Transient receptor potential channels TRPC1/TRPC6 regulate lamina cribrosa cell extracellular matrix gene transcription and proliferation.

The lamina cribrosa (LC) in glaucoma is with augmented production of extracellular matrix proteins (ECM) and connective tissue fibrosis. Fundamental pathological mechanisms for this fibrosis comprise fibrotic growth factors and oxidative stress. Transient receptor potential canonical channels (TRPC) channels play a key role in ECM fibrosis. Here, we study TRPC expression in glaucomatous LC cells, and investigate the role of TRPC in oxidative stress induced-profibrotic ECM gene transcription and cell proliferation in normal LC cells. Age-matched human LC cells (normal, n = 3 donors; glaucoma, n = 3 donors) were used. Hydrogen peroxide (H2O2, 100 µM), was used to induce oxidative stress in LC cells in the presence or absence of the pan TRPC inhibitor SKF96365 (10 µM) or knockdown of TRPC1/6 with siRNA. After treatments, ECM gene transcription, LC cell viability and proliferation and the phosphorylation of the transcription factor NFATc3, were measured using real time RT-PCR, colorimetric cell counting with the methyl-thiazolyl tetrazolium salt (MTS) assay, and Western immunoblotting, respectively. Results showed that TRPC1/C6 transcript and protein expression levels were significantly (p < 0.05) enhanced in glaucoma LC cells. Both SKF96365 and siRNA-TRPC1/C6 treatments significantly reduced the oxidative stress induced-ECM gene expression (transforming growth factor-ß1 (TGFß1), alpha smooth muscle actin (α-SMA), and collagen type 1A1 (Col1A1)), and cell proliferation in normal and glaucoma LC cells. Also, SKF96365 treatment inhibited the H2O2-induced NFATc3 protein dephosphorylation in LC cells. In conclusion, TRPC1/C6 expression is enhanced in glaucoma LC cells. These channels may contribute to oxidative stress-induced ECM gene transcription and cell proliferation in normal and glaucoma LC cells through Ca2+-NFATc3 signaling pathway mechanism. TRPC1 and TRPC6 channels could be important therapeutic targets to prevent ECM remodeling and fibrosis development in glaucoma optic neuropathy.

The Effect and Mechanism of TRPC1, 3, and 6 on the Proliferation, Migration, and Lumen Formation of Retinal Vascular Endothelial Cells Induced by High Glucose.

OBJECTIVE: Transient receptor potential canonical (TRPC) channels are involved in neovascularization repairing after vascular injury in many tissues. However, whether TRPCs play a regulatory role in the development of diabetic retinopathy (DR) has rarely been reported. In the present study, we selected TRPC1, 3, and 6 to determine their roles and mechanism in human retina vascular endothelial cells (HREC) under high glucose (HG) conditions. METHODS: HRECs were cultured in vitro under HG, hyper osmosis, and normal conditions. The expression of TRPC1, 3, and 6 in the cells at 24 and 48 h were detected by RT-polymerase chain reaction (PCR), Western blot and cell immunohistochemistry (IHC); In various concentrations, SKF96365 acted on HG cultured HRECs, the expression of vascular endothelial growth factor (VEGF) were detected by the same methods above; and the CCK-8, Transwell, cell scratch assay, and Matrigel assay were used to assess cell proliferation, migration, and lumen formation. RESULTS: The RT-PCR, Western blot, and IHC results showed that TRPC1 expression was increased, and TRPC6 mRNA expression was increased under high-glucose conditions. SKF96365 acted on HG cultured HRECs that VEGF expression was significantly decreased. The CCK-8 assay, Transwell assay, cell scratch assay, and Matrigel assay showed that cell proliferation, migration, and lumen formation were downregulated by SKF96365. CONCLUSION: HG can induce increased expression of TRPC1 and 6 in HRECs. Inhibition of the TRPC pathway not only can decrease VEGF expression but also can prevent proliferation, migration, and lumen formation of HRECs induced by HG. Inhibition of TRPC channels is expected to become a drug target for DR.

TRPC1 is a differential regulator of hypoxia-mediated events and Akt signalling in PTEN-deficient breast cancer cells.

