DHA induces Jurkat T-cell arrest in G2/M phase of cell cycle and modulates the plasma membrane expression of TRPC3/6 channels.

We investigated whether docosahexaenoic acid (DHA), a dietary n-3 fatty acid, modulates calcium (Ca2+) signaling and cell cycle progression in human Jurkat T-cells. Our study demonstrates that DHA inhibited Jurkat T-cell cycle progression by blocking their passage from S phase to G2/M phase. In addition, DHA decreased the plasma membrane expression of TRPC3 and TRPC6 calcium channels during T-cell proliferation. Interestingly, this fatty acid increased plasma membrane expression of TRPC6 after 24 h of mitogenic stimulation by phorbol-13-myristate-12-acetate (PMA) and ionomycin. These variations in the membrane expression of TRPC3 and TRPC6 channels were not directly correlated with the mRNA expression, indicating that it was a post-translational phenomenon. DHA increased free intracellular calcium concentrations, [Ca2+]i, via opening TRPC3 and TRPC6 channels. We conclude that the anti-proliferative effect of DHA might involve the modulation of TRPC3 and TRPC6 channels in human T-cells.

Expression of TRPC3 in cortical lesions from patients with focal cortical dysplasia.

Focal cortical dysplasia (FCD) is one of the main causes of medically intractable epilepsy. Some studies have reported that transient receptor potential canonical channel 3 (TRPC3) may play an important role in the occurrence of seizures. In this study, we investigated the expression patterns of TRPC3 in different types of FCD. Forty-five FCD specimens and 12 control samples from autopsies were used in our study. Western blotting, immunohistochemistry, and immunofluorescence staining were employed to detect protein expression and distribution. The amount of TRPC3 protein was markedly elevated in the FCD group. The immunohistochemistry results revealed that TRPC3 staining was strong in the malformed cells and microcolumns. Most of the TRPC3-positive cells were colabeled with glutamatergic and GABAergic markers. The overexpression and altered cellular distribution of TRPC3 in the FCD samples suggest that TRPC3 may be related to epileptogenesis in FCD.

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.

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.

Downregulation of TRPC6 expression is a critical molecular event during FK506 treatment for overactive bladder.

PURPOSE: It has been suggested that FK506 could improve some symptoms of OAB in both clinical settings and animal models; however, its mechanism of action is not well-understood. Here, we investigated the effect of FK506 on TRPC6 in bladder smooth muscle, and explored the possible involvement of TRPC6 in OAB. METHODS: FK506 was injected intraperitoneally into rats in which OAB was induced via BOO, and urodynamic indices were recorded. Rats and human bladder smooth muscle tissues with or without OAB were examined for TRPC6 expression by western blot, RT-PCR and IF staining. Cultured BSMCs were treated with PDGF, TRPC6 siRNAs and FK506. Then the TRPC6 expression and cellular proliferation were examined, and the Ca2+ influx and contractility of BSMCs were examined by time-lapse Ca2+ imaging and collagen gel contraction. Finally, IF and Co-IP were performed to test the effects of FK506 on NFAT translocation to the nucleus and the interaction of TRPC6 with FKBP12, respectively. RESULTS: FK506 improved urodynamic indices of OAB rats, and TRPC6 was expressed in rats and human bladder tissues. TRPC6 elevation in OAB rats was inhibited by FK506, and this inhibition coincided with improvements in urodynamic indices. PDGF enhanced TRPC6 expression, cellular proliferation, Ca2+ influx and contractility of BSMCs, and these effects were inhibited by TRPC6 siRNAs and FK506. FK506 inhibited NFAT translocation to the nucleus and disrupted the interaction of TRPC6 with FKBP12. CONCLUSIONS: Our results collectively indicate that FK506 may be used to treat OAB, and that TRPC6 may serve as an attractive target for therapeutic intervention in OAB.

TRPC channels mediated calcium entry is required for proliferation of human airway smooth muscle cells induced by nicotine-nAChR.

The present study was designed to explore the role of transient receptor potential canonical 3 (TRPC3) in nicotine-induced chronic obstructive pulmonary disease (COPD) and its underlying mechanism. In this study, the expression and localization of α5 nicotinic acetylcholine receptor (α5-nAchR) in lung tissues were determined by western blotting and immunohistochemistry. The quantitative real-time PCR (qRT-PCR) analysis was performed to examine the mRNA expression levels of α5-nAchR and TRPC3 in human airway smooth muscle cells (HASMCs). Cell viability was assessed by CCK-8 assay. Proliferation was detected by cell counting and EdU immunofluorescent staining. Fluorescence calcium imaging was carried out to measure cytosolic Ca2+ ([Ca2+]cyt) concentration. The results showed that the α5-nAchR and TRPC3 expressions were significantly up-regulated in lung tissues of COPD smokers. Nicotine promoted HASMC proliferation, which was accompanied by elevated α5-nAchR and TRPC3 expressions, basal [Ca2+]cyt, store-operated calcium entry (SOCE) and the rate of Mn2+ quenching in HASMCs. Further investigation indicated that nicotine-induced Ca2+ response and TRPC3 up-regulation was reversibly blocked by small interfering RNA (siRNA) suppression of α5-nAChR. The knockdown of TRPC3 blunted Ca2+ response and HASMC proliferation induced by nicotine. In conclusion, nicotine-induced HASMC proliferation was mediated by TRPC3-dependent calcium entry via α5-nAchR, which provided a potential target for treatment of COPD.

