the mechanism of ZnT-1 induction by DHP-3. A significant increase of ZnT-1 mRNA was observed 6h after DHP-3 treatment at concentrations over 0.5mM, and disappeared 24h after exposure. This induction pattern followed that of metal-responsive transcription factor 1 (MTF-1) mRNA, a metalloregulatory protein that serves as a major transcription factor of ZnT-1. Moreover, DHP-3 yielded transcriptional activation of MTF-1 in a luciferase reporter assay. The intracellular zinc content was unaffected by the compound; however, oxidative stress was observed in cells under the same conditions that activated the MTF-1 signaling pathway. These results suggest that DHP-3 is a novel ZnT-1 inducer and acts via activation of the MTF-1 signaling pathway. Additionally, the activation of MTF-1 by this compound likely occurs through oxidative stress.

Effects of prebiotic supplementation on the expression of proteins regulating iron absorption in anaemic growing rats.

Prebiotics may increase intestinal Fe absorption in anaemic growing rats. The present study evaluated the effects of high-performance (HP) inulin and oligofructose on factors that regulate Fe absorption in anaemic rats during the growth phase. Male Wistar rats aged 21 d of age were fed AIN-93G ration without Fe for 2 weeks to induce Fe-deficiency anaemia. The rats were fed on day 35 a control diet, or a diet with 10 % HP inulin, or a diet with 10 % oligofructose, without Fe supplementation. The animals were euthanised after 2 weeks, and segments of the duodenum, caecum, colon and liver were removed. The expression levels of proteins in the intestinal segments were assessed using Western blotting. The levels of serum, urine and liver hepcidin and the concentrations of IL-10, IL-6 and TNF-α in the caecum, colon and liver were measured using the ELISA test. HP inulin increased the expression of the divalent metal transporter 1 protein in the caecum by 162 % (P= 0·04), and the expression of duodenal cytochrome b reductase in the colon by 136 % (P= 0·02). Oligofructose decreased the expression of the protein ferroportin in the duodenum (P= 0·02), the concentrations of IL-10 (P= 0·044), IL-6 (P= 0·036) and TNF-α (P= 0·004) in the caecum, as well as the level of urinary hepcidin (P< 0·001). These results indicate that prebiotics may interfere with the expression of various intestinal proteins and systemic factors involved in the regulation of intestinal Fe absorption in anaemic rats during the growth phase.

High glucose level promotes migration behavior of breast cancer cells through zinc and its transporters.

BACKGROUND: The diabetes patients have been associated with an increased risk of mortality by breast cancer and there are difference between the breast cancer patients with diabetes, and their nondiabetic counterparts in the regimen choice and effects of breast cancer treatment. However, the pathophysiological relationships of diabetes and breast cancer have not yet been elucidated in detail. In this study, we investigate the breast cancer cell line, MCF-7 motility, which linked to invasion and metastasis, in high glucose level corresponding to hyperglycemia and the role of Zn and its transporter. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrated the significant motility of MCF-7 cultured in hyperglycemic level (25 mM glucose) in comparison to normal physiological glucose level (5.5 mM glucose). The other hand, the osmotic control medium, 5.5 mM glucose with 19.5 mM mannitol or fructose had no effect on migratory, suggesting that high glucose level promotes the migration of MCF-7. Moreover, the activity of intracellular Zn(2+) uptake significantly increased in high glucose-treated cells in comparison to 5.5 mM glucose, and the mRNA expression of zinc transporters, ZIP6 and ZIP10, was upregulated in 25 mM glucose-treated cells. The deficiency of ZIP6 or ZIP10 and intracellular Zn(2+) significantly inhibited the high migration activity in 25 mM glucose medium, indicating that Zn(2+) transported via ZIP6 and ZIP10 play an essential role in the promotion of cell motility by high glucose stimulation. CONCLUSION/SIGNIFICANCE: Zinc and its transporters, ZIP6 and ZIP10, are required for the motility stimulated with high glucose level. These findings provide the first evidence proposing the novel strategies for the diagnosis and therapy of breast cancer with hyperglycemia.

[The effect of Ca(2+)-induced opening of cyclosporine-sensitive pore on the oxygen consumption and functional state of rat liver mitochondria].

