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Objective To investigate the influence of transient receptor potential cation channel 6 (TRPC6) on renal tubulointerstitial inflammation in mice with diabetic kidney disease (DKD) and its mechanism. Methods A total of 36 male C57/BL6J mice, aged 6 weeks, were randomly divided into control group (group A), DKD model group (group B), DKD+normal saline intervention group (group C), DKD+mitophagy activator intervention group (group D), DKD+negative control lentivirus transfection group (group E), and DKD+TRPC6 knockdown lentivirus transfection group (group F), with 6 mice in each group. The mice in groups A and B were respectively given intraperitoneal injection of 0.1 mmol/L citrate buffer and 10 g/L streptozotocin (hereinafter referred to as administration); the mice in groups C and D were respectively given normal saline and 10 mmol/L urolithin A by gavage in addition to the treatment in group B; the mice in groups E and F were respectively injected with negative control lentivirus and TRPC6 knockdown lentivirus via the tail vein in addition to the treatment in group B. The levels of fasting blood glucose (FBG), urinary albumin-to-creatinine ratio (ACR), and blood urea nitrogen (BUN) were measured at week 12 after administration; PAS staining was used for the observation and scoring of renal tubular injury; RT-qPCR was used to measure the mRNA expression levels of inflammatory factors [interleukin-1β (IL-1β), monocyte chemoattractant protein-1 (MCP-1), and tumor necrosis factor-α (TNF-α)] in renal tissue; immunohistochemical staining was used to measure the protein expression level of TRPC6 in renal tissue, and Western blotting was used to measure the relative expression levels of TRPC6, LC3B, P62, PINK1, and Parkin in renal tissue; transmission electron microscopy was used to observe the change in the number of mitophagosomes in renal tubular cells of mice. HK-2 cells were divided into high glucose+TRPC6 siRNA transfection+DMSO intervention group (group G, treated with TRPC6 siRNA+35.0 mmol/L glucose+0.06% DMSO) and high glucose+TRPC6 siRNA transfection+mitophagy inhibitor intervention group (group H, treated with TRPC6 siRNA+35.0 mmol/L glucose+12 μmol/L oroxylin A), and then RT-qPCR was used to measure the mRNA expression levels of inflammatory factors (IL-1β, MCP-1, and TNF-α) in cells. Results At week 12 after administration, compared with group A, group B had significantly higher whole blood FBG, ACR, BUN, renal tubular injury score, mRNA expression levels of inflammatory factors, and protein expression levels of TRPC6 and P62 in renal tissue (t=2.77-13.61,P

Drosophila phototransduction is a model for signaling cascades that culminate in the activation of transient receptor potential (TRP) cation channels. TRP and TRPL are the canonical TRP (TRPC) channels that are regulated by light stimulation of rhodopsin and engagement of Gαq and phospholipase Cβ (PLC). Lipid metabolite(s) generated downstream of PLC are essential for the activation of the TRPC channels in photoreceptor cells. We sought to identify the key lipids produced subsequent to PLC stimulation that contribute to channel activation. Here, using genetics, lipid analysis, and Ca2+ imaging, we found that light increased the amount of an abundant endocannabinoid, 2-linoleoyl glycerol (2-LG), in vivo. The increase in 2-LG amounts depended on the PLC and diacylglycerol lipase encoded by norpA and inaE, respectively. This endocannabinoid facilitated TRPC-dependent Ca2+ influx in a heterologous expression system and in dissociated ommatidia from compound eyes. Moreover, 2-LG and mechanical stimulation cooperatively activated TRPC channels in ommatidia. We propose that 2-LG is a physiologically relevant endocannabinoid that activates TRPC channels in photoreceptor cells.