Hypoxia is a feature of the tumour microenvironment that promotes invasiveness, resistance to chemotherapeutics and cell survival. Our studies identify the transient receptor potential canonical-1 (TRPC1) ion channel as a key component of responses to hypoxia in breast cancer cells. This regulation includes control of specific epithelial to mesenchymal transition (EMT) events and hypoxia-mediated activation of signalling pathways such as activation of the EGFR, STAT3 and the autophagy marker LC3B, through hypoxia-inducible factor-1α (HIF1α)-dependent and -independent mechanisms. TRPC1 regulated HIF1α levels in PTEN-deficient MDA-MB-468 and HCC1569 breast cancer cell lines. This regulation arises from effects on the constitutive translation of HIF1α under normoxic conditions via an Akt-dependent pathway. In further support of the role of TRPC1 in EMT, its expression is closely associated with EMT- and metastasis-related genes in breast tumours, and is enhanced in basal B breast cancer cell lines. TRPC1 expression is also significantly prognostic for basal breast cancers, particularly those classified as lymph node positive. The defined roles of TRPC1 identified here could be therapeutically exploited for the control of oncogenic pathways in breast cancer cells.

Dopaminergic neurotoxins induce cell death by attenuating NF-κB-mediated regulation of TRPC1 expression and autophagy.

Alterations in Ca2+ homeostasis affect neuronal survival. However, the identity of Ca2+ channels and the mechanisms underlying neurotoxin-induced neuronal degeneration are not well understood. In this study, the dopaminergic neurotoxins 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenylpyridium ions (MPP+)/1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which mimic Parkinson's disease (PD), induced neuronal degeneration by decreasing store-mediated Ca2+ entry. The function of the transient receptor potential canonical (TRPC)-1 channel was decreased upon exposure to the neurotoxins, followed by a decrease in TRPC1 expression. Similar to neurotoxins, samples from patients with PD exhibited attenuated TRPC1 expression, which was accompanied by a decrease in autophagic markers and a subsequent increase in apoptosis markers. Furthermore, exposure to neurotoxins attenuated PKC phosphorylation, decreased expression of autophagic markers, and increased apoptosis in SHSY-5Y neuroblastoma cells, which was again dependent on TRPC1. Prolonged neurotoxin treatment attenuated the binding of NF-κB to the TRPC1 promoter, which resulted in a decrease in TRPC1 expression, thereby attenuating autophagy and activating cell death. Restoration of TRPC1 expression rescued the effects of the dopaminergic neurotoxins in neuroblastoma cells by increasing Ca2+ entry, restoring NF-κB activity, and promoting autophagy. Overall, these results suggest that dopaminergic neurotoxins initially decreased Ca2+ entry, which inhibited the binding of NF-κB to the TRPC1 promoter, thereby inhibiting TRPC1 expression and resulting in cell death by preventing autophagy.-Sukumaran, P., Sun, Y., Antonson, N., Singh, B. B. Dopaminergic neurotoxins induce cell death by attenuating NF-κB-mediated regulation of TRPC1 expression and autophagy.

TrpA1 Regulates Defecation of Food-Borne Pathogens under the Control of the Duox Pathway.

Pathogen expulsion from the gut is an important defense strategy against infection, but little is known about how interaction between the intestinal microbiome and host immunity modulates defecation. In Drosophila melanogaster, dual oxidase (Duox) kills pathogenic microbes by generating the microbicidal reactive oxygen species (ROS), hypochlorous acid (HOCl) in response to bacterially excreted uracil. The physiological function of enzymatically generated HOCl in the gut is, however, unknown aside from its anti-microbial activity. Drosophila TRPA1 is an evolutionarily conserved receptor for reactive chemicals like HOCl, but a role for this molecule in mediating responses to gut microbial content has not been described. Here we identify a molecular mechanism through which bacteria-produced uracil facilitates pathogen-clearing defecation. Ingestion of uracil increases defecation frequency, requiring the Duox pathway and TrpA1. The TrpA1(A) transcript spliced with exon10b (TrpA1(A)10b) that is present in a subset of midgut enteroendocrine cells (EECs) is critical for uracil-dependent defecation. TRPA1(A)10b heterologously expressed in Xenopus oocytes is an excellent HOCl receptor characterized with elevated sensitivity and fast activation kinetics of macroscopic HOCl-evoked currents compared to those of the alternative TRPA1(A)10a isoform. Consistent with TrpA1's role in defecation, uracil-excreting Erwinia carotovora showed higher persistence in TrpA1-deficient guts. Taken together, our results propose that the uracil/Duox pathway promotes bacteria expulsion from the gut through the HOCl-sensitive receptor, TRPA1(A)10b, thereby minimizing the chances that bacteria adapt to survive host defense systems.

