Flufenamic acid is a tool for investigating TRPC6-mediated calcium signalling in human conditionally immortalised podocytes and HEK293 cells

Mutations in the cation channel TRPC6 result in a renal-specific phenotype of familial nephrotic syndrome, affecting intracellular calcium ([Ca²⁺]_i) signalling in the glomerular podocyte. Tools to study native TRPC6 activity are scarce, although there has been recent success with flufenamic acid (FFA). We confirm the specificity of FFA for TRPC6 both in an artificial expression system and in a human conditionally immortalised podocyte cell line (ciPod).Cells were loaded with fura-2AM and changes in intracellular calcium ([Ca²⁺]_i) were calculated. 200 μM FFA induced an increase in [Ca²⁺]_i in HEK293 cells with native TRPC6 expression, which was enhanced by overexpression of TRPC6 and completely blocked in the absence of extracellular calcium. Expressed TRPC7 did not significantly affect the response to FFA whereas expressed TRPC3 reduced it. FFA also induced an increase ciPod in [Ca²⁺]_i, which was inhibited using SKF96365 and 2-APB, but not indomethacin. In ciPod, adenovirus (Ad-v) wild type (WT) TRPC6 increased [Ca²⁺]_i activity to FFA compared to native TRPC6, whereas activity was significantly reduced with Ad-v dominant negative (DN) TRPC6. The niflumic acid (NFA) induced increase in [Ca²⁺]_i in ciPod was not affected by Ad-v TRPC6 DN, and in HEK293 cells was not affected by WT TRPC6.In conclusion, FFA activates TRPC6 [Ca²⁺]_i signalling in both ciPod and HEK293 cells independently of TRPC3 and TRPC7, and independently of properties of the fenamate family.     

Reduction in TRPC4 expression specifically attenuates G-protein coupled receptor-stimulated increases in intracellular calcium in human myometrial cells

Canonical transient receptor potential (TRPC) proteins may play a role in regulating changes in intracellular calcium ([Ca²⁺]_i). Human myometrium expresses TRPC4, TRPC1 and TRPC6 mRNAs in greatest relative abundance. Contributions of TRPC4 to increases in [Ca²⁺]_i were assessed in PHM1–41 and primary human uterine smooth muscle (UtSMC) cells using short hairpin RNAs (shRNAs). Based on a reporter assay screen, one shRNA was selected to construct an adenoviral expression vector (TC4sh1). TC4sh1 induced both mRNA and protein TRPC4 knockdown in PHM1–41 cells without affecting expression of other TRPCs. Signal-regulated Ca²⁺ entry (SRCE), defined as a stimulus- and extracellular Ca²⁺-dependent increase in [Ca²⁺]_i, was measured in PHM1–41 cells treated with oxytocin (G-protein coupled receptor (GPCR)-stimulated), thapsigargin (store depletion-stimulated), and OAG (diacylglycerol-stimulated), using Fura-2. Cells infected with TC4sh1 exhibited attenuated oxytocin-, ATP- and PGF2α-mediated SRCE, but no change in thapsigargin- or OAG-stimulated SRCE. Similar results were obtained in primary uterine smooth muscle cells. Additionally, cells expressing TC4sh1 exhibited a significantly smaller increase in channel activity in response to oxytocin administration than did cells infected with empty virus. These data show that, in human myometrial cells, knockdown of endogenous TRPC4 specifically attenuates GPCR-stimulated, but not thapsigargin- or OAG-stimulated extracellular calcium-dependent increases in [Ca²⁺]_i. These data imply that, in this cellular context, the mechanisms regulating extracellular Ca²⁺-dependent increases in [Ca²⁺]_i are differentially affected by different signaling pathways.     

Discovery and characterization of a positive allosteric modulator of transient receptor potential canonical 6 (TRPC6) channels