N -Phenethyl-1-phenyl-pentan-3-amine (1) is a new compound synthesised as a simplified analogue of schwarzinicine A (2), a natural compound extracted from Ficus schwarzii. Compound 1 differs from compound 2 due to its structural simplification, featuring two phenyl rings without methoxy substitution, as opposed to compound 2 , which possesses three 3,4-dimethoxy aromatic rings. Our previous research findings highlighted the calcium-inhibitory effects of compound 2 , but the mechanism of action for compound 1 remains unexplored, serving as the primary focus of this study. Building upon our earlier research, this study aimed to elucidate compound 1 's calcium-modulating potential by using rat-isolated aortae in an organ bath set-up and HEK cells expressing hTRPC channels with the fluorometric assay to measure calcium influx. Compound 1 elicited a vasorelaxation response (E max 111.4%) similar to its parent compound 2 (E max 123.1%), and inhibited hTRPC3-, hTRPC4-, hTRPC5-, and hTRPC6-mediated calcium influx into HEK cells with IC 50 values of 6, 2, 2, 5 µM, respectively. Compound 1 has a similar pharmacological profile as its parent compound 2 , whereby it exerts a vasorelaxant effect by attenuating calcium influx and inhibits multiple TRPC channels. [Display omitted] • N -Phenethyl-1-phenyl-pentan-3-amine (1) is a analogue of Schwarzinicine A (2) that is extracted from Ficus schwarzii. • Compound 1 elicits a vasorelaxation response similar to its parent compound 2. • Both compounds inhibit calcium influx via hTRPC3-, hTRPC4-, hTRPC5-, and hTRPC6. [ABSTRACT FROM AUTHOR]

Drosophila TRP is a calcium-permeable cation channel essential for fly visual signal transduction. During phototransduction, Ca2+ mediates both positive and negative feedback regulation on TRP channel activity, possibly via binding to calmodulin (CaM). However, the molecular mechanism underlying Ca2+ modulated CaM/TRP interaction is poorly understood. Here, we discover an unexpected, Ca2+-dependent binding mode between CaM and TRP. The TRP tail contains two CaM binding sites (CBS1 and CBS2) separated by an ∼70-residue linker. CBS1 binds to the CaM N-lobe and CBS2 recognizes the CaM C-lobe. Structural studies reveal the lobe-specific binding of CaM to CBS1&2. Mutations introduced in both CBS1 and CBS2 eliminated CaM binding in full-length TRP, but surprisingly had no effect on the response to light under physiological conditions, suggesting alternative mechanisms governing Ca2+-mediated feedback on the channel activity. Finally, we discover that TRPC4, the closest mammalian paralog of Drosophila TRP, adopts a similar CaM binding mode.

Excessive collagen deposition by myofibroblasts during adverse cardiac remodeling leads to myocardial fibrosis that can compromise cardiac function. Unraveling the mechanisms underlying collagen gene expression in cardiac myofibroblasts is therefore an important clinical goal. The collagen receptors, discoidin domain receptor 2 (DDR2), a collagen-specific receptor tyrosine kinase, and integrin-β1, are reported to mediate tissue fibrosis. Here, we probed the role of DDR2-integrin-β1 cross-talk in the regulation of collagen α1(I) gene expression in angiotensin II (Ang II)-stimulated cardiac fibroblasts. Results from gene silencing/overexpression approaches, electrophoretic mobility shift assays, and ChIP revealed that DDR2 acts via extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase (ERK1/2 MAPK)-dependent transforming growth factor-β1 (TGF-β1) signaling to activate activator protein-1 (AP-1) that in turn transcriptionally enhances the expression of collagen-binding integrin-β1 in Ang II-stimulated cardiac fibroblasts. The DDR2-integrin-β1 link was also evident in spontaneously hypertensive rats and DDR2-knockout mice. Further, DDR2 acted via integrin-β1 to regulate α-smooth muscle actin (α-SMA) and collagen type I expression in Ang II-exposed cardiac fibroblasts. Downstream of the DDR2-integrin-β1 axis, α-SMA was found to regulate collagen α1(I) gene expression via the Ca2+ channel, transient receptor potential cation channel subfamily C member 6 (TRPC6), and the profibrotic transcription factor, Yes-associated protein (YAP). This finding indicated that fibroblast-to-myofibroblast conversion is mechanistically coupled to collagen expression. The observation that collagen receptor cross-talk underlies α-SMA-dependent collagen type I expression in cardiac fibroblasts expands our understanding of the complex mechanisms involved in collagen gene expression in the heart and may be relevant to cardiac fibrogenesis.

