Activation Of TRPV1 Channels Research Articles

Experimental spinal cord injury upregulates vagal nodose TRPV1 expression (902.4)

The gastrointestinal (GI) tract is critically dependent upon adequate blood flow. Spinal cord injury (SCI) produces dramatic GI and vascular dysregulation, including vascular hypotension and, possibly, visceral hypoperfusion. We have previously shown that T3‐SCI in rats results in diminished visceral blood flow, increased expression of inflammatory markers and consequent gastrointestinal dysmotility. Vagal afferent injury has been shown to induce changes in cholecystokinin (CCK) and transient receptor potential vanilloid type 1 receptor (TRPV1) expression within the nodose ganglion. We hypothesized that SCI leads to increased activation of TRPV1 channels along the vagus nerve.Nodose ganglia TRPV1 and CCK mRNA was quantified using qRT‐PCR, at 1‐day, 3‐days, and 7‐days post‐SCI and control. We used a superior mesenteric artery (SMA) occlusion model to show a worst case scenario of ischemia‐reperfusion to the GI tract. Our data show a significant increase in nodose TRPV1 and CCK of SCI and SMA rats compared to controls. The increased TRPV1 indicates GI inflammation and the increased CCK suggests that CCK increases as per vagal afferent injury. Our data present similarities to experimental ischemia‐reperfusion of the GI tract, in which SMA occlusion causes a dramatic inflammatory response within the GI tract. We propose that our model of SCI represents a continuum of GI ischemia and that the increase in inflammatory mediators and TRPV1 activation may play a role in the post‐SCI pathophysiology of gastric vagal afferents.Grant Funding Source: Supported by: NINDS 49177

LE135, a retinoid acid receptor antagonist, produces pain through direct activation of TRP channels

Retinoids, through their activation of retinoic acid receptors (RARs) and retinoid X receptors, regulate diverse cellular processes, and pharmacological intervention in their actions has been successful in the treatment of skin disorders and cancers. Despite the many beneficial effects, administration of retinoids causes irritating side effects with unknown mechanisms. Here, we demonstrate that LE135 [4-(7,8,9,10-tetrahydro-5,7,7,10,10-pentamethyl-5H-benzo[e]naphtho[2,3-b][1,4]diazepin-13-yl)benzoic acid], a selective antagonist of RARβ , is a potent activator of the capsaicin (TRPV1) and wasabi (TRPA1) receptors, two critical pain-initiating cation channels. We performed to investigate the excitatory effects of LE135 on TRPV1 and TRPA1 channels expressed in HEK293T cells and in dorsal root ganglia neurons with calcium imaging and patch-clamp recordings. We also used site-directed mutagenesis of the channels to determine the structural basis of LE135-induced activation of TRPV1 and TRPA1 channels and behavioural testing to examine if pharmacological inhibition and genetic deletion of the channels affected LE135-evoked pain-related behaviours. LE135 activated both the capsaicin receptor (TRPV1) and the allyl isothiocyanate receptor (TRPA1) heterologously expressed in HEK293T cells and endogenously expressed by sensory nociceptors. Mutations disrupting the capsaicin-binding site attenuated LE135 activation of TRPV1 channels and a single mutation (K170R) eliminated TRPA1 activity evoked by LE135. Intraplantar injection of LE135 evoked pain-related behaviours. Both TRPV1 and TRPA1 channels were involved in LE135-elicited pain-related responses, as shown by pharmacological and genetic ablation studies. This blocker of retinoid acid signalling also exerted non-genomic effects through activating the pain-initiating TRPV1 and TRPA1 channels.

H2 S modulates duodenal motility in male rats via activating TRPV1 and K(ATP) channels.

