Inhibition of endothelial cell Ca²? entry and transient receptor potential channels by Sigma-1 receptor ligands.
ABSTRACT: BACKGROUND AND PURPOSE:The Sigma-1 receptor (Sig1R) impacts on calcium ion signalling and has a plethora of ligands. This study investigated Sig1R and its ligands in relation to endogenous calcium events of endothelial cells and transient receptor potential (TRP) channels. EXPERIMENTAL APPROACH:Intracellular calcium and patch clamp measurements were made from human saphenous vein endothelial cells and HEK 293 cells expressing exogenous human TRPC5, TRPM2 or TRPM3. Sig1R ligands were applied and short interfering RNA was used to deplete Sig1R. TRP channels tagged with fluorescent proteins were used for subcellular localization studies. KEY RESULTS:In endothelial cells, 10-100 ?M of the Sig1R antagonist BD1063 inhibited sustained but not transient calcium responses evoked by histamine. The Sig1R agonist 4-IBP and related antagonist BD1047 were also inhibitory. The Sig1R agonist SKF10047 had no effect. Sustained calcium entry evoked by VEGF or hydrogen peroxide was also inhibited by BD1063, BD1047 or 4-IBP, but not SKF10047. 4-IBP, BD1047 and BD1063 inhibited TRPC5 or TRPM3, but not TRPM2. Inhibitory effects of BD1047 were rapid in onset and readily reversed on washout. SKF10047 inhibited TRPC5 but not TRPM3 or TRPM2. Depletion of Sig1R did not prevent the inhibitory actions of BD1063 or BD1047 and Sig1R did not co-localize with TRPC5 or TRPM3. CONCLUSIONS AND IMPLICATIONS:The data suggest that two types of Sig1R ligand (BD1047/BD1063 and 4-IBP) are inhibitors of receptor- or chemically activated calcium entry channels, acting relatively directly and independently of the Sig1R. Chemical foundations for TRP channel inhibitors are suggested.
Project description:The aim of this study was to generate new insight into chemical regulation of transient receptor potential (TRP) channels with relevance to glucose homeostasis and the metabolic syndrome. Human TRP melastatin 2 (TRPM2), TRPM3, and TRP canonical 5 (TRPC5) were conditionally overexpressed in human embryonic kidney 293 cells and studied by using calcium-measurement and patch-clamp techniques. Rosiglitazone and other peroxisome proliferator-activated receptor-? (PPAR-?) agonists were investigated. TRPM2 was unaffected by rosiglitazone at concentrations up to 10 ?M but was inhibited completely at higher concentrations (IC(50), ?22.5 ?M). TRPM3 was more potently inhibited, with effects occurring in a biphasic concentration-dependent manner such that there was approximately 20% inhibition at low concentrations (0.1-1 ?M) and full inhibition at higher concentrations (IC(50), 5-10 ?M). PPAR-? antagonism by 2-chloro-5-nitrobenzanilide (GW9662) did not prevent inhibition of TRPM3 by rosiglitazone. TRPC5 was strongly stimulated by rosiglitazone at concentrations of ?10 ?M (EC(50), ?30 ?M). Effects on TRPM3 and TRPC5 occurred rapidly and reversibly. Troglitazone and pioglitazone inhibited TRPM3 (IC(50), 12 ?M) but lacked effect on TRPC5, suggesting no relevance of PPAR-? or the thiazolidinedione moiety to rosiglitazone stimulation of TRPC5. A rosiglitazone-related but nonthiazolidinedione PPAR-? agonist, N-(2-benzoylphenyl)-O-[2-(methyl-2-pyridinylamino)ethyl]-l-tyrosine (GW1929), was a weak stimulator of TRPM3 and TRPC5. The natural PPAR-? agonist 15-deoxy prostaglandin J(2), had no effect on TRPM3 or TRPC5. The data suggest that rosiglitazone contains chemical moieties that rapidly, strongly, and differentially modulate TRP channels independently of PPAR-?, potentially contributing to biological consequences of the agent and providing the basis for novel TRP channel pharmacology.