Increased size and cellularity of advanced atherosclerotic lesions in mice with endothelial overexpression of the human TRPC3 channel.

In previous in vitro studies, we showed that Transient Receptor Potential Canonical 3 (TRPC3), a calcium-permeable, nonselective cation channel endowed with high constitutive function, is an obligatory component of the inflammatory signaling that controls expression of the vascular cell adhesion molecule-1 (VCAM-1) and monocyte adhesion to coronary artery endothelial cells. Also, TRPC3 expression in these cells was found to be up-regulated by proatherogenic factors, which enhanced inflammation and VCAM-1 expression. However, it remained to be determined whether these in vitro findings were of relevance to atherosclerotic lesion development in vivo. To answer this important question in the present work, we generated mice with endothelial-specific overexpression of human TRPC3 in an Apoe knockout background (TgEST3ApoeKO) and examined lesions in the aortic sinus following 10 and 16 wk on a high-fat diet. No significant differences were found in size or complexity of early stage lesions (10 wk). However, advanced plaques (16 wk) from TgEST3ApoeKO mice exhibited a significant increase in size and macrophage content compared with nontransgenic littermate controls. Remarkably, this change was correlated with increased VCAM-1 and phospho-IkBα immunoreactivity along the endothelial lining of lesions from transgenic animals compared with controls. These findings validate the in vivo relevance of previous in vitro findings and represent, to our knowledge, the first in vivo evidence for a proatherogenic role of endothelial TRPC3.

Increased CaSR and TRPC6 pulmonary vascular expression in the nitrofen-induced model of congenital diaphragmatic hernia.

AIMS AND OBJECTIVES: The high morbidity and mortality rates in congenital diaphragmatic hernia (CDH) are attributed primarily to severe lung hypoplasia and/or persistent pulmonary hypertension (PPH). PPH in CDH is characterized by abnormal vascular remodeling with thickening of medial and adventitial layers and extension of smooth muscle into previously nonmuscularized arteries. Excessive proliferation of pulmonary arterial smooth muscle cells (PASMC) is an important contributor to the concentric pulmonary arterial remodeling. An increase in cytosolic-free Ca2+ concentration in PASMC is a major trigger for pulmonary vasoconstriction and a key stimulus for PASMC proliferation and migration. Calcium-sensing receptor (CaSR), a member of the G-protein coupled receptor family, is activated by cations (e.g., Ca2+, Mg2+) and polyamines. Under normal physiological conditions, the expression levels of CaSR in the pulmonary vasculature are very low. Canonical transient receptor potential channels (TRPCs) constitute a series of nonselective cation channels with variable degree of Ca2+ selectivity. TRPC6 has been reported to play a crucial role in the regulation of neo-muscularization, vasoreactivity, and vasomotor tone in the pulmonary vasculature. We hypothesized that CaSR and TRPC6 expression is upregulated in the pulmonary vasculature of nitrofen-induced CDH rats. MATERIALS AND METHODS: Following ethical approval (REC1103), time-pregnant Sprague Dawley rats received nitrofen or vehicle on gestational day (D) 9. D21 fetuses were divided into CDH and control (n = 12). Quantitative real-time polymerase chain reaction (QRT-PCR), western blotting, and confocal-immunofluorescence microscopy were performed to detect lung gene and protein expression of CaSR and TRPC6. RESULTS: QRT-PCR and western blot analysis revealed that CaSR and TPRC6 expression was significantly increased in the CDH group compared to controls (p < 0.05). Confocal-immunofluorescence microscopy revealed that CaSR and TRPC6 lung expression was markedly increased in CDH group compared to controls. CONCLUSION: Increased CaSR and TRPC6 expression in CDH lung suggests that CaSR interacting with TRPC6 may contribute to abnormal vascular remodeling resulting in pulmonary vasoconstriction and development of PPH.

Transient receptor potential canonical 5 (TRPC5) protects against pain and vascular inflammation in arthritis and joint inflammation.