The non-selective second messenger-gated cation channel TRPC6 (transient receptor potential canonical 6) is activated by diacylglycerols (DAG) in a PKC-independent manner and plays important roles in a variety of physiological processes and diseases. In order to facilitate novel therapies, the development of potent inhibitors as well as channel-activating agents is of great interest. The screening of a chemical library, comprising about 17,000 small molecule compounds, revealed an agent, which induced increases in intracellular Ca²⁺ concentrations ([Ca²⁺]_i) in a concentration-dependent manner (EC₅₀ = 2.37 ± 0.25 μM) in stably TRPC6-expressing HEK293 cells. This new compound (C20) selectively acts on TRPC6, unlike OAG (1-oleoyl-1-acetyl-sn-glycerol), which also activates PKC and does not discriminate between TRPC6 and the closely related channels TRPC3 and TRPC7. Further evaluation by Ca²⁺ assays and electrophysiological studies revealed that C20 rather operated as an enhancer of channel activation than as an activator by itself and led to the assumption that the compound C20 is an allosteric modulator of TRPC6, enabling low basal concentrations of DAG to induce activation of the ion channel. Furthermore, C20 was tested in human platelets that express TRPC6. A combined activation of TRPC6 with C20 and OAG elicited a robust increase in [Ca²⁺]_i in human platelets. This potentiated channel activation was sensitive to TRPC6 channel blockers. To achieve sufficient amounts of C20 for biological studies, we applied a one-pot synthesis strategy. With regard to studies in native systems, the sensitizing ability of C20 can be a valuable pharmacological tool to selectively exaggerate TRPC6-dependent signals.     

Roles of transient receptor potential canonical (TRPC) channels and reverse-mode Na/Ca exchanger on cell proliferation in human cardiac fibroblasts: Effects of transforming growth factor β1

Expression of transient receptor potential canonical channels (TRPC) and the effects of transforming growth factor-β1 (TGF-β1) on Ca²⁺ signals and fibroblast proliferation were investigated in human cardiac fibroblasts. The conventional and quantitative real-time RT-PCR, western blot, immunocytochemical analysis, and intracellular Ca²⁺ concentration [Ca²⁺]_i measurement were applied. Cell proliferation and cell cycle progression were assessed using MTT assays and fluorescence activated cell sorting. Human cardiac fibroblasts have the expression of TRPC1,3,4,6 mRNA and proteins. 1-oleoyl-2-acetyl-sn-glycerol (OAG) and thapsigargin induced extracellular Ca²⁺-mediated [Ca²⁺]_i rise. siRNA for knock down of TRPC6 reduced OAG-induced Ca²⁺ entry. Hyperforin as well as angiotensin II (Ang II) induced Ca²⁺ entry. KB-R7943, a reverse-mode Na⁺/Ca²⁺ exchanger (NCX) inhibitor, and/or replacement of Na⁺ with NMDG⁺ inhibited thapsigargin-, OAG- and Ang II-induced Ca²⁺ entry. Treatment with TGF-β1 increased thapsigargin-, OAG- and Ang II-induced Ca²⁺ entry with an enhancement of TRPC1,6 protein expression, suppressed by KB-R7943. TGF-β1 and AngII promoted cell cycle progression from G0/G1 to S/G2/M and cell proliferation. A decrease of the extracellular Ca²⁺ and KB-R7943 suppressed it. Human cardiac fibroblasts contain several TRPC-mediated Ca²⁺ influx pathways, which activate the reverse-mode NCX. TGF-β1 enhances the Ca²⁺ influx pathways requiring Ca²⁺ signals for its effect on fibroblast proliferation.     

Distinct Contributions of Orai1 and TRPC1 to Agonist-Induced [Ca2+]i Signals Determine Specificity of Ca2+-Dependent Gene Expression

Regulation of critical cellular functions, including Ca²⁺-dependent gene expression, is determined by the temporal and spatial aspects of agonist-induced Ca²⁺ signals. Stimulation of cells with physiological concentrations of agonists trigger increases [Ca²⁺]_i due to intracellular Ca²⁺ release and Ca²⁺ influx. While Orai1-STIM1 channels account for agonist-stimulated [Ca²⁺]_i increase as well as activation of NFAT in cells such as lymphocytes, RBL and mast cells, both Orai1-STIM1 and TRPC1-STIM1 channels contribute to [Ca²⁺]_i increases in human submandibular gland (HSG) cells. However, only Orai1-mediated Ca²⁺ entry regulates the activation of NFAT in HSG cells. Since both TRPC1 and Orai1 are activated following internal Ca²⁺ store depletion in these cells, it is not clear how the cells decode individual Ca²⁺ signals generated by the two channels for the regulation of specific cellular functions. Here we have examined the contributions of Orai1 and TRPC1 to carbachol (CCh)-induced [Ca²⁺]_i signals and activation of NFAT in single cells. We report that Orai1-mediated Ca²⁺ entry generates [Ca²⁺]_i oscillations at different [CCh], ranging from very low to high. In contrast, TRPC1-mediated Ca²⁺ entry generates sustained [Ca²⁺]_i elevation at high [CCh] and contributes to frequency of [Ca²⁺]_i oscillations at lower [agonist]. More importantly, the two channels are coupled to activation of distinct Ca²⁺ dependent gene expression pathways, consistent with the different patterns of [Ca²⁺]_i signals mediated by them. Nuclear translocation of NFAT and NFAT-dependent gene expression display “all-or-none” activation that is exclusively driven by local [Ca²⁺]_i generated by Orai1, independent of global [Ca²⁺]_i changes or TRPC1-mediated Ca²⁺ entry. In contrast, Ca²⁺ entry via TRPC1 primarily regulates NFκB-mediated gene expression. Together, these findings reveal that Orai1 and TRPC1 mediate distinct local and global Ca²⁺ signals following agonist stimulation of cells, which determine the functional specificity of the channels in activating different Ca²⁺-dependent gene expression pathways.     