H2 S induces vasodilatation by opening KATP channels but it may also affect other ion channels. The aim of this study was to investigate the effect of H2 S on intestinal motility in rats and its underlying mechanism. The tension of intestinal muscle strips, afferent firing of intestinal mesenteric nerves, length of duodenal smooth muscle cells and whole-cell membrane potential of dorsal root ganglion (DRG) neurons were monitored. H2 S-producing enzymes were located by immunofluorescence staining. NaHS exerted early transient excitation and late long-lasting inhibition on the intestinal contraction. The excitation was attenuated by TRPV1 antagonists capsazepine, A784168, SB-366791 and NK1 receptor antagonist L703606, while the inhibition was attenuated by glibenclamide. NaHS increased duodenal afferent nerve firing and depolarized DRG neurons. These effects were reduced by capsazepine and A784168. NaHS relaxed isolated duodenal smooth muscle cells. The KATP channels were expressed in smooth muscle cells. Cystathionine β-synthase and cystathionine γ-lyase were expressed in rat duodenal myenteric neurons. L-cysteine and S-adenosyl-L-methionine increased the contraction of duodenal muscle strips, an effect attenuated by capsazepine and L703606. NaHS induces biphasic effects on intestinal motility in rats while endogenous H2 S only exerts an excitatory effect. This transient excitatory effect might be mediated by activation of TRPV1 channels in sensory nerve terminals with the consequent release of substance P. The long-lasting inhibitory effect might be mediated by activation of KATP channels in the smooth muscle cells. These findings reveal a novel mechanism for the excitatory effect of H2 S on gastrointestinal motility.

Structural Characterization of Double-Knot Toxin, an Activator of TRPV1 Channels

Venom from poisonous organisms is a rich source of peptide toxins interacting with different ion channels proteins. These peptide toxins modulate ion channels by different mechanisms, and have been widely used as tools for investigating ion channel mechanisms. Double-knot toxin (DkTx) is a novel peptide toxin that activates TRPV1 channels, and contains two inhibitory cysteine knot (ICK) motifs, as its name suggests. Previous studies show that DkTx activates TRPV1 channels, and suggest that the avidity of the toxin (slow unbinding) arises from its bivalent nature. Here we use solid-phase peptide synthesis to individually produce the two knots of DkTx (K1 and K2), fold each in vitro, and find that they exhibit different binding affinities for the channel even though they share high sequence homology. As a first step toward understanding the structural and functional relationship of DkTx binding to TRPV1 channels, we determined solution structures of each knot in using NMR. The structures show that DkTx is composed of two notably amphipathic ICK motifs (each with two beta-strands) that are connected by a flexible linker, and that K2 has a larger hydrophobic surface compared to K1. In addition, the single conserved Trp residue in each knot show different orientations, with that in K1 exhibiting greater solvent exposure. Interestingly, using intrinsic Trp fluorescence, we observe strong partitioning of DkTx and K1, but see no evidence of membrane partitioning for K2. We also made a series of K1/K2 chimeras, and identified variant residues in two loops and the C-terminus that are responsible for the higher activity of K2. From these results we propose that membrane interactions are involved in the mechanisms of DkTx activation of TRPV1, and identify surfaces of the two knots that likely involved in binding to the channel.

Proton Activation of TRPV1 Channels

Proton is the best-known endogenous ligand for TRPV1, a pain-sensing polymodal receptor. Extracellular acidification (e.g., due to inflammation, tissue damage and ischemia) strongly activates the channel. However, the underlying molecular mechanism remains largely unknown. Outside-out patch-clamp recordings at room temperature from HEK293 cells showed that proton induces mTRPV1 currents with an apparent EC50 of 1.3 μM (pH 5.9), in the same range as the apparent EC50 for capsaicin (of 1.0 μM). Proton exhibits two effects on TRPV1: binding of proton to extracellular sites promotes channel activation with an apparent EC50 of 1.7 μM (pH 5.8), whereas proton inhibits ion permeation with an apparent IC50 of 2.1 μM (pH 5.7). Combination of these two effects yields a prominent off response upon removal of acidic solutions, during which the channel current increases abruptly and then slowly decays back to the baseline. Proton potentiation of gating also strongly affects TRPV1 activation by capsaicin, voltage, and heat, causing substantial shifts in their dynamic ranges. Synergistic effects among multiple stimuli allow mild acidification to induce strong TRPV1 activation that may contribute to painful sensation.