Williams syndrome (WS) and 7q11.23 duplication syndrome (Dup7q11.23) are neurodevelopmental disorders caused by the deletion and duplication, respectively, of ~ 25 protein-coding genes on chromosome 7q11.23. The general transcription factor 2I (GTF2I, protein TFII-I) is one of these proteins and has been implicated in the neurodevelopmental phenotypes of WS and Dup7q11.23. Here, we investigated the effect of copy number alterations in Gtf2i on neuronal maturation and intracellular calcium entry mechanisms known to be associated with this process. Mice with a single copy of Gtf2i (Gtf2i+/Del) had increased axonal outgrowth and increased TRPC3-mediated calcium entry upon carbachol stimulation. In contrast, mice with 3 copies of Gtf2i (Gtf2i+/Dup) had decreases in axon outgrowth and in TRPC3-mediated calcium entry. The underlying mechanism was that TFII-I did not affect TRPC3 protein expression, while it regulated TRPC3 membrane translocation. Together, our results provide novel functional insight into the cellular mechanisms that underlie neuronal maturation in the context of the 7q11.23 disorders.

The human transient receptor potential canonical 5 (TRPC5) is a calcium‐permeable, nonselective cation channel expressed in the central and peripheral nervous system and also in other tissues such as the kidney, synovium, and odontoblasts. TRPC5 has been recently confirmed to play a key role in spontaneous, inflammatory mechanical, and cold pain. Although TRPC5 activation is known to be cold sensitive, it is unclear whether this property is intrinsic to the channel protein and whether or to what extent it may be determined by the cellular environment. In this study, we explored the cold sensitivity of human TRPC5 at the single‐channel level using transiently transfected HEK293T cells. Upon decreasing the temperature, the channel demonstrated prolonged mean open dwell times and a robust increase in the open probability (Po), whereas the amplitude of unitary currents decreased ~1.5‐fold per 10°C of temperature difference. In the absence of any agonists, the temperature dependence of Po was sigmoidal, with a steep slope within the temperature range of 16°C–11°C, and exhibited saturation below 8–5°C. Thermodynamic analysis revealed significant changes in enthalpy and entropy, suggesting that substantial conformational changes accompany cold‐induced gating. The mutant channel T970A, in which the regulation downstream of G‐protein coupled receptor signaling was abrogated, exhibited higher basal activity at room temperature and a less steep temperature response profile, with an apparent threshold below 22°C. An even more pronounced decrease in the activation threshold was observed in a mutant that disrupted the electrostatic interaction of TRPC5 with the endoplasmic reticulum calcium sensor stromal interaction molecule 1. Thus, TRPC5 exhibits features of an intrinsically cold‐gated channel; its sensitivity to cold tightly depends on the phosphorylation status of the protein and intracellular calcium homeostasis. [ABSTRACT FROM AUTHOR]

The SMOOTHENED inhibitor vismodegib is FDA approved for advanced basal cell carcinoma (BCC), and shows promise in clinical trials for SONIC HEDGEHOG (SHH)-subgroup medulloblastoma (MB) patients. Clinical experience with BCC patients shows that continuous exposure to vismodegib is necessary to prevent tumor recurrence, suggesting the existence of a vismodegib-resistant reservoir of tumor-propagating cells. We isolated such tumor-propagating cells from a mouse model of SHH-subgroup MB and grew them as sphere cultures. These cultures were enriched for the MB progenitor marker SOX2 and formed tumors in vivo. Moreover, while their ability to self-renew was resistant to SHH inhibitors, as has been previously suggested, this self-renewal was instead WNT-dependent. We show here that loss of Trp53 activates canonical WNT signaling in these SOX2-enriched cultures. Importantly, a small molecule WNT inhibitor was able to reduce the propagation and growth of SHH-subgroup MB in vivo, in an on-target manner, leading to increased survival. Our results imply that the tumor-propagating cells driving the growth of bulk SHH-dependent MB are themselves WNT dependent. Further, our data suggest combination therapy with WNT and SHH inhibitors as a therapeutic strategy in patients with SHH-subgroup MB, in order to decrease the tumor recurrence commonly observed in patients treated with vismodegib.