Diacylglycerol-containing oleic acid induces increases in [Ca]i via TRPC3/6 channels in human T-cells

Though most of the studies have focused on the effects of free fatty acids on T-cell activation, fatty acids incorporated into plasma membrane phospholipids may also affect cell signaling via diacylglycerol (DAG), generally produced by phospholipid hydrolysis. In the present study, we have synthesized a DAG-containing oleic acid and studied its implication in the modulation of calcium signaling in human Jurkat T-cells. 1-palmitoyl-2-oleoyl-sn-glycerol (POG) induced a dose-dependent increase in [Ca²⁺]_i. This effect was due to the presence of oleic acid at the sn-2 position as no differences were observed between POG and 1-stearoly-2-oleoyl-sn-glycerol (SOG). However, the substitution of oleic acid with arachidonic acid at the sn-2 position of the DAG moiety exerted a different response on the increases in [Ca²⁺]_i in these cells. POG-evoked increases in [Ca²⁺]_i were not due to its metabolites. Furthermore, POG-induced increases in [Ca²⁺]_i were due to the opening of TRPC3/TRPC6 channels as silencing of TRPC3 and TRPC6 genes by shRNA abolished calcium entry. Moreover, disruption of lipid rafts with methyl-β-cyclodextrin completely abolished POG-evoked increases in [Ca²⁺]_i. In conclusion, our results demonstrate that oleic acid can influence T-lymphocyte functions, in the conjugated form of DAG, via opening TRPC3/6 channels.     

TRPC6 inhibited NMDA receptor activities and protected neurons from ischemic excitotoxicity

Excitotoxicity induced by NMDA receptor‐mediated intracellular Ca²⁺ ([Ca²⁺]_i) overload is a major cause of delayed neuronal death in cerebral ischemia. Transient receptor potential canonical (TRPC) 6 protects neurons from ischemic brain damage. However, the mechanisms by which TRPC6 protects neurons are largely unknown. Here, we reported that TRPC6 suppressed the [Ca²⁺]_i elevation induced by NMDA and protected neurons from excitotoxicity. Over‐expressing or down‐regulating TRPC6 suppressed or aggravated Ca²⁺ overload under excitotoxicity, respectively. TRPC6 protected cultured neurons from damage caused by NMDA toxicity or oxygen glucose deprivation (OGD). Moreover, the infarct volume in TRPC6 transgenic (Tg) mice was smaller than that in wild‐type (WT) littermates. The TRPC6 Tg mice had better behavior performance and lower mortality than their WT littermates. Thus, TRPC6 inhibited NMDA receptor‐triggered neurotoxicity and protected neurons from ischemic brain damage. Increase in TRPC6 activity could be a potential strategy for stroke prevention and therapy.     

Palmitate induces human glomerular mesangial cells fibrosis through CD36-mediated transient receptor potential canonical channel 6/nuclear factor of activated T cell 2 activation