Monoacylglycerols Activate TRPV1 – A Link between Phospholipase C and TRPV1

Phospholipase C-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate generates diacylglycerol, inositol 1,4,5-trisphosphate and protons, all of which can regulate TRPV1 activity via different mechanisms. Here we explored the possibility that the diacylglycerol metabolites 2-arachidonoylglycerol and 1-arachidonoylglycerol, and not metabolites of these monoacylglycerols, activate TRPV1 and contribute to this signaling cascade. 2-Arachidonoylglycerol and 1-arachidonoylglycerol activated native TRPV1 on vascular sensory nerve fibers and heterologously expressed TRPV1 in whole cells and inside-out membrane patches. The monoacylglycerol lipase inhibitors methylarachidonoyl-fluorophosphonate and JZL184 prevented the metabolism of deuterium-labeled 2-arachidonoylglycerol and deuterium-labeled 1-arachidonoylglycerol in arterial homogenates, and enhanced TRPV1-mediated vasodilator responses to both monoacylglycerols. In mesenteric arteries from TRPV1 knock-out mice, vasodilator responses to 2-arachidonoylglycerol were minor. Bradykinin and adenosine triphosphate, ligands of phospholipase C-coupled membrane receptors, increased the content of 2-arachidonoylglycerol in dorsal root ganglia. In HEK293 cells expressing the phospholipase C-coupled histamine H1 receptor, exposure to histamine stimulated the formation of 2-AG, and this effect was augmented in the presence of JZL184. These effects were prevented by the diacylglycerol lipase inhibitor tetrahydrolipstatin. Histamine induced large whole cell currents in HEK293 cells co-expressing TRPV1 and the histamine H1 receptor, and the TRPV1 antagonist capsazepine abolished these currents. JZL184 increased the histamine-induced currents and tetrahydrolipstatin prevented this effect. The calcineurin inhibitor ciclosporin and the endogenous “entourage” compound palmitoylethanolamide potentiated the vasodilator response to 2-arachidonoylglycerol, disclosing TRPV1 activation of this monoacylglycerol at nanomolar concentrations. Furthermore, intracerebroventricular injection of JZL184 produced TRPV1-dependent antinociception in the mouse formalin test. Our results show that intact 2-arachidonoylglycerol and 1-arachidonoylglycerol are endogenous TRPV1 activators, contributing to phospholipase C-dependent TRPV1 channel activation and TRPV1-mediated antinociceptive signaling in the brain.

TRPV4 channel activation leads to endothelium-dependent relaxation mediated by nitric oxide and endothelium-derived hyperpolarizing factor in rat pulmonary artery

The purpose of the present study was to characterize TRPV4 channels in the rat pulmonary artery and examine their role in endothelium-dependent relaxation. Tension, Real-Time polymerase chain reaction (Real-Time PCR) and Western blot experiments were conducted on left and right branches of the main pulmonary artery from male Wistar rats. TRPV4 channel agonist GSK1016790A (GSK) caused concentration-related robust relaxation (Emax 88.6±5.5%; pD2 8.7±0.2) of the endothelium-intact pulmonary artery. Endothelium-denudation nearly abolished the relaxation (Emax 5.6±1.3%) to GSK. TRPV4 channel selective antagonist HC067047 significantly attenuated GSK-induced relaxation (Emax 56.2±6.6% vs. control Emax 87.9±3.3%) in endothelium-intact vessels, but had no effect on either ACh-induced endothelium-dependent or SNP-induced endothelium-independent relaxations. GSK-induced relaxations were markedly inhibited either in the presence of NO synthase inhibitor L-NAME (Emax 8.5±2.7%) or sGC inhibitor ODQ (Emax 28.1±5.9%). A significant portion (Emax 30.2±4.4%) of endothelium-dependent relaxation still persisted in the combined presence of L-NAME and cyclooxygenase inhibitor indomethacin. This EDHF-mediated relaxation was sensitive to inhibition by 60mM K+ depolarizing solution or K+ channel blockers apamin (SKCa; KCa2.3) and TRAM-34 (IKCa; KCa3.1). GSK (10−10−10−7M) caused either modest decrease or increase in the basal tone of endothelium-intact or denuded rings, respectively. We found a greater abundance (>1.5 fold) of TRPV4 mRNA and protein expressions in endothelium-intact vs. denuded vessels, suggesting the presence of this channel in pulmonary endothelial and smooth muscle cells as well. The present study demonstrated that NO and EDHF significantly contributed to TRPV4 channel-mediated endothelium-dependent relaxation of the rat pulmonary artery.