The effect of Ca(2+)-induced opening of cyclosporine-sensitive pore (mitochondrial permeability transition pore, MPTP) on the oxygen consumption and mitochondrial functional state was studied in the rat liver mitochondria. It was shown that, with the use of glutamate as oxidation substrate, in the absence of depolarization MPTP opening results in the increase of steady state respiration rate because of the activation of cyclosporine-sensitive Ca2+/H(+)-exchange and Ca2+ cycling, which was supported by the simultaneous work of MPTP and Ca(2+)-uniporter. With the aid of selective blockers, cyclosporine A and ruthenium red, it was shown that MPTP and Ca(2+)-uniporter contribute equally to the Ca(2+)-cycling and mitochondrial respiration. It was shown that bioenergetic effects of MPTP opening under steady state conditions (increase in the oxygen consumption rate under substrate oxidation without ADP, decrease in respiratory control ratio as well as the effectiveness of ATP synthesis, P/O) are close to the functional alterations, which result from the increase of endogenous proton conductance of mitochondrial membrane. Uncoupling effect of MPTP opening, by itself, had no effect on phosphorylation rate, which remains relatively stable because the fall of P/O is compensated by the activation of respiratory chain and the increase in the rate of state 3 respiration. It was concluded that under physiologically normal conditions MPTP might function as the endogenous mechanism of mild uncoupling of respiratory chain.

Targeting the hepcidin-ferroportin axis to develop new treatment strategies for anemia of chronic disease and anemia of inflammation.

Anemia of chronic disease (ACD) or anemia of inflammation is prevalent in patients with chronic infection, autoimmune disease, cancer, and chronic kidney disease. ACD is associated with poor prognosis and lower quality of life. Management of ACD using intravenous iron and erythropoiesis stimulating agents are ineffective for some patients and are not without adverse effects, driving the need for new alternative therapies. Recent advances in our understanding of the molecular mechanisms of iron regulation reveal that increased hepcidin, the iron regulatory hormone, is a key factor in the development of ACD. In this review, we will summarize the role of hepcidin in iron homeostasis, its contribution to the pathophysiology of ACD, and novel strategies that modulate hepcidin and its target ferroportin for the treatment of ACD.

Molecular and biophysical basis of glutamate and trace metal modulation of voltage-gated Ca(v)2.3 calcium channels.

Here, we describe a new mechanism by which glutamate (Glu) and trace metals reciprocally modulate activity of the Ca(v)2.3 channel by profoundly shifting its voltage-dependent gating. We show that zinc and copper, at physiologically relevant concentrations, occupy an extracellular binding site on the surface of Ca(v)2.3 and hold the threshold for activation of these channels in a depolarized voltage range. Abolishing this binding by chelation or the substitution of key amino acid residues in IS1-IS2 (H111) and IS2-IS3 (H179 and H183) loops potentiates Ca(v)2.3 by shifting the voltage dependence of activation toward more negative membrane potentials. We demonstrate that copper regulates the voltage dependence of Ca(v)2.3 by affecting gating charge movements. Thus, in the presence of copper, gating charges transition into the "ON" position slower, delaying activation and reducing the voltage sensitivity of the channel. Overall, our results suggest a new mechanism by which Glu and trace metals transiently modulate voltage-dependent gating of Ca(v)2.3, potentially affecting synaptic transmission and plasticity in the brain.

Diclofenac-induced stimulation of SMCT1 (SLC5A8) in a heterologous expression system: a RPE specific phenomenon.