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.

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.

Role of canonical transient receptor potential channel-3 in acetylcholine-induced mouse airway smooth muscle cell proliferation.

AIMS: Canonical transient receptor potential channel-3 (TRPC3)-encoded Ca2+-permeable nonselective cation channel (NSCC) has been proven to be an important native constitutively active channel in airway smooth muscle cell (ASMC), which plays significant roles in physiological and pathological conditions by controlling Ca2+ homeostasis in ASMC. Acetylcholine (ACh) is generally accepted as a contractile parasympathetic neurotransmitter in the airway. Recently studies have revealed the pathological role of ACh in airway remodeling, however, the mechanisms remain unclear. Here, we investigated the role of TRPC3 in ACh-induced ASMC proliferation. MATERIALS AND METHODS: Primary mouse ASMCs were cultured with or without ACh treatment, then cell viability, TRPC3 expression, NSCC currents and [Ca2+]i changes were examined by MTT assay, cell counting, Western blotting, standard whole-cell patch clamp recording and calcium imaging, respectively. Small interfering RNA (siRNA) technology was used to confirm the contribution of TRPC3 to ACh-induced ASMC proliferation. KEY FINDINGS: TRPC3 blocker Gd3+, antibody or siRNA largely inhibited ACh-induced up-regulation of TRPC3 protein, enhancement of NSCC currents, resting [Ca2+]i and KCl-induced changes in [Ca2+]i, eventually inhibiting ACh-induced ASMC proliferation. SIGNIFICANCE: Our data suggested ACh could induce ASMC proliferation, and TRPC3 may be involved in ACh-induced ASMC proliferation that occurs with airway remodeling.

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.

Hyperforin/HP-ß-Cyclodextrin Enhances Mechanosensitive Ca2+ Signaling in HaCaT Keratinocytes and in Atopic Skin Ex Vivo Which Accelerates Wound Healing.

Cutaneous wound healing is accelerated by mechanical stretching, and treatment with hyperforin, a major component of a traditional herbal medicine and a known TRPC6 activator, further enhances the acceleration. We recently revealed that this was due to the enhancement of ATP-Ca2+ signaling in keratinocytes by hyperforin treatment. However, the low aqueous solubility and easy photodegradation impede the topical application of hyperforin for therapeutic purposes. We designed a compound hydroxypropyl-ß-cyclodextrin- (HP-ß-CD-) tetracapped hyperforin, which had increased aqueous solubility and improved photoprotection. We assessed the physiological effects of hyperforin/HP-ß-CD on wound healing in HaCaT keratinocytes using live imaging to observe the ATP release and the intracellular Ca2+ increase. In response to stretching (20%), ATP was released only from the foremost cells at the wound edge; it then diffused to the cells behind the wound edge and activated the P2Y receptors, which caused propagating Ca2+ waves via TRPC6. This process might facilitate wound closure, because the Ca2+ response and wound healing were inhibited in parallel by various inhibitors of ATP-Ca2+ signaling. We also applied hyperforin/HP-ß-CD on an ex vivo skin model of atopic dermatitis and found that hyperforin/HP-ß-CD treatment for 24 h improved the stretch-induced Ca2+ responses and oscillations which failed in atopic skin.

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.

Transient Receptor Potential-canonical 1 is Essential for Environmental Enrichment-Induced Cognitive Enhancement and Neurogenesis.

Transient receptor potential-canonical 1 (TRPC1) plays a crucial role in neuronal survival, nerve regeneration, and protects neurons from neurotoxic injury, but it is not reported whether or how TRPC1 may affect learning and memory. Here, we found that TRPC1 knockout did not significantly affect the spatial learning and memory ability when the mice were housed in standard cages (SC). Interestingly, after the mice were exposed to environmental enrichment (EE) for 4 weeks, TRPC1 knockout abolished the EE-induced spatial memory enhancement, LTP induction, and neurogenesis in hippocampal DG subset. By stereotaxic infusion of the recombinant adeno-associated viruses (rAAV)-TRPC1 into the hippocampal DG subsets bilaterally, we observed that the EE-associated neurogenesis, LTP induction and the cognitive enhancement were efficiently rescued in TRPC1 knockout mice. EE increased the phosphorylation levels of ERK, p38, and cyclic AMP response element-binding protein (CREB) in wild-type mice, whereas the activation of ERK and CREB was not seen in TRPC1 knockout mice, and the phosphorylation of p38 was same in EE-TRPC1-/- and WT-EE. Finally, EE increased TRPC1 expression and overexpression of TRPC1 increased neurogenesis and activated ERK/CREB pathway in the wild-type mice. These findings suggest that TRPC1 is indispensable for the EE-induced hippocampal neurogenesis and cognitive enhancement.