TRPP2 and TRPV4 Form an EGF-Activated Calcium Permeable Channel at the Apical Membrane of Renal Collecting Duct Cells

ObjectiveRegulation of apical calcium entry is important for the function of principal cells of the collecting duct. However, the molecular identity and the regulators of the transporter/channel, which is responsible for apical calcium entry and what factors regulate the calcium conduction remain unclear.Methods and ResultsWe report that endogenous TRPP2 and TRPV4 assemble to form a 23-pS divalent cation-permeable non-selective ion channel at the apical membrane of renal principal cells of the collecting duct. TRPP2\\TRPV4 channel complex was identified by patch-clamp, immunofluorescence and co-immunprecipitation studies in both principal cells that either possess normal cilia (cilia (+)) or in which cilia are absent (cilia (-)). This channel has distinct biophysical and pharmacological and regulatory profiles compared to either TRPP2 or TRPV4 channels. The rate of occurrence detected by patch clamp was higher in cilia (-) compared to cilia (+) cells. In addition, shRNA knockdown of TRPP2 increased the prevalence of TRPV4 channel activity while knockdown of TRPV4 resulted in TRPP2 activity and knockdown of both proteins vastly decreased the 23-pS channel activity. Epidermal growth factor (EGF) stimulated TRPP2\\TRPV4 channel through the EGF receptor (EGFR) tyrosine kinase-dependent signaling. With loss of cilia, apical EGF treatment resulted in 64-fold increase in channel activity in cilia (-) but not cilia (+) cells. In addition EGF increased cell proliferation in cilia (-) cell that was dependent upon TRPP2\\TRPV4 channel mediated increase in intracellular calcium.ConclusionWe conclude that in the absence of cilia, an EGF activated TRPP2\\TRPV4 channel may play an important role in increased cell proliferation and cystogenesis.

Serotonin receptors take the TRiPV4 highway in chronic hypoxic pulmonary hypertension. Focus on “TRPV4 channel contributes to serotonin-induced pulmonary vasoconstriction and the enhanced vascular reactivity in chronic hypoxic pulmonary hypertension”

pulmonary arterial hypertension (PAH) is a rare human disease displaying a very poor prognosis of survival. The chronic increase in pulmonary vascular resistance associated with PAH eventually leads to right ventricular hypertrophy and failure, and ultimately death. It is well accepted that the

Arachidonic Acid–Induced Dilation in Human Coronary Arterioles: Convergence of Signaling Mechanisms on Endothelial TRPV4‐Mediated Ca 2+ Entry