SMCT1 is a Na(+)-coupled monocarboxylate transporter expressed in a variety of tissues including kidney, thyroid, small intestine, colon, brain, and retina. We found recently that several non-steroidal anti-inflammatory drugs (NSAIDs) inhibit the activity of SMCT1. Here we evaluated the effect of diclofenac, also a NSAID, on SMCT1. SMCT1 cDNA was expressed heterologously in the human retinal pigment epithelial cell lines HRPE and ARPE-19, the human mammary epithelial cell line MCF7, and in Xenopus laevis oocytes. Transport was monitored by substrate uptake and substrate-induced currents. Na(+)-dependent uptake/current was considered as SMCT1 activity. The effect of diclofenac was evaluated for specificity, dose-response, and influence on transport kinetics. To study the specificity of the diclofenac effect, we evaluated the influence of this NSAID on the activity of several other cloned transporters in mammalian cells under identical conditions. In contrast to several NSAIDs that inhibited SMCT1, diclofenac stimulated SMCT1 when expressed in HRPE and ARPE-19 cells. The stimulation was marked, ranging from 2- to 5-fold depending on the concentration of diclofenac. The stimulation was associated with an increase in the maximal velocity of the transport system as well as with an increase in substrate affinity. The observed effect on SMCT1 was selective because the activity of several other cloned transporters, when expressed in HRPE cells and studied under identical conditions, was not affected by diclofenac. Interestingly, the stimulatory effect on SMCT1 observed in HRPE and ARPE-19 cells was not evident in MCF7 cells nor in the X. laevis expression system, indicating that SMCT1 was not the direct target for diclofenac. The RPE-specific effect suggests that the target of diclofenac that mediates the stimulatory effect is expressed in RPE cells but not in MCF7 cells or in X. laevis oocytes. Since SMCT1 is a concentrative transporter for metabolically important compounds such as pyruvate, lactate, beta-hydroxybutyrate, and nicotinate, the stimulation of its activity by diclofenac in RPE cells has biological and clinical significance.

Calmodulin and calcium interplay in the modulation of TRPC5 channel activity. Identification of a novel C-terminal domain for calcium/calmodulin-mediated facilitation.

TRPC5 forms Ca2+-permeable nonselective cation channels important for neurite outgrowth and growth cone morphology of hippocampal neurons. Here we studied the activation of mouse TRPC5 expressed in Chinese hamster ovary and human embryonic kidney 293 cells by agonist stimulation of several receptors that couple to the phosphoinositide signaling cascade and the role of calmodulin (CaM) on the activation. We showed that exogenous application of 10 microM CaM through patch pipette accelerated the agonist-induced channel activation by 2.8-fold, with the time constant for half-activation reduced from 4.25 +/- 0.4 to 1.56 +/- 0.85 min. We identified a novel CaM-binding site located at the C terminus of TRPC5, 95 amino acids downstream from the previously determined common CaM/IP3R-binding (CIRB) domain for all TRPC proteins. Deletion of the novel CaM-binding site attenuated the acceleration in channel activation induced by CaM. However, disruption of the CIRB domain from TRPC5 rendered the channel irresponsive to agonist stimulation without affecting the cell surface expression of the channel protein. Furthermore, we showed that high (>5 microM) intracellular free Ca2+ inhibited the current density without affecting the time course of TRPC5 activation by receptor agonists. These results demonstrated that intracellular Ca2+ has dual and opposite effects on the activation of TRPC5. The novel CaM-binding site is important for the Ca2+/CaM-mediated facilitation, whereas the CIRB domain is critical for the overall response of receptor-induced TRPC5 channel activation.

Capsaicin augments synaptic transmission in the rat medial preoptic nucleus.

The medial preoptic nucleus (MPN) is the major nucleus of the preoptic area (POA), a hypothalamic area involved in the regulation of body-temperature. Injection of capsaicin into this area causes hypothermia in vivo. Capsaicin also causes glutamate release from hypothalamic slices. However, no data are available on the effect of capsaicin on synaptic transmission within the MPN. Here, we have studied the effect of exogenously applied capsaicin on spontaneous synaptic activity in hypothalamic slices of the rat. Whole-cell patch-clamp recordings were made from visually identified neurons located in the MPN. In a subset of the studied neurons, capsaicin enhanced the frequency of spontaneous glutamatergic EPSCs. Remarkably, capsaicin also increased the frequency of GABAergic IPSCs, an effect that was sensitive to removal of extracellular calcium, but insensitive to tetrodotoxin. This suggests an action of capsaicin at presynaptic GABAergic terminals. In contrast to capsaicin, the TRPV4 agonist 4alpha-PDD did not affect GABAergic IPSCs. Our results show that capsaicin directly affects synaptic transmission in the MPN, likely through actions at presynaptic terminals as well as on projecting neurons. Our data add to the growing evidence that capsaicin receptors are not only expressed in primary afferent neurons, but also contribute to synaptic processing in some CNS regions.