BackgroundOur previous study suggested that palmitate (PA) induces human glomerular mesangial cells (HMCs) fibrosis. However, the mechanism is not fully understood. Recent studies suggested that transient receptor potential canonical channel 6 (TRPC6)/nuclear factor of activated T cell 2 (NFAT2) played an important role in renal fibrosis. Moreover, cluster of differentiation 36 (CD36) regulated the synthesis of TPRC6 agonist diglyceride. In the present study, we investigated whether PA induced HMCs fibrosis via TRPC6/NFAT2 mediated by CD36.MethodsA type 2 diabetic nephropathy (DN) model was established in Sprague Dawley rats, and HMCs were stimulated with PA. Lipid accumulation and free fatty acid (FFA) uptake were measured. The expression levels of TGF-β1, p-Smad2/3, FN, TRPC6, NFAT2 and CD36 were evaluated. The intracellular calcium concentration ([Ca²⁺]i) was assessed.ResultsFFA were elevated in type 2 DN rats with kidney fibrosis in addition to NFAT2 and CD36 expression. In vitro, PA induced HMCs fibrosis, [Ca²⁺]_i elevation and NFAT2 activation. SKF96365 or TRPC6-siRNA could attenuate PA-induced HMCs damage. By contrast, the TRPC6 activator showed the opposite effect. Moreover, NFAT2-siRNA also suppressed PA-induced HMCs fibrosis. CD36 knockdown inhibited the PA-induced [Ca²⁺]_i elevation and NFAT2 expression. In addition, long-term treatment with PA decreased TRPC6 expression in HMCs.ConclusionThe results of this study demonstrated that PA could induce the activation of the [Ca²⁺]_i/NFAT2 signaling pathway through TRPC6, which led to HMCs fibrosis. Although activation of TRPC6 attributed to CD36-mediated lipid deposition, long-term stimulation of PA may lead to negative feedback on the expression of TPRC6.     

Calcium-sensing receptor activation contributed to apoptosis stimulates TRPC6 channel in rat neonatal ventricular myocytes

Capacitative calcium entry (CCE) refers to the influx of calcium through plasma membrane channels activated on depletion of endoplasmic sarcoplasmic/reticulum (ER/SR) Ca²⁺ stores, which is performed mainly by the transient receptor potential (TRP) channels. TRP channels are expressed in cardiomyocytes. Calcium-sensing receptor (CaR) is also expressed in rat cardiac tissue and plays an important role in mediating cardiomyocyte apoptosis. However, there are no data regarding the link between CaR and TRP channels in rat heart. In this study, in rat neonatal myocytes, by Ca²⁺ imaging, we found that the depletion of ER/SR Ca²⁺ stores by thapsigargin (TG) elicited a transient rise in cytoplasmic Ca²⁺ ([Ca²⁺]_i), followed by sustained increase depending on extracellular Ca²⁺. But, TRP channels inhibitor (SKF96365), not L-type channels or the Na⁺/Ca²⁺ exchanger inhibitors, inhibited [Ca²⁺]_i relatively high. Then, we found that the stimulation of CaR with its activator gadolinium chloride (GdCl₃) or by an increased extracellular Ca²⁺([Ca²⁺]_o) increased the concentration of intracelluar Ca²⁺, whereas, the sustained elevation of [Ca²⁺]_i was reduced in the presence of SKF96365. Similarly, the duration of [Ca²⁺]_i increase was also shortened in the absence of extracellular Ca²⁺. Western blot analysis showed that GdCl₃ increased the expression of TRPC6, which was reversed by SKF96365. Additionally, SKF96365 reduced cardiomyocyte apoptosis induced by GdCl₃. Our results suggested that CCE exhibited in rat neonatal myocytes and CaR activation induced Ca²⁺-permeable cationic channels TRPCs to gate the CCE, for which TRPC6 was one of the most likely candidates. TRPC6 channel was functionally coupled with CaR to enhance the cardiomyocyte apoptosis.     

Calcium entry via TRPC6 mediates albumin overload-induced endoplasmic reticulum stress and apoptosis in podocytes

Albumin, which is the most abundant component of urine proteins, exerts injurious effects on renal cells in chronic kidney diseases. However, the toxicity of albumin to podocytes is not well elucidated. Here, we show that a high concentration of albumin triggers intracellular calcium ([Ca²⁺]_i) increase through mechanisms involving the intracellular calcium store release and extracellular calcium influx in conditionally immortalized podocytes. The canonical transient receptor potential-6 (TRPC6) channel, which is associated with a subset of familial forms of focal segmental glomerulosclerosis (FSGS) and several acquired proteinuric kidney diseases, was shown to be one of the important Ca²⁺ permeable ion channels in podocytes. Therefore we explored the role of TRPC6 on albumin-induced functional and structural changes in podocytes. It was found that albumin-induced increase in [Ca²⁺]_i was blocked by TRPC6 siRNA or SKF-96365, a blocker of TRP cation channels. Long-term albumin exposure caused an up-regulation of TRPC6 expression in podocytes, which was inhibited by TRPC6 siRNA. Additionally, the inhibition of TRPC6 prevented the F-actin cytoskeleton disruption that is induced by albumin overload. Moreover, albumin overload induced expression of the endoplasmic reticulum (ER) stress protein GRP78, led to caspase-12 activation and ultimately podocyte apoptosis, all of which were abolished by the knockdown of TRPC6 using TRPC6 siRNA. These results support the view that albumin overload may induce ER stress and the subsequent apoptosis in podocytes via TRPC6-mediated Ca²⁺ entry.     