BackgroundArachidonic acid (AA) and/or its enzymatic metabolites are important lipid mediators contributing to endothelium‐derived hyperpolarizing factor (EDHF)–mediated dilation in multiple vascular beds, including human coronary arterioles (HCAs). However, the mechanisms of action of these lipid mediators in endothelial cells (ECs) remain incompletely defined. In this study, we investigated the role of the transient receptor potential vanilloid 4 (TRPV4) channel in AA‐induced endothelial Ca2+ response and dilation of HCAs.Methods and ResultsAA induced concentration‐dependent dilation in isolated HCAs. The dilation was largely abolished by the TRPV4 antagonist RN‐1734 and by inhibition of endothelial Ca2+‐activated K+ channels. In native and TRPV4‐overexpressing human coronary artery ECs (HCAECs), AA increased intracellular Ca2+ concentration ([Ca2+]i), which was mediated by TRPV4‐dependent Ca2+ entry. The AA‐induced [Ca2+]i increase was inhibited by cytochrome P450 (CYP) inhibitors. Surprisingly, the CYP metabolites of AA, epoxyeicosatrienoic acids (EETs), were much less potent activators of TRPV4, and CYP inhibitors did not affect EET production in HCAECs. Apart from its effect on [Ca2+]i, AA induced endothelial hyperpolarization, and this effect was required for Ca2+ entry through TRPV4. AA‐induced and TRPV4‐mediated Ca2+ entry was also inhibited by the protein kinase A inhibitor PKI. TRPV4 exhibited a basal level of phosphorylation, which was inhibited by PKI. Patch‐clamp studies indicated that AA activated TRPV4 single‐channel currents in cell‐attached and inside‐out patches of HCAECs.ConclusionsAA dilates HCAs through a novel mechanism involving endothelial TRPV4 channel‐dependent Ca2+ entry that requires endothelial hyperpolarization, PKA‐mediated basal phosphorylation of TRPV4, and direct activation of TRPV4 channels by AA.

Zingerone enhances glutamatergic spontaneous excitatory transmission by activating TRPA1 but not TRPV1 channels in the adult rat substantia gelatinosa

Transient receptor potential (TRP) channels are thought to play a role in regulating nociceptive transmission to spinal substantia gelatinosa (SG) neurons. It remains to be unveiled whether the TRP channels in the central nervous system are different in property from those involved in receiving nociceptive stimuli in the peripheral nervous system. We examined the effect of the vanilloid compound zingerone, which activates TRPV1 channels in the cell body of a primary afferent neuron, on glutamatergic excitatory transmission in the SG neurons of adult rat spinal cord slices by using the whole cell patch-clamp technique. Bath-applied zingerone reversibly and concentration-dependently increased spontaneous excitatory postsynaptic current (EPSC) frequency. This effect was accompanied by an inward current at -70 mV that was resistant to glutamate receptor antagonists. These zingerone effects were repeated and persisted in Na(+)-channel blocker tetrodotoxin-, La(3+)-, or IP3-induced Ca(2+)-release inhibitor 2-aminoethoxydiphenyl borate-containing or Ca(2+)-free Krebs solution. Zingerone activity was resistant to the selective TRPV1 antagonist capsazepine but sensitive to the nonselective TRP antagonist ruthenium red, the TRPA1 antagonist HC-030031, and the Ca(2+)-induced Ca(2+)-release inhibitor dantrolene. TRPA1 agonist allyl isothiocyanate but not capsaicin inhibited the facilitatory effect of zingerone. On the other hand, zingerone reduced monosynaptically evoked EPSC amplitudes, as did TRPA1 agonists. Like allyl isothiocyanate, zingerone enhanced GABAergic spontaneous inhibitory transmission in a manner sensitive to tetrodotoxin. We conclude that zingerone presynaptically facilitates spontaneous excitatory transmission, probably through Ca(2+)-induced Ca(2+)-release mechanisms, and produces a membrane depolarization in SG neurons by activating TRPA1 but not TRPV1 channels.

Multiscale computational models of microvascular reactivity from ion channels to intercellular signaling

Detailed compartmental, continuous, and multicellular computational models of smooth muscle (SM) and endothelial cell (EC) electrophysiology were developed and validated from patch clamp and calcium (Ca2+) imaging data. These models examine localized Ca2+ events from individual ion channels, role of signaling microdomains, effect of ion channel distribution, and homo‐ and hetero‐cellular signaling in regulation of vascular tone. Activation of TRPV4 channels in EC generates sparklets that activate colocalized IKCa channels and EDHF. EDHF is transmitted robustly by hyperpolarizing current through gap junctions, but less so by extracellular potassium. During SM stimulation, diffusion of IP3, and to smaller degree Ca2+, from SM to EC projections induces localized Ca2+ events and EDHF, which closes the myoendothelial feedback loop. Spatial heterogeneities in Ca2+ handling components generate oscillatory Ca2+ waves. In vasomotion, Ca2+ oscillations in SM can be synchronized by electrical or IP3 coupling through SM gap junctions. Conducted relaxation is mediated mainly by electrotonic spread of local hyperpolarization along the endothelium, but EC Ca2+ spread from the stimulus site and facilitation mechanism along the conduction can amplify the response. In conclusion, vasoreactivity involves complex signaling, which can be analyzed with the aid of computational models. Supported by NIH SC1HL95101.