2-aminoethoxydiphenyl borate is a common activator of TRPV1, TRPV2, and TRPV3.

The transient receptor potential (TRP) superfamily contains a large number of proteins encoding cation permeable channels that are further divided into TRPC (canonical), TRPM (melastatin), and TRPV (vanilloid) subfamilies. Among the six TRPV members, TRPV1, TRPV2, TRPV3, and TRPV4 form heat-activated cation channels, which serve diverse functions ranging from nociception to osmolality regulation. Although chemical activators for TRPV1 and TRPV4 are well documented, those for TRPV2 and TRPV3 are lacking. Here we show that in the absence of other stimuli, 2-aminoethoxydiphenyl borate (2APB) activates TRPV1, TRPV2, and TRPV3, but not TRPV4, TRPV5, and TRPV6 expressed in HEK293 cells. In contrast, 2APB inhibits the activity of TRPC6 and TRPM8 evoked by 1-oleolyl-2-acetyl-sn-glycerol and menthol, respectively. In addition, low levels of 2APB strongly potentiate the effect of capsaicin, protons, and heat on TRPV1 as well as that of heat on TRPV3 expressed in Xenopus oocytes. In dorsal root ganglia neurons, supra-additive stimulations were evoked by 2APB and capsaicin or 2APB and acid. Our data suggest the existence of a common activation mechanism for TRPV1, TRPV2, and TRPV3 that may serve as a therapeutic target for pain management and treatment for diseases caused by hypersensitivity and temperature misregulation.

TRPV4 calcium entry channel: a paradigm for gating diversity.

The vanilloid receptor-1 (VR1, now TRPV1) was the founding member of a subgroup of cation channels within the TRP family. The TRPV subgroup contains six mammalian members, which all function as Ca2+ entry channels gated by a variety of physical and chemical stimuli. TRPV4, which displays 45% sequence identity with TRPV1, is characterized by a surprising gating promiscuity: it is activated by hypotonic cell swelling, heat, synthetic 4alpha-phorbols, and several endogenous substances including arachidonic acid (AA), the endocannabinoids anandamide and 2-AG, and cytochrome P-450 metabolites of AA, such as epoxyeicosatrienoic acids. This review summarizes data on TRPV4 as a paradigm of gating diversity in this subfamily of Ca2+ entry channels.

Cell swelling, heat, and chemical agonists use distinct pathways for the activation of the cation channel TRPV4.

TRPV4 is a Ca(2+)- and Mg(2+)-permeable cation channel within the vanilloid receptor subgroup of the transient receptor potential (TRP) family, and it has been implicated in Ca(2+)-dependent signal transduction in several tissues, including brain and vascular endothelium. TRPV4-activating stimuli include osmotic cell swelling, heat, phorbol ester compounds, and 5',6'-epoxyeicosatrienoic acid, a cytochrome p450 epoxygenase metabolite of arachidonic acid (AA). It is presently unknown how these distinct activators converge on opening of the channel. Here, we demonstrate that blockers of phospholipase A(2) (PLA(2)) and cytochrome p450 epoxygenase inhibit activation of TRPV4 by osmotic cell swelling but not by heat and 4alpha-phorbol 12,13-didecanoate. Mutating a tyrosine residue (Tyr-555) in the N-terminal part of the third transmembrane domain to an alanine strongly impairs activation of TRPV4 by 4alpha-phorbol 12,13-didecanoate and heat but has no effect on activation by cell swelling or AA. We conclude that TRPV4-activating stimuli promote channel opening by means of distinct pathways. Cell swelling activates TRPV4 by means of the PLA(2)-dependent formation of AA, and its subsequent metabolization to 5',6'-epoxyeicosatrienoic acid by means of a cytochrome p450 epoxygenase-dependent pathway. Phorbol esters and heat operate by means of a distinct, PLA(2)- and cytochrome p450 epoxygenase-independent pathway, which critically depends on an aromatic residue at the N terminus of the third transmembrane domain.