Diacylglycerol analogues activate second messenger-operated calcium channels exhibiting TRPC-like properties in cortical neurons

The lipid diacylglycerol (DAG) analogue 1‐oleoyl‐2‐acetyl‐sn‐glycerol (OAG) was used to verify the existence of DAG‐sensitive channels in cortical neurons dissociated from E13 mouse embryos. Calcium imaging experiments showed that OAG increased the cytosolic concentration of Ca²⁺ ([Ca²⁺]i) in nearly 35% of the KCl‐responsive cells. These Ca²⁺ responses disappeared in a Ca²⁺‐free medium supplemented with EGTA. Mn²⁺ quench experiments showed that OAG activated Ca²⁺‐conducting channels that were also permeant to Ba²⁺. The OAG‐induced Ca²⁺ responses were unaffected by nifedipine or omega‐conotoxin GVIA (Sigma‐Aldrich, Saint‐Quentin Fallavier, France) but blocked by 1‐[β‐(3‐(4‐Methoxyphenyl)propoxy)‐4‐methoxyphenethyl]‐1H‐imidazole hydrochloride (SKF)‐96365 and Gd³⁺. Replacing Na⁺ ions with N‐methyl‐d ‐glucamine diminished the amplitude of the OAG‐induced Ca²⁺ responses showing that the Ca²⁺ entry was mediated via Na⁺‐dependent and Na⁺‐independent mechanisms. Experiments carried out with the fluorescent Na⁺ indicator CoroNa Green showed that OAG elevated [Na⁺]i. Like OAG, the DAG lipase inhibitor RHC80267 increased [Ca²⁺]i but not the protein kinase C activator phorbol 12‐myristate 13‐acetate. Moreover, the OAG‐induced Ca²⁺ responses were not regulated by protein kinase C activation or inhibition but they were augmented by flufenamic acid which increases currents through C‐type transient receptor potential protein family (TRPC) 6 channels. In addition, application of hyperforin, a specific activator of TRPC6 channels, elevated [Ca²⁺]i. Whole‐cell patch‐clamp recordings showed that hyperforin activated non‐selective cation channels. They were blocked by SKF‐96365 but potentiated by flufenamic acid. Altogether, our data show the presence of hyperforin‐ and OAG‐sensitive Ca²⁺‐permeable channels displaying TRPC6‐like properties. This is the first report revealing the existence of second messenger‐operated channels in cortical neurons.     

Suppression of Rad leads to arrhythmogenesis via PKA-mediated phosphorylation of ryanodine receptor activity in the heart

Ras-related small G-protein Rad plays a critical role in generating arrhythmias via regulation of the L-type Ca²⁺ channel (LTCC). The aim was to demonstrate the role of Rad in intracellular calcium homeostasis by cardiac-Specific dominant-negative suppression of Rad. Transgenic (TG) mice overexpressing dominant-negative mutant Rad (S105N Rad TG) were generated. To measure intracellular Ca²⁺ concentration ([Ca²⁺]_i), we recorded [Ca²⁺]_i transients and Ca²⁺ sparks from isolated cardiomyocytes using confocal microscopy. The mean [Ca²⁺]_i transient amplitude was significantly increased in S105N Rad TG cardiomyocytes, compared with control littermate mouse cells. The frequency of Ca²⁺ sparks was also significantly higher in TG cells than in control cells, although there were no significant differences in amplitude. The sarcoplasmic reticulum Ca²⁺ content was not altered in the S105N Rad TG cells, as assessed by measuring caffeine-induced [Ca²⁺]_i transient. In contrast, phosphorylation of Ser²⁸⁰⁹ on the cardiac ryanodine receptor (RyR2) was significantly enhanced in TG mouse hearts compared with controls. Additionally, the Rad-mediated RyR2 phosphorylation was regulated via a direct interaction of Rad with protein kinase A (PKA).     