Project description:Isoform-specific ion channel blockers are useful for target validation in drug discovery and can provide the basis for new therapeutic agents and aid in determination of physiological functions of ion channels. The aim of this study was to generate a specific blocker of human TRPM3 channels as a tool to help investigations of this member of the TRP cationic channel family.A polyclonal antibody (TM3E3) was made to a conserved peptide of the third extracellular (E3) loop of TRPM3 and tested for binding and functional effect. Studies of channel activity were made by whole-cell planar patch-clamp and fura-2 intracellular Ca(2+) measurement.Ionic current mediated by TRPM3 was inhibited partially by TM3E3 over a period of 5-10 min. Ca(2+) entry in TRPM3-expressing cells was also partially inhibited by TM3E3 in a peptide-specific manner and independently of the type of agonist used to activate TRPM3. TM3E3 had no effect on TRPC5, TRPV4, TRPM2 or an endogenous ATP response.The data show the successful development of a specific TRPM3 inhibitor and give further confidence in E3 targeting as an approach to producing isoform-specific ion channel blockers.
Project description:TRPC5 is a mammalian homologue of the Drosophila Transient Receptor Potential (TRP) channel and has expression and functions in the cardiovascular and nervous systems. It forms a calcium-permeable cation channel that can be activated by a variety of signals including carbachol (acting at muscarinic receptors), lanthanides (e.g. Gd3+) and phospholipids (e.g. lysophosphatidylcholine: LPC). Here we report the effects of inhalational (halothane and chloroform) and intravenous (propofol) general anaesthetics upon TRPC5.Human TRPC5 channels were expressed in HEK 293 cells and studied using fura-2 and patch-clamp recording to measure intracellular calcium and membrane currents respectively at room temperature. Human TRPM2 channels were studied for comparison.TRPC5 activation by carbachol, Gd3+ or LPC was inhibited by halothane and chloroform at > or =0.1 and 0.2 mM respectively. Neither agent inhibited TRPM2. Propofol had an initial stimulatory effect on TRPC5 (evident in patch-clamp recordings only) and an inhibitory effect at > or =10 microM. TRPM2 was not affected by propofol. Propofol inhibited activation of TRPC5 by Gd3+ but not LPC, suggesting the effect was not directly on the channel. Propofol's anti-oxidant property was not necessary for its inhibitory effect because di-isopropyl benzene, a propofol analogue that lacks the hydroxyl group, also inhibited TRPC5.The data show the sensitivity of TRPC5 channel to general anaesthetics and suggest that some of the effects could have clinical relevance. The effects may be explained in part by the sensitivity of the channel to biophysical properties of the lipid bilayer.
Project description:Members of the TRP superfamily of ion channels mediate mechanosensation in some organisms, and have been suggested as candidates for the mechanotransduction channel in vertebrate hair cells. Some TRP channels can be ruled out based on lack of an inner ear phenotype in knockout animals or pore properties not similar to the hair-cell channel. Such studies have excluded Trpv4, Trpa1, Trpml3, Trpm1, Trpm3, Trpc1, Trpc3, Trpc5, and Trpc6. However, others remain reasonable candidates. We used data from an RNA-seq analysis of gene expression in hair cells as well as data on TRP channel conductance to narrow the candidate group. We then characterized mice lacking functional Trpm2, Pkd2, Pkd2l1, Pkd2l2 and Pkd1l3, using scanning electron microscopy, auditory brainstem response, permeant dye accumulation, and single-cell electrophysiology. In all of these TRP-deficient mice, and in double and triple knockouts, mechanotransduction persisted. Together with published studies, these results argue against the participation of any of the 33 mouse TRP channels in hair cell transduction.
Project description:BACKGROUND AND PURPOSE: Fenamates are N-phenyl-substituted anthranilic acid derivatives clinically used as non-steroid anti-inflammatory drugs in pain treatment. Reports describing fenamates as tools to interfere with cellular volume regulation attracted our attention based on our interest in the role of the volume-modulated transient receptor potential (TRP) channels TRPM3 and TRPV4. EXPERIMENTAL APPROACH: Firstly, we measured the blocking potencies and selectivities of fenamates on TRPM3 and TRPV4 as well as TRPC6 and TRPM2 by Ca(2+) imaging in the heterologous HEK293 cell system. Secondly, we further investigated the effects of mefenamic acid on cytosolic Ca(2+) and on the membrane voltage in single HEK293 cells that exogenously express TRPM3. Thirdly, in insulin-secreting INS-1E cells, which endogenously express TRPM3, we validated the effect of mefenamic acid on cytosolic Ca(2+) and insulin secretion. KEY RESULTS: We identified and characterized mefenamic acid as a selective and potent TRPM3 blocker, whereas other fenamate structures non-selectively blocked TRPM3, TRPV4, TRPC6 and TRPM2. CONCLUSIONS AND IMPLICATIONS: This study reveals that mefenamic acid selectively inhibits TRPM3-mediated calcium entry. This selectivity was further confirmed using insulin-secreting cells. K(ATP) channel-dependent increases in cytosolic Ca(2+) and insulin secretion were not blocked by mefenamic acid, but the selective stimulation of TRPM3-dependent Ca(2+) entry and insulin secretion induced by pregnenolone sulphate were inhibited. However, the physiological regulator of TRPM3 in insulin-secreting cells remains to be elucidated, as well as the conditions under which the inhibition of TRPM3 can impair pancreatic ?-cell function. Our results strongly suggest mefenamic acid is the most selective fenamate to interfere with TRPM3 function.