Role of localized calcium events in regulation of vascular tone: A theoretical investigation

Localized calcium (Ca2+) events like sparks, puffs, pulsars and sparklets have been identified in the vascular cells. They have been suggested to play an important role in modulation of vessel tone. Theoretical modeling can provide quantitative insights and improve the fundamental understanding of the generation of these localized Ca2+ events. Hence we develop detailed finite element cellular models of Ca2+ dynamics and membrane electrophysiology in smooth muscle and endothelial cells. These models are integrated and modified to include myoendothelial projections with localized IP3 receptors, intermediate conductance Ca2+ activated potassium channels (IKCa) and TRPV4 channels. Activation of TRPV4 channels resulted in localized Ca2+ signals (sparklets). A single TRPV4 channel activation in absence of other channels and pumps resulted in localized calcium increase >;100nM and a spatial spread of ~ 6μm. The parameters of sparklets are affected by variety of factors of which open time of the channel, cooperative gating of the channels, diffusion coefficient of calcium and channel current can be the most important. The localized Ca2+ increase can activate IKCa channels and cause endothelium‐derived hyperpolarizing response modulating the vascular tone. The study provides a novel theoretical tool to examine the mechanisms behind localized calcium events and the regulation of vascular tone.(Supported by NIH SC1HL95101).

Activation and desensitization of TRPV1 channels in sensory neurons by the PPARα agonist palmitoylethanolamide

Palmitoylethanolamide (PEA) is an endogenous fatty acid amide displaying anti-inflammatory and analgesic actions. To investigate the molecular mechanism responsible for these effects, the ability of PEA and of pain-inducing stimuli such as capsaicin (CAP) or bradykinin (BK) to influence intracellular calcium concentrations ([Ca²⁺](i)) in peripheral sensory neurons, has been assessed in the present study. The potential involvement of the transcription factor PPARα and of TRPV1 channels in PEA-induced effects was also studied. [Ca²⁺](i) was evaluated by single-cell microfluorimetry in differentiated F11 cells. Activation of TRPV1 channels was assessed by imaging and patch-clamp techniques in CHO cells transiently-transfected with rat TRPV1 cDNA. In F11 cells, PEA (1-30 μM) dose-dependently increased [Ca²⁺](i). The TRPV1 antagonists capsazepine (1 μM) and SB-366791 (1 μM), as well as the PPARα antagonist GW-6471 (10 μM), inhibited PEA-induced [Ca²⁺](i) increase; blockers of cannabinoid receptors were ineffective. PEA activated TRPV1 channels heterologously expressed in CHO cells; this effect appeared to be mediated at least in part by PPARα. When compared with CAP, PEA showed similar potency and lower efficacy, and caused stronger TRPV1 currents desensitization. Sub-effective PEA concentrations, closer to those found in vivo, counteracted CAP- and BK-induced [Ca²⁺](i) transients, as well as CAP-induced TRPV1 activation. Activation of PPARα and TRPV1 channels, rather than of cannabinoid receptors, largely mediate PEA-induced [Ca²⁺](i) transients in sensory neurons. Differential TRPV1 activation and desensitization by CAP and PEA might contribute to their distinct pharmacological profile, possibly translating into potentially relevant clinical differences.