Effects of chronic treatment with fluoxetine on receptor-stimulated increase of [Ca2+]i in astrocytes mimic those of acute inhibition of TRPC1 channel activity

Primary cultures of mouse astrocytes were used to investigate effects by chronic treatment (3-21 days) with fluoxetine (0.5-10 μM) on capacitative Ca²⁺ influx after treatment with the SERCA inhibitor thapsigargin and on receptor agonist-induced increases in free cytosolic Ca²⁺ concentration [Ca²⁺]_i, determined with Fura-2. The agonists were the 5-HT_2B agonist fluoxetine, the α₂-adrenergic agonist dexmedetomidine, and ryanodine receptor (RyR) and IP₃ receptor (IP₃R) agonists. In untreated sister cultures each agonist distinctly increased [Ca²⁺]_i, but in cultures treated for sufficient length of time or with sufficiently high doses of fluoxetine, acute administration of fluoxetine, dexmedetomidine, or RyR or IP₃R agonists elicited reduced, in some cases abolished, effects. Capacitative Ca²⁺ entry, meditated by TRPC1 channels, was sufficiently inhibited to cause a depletion of Ca²⁺ stores, which could explain the reduced agonist effects. All effects of chronic fluoxetine administration could be replicated by TRPC1 channel antibody or siRNA. Since increases in astrocytic [Ca²⁺]_i regulate release of gliotransmitters, these effects may have profound effects on brain function. They may be important for therapeutic effects of all 5 conventional ‘serotonin-specific reuptake inhibitors’ (SSRIs), which at concentrations used therapeutically (∼1 μM) share other of fluoxetine's chronic effects (Zhang et al., Neuron Glia Biol. 16 (2010) 1-13).     

Effects of intratracheal administration of nuclear factor-kappaB decoy oligodeoxynucleotides on long-term cigarette smoke-induced lung inflammation and pathology in mice

Background

Sildenafil, a potent phosphodiesterase type 5 (PDE5) inhibitor, has been proposed as a treatment for pulmonary arterial hypertension (PAH). The mechanism of its anti-proliferative effect on pulmonary artery smooth muscle cells (PASMC) is unclear. Nuclear translocation of nuclear factor of activated T-cells (NFAT) is thought to be involved in PASMC proliferation and PAH. Increase in cytosolic free [Ca²⁺] ([Ca²⁺]_i) is a prerequisite for NFAT nuclear translocation. Elevated [Ca²⁺]_i in PASMC of PAH patients has been demonstrated through up-regulation of store-operated Ca²⁺ channels (SOC) which is encoded by the transient receptor potential (TRP) channel protein. Thus we investigated if: 1) up-regulation of TRPC1 channel expression which induces enhancement of SOC-mediated Ca²⁺ influx and increase in [Ca²⁺]_i is involved in hypoxia-induced PASMC proliferation; 2) hypoxia-induced promotion of [Ca²⁺]_i leads to nuclear translocation of NFAT and regulates PASMC proliferation and TRPC1 expression; 3) the anti-proliferative effect of sildenafil is mediated by inhibition of this SOC/Ca²⁺/NFAT pathway.

Methods

Human PASMC were cultured under hypoxia (3% O₂) with or without sildenafil treatment for 72 h. Cell number and cell viability were determined with a hemocytometer and MTT assay respectively. [Ca²⁺]_i was measured with a dynamic digital Ca²⁺ imaging system by loading PASMC with fura 2-AM. TRPC1 mRNA and protein level were detected by RT-PCR and Western blotting respectively. Nuclear translocation of NFAT was determined by immunofluoresence microscopy.

Results

Hypoxia induced PASMC proliferation with increases in basal [Ca²⁺]_i and Ca²⁺ entry via SOC (SOCE). These were accompanied by up-regulation of TRPC1 gene and protein expression in PASMC. NFAT nuclear translocation was significantly enhanced by hypoxia, which was dependent on SOCE and sensitive to SOC inhibitor SKF96365 (SKF), as well as cGMP analogue, 8-brom-cGMP. Hypoxia-induced PASMC proliferation and TRPC1 up-regulation were inhibited by SKF and NFAT blocker (VIVIT and Cyclosporin A). Sildenafil treatment ameliorated hypoxia-induced PASMC proliferation and attenuated hypoxia-induced enhancement of basal [Ca²⁺]_i, SOCE, up-regulation of TRPC1 expression, and NFAT nuclear translocation.

Conclusion

The SOC/Ca²⁺/NFAT pathway is, at least in part, a downstream mediator for the anti-proliferative effect of sildenafil, and may have therapeutic potential for PAH treatment.     