Avoidance of noxious ambient heat is crucial for survival. A well-known phenomenon is that animals are sensitive to the rate of temperature change. However, the cellular and molecular underpinnings through which animals sense and respond much more vigorously to fast temperature changes are unknown. Using Drosophila larvae, we found that nociceptive rolling behavior was triggered at lower temperatures and at higher frequencies when the temperature increased rapidly. We identified neurons in the brain that were sensitive to the speed of the temperature increase rather than just to the absolute temperature. These cellular and behavioral responses depended on the TRPA1 channel, whose activity responded to the rate of temperature increase. We propose that larvae use low-threshold sensors in the brain to monitor rapid temperature increases as a protective alert signal to trigger rolling behaviors, allowing fast escape before the temperature of the brain rises to dangerous levels.

Patch clamp studies from dorsal root ganglia (DRGs) neurons have increased our understanding of the peripheral nervous system. Currently, the majority of recordings are conducted on dissociated DRG neurons, which is a standard preparation for most laboratories. Neuronal properties, however, can be altered by axonal injury resulting from enzyme digestion used in acquiring dissociated neurons. Further, dissociated neuron preparations cannot fully represent the microenvironment of the DRG since loss of contact with satellite glial cells that surround the primary sensory neurons is an unavoidable consequence of this method. To overcome the limitations in using conventional dissociated DRG neurons for patch clamp recordings, in this report we describe a method to prepare intact DRGs and conduct patch clamp recordings on individual primary sensory neurons ex vivo. This approach permits the fast and straightforward preparation of intact DRGs, mimicking in vivo conditions by keeping DRG neurons associated with their surrounding satellite glial cells and basement membrane. Furthermore, the method avoids axonal injury from manipulation and enzyme digestion such as when dissociating DRGs. This ex vivo preparation can additionally be used to study the interaction between primary sensory neurons and satellite glial cells.

Hunger is a complex motivational state that drives multiple behaviors. The sensation of hunger is caused by an imbalance between energy intake and expenditure. One immediate response to hunger is increased food consumption. Hunger also modulates behaviors related to food seeking such as increased locomotion and enhanced sensory sensitivity in both insects and vertebrates. In addition, hunger can promote the expression of food-associated memory. Although progress is being made, how hunger is represented in the brain and how it coordinates these behavioral responses is not fully understood in any system. Here, we use Drosophila melanogaster to identify neurons encoding hunger. We found a small group of neurons that, when activated, induced a fed fly to eat as though it were starved, suggesting that these neurons are downstream of the metabolic regulation of hunger. Artificially activating these neurons also promotes appetitive memory performance in sated flies, indicating that these neurons are not simply feeding command neurons but likely play a more general role in encoding hunger. We determined that the neurons relevant for the feeding effect are serotonergic and project broadly within the brain, suggesting a possible mechanism for how various responses to hunger are coordinated. These findings extend our understanding of the neural circuitry that drives feeding and enable future exploration of how state influences neural activity within this circuit.

The Drosophila mushroom bodies are critical association areas whose role in olfactory associative learning has been well characterized. Recent behavioral studies using a taste association paradigm revealed that gustatory conditioning also requires the mushroom bodies (Masek and Scott, 2010; Keene and Masek, 2012). Here, we examine the representations of tastes and the neural sites for taste associations in the mushroom bodies. Using molecular genetic approaches to target different neuronal populations, we find that the gamma lobes of the mushroom bodies and a subset of dopaminergic input neurons are required for taste associative learning. Monitoring responses to taste compounds in the mushroom body calyx with calcium imaging reveals sparse, taste-specific and organ-specific activation in the Kenyon cell dendrites of the main calyx and the dorsal accessory calyx. Our work provides insight into gustatory representations in the mushroom bodies, revealing the essential role of gustatory inputs not only as rewards and punishments but also as adaptive cues.