OBJECTIVE: Transient receptor potential canonical 5 (TRPC5) is functionally expressed on a range of cells including fibroblast-like synoviocytes, which play an important role in arthritis. A role for TRPC5 in inflammation has not been previously shown in vivo. We investigated the contribution of TRPC5 in arthritis. METHODS: Male wild-type and TRPC5 knockout (KO) mice were used in a complete Freund's adjuvant (CFA)-induced unilateral arthritis model, assessed over 14 days. Arthritis was determined by measurement of knee joint diameter, hindlimb weightbearing asymmetry and pain behaviour. Separate studies involved chronic pharmacological antagonism of TRPC5 channels. Synovium from human postmortem control and inflammatory arthritis samples were investigated for TRPC5 gene expression. RESULTS: At baseline, no differences were observed. CFA-induced arthritis resulted in increased synovitis in TRPC5 KO mice assessed by histology. Additionally, TRPC5 KO mice demonstrated reduced ispilateral weightbearing and nociceptive thresholds (thermal and mechanical) following CFA-induced arthritis. This was associated with increased mRNA expression of inflammatory mediators in the ipsilateral synovium and increased concentration of cytokines in synovial lavage fluid. Chronic treatment with ML204, a TRPC5 antagonist, augmented weightbearing asymmetry, secondary hyperalgesia and cytokine concentrations in the synovial lavage fluid. Synovia from human inflammatory arthritis demonstrated a reduction in TRPC5 mRNA expression. CONCLUSIONS: Genetic deletion or pharmacological blockade of TRPC5 results in an enhancement in joint inflammation and hyperalgesia. Our results suggest that activation of TRPC5 may be associated with an endogenous anti-inflammatory/analgesic pathway in inflammatory joint conditions.

High Glucose Enhances Isoflurane-Induced Neurotoxicity by Regulating TRPC-Dependent Calcium Influx.

Isoflurane is a commonly used inhalational anesthetic that can induce neurotoxicity via elevating cytosolic calcium (Ca2+). High glucose regulates the expression of a family of non-selective cation channels termed transient receptor potential canonical (TRPC) channels that may contribute to Ca2+ influx. In the present study, we investigated whether high glucose enhances isoflurane-induced neurotoxicity by regulating TRPC-dependent Ca2+ influx. First, we evaluated toxic damage in mice primary cultured hippocampal neurons and human neuroblastoma cells (SH-SY5Y cells) after hyperglycemia and isoflurane exposure. Next, we investigated cytosolic Ca2+ concentrations, TRPC mRNA expression levels and tested the effect of the TRPC channel blocker SKF96365 on cytosolic Ca2+ levels in cells treated with high glucose or/and isoflurane. Finally, we employed knocked down TRPC6 to demonstrate the role of TRPC in high glucose-mediated enhancement of isoflurane-induced neurotoxicity. The results showed that high glucose could enhance isoflurane-induecd toxic damage in primary hippocampal neurons and SH-SY5Y cells. High glucose enhanced the isoflurane-induced increase of cytosolic Ca2+ in SH-SY5Y cells. High glucose elevated TRPC mRNA expression, especially that of TRPC6. SKF96365 and knock down of TRPC6 were able to inhibit the high glucose-induced increase of cytosolic Ca2+ and decrease isoflurane-induced neurotoxicity in SH-SY5Y cells cultured with high glucose. Our findings indicate that high glucose could elevate TRPC expression, thus increasing Ca2+ influx and enhancing isoflurane-induced neurotoxicity.

TRPC3 amplifies B-cell receptor-induced ERK signalling via protein kinase D-dependent Rap1 activation.

Sustained activation of extracellular-signal-regulated kinase (ERK) has an important role in the decision regarding the cell fate of B-lymphocytes. Recently, we demonstrated that the diacylglycerol-activated non-selective cation channel canonical transient receptor potential 3 (TRPC3) is required for the sustained ERK activation induced by the B-cell receptor. However, the signalling mechanism underlying TRPC3-mediated ERK activation remains elusive. In the present study, we have shown that TRPC3 mediates Ca(2+) influx to sustain activation of protein kinase D (PKD) in a protein kinase C-dependent manner in DT40 B-lymphocytes. The later phase of ERK activation depends on the small G-protein Rap1, known as a downstream target of PKD, whereas the earlier phase of ERK activation depends on the Ras protein. It is of interest that sustained ERK phosphorylation is required for the full induction of the immediate early gene Egr-1 (early growth response 1). These results suggest that TRPC3 reorganizes the BCR signalling complex by switching the subtype of small G-proteins to sustain ERK activation in B-lymphocytes.