Project description:1 2-aminoethoxydiphenyl borate (2-APB) has been widely used to examine the roles of inositol 1,4,5-trisphosphate receptors (IP3Rs) and store-operated Ca2+ entry and is an emerging modulator of cationic channels encoded by transient receptor potential (TRP) genes. 2 Using Ca2+-indicator dye and patch-clamp recording we first examined the blocking effect of 2-APB on human TRPC5 channels expressed in HEK-293 cells. 3 The concentration-response curve has an IC50 of 20 microM and slope close to 1.0, suggesting one 2-APB molecule binds per channel. The blocking effect is not shared by other Ca2+ channel blockers including methoxyverapamil, nifedipine, N-propargylnitrendipine, or berberine. 4 In whole-cell and excised membrane patch recordings, 2-APB acts from the extracellular but not intracellular face of the membrane. 5 Block of TRPC5 by 2-APB is less at positive voltages, suggesting that it enters the electric field or acts by modulating channel gating. 6 2-APB also blocks TRPC6 and TRPM3 expressed in HEK-293 cells, but not TRPM2. 7 Block of TRP channels by 2-APB may be relevant to cell proliferation because 2-APB has a greater inhibitory effect on proliferation in cells overexpressing TRPC5. 8 Our data indicate a specific and functionally important binding site on TRPC5 that enables block by 2-APB. The site is only available via an extracellular route and the block shows mild voltage-dependence.
Project description:Transient receptor potential canonical 5 (TRPC5) channels are widely expressed, including in the CNS, where they potentiate fear responses. They also contribute to other non-selective cation channels that are stimulated by G-protein-coupled receptor agonists and lipid and redox factors. Steroids are known to modulate fear and anxiety states, and we therefore investigated whether TRPC5 exhibited sensitivity to steroids.Human TRPC5 channels were conditionally expressed in HEK293 cells and studied using intracellular Ca2+ measurement, whole-cell voltage-clamp and excised patch techniques. For comparison, control experiments were performed with cells lacking TRPC5 channels or expressing another TRP channel, TRPM2. Native TRPC channel activity was recorded from vascular smooth muscle cells.Extracellular application of pregnenolone sulphate, pregnanolone sulphate, pregnanolone, progesterone or dihydrotestosterone inhibited TRPC5 activity within 1-2min. Dehydroepiandrosterone sulphate or 17?-oestradiol had weak inhibitory effects. Pregnenolone, and allopregnanolone, a progesterone metabolite and stereo-isomer of pregnanolone, all had no effects. Progesterone was the most potent of the steroids, especially against TRPC5 channel activity evoked by sphingosine-1-phosphate. In outside-out patch recordings, bath-applied progesterone and dihydrotestosterone had strong and reversible effects, suggesting relatively direct mechanisms of action. Progesterone inhibited native TRPC5-containing channel activity, evoked by oxidized phospholipid.Our data suggest that TRPC5 channels are susceptible to relatively direct and rapid stereo-selective steroid modulation, leading to channel inhibition. The study adds to growing appreciation of TRP channels as non-genomic steroid sensors.