Daily rhythms in Drosophila under semi-natural conditions (or SN) have received much recent attention. One of the striking differences in the behaviour of wild type flies under SN is the presence of an additional peak of activity in the middle of the day. This is referred to as the afternoon peak (A-peak) and is absent under standard laboratory regimes using gated light and temperature cues. Although previous reports identified the physical factors that contribute towards the A-peak there is no evidence for underlying molecular mechanisms or pathways that control A-peak. We report that the A-peak is mediated by thermosensitive dTRPA1 (drosophila Transient Receptor Potential- A1) ion channels as this peak is absent in dTRPA1 null mutants. Further, when natural cycles of light and temperature are simulated in the lab, we find that the amplitude of the A-peak is dTRPA1-dependent. Although a few circadian neurons express dTRPA1, we show that modulation of A-peak is primarily influenced by non-CRY dTRPA1 expressing neurons. Hence, we propose that A-peak of activity observed under SN is a temperature sensitive response in flies that is elicited through dTRPA1 receptor signalling.

Objectives A previous in vitro study reported that the monoterpene oxide 1,8-cineole (cineole) attenuates neuronal caused by oxygen--glucose deprivation/reoxygenation in culture. However, to date, there is no in vivo evidence showing neuroprotective effects of cineole against stroke. This study aimed to investigate whether cineole attenuates cerebral ischaemic damage in rats. Methods A rat model of middle cerebral artery occlusion (MCAO) followed by 24 h reperfusion was applied. Male rats were treated with oral cineole (100 mg/kg) for 7 consecutive days, then subjected to MCAO surgery. Infarct volume, neurologic deficits, apoptosis and expression levels of all-spectrin breakdown products of 145 kDa (SBDP145), transient receptor potential canonical (subtype) 6 (TRPC6) and phosphorylated CREB (p-CREB) were measured in ischaemic brain tissues. Key findings Cineole treatment significantly reduced infarct volume, neurological dysfunction, neuronal apoptosis, SBDP145 formation and TRPC6 degradation and enhanced p-CREB expression in MCAO rats compared with vehicle treatment. These neuroprotective effects were markedly suppressed by pharmacological inhibition of MEK or CaMKIV signalling. Conclusions Our study provides in vivo evidence demonstrating that cineole pretreatment attenuates ischaemic stroke-induced brain damage, mainly through blocking calpain-induced TRPC6 degradation and activating CREB via MEK/CREB and CaMKIV/CREB signalling pathways. [ABSTRACT FROM AUTHOR]

Odorant cues are recognized by receptors expressed on olfactory sensory neurons, the primary sensory neurons of the olfactory epithelium. Odorant receptors typically obey the "one receptor, one neuron" rule, in which the receptive field of the olfactory neuron is determined by the singular odorant receptor that it expresses. Odor-evoked receptor activity across the population of olfactory neurons is then interpreted by the brain to identify the molecular nature of the odorant stimulus. In the present study, we characterized the properties of a C family G-protein-coupled receptor that, unlike most other odorant receptors, is expressed in a large population of microvillous sensory neurons in the zebrafish olfactory epithelium and the mouse vomeronasal organ. We found that this receptor, OlfCc1 in zebrafish and its murine ortholog Vmn2r1, is a calcium-dependent, low-sensitivity receptor specific for the hydrophobic amino acids isoleucine, leucine, and valine. Loss-of-function experiments in zebrafish embryos demonstrate that OlfCc1 is required for olfactory responses to a diverse mixture of polar, nonpolar, acidic, and basic amino acids. OlfCc1 was also found to promote localization of other OlfC receptor family members to the plasma membrane in heterologous cells. Together, these results suggest that the broadly expressed OlfCc1 is required for amino acid detection by the olfactory system and suggest that it plays a role in the function and/or intracellular trafficking of other olfactory and vomeronasal receptors with which it is coexpressed.