Synaptopodin Limits TRPC6 Podocyte Surface Expression and Attenuates Proteinuria.

Gain-of-function mutations of classic transient receptor potential channel 6 (TRPC6) were identified in familial FSGS, and increased expression of wild-type TRPC6 in glomeruli is observed in several human acquired proteinuric diseases. Synaptopodin, an actin binding protein that is important in maintaining podocyte function, is downregulated in various glomerular diseases. Here, we investigated whether synaptopodin maintains podocyte function by regulating podocyte surface expression and activity of TRPC6. We show indirect interaction and nonrandom association of synaptopodin and TRPC6 in podocytes. Knockdown of synaptopodin in cultured mouse podocytes increased the expression of TRPC6 at the plasma membrane, whereas overexpression of synaptopodin decreased it. Mechanistically, synaptopodin-dependent TRPC6 surface expression required functional actin and microtubule cytoskeletons. Overexpression of wild-type or FSGS-inducing mutant TRPC6 in synaptopodin-depleted podocytes enhanced TRPC6-mediated calcium influx and induced apoptosis. In vivo, knockdown of synaptopodin also caused increased podocyte surface expression of TRPC6. Administration of cyclosporin A, which stabilizes synaptopodin, reduced LPS-induced proteinuria significantly in wild-type mice but to a lesser extent in TRPC6 knockout mice. Furthermore, administration of cyclosporin A reversed the LPS-induced increase in podocyte surface expression of TRPC6 in wild-type mice. Our findings suggest that alteration in synaptopodin levels under disease conditions may modify intracellular TRPC6 channel localization and activity, which further contribute to podocyte dysfunction. Reducing TRPC6 surface levels may be a new approach to restoring podocyte function.

The involvement of transient receptor potential canonical type 1 in skeletal muscle regrowth after unloading-induced atrophy.

KEY POINTS: Decreased mechanical loading results in skeletal muscle atrophy. The transient receptor potential canonical type 1 (TRPC1) protein is implicated in this process. Investigation of the regulation of TRPC1 in vivo has rarely been reported. In the present study, we employ the mouse hindlimb unloading and reloading model to examine the involvement of TRPC1 in the regulation of muscle atrophy and regrowth, respectively. We establish the physiological relevance of the concept that manipulation of TRPC1 could interfere with muscle regrowth processes following an atrophy-inducing event. Specifically, we show that suppressing TRPC1 expression during reloading impairs the recovery of the muscle mass and slow myosin heavy chain profile. Calcineurin appears to be part of the signalling pathway involved in the regulation of TRPC1 expression during muscle regrowth. These results provide new insights concerning the function of TRPC1. Interventions targeting TRPC1 or its downstream or upstream pathways could be useful for promoting muscle regeneration. ABSTRACT: Decreased mechanical loading, such as bed rest, results in skeletal muscle atrophy. The functional consequences of decreased mechanical loading include a loss of muscle mass and decreased muscle strength, particularly in anti-gravity muscles. The purpose of this investigation was to clarify the regulatory role of the transient receptor potential canonical type 1 (TRPC1) protein during muscle atrophy and regrowth. Mice were subjected to 14 days of hindlimb unloading followed by 3, 7, 14 and 28 days of reloading. Weight-bearing mice were used as controls. TRPC1 expression in the soleus muscle decreased significantly and persisted at 7 days of reloading. Small interfering RNA (siRNA)-mediated downregulation of TRPC1 in weight-bearing soleus muscles resulted in a reduced muscle mass and a reduced myofibre cross-sectional area (CSA). Microinjecting siRNA into soleus muscles in vivo after 7 days of reloading provided further evidence for the role of TRPC1 in regulating muscle regrowth. Myofibre CSA, as well as the percentage of slow myosin heavy chain-positive myofibres, was significantly lower in TRPC1-siRNA-expressing muscles than in control muscles after 14 days of reloading. Additionally, inhibition of calcineurin (CaN) activity downregulated TRPC1 expression in both weight-bearing and reloaded muscles, suggesting a possible association between CaN and TRPC1 during skeletal muscle regrowth.