Localization and expression patterns of TRP channels in submandibular gland development.

OBJECTIVE: Expression of Transient receptor potential (TRP) channels: TRP canonical (TRPC)1, TRP vanilloid (TRPV)3, TRPV4 and TRP melastatin (TRPM)8 in adult rat salivary gland has recently been reported. The authors investigated expression of these TRP channels in the submandibular gland during early developmental stage in which the cell constitution is different, and discussed the function of TRP in the submandibular gland in early development. DESIGN: Using rat submandibular gland at embryonic days (E)18 and E20 and postnatal days (PN)0 and PN5 and PN28, expression of TRPV3, TRPV4, TRPC1 and TRPM8 was investigated using real-time polymerase chain reaction (RT-PCR) and immunohistochemistry. RESULTS: All TRP channels were expressed in cells constituting the submandibular gland in early developmental stage, but an increase in the expression level at PN5 on RT-PCR was significant compared with those at E18, PN0 and PN28 in TRPC1 and TRPV4 channels, whereas an increase was observed but not significant in the others. On immunohistochemical staining at PN5, whereas strong reactions of anti-TRPM8 antibody, anti-TRPV3 and anti-TRPV4 antibodies were observed in cells which proliferated from a terminal portion of cells arranged tubular structure which previously constituted mostly the submandibular gland. CONCLUSION: It was clarified that TRP channels are expressed in the rat submandibular gland in early developmental stage although cells constituting the submandibular gland are different from those in adult animals, suggesting that these TRP channels are involved in cell differentiation in at PN5 into the adult submandibular gland during early development.

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.

Cannabinoids increase mechanosensitivity of trigeminal ganglion neurons innervating the inner walls of rat anterior chambers via activation of TRPA1.

Our previous study found that some trigeminal ganglion (TG) nerve endings in the inner walls of rat anterior chambers were mechanosensitive, and transient receptor potential ankyrin 1 (TRPA1) was an essential mechanosensitive channel in the membrane. To address the effect of cannabinoids on the mechanosensitive TG nerve endings in the inner walls of anterior chambers of rat eye, we investigated the effect of the (R)-(+)-WIN55, 212-2 mesylate salt (WIN), a synthetic cannabinoid on their cell bodies in vitro. Rat TG neurons innervating the inner walls of the anterior chambers were labeled by 1,1'-dilinoleyl-3,3,3',3'-tetramethylindocarbocyanine, 4-chlorobenzenesulfona (FAST DiI). Whole cell patch clamp was performed to record the currents induced by drugs and mechanical stimulation. Mechanical stimulation was applied to the neurons by buffer ejection. WIN evoked inward currents via TRPA1 activation in FAST DiI-labeled TG neurons. WIN enhanced mechanosensitive currents via TRPA1 activation in FAST DiI-labeled TG neurons. Our results indicate that cannabinoids can enhance the mechanosensitivity of TG endings in the inner walls of anterior chambers of rat eye via TRPA1 activation.

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.

Astragaloside IV prevents high glucose-induced podocyte apoptosis via downregulation of TRPC6.

Diabetic nephropathy (DN) is one of the most important causes of end­stage renal disease. Astragaloside IV (AS-IV) is a saponin isolated from Astragalus membranaceus, which possesses various pharmacological activities. AS­IV prevents podocyte apoptosis and ameliorates renal injury in DN; however, few studies have focused on its effects on ion channels. The transient receptor potential channel 6 (TRPC6) is an important Ca2+­permeable ion channel in podocytes, which is involved in high glucose (HG)-induced podocyte apoptosis. The aim of the present study was to investigate whether AS­IV prevented HG­induced podocyte apoptosis via TRPC6. Cultured podocytes were pre­treated with 10, 20 or 40 µM AS­IV for 1 h prior to HG exposure for 24 h. Apoptosis, cell viability, expression of TRPC6, nuclear factor of activated T cells (NFAT2) and B­cell lymphoma 2­associated X protein (Bax), as well as the intracellular Ca2+ concentration were subsequently analyzed. The results indicated that HG induced podocyte apoptosis and upregulation of TRPC6, and increased intracellular Ca2+. Furthermore, enhanced NFAT2 and Bax expression was detected. Conversely, AS­IV protected HG­induced podocyte apoptosis, downregulated TRPC6 expression and suppressed intracellular Ca2+ in HG-stimulated podocytes. AS­IV also suppressed NFAT2 and Bax expression. These results suggest that AS­IV may prevent HG-induced podocyte apoptosis via downregulation of TRPC6, which is possibly mediated via the calcineurin/NFAT signaling pathway.

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.

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.