We recorded the activity of single mechanosensitive (MS) ion channels from membrane patches on single muscle fibers isolated from mice. We investigated the actions of various TRP (transient receptor potential) channel blockers on MS channel activity. 2-aminoethoxydiphenyl borate (2-APB) neither inhibited nor facilitated single channel activity at submillimolar concentrations. The absence of an effect of 2-APB indicates MS channels are not composed purely of TRPC or TRPV1, 2 or 3 proteins. Exposing patches to 1-oleolyl-2-acetyl-sn-glycerol (OAG), a potent activator of TRPC channels, also had no effect on MS channel activity. In addition, flufenamic acid and spermidine had no effect on the activity of single MS channels. By contrast, SKF-96365 and ruthenium red blocked single-channel currents at micromolar concentrations. SKF-96365 produced a rapid block of the open channel current. The blocking rate depended linearly on blocker concentration, while the unblocking rate was independent of concentration, consistent with a simple model of open channel block. A fit to the concentration-dependence of block gave k(on) = 13 x 10 ( 6) M (-1) s (-1) and k(off) = 1609 sec (-1) with K(D) = ~124 µM. Block by ruthenium red was complex, involving both reduction of the amplitude of the single-channel current and increased occupancy of subconductance levels. The reduction in current amplitude with increasing concentration of ruthenium red gave a K(D) = ~49 µM. The high sensitivity of MS channels to block by ruthenium red suggests MS channels in skeletal muscle contain TRPV subunits. Recordings from skeletal muscle isolated from TRPV4 knockout mice failed to show MS channel activity, consistent with a contribution of TRPV4. In addition, exposure to hypo-osmotic solutions increases opening of MS channels in muscle. Our results provide evidence TRPV4 contributes to MS channels in skeletal muscle.

The hereditary cerebellar ataxias are a diverse group of neurodegenerative disorders primarily characterised by loss of balance and coordination due to dysfunction of the cerebellum and its associated pathways. Although many genetic mutations causing inherited cerebellar ataxia have been identified, a significant percentage of patients remain whose cause is unknown. The transient receptor potential (TRP) family member TRPC3 is a non-selective cation channel linked to key signalling pathways that are affected in cerebellar ataxia. Furthermore, genetic mouse models of TRPC3 dysfunction display cerebellar ataxia, making the TRPC3 gene an excellent candidate for screening ataxic patients with unknown genetic aetiology. Here, we report a genetic screen for TRPC3 mutations in a cohort of 98 patients with genetically undefined late-onset cerebellar ataxia and further ten patients with undefined episodic ataxia. We identified a number of variants but no causative mutations in TRPC3. Our findings suggest that mutations in TRPC3 do not significantly contribute to the cause of late-onset and episodic human cerebellar ataxias.

Background/Aims To investigate an effect of ML204 (an inhibitor of transient receptor potential canonical 4 and 5 [TRPC4/5] channels) on interstitial cells of Cajal (ICCs) and therefore determine whether TRPC4/5 channels act on ICC-generated pacemaker activity. Methods We enforced whole cell patch clamp analysis, measurements of the intracellular Ca2+ concentration, and reverse transcription polymerase chain reaction to determine the effect of ML204 (10 μM) or englerin A (a selective activator of TRPC4/5 channeles, 10 μM) and the existence of TRPC4/5 in mouse small intestinal ICC. Results Treatment of ICCs with ML204 or englerin A caused the membrane potentials to depolarize. This depolarization effect of membrane potentials by ML204 in ICCs was observed to be concentration-dependent. After treating Ca2+- and Na+-free solutions or flufenamic acid (a non-selective cation channel blocker), the pacemaker potentials in the ICCs were abolished. A specific anoctamin 1 channel blocker did not have any effect on the pacemaker activity in ML204-untreated control cells; however, they blocked ML204-induced pacemaker activity in ICCs. Specific primers designed against TRPC4 and TRPC5 detected the presence of TRPC4/5 in small intestinal ICCs, and the application of ML204 increased raise the frequency of Ca2+ oscillations in ICCs, as assessed using Fluo-4 AM. Conclusion The results implied that ML204 could not inhibit the pacemaker activity but depolarized the membrane potential of ICCs by regulating intracellular Ca2+ oscillations and anoctamin 1 channels. [ABSTRACT FROM AUTHOR]