TRPV1 Ion Channel Activation Is Enhanced by Bradykinin in Sensory Neuronal Cells

Capsaicin (CAP) induces increase in cytosolic calcium, [Ca2+]i, in sensory neurons (CATH.a) through either L-/N-type calcium channels or TRPV1 channels. Activation of TRPV1 channels by protein kinases (e.g., PKCs) allows their integration with membrane-associated G-protein coupled receptors (GPCRs). Bradykinin (BK) induces activation of guanine nucleotide-binding proteins subunit-α class-q/11 (Gq/11α-protein) which activates PKC. The objective of this study was to determine whether transactivation of GPCR pathways in sensory neurons co-expressing BK2 receptors (BK2R) and TRPV1 caused hyper-reactivity of TRPV1 channels in response to BK.

Retinal cell death induced by TRPV1 activation involves NMDA signaling and upregulation of nitric oxide synthases.

The activation of the transient receptor potential vanilloid type 1 channel (TRPV1) has been correlated with oxidative and nitrosative stress and cell death in the nervous system. Our previous results indicate that TRPV1 activation in the adult retina can lead to constitutive and inducible nitric oxide synthase-dependent protein nitration and apoptosis. In this report, we have investigated the potential effects of TRPV1 channel activation on nitric oxide synthase (NOS) expression and function, and the putative participation of ionotropic glutamate receptors in retinal TRPV1-induced protein nitration, lipid peroxidation, and DNA fragmentation. Intravitreal injections of the classical TRPV1 agonist capsaicin up-regulated the protein expression of the inducible and endothelial NOS isoforms. Using 4,5-diaminofluorescein diacetate for nitric oxide (NO) imaging, we found that capsaicin also increased the production of NO in retinal blood vessels. Processes and perikarya of TRPV1-expressing neurons in the inner nuclear layer of the retina were found in the vicinity of nNOS-positive neurons, but those two proteins did not colocalize. Retinal explants exposed to capsaicin presented high protein nitration, lipid peroxidation, and cell death, which were observed in the inner nuclear and plexiform layers and in ganglion cells. This effect was partially blocked by AP-5, a NMDA glutamate receptor antagonist, but not by CNQX, an AMPA/kainate receptor antagonist. These data support a potential role for TRPV1 channels in physiopathological retinal processes mediated by NO, which at least in part involve glutamate release.

The monoterpene (–)‐carvone: A novel agonist of TRPV1 channels

(-)-Carvone is an antinociceptive monoterpene found as the main active constituent of essential oils obtained from plants of the genus Mentha. Here, we have investigated the pharmacology of this monoterpene in dorsal root ganglia (DRG) neurons and TRPV1-expressing HEK293 cells. (-)-carvone at pharmacological active concentrations did not reveal significant cytotoxicity to the cells used in this study, as investigated by neutral red and propidium iodide flow cytometry assays. In calcium imaging experiments 1 mM (-)-carvone increased the cytosolic calcium levels in DRG neurons from 120.6 ± 5.0 nM (basal) to 310.7 ± 23.1 nM (P < 0.05). These effects were completely abolished when neurons were preincubated with calcium-free bath solution or ruthenium-red (5 µM) and capsazepine (10 µM), suggesting the possibility of TRPV1 channel-activation by (-)-carvone. Activity of (-)-carvone on TRPV1 channels was further investigated in HEK293 cells expressing recombinant human TRPV1 channels revealing dose-dependent calcium transients with an EC(50) of 1.3 ± 0.2 mM (Hill coefficient = 2.5). In conclusion, we show for the first time the ability of (-)-carvone to induce increases in cytosolic calcium concentration through TRPV1 activation.