Project description:Pathogenesis of HIV-associated neurocognitive disorders (HAND) is mediated through the infiltration of perivascular macrophages into the brain with the secretion of viral, neurotoxic and inflammatory proteins. One of these proteins is cathepsin B (CATB), a lysosomal cysteine protease that induces neuronal apoptosis, and increases in plasma and cerebrospinal fluid from HIV-1 infected patients (Cantres-Rosario et al. AIDS 27(3):347-356, 2013). Cocaine further potentiates CATB neurotoxicity in vitro and in vivo (Zenón et al. J NeuroImmune Pharmacol 9(5):703-715, 2014). Modulation of sigma-1 (Sig1R) by cocaine increases oxidative species, cytokines and other factors that promote lysosomal disruption. However, the role of Sig1R in CATB secretion and HIV-1 replication in macrophages exposed to cocaine is unknown. We hypothesized that pharmacological modulation of Sig1R would alter CATB secretion from HIV-1 infected macrophages in vitro and in vivo. To test our hypothesis, monocyte derived-macrophages (MDM) from HIV-1 seronegative donors were isolated, infected with HIV-1ADA, and pretreated with Sig1R antagonist (BD1047) or Sig1R agonist (PRE-084) prior to cocaine exposure and followed for 3,6,9 and 11 days post-infection (dpi). Experiments in vivo were conducted using the HIV encephalitis mouse model (HIVE) with BD1047 treatments prior to cocaine for 14 days. Results demonstrate that in presence of cocaine, BD1047 decreases CATB secretion at 11 dpi, while PRE-084 did not have an effect. In the mouse model, BD1047 treatment prior to cocaine decreased CATB expression, cleaved caspase-3 an p24 antigen levels, reduced astrocytosis, but did not increase MAP-2 or synaptophysin. Results demonstrate that Sig1R plays a role in the modulation of CATB levels in HIV-1 infected MDM exposed to cocaine in vitro and in vivo. Graphical Abstract ?.
Project description:Thermally activated ion channels are known to detect the entire thermal range from extreme heat (TRPV2), painful heat (TRPV1, TRPM3 and ANO1), non-painful warmth (TRPV3 and TRPV4) and non-painful coolness (TRPM8) through to painful cold (TRPA1). Genetic deletion of each of these ion channels, however, has only modest effects on thermal behaviour in mice, with the exception of TRPM8, the deletion of which has marked effects on the perception of moderate coolness in the range 10-25?°C. The molecular mechanism responsible for detecting non-painful warmth, in particular, is unresolved. Here we used calcium imaging to identify a population of thermally sensitive somatosensory neurons which do not express any of the known thermally activated TRP channels. We then used a combination of calcium imaging, electrophysiology and RNA sequencing to show that the ion channel generating heat sensitivity in these neurons is TRPM2. Autonomic neurons, usually thought of as exclusively motor, also express TRPM2 and respond directly to heat. Mice in which TRPM2 had been genetically deleted showed a striking deficit in their sensation of non-noxious warm temperatures, consistent with the idea that TRPM2 initiates a 'warm' signal which drives cool-seeking behaviour.
Project description:BACKGROUND: Somatosensory nerve fibres arising from cell bodies within the trigeminal ganglia (TG) in the head and from a string of dorsal root ganglia (DRG) located lateral to the spinal cord convey endogenous and environmental stimuli to the central nervous system. Although several members of the transient receptor potential (TRP) superfamily of cation channels have been implicated in somatosensation, the expression levels of TRP channel genes in the individual sensory ganglia have never been systematically studied. RESULTS: Here, we used quantitative real-time PCR to analyse and compare mRNA expression of all TRP channels in TG and individual DRGs from 27 anatomically defined segments of the spinal cord of the mouse. At the mRNA level, 17 of the 28 TRP channel genes, TRPA1, TRPC1, TRPC3, TRPC4, TRPC5, TRPM2, TRPM3, TRPM4, TRPM5, TRPM6, TRPM7, TRPM8, TRPV1, TRPV2, TRPV4, TRPML1 and TRPP2, were detectable in every tested ganglion. Notably, four TRP channels, TRPC4, TRPM4, TRPM8 and TRPV1, showed statistically significant variation in mRNA levels between DRGs from different segments, suggesting ganglion-specific regulation of TRP channel gene expression. These ganglion-to-ganglion differences in TRP channel transcript levels may contribute to the variability in sensory responses in functional studies. CONCLUSIONS: We developed, compared and refined techniques to quantitatively analyse the relative mRNA expression of all TRP channel genes at the single ganglion level. This study also provides for the first time a comparative mRNA distribution profile in TG and DRG along the entire vertebral column for the mammalian TRP channel family.