Animals sense changes in ambient temperature irrespective of whether core body temperature is internally maintained (homeotherms) or subject to environmental variation (poikilotherms). Here we show that a cold-sensitive ion channel, TRPM8, displays dramatically different thermal activation ranges in frogs versus mammals or birds, consistent with variations in these species' cutaneous and core body temperatures. Thus, somatosensory receptors are not static through evolution, but show functional diversity reflecting the characteristics of an organism's ecological niche.

Objective: To characterize the calcium influx pathways implicated in the sustained elevation of endothelial intracellular calcium concentration, required for the synthesis and release of relaxing factors. Methods: We evaluated the effect of the newly synthesized pyrazole derivatives, described as selective inhibitors for ORAI (BTP2/Pyr2 and Pyr6) and TRPC3 (Pyr3 and Pyr10) channels, upon endothelium- and extracellular calcium-dependent relaxations stimulated by acetylcholine and thapsigargin, in pre-constricted rat thoracic aortic rings. Results: Acetylcholine and thapsigargin responses were completely reverted by Pyr2 and Pyr6 (1 to 3µM). Pyr3 (0.3 to 3µM) caused a rapid reversal of acetylcholine (6.2±0.08mg.s-1) and thapsigargin (3.9±0.25mg.s-1) relaxations, whereas the more selective TRPC3 blocker Pyr10 (1 to 3µM) had no effect. The recently described TRPC4/5 selective blocker, ML204 (1 to 3µM), reverted completely acetylcholine relaxations, but minimally thapsigargin induced ones. Noteworthy, relaxations elicited by GSK1016790A (TRPV4 agonist) were unaffected by pyrazole compounds or ML204. After Pyr2 and Pyr6 pre-incubation, acetylcholine and thapsigargin evoked transient relaxations similar in magnitude and kinetics to those observed in the absence of extracellular calcium. Sodium nitroprusside relaxations as well as phenylephrine-induced contractions (denuded aorta) were not affected by any of pyrazole compounds (1 to 3µM). Conclusion: These observations revealed a previously unrecognized complexity in rat aorta endothelial calcium influx pathways, which result in production and release of nitric oxide. Pharmacologically distinguishable pathways mediate acetylcholine (ORAI/TRPC other than TRPC3/TRPC4 calcium-permeable channels) and thapsigargin (TRPC4 not required) induced calcium influx. [ABSTRACT FROM AUTHOR]

Cardiac hypertrophy, induced by neurohumoral factors, including angiotensin II and endothelin-1, is a major predisposing factor for heart failure. These ligands can induce hypertrophic growth of neonatal rat cardiomyocytes (NRCMs) mainly through Ca2+-dependent calcineurin/nuclear factor of activated T cell (NFAT) signaling pathways activated by diacylglycerol-activated transient receptor potential canonical 3 and 6 (TRPC3/6) heteromultimer channels. Although extracellular nucleotide, adenosine 5′-triphosphate (ATP), is also known as most potent Ca2+-mobilizing ligand that acts on purinergic receptors, ATP never induces cardiomyocyte hypertrophy. Here we show that ATP-induced production of nitric oxide (NO) negatively regulates hypertrophic signaling mediated by TRPC3/6 channels in NRCMs. Pharmacological inhibition of NO synthase (NOS) potentiated ATP-induced increases in NFAT activity, protein synthesis, and transcriptional activity of brain natriuretic peptide. ATP significantly increased NO production and protein kinase G (PKG) activity compared to angiotensin II and endothelin-1. We found that ATP-induced Ca2+ signaling requires inositol 1,4,5-trisphosphate (IP3) receptor activation. Interestingly, inhibition of TRPC5, but not TRPC6 attenuated ATP-induced activation of Ca2+/NFAT-dependent signaling. As inhibition of TRPC5 attenuates ATP-stimulated NOS activation, these results suggest that NO-cGMP-PKG axis activated by IP3-mediated TRPC5 channels underlies negative regulation of TRPC3/6-dependent hypertrophic signaling induced by ATP stimulation.