Transient receptor potential vanilloid 2-mediated shear-stress responses in C2C12 myoblasts are regulated by serum and extracellular matrix.

The developmental sensitivity of skeletal muscle to mechanical forces is unparalleled in other tissues. Calcium entry via reputedly mechanosensitive transient receptor potential (TRP) channel classes has been shown to play an essential role in both the early proliferative stage and subsequent differentiation of skeletal muscle myoblasts, particularly TRP canonical (TRPC) 1 and TRP vanilloid (TRPV) 2. Here we show that C2C12 murine myoblasts respond to fluid flow-induced shear stress with increments in cytosolic calcium that are largely initiated by the mechanosensitive opening of TRPV2 channels. Response to fluid flow was augmented by growth in low extracellular serum concentration (5 vs. 20% fetal bovine serum) by greater than 9-fold and at 18 h in culture, coincident with the greatest TRPV2 channel expression under identical conditions (P < 0.02). Fluid flow responses were also enhanced by substrate functionalization with laminin, rather than with fibronectin, agreeing with previous findings that the gating of TRPV2 is facilitated by laminin. Fluid flow-induced calcium increments were blocked by ruthenium red (27%) and SKF-96365 (38%), whereas they were unaltered by 2-aminoethoxydiphenyl borate, further corroborating that TRPV2 channels play a predominant role in fluid flow mechanosensitivity over that of TRPC1 and TRP melastatin (TRPM) 7.

Increased expression of transient receptor potential canonical 6 (TRPC6) in differentiating human megakaryocytes.

Members of the transient receptor potential (TRP) family of cation conducting channels are found in several tissues and cell types where they have different physiological functions. The canonical TRP channel 6 (TRPC6) is present on the platelet membrane and appears to participate in calcium influx during platelet activation. However, limited information is available on the importance of TRPC channels in megakaryocytes (MKs), the precursor cells of platelets. We determined the mRNA and protein expression of TRPC family members and investigated the role of TRPC6 for proliferation and differentiation of human MKs derived from CD34+ progenitor cells. TRPC6 transcripts were highly expressed during the differentiation of MKs and TRPC6 protein was detectable in MK cytoplasm by confocal staining. TRPC6 channel activity was modulated by pharmacological approaches using flufenamic acid (FFA) for activation and SKF96365 for inhibition. Upon FFA stimulation in MKs, an increase in intracellular calcium was observed, which was blocked by SKF96365 at 10 µM concentration. Incubation of MKs with SKF96365 resulted in a reduction in thrombopoietin-stimulated cell proliferation. Our results suggest a role of TRPC6 in calcium homeostasis during MK development, particularly for cell proliferation.

Schisandrin B inhibits the proliferation of airway smooth muscle cells via microRNA-135a suppressing the expression of transient receptor potential channel 1.

Airway smooth muscle cell (ASMC) was known to involve in the pathophysiology of asthma. Schisandrin B was reported to have anti-asthmatic effects in a murine asthma model. However, the molecular mechanism involving in the effect of Schisandrin B on ASMCs remains poorly understood. Sprague-Dawley rats were divided into three groups: rats as the control (Group 1), sensitized rats (Group 2), sensitized rats and intragastric-administrated Schisandrin B (Group 3). The expression of miR-135a and TRPC1 was detected in the rats from three groups. Platelet-derived growth factor (PDGF)-BB was used to induce the proliferation of isolated ASMCs, and the expression of miR-135a and TRPC1 was detected in PDGF-BB-treated ASMCs. Cell viability was examined in ASMCs transfected with miR-135a inhibitor or si-TRPC1. The expression of TRPC1 was examined in A10 cells pretreated with miR-135a inhibitor or miR-135a mimic. In this study, we found that Schisandrin B attenuated the inspiratory and expiratory resistances in sensitized rats. Schisandrin B upregulated the mRNA level of miR-135a and decreased the expression of TRPC1 in sensitized rats. In addition, Schisandrin B reversed the expression of miR-135a and TRPC1 in PDGF-BB-induced ASMCs. Si-TRPC1 abrogated the increasing proliferation of ASMCs induced by miR-135a inhibitor. We also found that miR-135a regulated the expression of TRPC1 in the A10 cells. These results demonstrate that Schisandrin B inhibits the proliferation of ASMCs via miR-135a suppressing the expression of TRPC1.