Effect of tetramethylpyrazine (TMP) on Ca2+ signal transduction and cell viability in a model of renal tubular cells

Tetramethylpyrazine (TMP) is a compound purified from herb. Its effect on Ca²⁺ concentrations ([Ca²⁺]_i) in renal cells is unclear. This study examined whether TMP altered Ca²⁺ signaling in Madin‐Darby canine kidney (MDCK) cells. TMP at 100–800 μM induced [Ca²⁺]_i rises, which were reduced by Ca²⁺ removal. TMP induced Mn²⁺ influx implicating Ca²⁺ entry. TMP‐induced Ca²⁺ entry was inhibited by 30% by modulators of protein kinase C (PKC) and store‐operated Ca²⁺ channels. Treatment with the endoplasmic reticulum Ca²⁺ pump inhibitor 2,5‐di‐tert‐butylhydroquinone (BHQ) inhibited 93% of TMP‐evoked [Ca²⁺]_i rises. Treatment with TMP abolished BHQ‐evoked [Ca²⁺]_i rises. Inhibition of phospholipase C (PLC) abolished TMP‐induced responses. TMP at 200–1000 μM decreased viability, which was not reversed by pretreatment with the Ca²⁺ chelator 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid‐acetoxymethyl ester. Together, in MDCK cells, TMP induced [Ca²⁺]_i rises by evoking PLC‐dependent Ca²⁺ release from endoplasmic reticulum and Ca²⁺ entry via PKC‐sensitive store‐operated Ca²⁺ entry. TMP also caused Ca²⁺‐independent cell death.     

Phorbol esters and diacylglycerol inhibit vasopressin-induced increases in cytoplasmic-free Ca and Ca efflux in Swiss 3T3 cells

Vasopressin increased intracellular free calcium concentration [Ca²⁺]_i in quin-2-loaded quiescent Swiss 3T3 cells. This effect of vasopressin was rapidly inhibited by biologically active tumour promoters including phorbol dibutyrate (PBt₂) and by the synthetic diacylglycerol 1-oleoyl-2-acetyl-glycerol (OAG). Prolonged pretreatment of Swiss 3T3 cells with PBt₂ causes a loss of protein kinase C activity (Rodriguez-Pena & Rozengurt, Biochem biophys res commun 120 (1984) 1053) [28]. This pretreatment abolished the inhibition by PBt₂ or OAG of vasopressin-mediated increases in Ca²⁺]_i. Vasopressin also stimulated ⁴⁵Ca²⁺ efflux from cells pre-loaded with the isotope. This effect of the hormone was also inhibited by PBt₂. Prolonged pretreatment with PBt₂ prevented the inhibition of vasopressin-stimulated ⁴⁵Ca²⁺ release by PBt₂. Thus, protein kinase C stimulation inhibits vasopressin-mediated increases in [Ca²⁺]_i and ⁴⁵Ca²⁺ efflux apparently by blocking the increased release of Ca²⁺ from an intracellular store caused by the hormone. These findings suggest that activation of protein kinase C may act as a feedback inhibitor to modulate ligand-mediated increases in [Ca²⁺]_i.     

Sarafotoxin-Induced Calcium Mobilization in Cultured Dog Tracheal Smooth Muscle Cells

Abstract

Sarafotoxin b (S6b) -induced changes in intracellular Ca[²⁺] concentration ([Ca ²⁺]i) were monitored in cultured canine tracheal smooth muscle cells (TSMCs) by a fluorescent Ca²⁺ indicator fura-2. S6b elicited an initial transient peak followed by a sustained elevation of [Ca²⁺]_i. BQ-123, an endothelin (ET_A) eceptor antagonist, had a high affinity to block the rise [Ca²⁺]_i response to S6b. In the absence of external Ca²⁺, only an initial transient peak of [Ca²⁺]_i was seen, the sustained elevation of [Ca²⁺]_i could then be evoked by addition of 1.8 mM [Ca²⁺] Ca²⁺ influx was required for the changes of [Ca²⁺]_i, since the Ca²⁺-channel blockers, diltiazem, verapamil, an& Nip+, decreased both the initial and sustained elevation of [Ca2+Ii in response to S6b. TSMCs pretreated with phorbol 12-myristate 13- acetate (PMA, 1 (M) for 30 min attenuated Ca²⁺ mobilization induced by S6b, w ich was reversed by stauros orine, a protein kinase C (PKC) inhibitor. The change of [Ca2P] + induced by S6b was attenuated by cholera toxin pretreatmenk, but not by pertussis toxin. These data demonstrate that the initial detectable increase in [Ca2+Ii stimulated by S6b is due to the activation of ETA receptors and subsequent release of Ca²⁺ internal stores, whereas the contribution of external Ca²⁺ follows and partially involves a diltiazem- and verapamil-sensitive process. The inhibition of PMA on S6b-induced Ca²⁺ mobilization was inversely correlated with membraneous PKC activity.     