Cardiac hypertrophy, induced by neurohumoral factors, including angiotensin II and endothelin-1, is a major predisposing factor for heart failure. These ligands can induce hypertrophic growth of neonatal rat cardiomyocytes (NRCMs) mainly through Ca2C-dependent calcineurin/nuclear factor of activated T cell (NFAT) signaling pathways activated by diacylglycerol-activated transient receptor potential canonical 3 and 6 (TRPC3/6) heteromultimer channels. Although extracellular nucleotide, adenosine 50-triphosphate (ATP), is also known as most potent Ca2C-mobilizing ligand that acts on purinergic receptors, ATP never induces cardiomyocyte hypertrophy. Here we show that ATP-induced production of nitric oxide (NO) negatively regulates hypertrophic signaling mediated by TRPC3/6 channels in NRCMs. Pharmacological inhibition of NO synthase (NOS) potentiated ATP-induced increases in NFAT activity, protein synthesis, and transcriptional activity of brain natriuretic peptide. ATP significantly increased NO production and protein kinase G (PKG) activity compared to angiotensin II and endothelin-1. We found that ATP-induced Ca2C signaling requires inositol 1,4,5-trisphosphate (IP3) receptor activation. Interestingly, inhibition of TRPC5, but not TRPC6 attenuated ATP-induced activation of Ca2C/NFAT-dependent signaling. As inhibition of TRPC5 attenuates ATP-stimulated NOS activation, these results suggest that NO-cGMP-PKG axis activated by IP3-mediated TRPC5 channels underlies negative regulation of TRPC3/6-dependent hypertrophic signaling induced by ATP stimulation. [ABSTRACT FROM AUTHOR]

Objective Diabetic retinopathy (DR) is induced by an accumulation of reactive metabolites such as ROS, RNS, and RCS species, which were reported to modulate the activity of cation channels of the TRPC family. In this study, we use Trpc1/4/5/6 −/− compound knockout mice to analyze the contribution of these TRPC proteins to diabetic retinopathy. Methods We used Nanostring- and qPCR-based analysis to determine mRNA levels of TRPC channels in control and diabetic retinae and retinal cell types. Chronic hyperglycemia was induced by Streptozotocin (STZ) treatment. To assess the development of diabetic retinopathy, vasoregression, pericyte loss, and thickness of individual retinal layers were analyzed. Plasma and cellular methylglyoxal (MG) levels, as well as Glyoxalase 1 (GLO1) enzyme activity and protein expression, were measured in WT and Trpc1/4/5/6 −/− cells or tissues. MG-evoked toxicity in cells of both genotypes was compared by MTT assay. Results We find that Trpc1/4/5/6 −/− mice are protected from hyperglycemia-evoked vasoregression determined by the formation of acellular capillaries and pericyte drop-out. In addition, Trpc1/4/5/6 −/− mice are resistant to the STZ-induced reduction in retinal layer thickness. The RCS metabolite methylglyoxal, which represents a key mediator for the development of diabetic retinopathy, was significantly reduced in plasma and red blood cells (RBCs) of STZ-treated Trpc1/4/5/6 −/− mice compared to controls. GLO1 is the major MG detoxifying enzyme, and its activity and protein expression were significantly elevated in Trpc1/4/5/6 -deficient cells, which led to significantly increased resistance to MG toxicity. GLO1 activity was also increased in retinal extracts from Trpc1/4/5/6 −/− mice. The TRPCs investigated here are expressed at different levels in endothelial and glial cells of the retina. Conclusion The protective phenotype in diabetic retinopathy observed in Trpc1/4/5/6 −/− mice is suggestive of a predominant action of TRPCs in Müller cells and microglia because of their central position in the retention of a proper homoeostasis of the neurovascular unit. [ABSTRACT FROM AUTHOR]