TRPV4 is a widely expressed cation channel of the 'transient receptor potential' (TRP) family that is related to the vanilloid receptor VR1 (TRPV1). It functions as a Ca2+ entry channel and displays remarkable gating promiscuity by responding to both physical stimuli (cell swelling, innoxious heat) and the synthetic ligand 4alphaPDD. An endogenous ligand for this channel has not yet been identified. Here we show that the endocannabinoid anandamide and its metabolite arachidonic acid activate TRPV4 in an indirect way involving the cytochrome P450 epoxygenase-dependent formation of epoxyeicosatrienoic acids. Application of 5',6'-epoxyeicosatrienoic acid at submicromolar concentrations activates TRPV4 in a membrane-delimited manner and causes Ca2+ influx through TRPV4-like channels in vascular endothelial cells. Activation of TRPV4 in vascular endothelial cells might therefore contribute to the relaxant effects of endocannabinoids and their P450 epoxygenase-dependent metabolites on vascular tone.

Summary Abnormal tumor vessels promote metastasis and impair chemotherapy. Hence, tumor vessel normalization (TVN) is emerging as anti-cancer treatment. Here, we show that tumor endothelial cells (ECs) have a hyper-glycolytic metabolism, shunting intermediates to nucleotide synthesis. EC haplo-deficiency or blockade of the glycolytic activator PFKFB3 did not affect tumor growth, but reduced cancer cell invasion, intravasation and metastasis by normalizing tumor vessels, which improved vessel maturation and perfusion. Mechanistically, PFKFB3 inhibition tightened the vascular barrier by reducing VE-cadherin endocytosis in ECs, and rendering pericytes more quiescent and adhesive (via upregulation of N-cadherin) through glycolysis reduction; it also lowered the expression of cancer cell adhesion molecules in ECs by decreasing NF-κB signaling. PFKFB3-blockade treatment also improved chemotherapy of primary and metastatic tumors.

Transient receptor potential melastatin-3 (TRPM3) is a broadly expressed Ca(2+)-permeable nonselective cation channel. Previous work has demonstrated robust activation of TRPM3 by the neuroactive steroid pregnenolone sulfate (PS), but its in vivo gating mechanisms and functions remained poorly understood. Here, we provide evidence that TRPM3 functions as a chemo- and thermosensor in the somatosensory system. TRPM3 is molecularly and functionally expressed in a large subset of small-diameter sensory neurons from dorsal root and trigeminal ganglia, and mediates the aversive and nocifensive behavioral responses to PS. Moreover, we demonstrate that TRPM3 is steeply activated by heating and underlies heat sensitivity in a subset of sensory neurons. TRPM3-deficient mice exhibited clear deficits in their avoidance responses to noxious heat and in the development of inflammatory heat hyperalgesia. These experiments reveal an unanticipated role for TRPM3 as a thermosensitive nociceptor channel implicated in the detection of noxious heat.

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 (PLA2) and cytochrome P450 epoxygenase inhibit activation of TRPV4 by osmotic cell swelling but not by heat and 4␣-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 4␣-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.T he TRPV subfamily of the transient receptor potential (TRP) family of cation channels consists of at least six mammalian channels homologous to the vanilloid receptor (for a unifying nomenclature, see ref. 1). The TRPV channels are activated by a variety of signals, including chemical and thermal stimuli, cell swelling, low intracellular Ca 2ϩ , and endogenous or synthetic ligands (2-10). Members of this subfamily contain a hydrophobic core region comprising six putative transmembrane segments (TM1-TM6), a pore-loop region between TM5 and TM6, a cytoplasmic N terminus with three to six ankyrin repeats, and a cytoplasmic C terminus (1, 3). The TRPV subfamily can be subdivided into two groups. One group is formed by TRPV1-TRPV4, which display a moderate Ca 2ϩ selectivity (P Ca ͞P Na Ͻ 10, in which P is permeability), a weak field-strength monovalent cation permeability sequence, and steep temperature dependence (5,6,(11)(12)(13)(14)(15)(16). The second group is formed by TRPV5 and TRPV6, which are highly Ca 2ϩ selective (P Ca ͞P Na Ͼ 100) and display a permeability sequence for monovalent cations consistent with a strong field-strength binding site but show little temperature dependence (10,17,18).TRPV4 was identified originally as a channel activated by hypotonic cell swelling (11,13,19), but later reports show that it can be activated also by synthetic agonists, such as the phorbol ester 4␣-phorbol 12,13-didecanoate (4␣-PDD) (5), temperatures Ͼ27°C (6, 20), and acidic pH and citrate (ref. 21; see also ref. 5). Moreover, rec...

We have studied activation by phorbol derivatives of TRPV4 channels, the human VRL-2, and murine TRP12 channels, which are highly homologous to the human VR-OAC, and the human and murine OTRPC4 channel. ] i inhibits the channel with an IC 50 of 406 nM. Ruthenium Red at a concentration of 1 M completely blocks inward currents at ؊80 mV but has a smaller effect on outward currents likely indicating a voltage dependent channel block. We concluded that the phorbol derivatives activate TRPV4 (VR-OAC, VRL-2, OTRPC4, TRP12) independently from protein kinase C, in a manner consistent with direct agonist gating of the channel.