Methylmercury-Induced Elevations in Intrasynaptosomal Zinc Concentrations: An 19F-NMR Study

Abstract: Methylmercury (MeHg) increases the concentration of intracellular Ca²⁺ ([Ca²⁺]_i) and another endogenous polyvalent cation in both synaptosomes and NG108-15 cells. In synaptosomes, the elevation in [Ca²⁺]_i was strictly dependent on extracellular Ca²⁺ (Ca²⁺_e); similarly, in NG108-15 cells, a component of the elevations in [Ca²⁺]_i was Ca²⁺_e dependent. The MeHg-induced elevations in endogenous polyvalent cation concentration were independent of Ca²⁺_e in synaptosomes and NG108-15 cells. The pattern of alterations in fura-2 fluorescence suggested the endogenous polyvalent cation may be Zn²⁺. Using ¹⁹F-NMR spectroscopy of rat cortical synaptosomes loaded with the fluorinated chelator 1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N′,N′-tetraacetic acid (5F-BAPTA), we have determined unambiguously that MeHg increases the free intrasynaptosomal Zn²⁺ concentration ([Zn²⁺]_i). In buffer containing 200 µM EGTA to prevent the Ca²⁺_e-dependent elevations in [Ca²⁺]_i, the [Zn²⁺]_i was 1.37 ± 0.20 nM; following a 40-min exposure to MeHg-free buffer [Zn²⁺]_i was 1.88 ± 0.53 nM. Treatment of synaptosomes for 40 min with 125 µM MeHg yielded [Zn²⁺]_i of 2.69 ± 0.55 nM, whereas 250 µM MeHg significantly elevated [Zn²⁺]_i to 3.99 ± 0.68 nM. No Zn²⁺ peak was observed in synaptosomes treated with the cell-permeant heavy metal chelator N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN, 100 µM) following 250 µM MeHg exposure. [Ca²⁺]_i in buffer containing 200 µM EGTA was 338 ± 26 nM and was 370 ± 64 nM following an additional 40-min exposure to MeHg-free buffer. [Ca²⁺]_i was 498 ± 28 or 492 ± 53 nM during a 40-min exposure to 125 or 250 µM MeHg, respectively. None of the values of [Ca²⁺]_i differed significantly from either pretreatment levels or buffer-treated controls.     

Carvacrol-induced [Ca2+]i rise and apoptosis in human glioblastoma cells

AimsThis study examined whether the essential oil component carvacrol altered cytosolic free Ca²⁺ level ([Ca²⁺]_i) and viability in human glioblastoma cells.Main methodsThe Ca²⁺-sensitive fluorescent dye fura-2 was applied to measure [Ca²⁺]_i. Cell viability was measured by detecting reagent WST-1. Apoptosis and reactive oxygen species (ROS) were detected by flow cytometry.Key findingsCarvacrol at concentrations of 400–1000 μM induced a [Ca²⁺]_i rise in a concentration-dependent fashion. The response was decreased partially by removal of extracellular Ca²⁺. Carvacrol-induced Ca²⁺ signal was not altered by nifedipine, econazole, SK&;F96365, and protein kinase C activator phorbol myristate acetate (PMA), but was inhibited by the protein kinase C inhibitor GF109203X. When extracellular Ca²⁺ was removed, incubation with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) abolished carvacrol-induced [Ca²⁺]_i rise. Incubation with carvacrol also abolished thapsigargin or BHQ-induced [Ca²⁺]_i rise. Inhibition of phospholipase C with U73122 abolished carvacrol-induced [Ca²⁺]_i rise. At concentrations of 200–800 μM, carvacrol killed cells in a concentration-dependent manner. This cytotoxic effect was not changed by chelating cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N–-tetraacetic acid/acetoxy methyl (BAPTA/AM). Annexin V/propidium iodide staining data suggest that carvacrol (200, 400 and 600 μM) induced apoptosis in a concentration-dependent manner. At concentrations of 200, 400 and 600 μM, carvacrol induced production of ROS.SignificanceIn human glioblastoma cells, carvacrol induced a [Ca²⁺]_i rise by inducing phospholipase C-dependent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ entry via protein kinase C-sensitive, non store-operated Ca²⁺ channels. Carvacrol induced cell death that might involve ROS-mediated apoptosis.