Effects of ganoderic acids on epileptiform discharge hippocampal neurons: insights from alterations of BDNF,TRPC3 and apoptosis.

Recently, Ganoderma lucidum spores (GLS) have shown anti-epileptic effects. However, there are no reports on the anti-epileptic effects of its chemical constituents ganoderic acids (GAs), and more research is needed to better understand the mechanism of GLS activity. In this work, rat primary hippocampal neurons in an in vitro model were used to assess the intervention effects of GAs on epileptiform discharge hippocampal neurons and expression of both BDNF and TRPC3, with the aid of immunofluorescence, MTT method and flow cytometry. It was found that BDNF and TRPC3 are expressed in all cells and were mainly localized in the cytoplasm. The fluorescence intensities of BDNF and TRPC3 in GAs groups were higher than those of normal control and model groups, especially at 80 µg/ml (P < 0.05). The apoptosis rate of neurons was inversely proportional to BDNF and TRPC3 changes (P < 0.01). Therefore, BDNF and TRPC3 should be involved in the occurrence and development of epilepsy. GAs might indirectly inhibit mossy fiber sprouting and adjust the synaptic reconstructions by promoting the expression of BDNF and TRPC3. Besides, GAs could exert a protective effect on hippocampal neurons by promoting neuronal survival and the recovery of injured neurons.

Decreased anxiety-like behavior and Gαq/11-dependent responses in the amygdala of mice lacking TRPC4 channels.

Transient receptor potential (TRP) channels are abundant in the brain where they regulate transmission of sensory signals. The expression patterns of different TRPC subunits (TRPC1, 4, and 5) are consistent with their potential role in fear-related behaviors. Accordingly, we found recently that mutant mice lacking a specific TRP channel subunit, TRPC5, exhibited decreased innate fear responses. Both TRPC5 and another member of the same subfamily, TRPC4, form heteromeric complexes with the TRPC1 subunit (TRPC1/5 and TRPC1/4, respectively). As TRP channels with specific subunit compositions may have different functional properties, we hypothesized that fear-related behaviors could be differentially controlled by TRPCs with distinct subunit arrangements. In this study, we focused on the analysis of mutant mice lacking the TRPC4 subunit, which, as we confirmed in experiments on control mice, is expressed in brain areas implicated in the control of fear and anxiety. In behavioral experiments, we found that constitutive ablation of TRPC4 was associated with diminished anxiety levels (innate fear). Furthermore, knockdown of TRPC4 protein in the lateral amygdala via lentiviral-mediated gene delivery of RNAi mimicked the behavioral phenotype of constitutive TRPC4-null (TRPC4(-/-)) mouse. Recordings in brain slices demonstrated that these behavioral modifications could stem from the lack of TRPC4 potentiation in neurons in the lateral nucleus of the amygdala through two Gαq/11 protein-coupled signaling pathways, activated via Group I metabotropic glutamate receptors and cholecystokinin 2 receptors, respectively. Thus, TRPC4 and the structurally and functionally related subunit, TRPC5, may both contribute to the mechanisms underlying regulation of innate fear responses.

Upregulated expression of STIM2, TRPC6, and Orai2 contributes to the transition of pulmonary arterial smooth muscle cells from a contractile to proliferative phenotype.