Modeling Temperature-Dependent Ion Channel Protein Structural Changes with Rosetta

Temperature-sensing ion channels are thought to adopt different conformations at varying temperatures, driven by a significant difference in free energy between the closed and open states. In support of this notion, we previously observed with site-directed fluorescence recordings that pore region undergoes substantial structural rearrangements during the heat activation of TRPV1 channels. Temperature-driven structural changes have also been suggested in other protein regions and channel types. To reveal such structural changes, we are exploring the Rosetta modeling method to predict channel protein structural differences at two different temperatures.Rosetta is a de novo/comparative protein structure modeling algorithm. As one of the top-performing programs for protein structure prediction, it has predicted protein structures with high backbone accuracies. As a test case, we modeled the N-domain of Troponin C (NTnC) at both 4®ƒ and 30®ƒ, for which NMR structures are known. Rosetta-predicted structures of NTnC align with the NMR-determined structures at these two temperatures with ∼ 3 A backbone root mean square deviation. Our approach should be generally applicable to modeling temperature-dependent protein conformational rearrangements.

High Yield Production and Refolding of the Double-Knot Toxin, an Activator of TRPV1 Channels

A unique peptide toxin, named double-knot toxin (DkTx), was recently purified from the venom of the tarantula Ornithoctonus huwena and was found to stably activate TRPV1 channels by targeting the outer pore domain. DkTx has been shown to consist of two inhibitory cysteine-knot (ICK) motifs, referred to as K1 and K2, each containing six cysteine residues. Beyond this initial characterization, however, the structural and functional details about DkTx remains elusive in large part due to the lack of a high yielding methodology for the synthesis and folding of this cysteine-rich peptide. Here, we overcome this obstacle by generating pure DkTx in quantities sufficient for structural and functional analyses. Our methodology entails expression of DkTx in E. coli followed by oxidative folding of the isolated linear peptide. Upon screening of various oxidative conditions for optimizing the folding yield of the toxin, we observed that detergents were required for efficient folding of the linear peptide. Our synthetic DkTx co-eluted with the native toxin on HPLC, and irreversibly activated TRPV1 in a manner identical to native DkTx. Interestingly, we find that DkTx has two interconvertible conformations present in a 1∶6 ratio at equilibrium. Kinetic analysis of DkTx folding suggests that the K1 and K2 domains influence each other during the folding process. Moreover, the CD spectra of the toxins shows that the secondary structures of K1 and K2 remains intact even after separating the two knots. These findings provide a starting point for detailed studies on the structural and functional characterization of DkTx and utilization of this toxin as a tool to explore the elusive mechanisms underlying the polymodal gating of TRPV1.

Multiple mechanisms involved in the large-spectrum therapeutic potential of cannabidiol in psychiatric disorders

Cannabidiol (CBD) is a major phytocannabinoid present in the Cannabis sativa plant. It lacks the psychotomimetic and other psychotropic effects that the main plant compound Δ(9)-tetrahydrocannabinol (THC) being able, on the contrary, to antagonize these effects. This property, together with its safety profile, was an initial stimulus for the investigation of CBD pharmacological properties. It is now clear that CBD has therapeutic potential over a wide range of non-psychiatric and psychiatric disorders such as anxiety, depression and psychosis. Although the pharmacological effects of CBD in different biological systems have been extensively investigated by in vitro studies, the mechanisms responsible for its therapeutic potential are still not clear. Here, we review recent in vivo studies indicating that these mechanisms are not unitary but rather depend on the behavioural response being measured. Acute anxiolytic and antidepressant-like effects seem to rely mainly on facilitation of 5-HT1A-mediated neurotransmission in key brain areas related to defensive responses, including the dorsal periaqueductal grey, bed nucleus of the stria terminalis and medial prefrontal cortex. Other effects, such as anti-compulsive, increased extinction and impaired reconsolidation of aversive memories, and facilitation of adult hippocampal neurogenesis could depend on potentiation of anandamide-mediated neurotransmission. Finally, activation of TRPV1 channels may help us to explain the antipsychotic effect and the bell-shaped dose-response curves commonly observed with CBD. Considering its safety profile and wide range of therapeutic potential, however, further studies are needed to investigate the involvement of other possible mechanisms (e.g. inhibition of adenosine uptake, inverse agonism at CB2 receptor, CB1 receptor antagonism, GPR55 antagonism, PPARγ receptors agonism, intracellular (Ca(2+)) increase, etc.), on CBD behavioural effects.