Pulmonary arterial hypertension (PAH) is a progressive disease that, if left untreated, eventually leads to right heart failure and death. Elevated pulmonary arterial pressure (PAP) in patients with PAH is mainly caused by an increase in pulmonary vascular resistance (PVR). Sustained vasoconstriction and excessive pulmonary vascular remodeling are two major causes for elevated PVR in patients with PAH. Excessive pulmonary vascular remodeling is mediated by increased proliferation of pulmonary arterial smooth muscle cells (PASMC) due to PASMC dedifferentiation from a contractile or quiescent phenotype to a proliferative or synthetic phenotype. Increased cytosolic Ca(2+) concentration ([Ca(2+)]cyt) in PASMC is a key stimulus for cell proliferation and this phenotypic transition. Voltage-dependent Ca(2+) entry (VDCE) and store-operated Ca(2+) entry (SOCE) are important mechanisms for controlling [Ca(2+)]cyt. Stromal interacting molecule proteins (e.g., STIM2) and Orai2 both contribute to SOCE and we have previously shown that STIM2 and Orai2, specifically, are upregulated in PASMC from patients with idiopathic PAH and from animals with experimental pulmonary hypertension in comparison to normal controls. In this study, we show that STIM2 and Orai2 are upregulated in proliferating PASMC compared with contractile phenotype of PASMC. Additionally, a switch in Ca(2+) regulation is observed in correlation with a phenotypic transition from contractile PASMC to proliferative PASMC. PASMC in a contractile phenotype or state have increased VDCE, while in the proliferative phenotype or state PASMC have increased SOCE. The data from this study indicate that upregulation of STIM2 and Orai2 is involved in the phenotypic transition of PASMC from a contractile state to a proliferative state; the enhanced SOCE due to upregulation of STIM2 and Orai2 plays an important role in PASMC proliferation.

Antialbuminuric actions of calcilytics in the remnant kidney.

Hyperphosphatemia accelerates the progression of chronic kidney diseases. In the present study, the effects of ronacaleret, a calcilytic agent, on renal injury were assessed in the following four groups of rats: 5/6-nephrectomized Wistar rats as a control (C group), rats treated with ronacaleret (3 mg·kg(-1)·day(-1); R group), rats treated with calcitriol (30 ng·kg(-1)·day(-1); V group), and rats treated with both ronacaleret and calcitriol (R + V group). Three months later, rats were euthanized under anesthesia, and the remnant kidneys were harvested for analysis. Albuminuria was lower in the R and V groups than in the C group (P < 0.05). Creatinine clearance was elevated in the R and V groups compared with the C group (P < 0.05). Serum Ca(2+) and renal ANG II were higher in the R + V group than in the C group (P < 0.05 for each), and serum phosphate was reduced in the R group compared with the C group (P < 0.05). Fibroblast growth factor-23 was lower in the R group and higher in the V and R + V groups than in the C group. However, parathyroid hormone did not differ significantly among the four groups. Renal klotho expression was elevated in the R and V groups compared with the C group (P < 0.05). The present data indicate that ronacaleret preserves klotho expression and renal function with reductions in serum phosphate and albuminuria in 5/6-nephrectomized rats. Our findings demonstrate that vitamin D prevents declines in klotho expression and renal function, suppressing albuminuria.

Glucose specifically regulates TRPC6 expression in the podocyte in an AngII-dependent manner.

Slit diaphragm and podocyte damage is crucial in the pathogenesis of proteinuria in diabetic nephropathy (DNP). Gain-of-function mutations in TRPC6, a slit diaphragm-associated ion channel, cause glomerulosclerosis; TRPC6 expression is increased in acquired glomerular disease. Hyperglycemia and high intrarenal angiotensin II (AngII) levels could contribute to podocyte injury in DNP. We determined whether glucose regulates TRPC6 expression and TRPC6-mediated Ca(2+) influx into the podocyte and whether these effects are AngII dependent. High glucose levels increased TRPC6 mRNA and protein expression in cultured podocytes; however, TRPC1 and TRPC5 mRNA expression was unaltered. AngII and inducing podocyte injury also specifically increased TRPC6 expression. Angiotensin receptor blockade and inhibition of local AngII production through angiotensin-converting enzyme inhibition prevented glucose-mediated increased TRPC6 expression. In addition, high glucose concentration pretreatment enhanced Ca(2+) influx in podocytes, which was prevented by concomitant angiotensin receptor blockade application and TRPC6 knockdown. Studies with a TRPC6 luciferase promoter construct demonstrated a glucose concentration-dependent effect on TRPC6 promoter activity. In vivo, podocyte TRPC6 protein expression was increased in proteinuric streptozotocin-induced diabetic rats. These data suggest that glucose can activate a local renin-angiotensin system in the podocyte, leading to increased TRPC6 expression, which enhances TRPC6-mediated Ca(2+) influx. Regulation of TRPC6 expression could be an important factor in podocyte injury due to chronic hyperglycemia and the antiproteinuric effect of angiotensin receptor blockade or angiotensin-converting enzyme inhibition in DNP.