Increase In Cytosolic Free Ca2 Research Articles

Silver nanoparticles stimulate 5-Fluorouracil-induced colorectal cancer cells to kill through the upregulation TRPV1-mediated calcium signaling pathways.

The involvement of the TRP vanilloid 1 (TRPV1)cation channel on the 5-Fluorouracil (5-FU)-caused Ca2+ signals through the activation of the apoptotic signaling pathway and stimulating the mitochondrial Ca2+ and Zn2+ accumulation-induced reactive oxygen species (ROS) productions in several cancer cells, except the colorectal cancer (HT-29) cell line, was recently reported. I aimed to investigate the action of silver nanoparticles (SiNPs) and 5-FU incubations through the activation of TRPV1 on ROS, apoptosis, and cell death in the HT-29 cell line. The cells were divided into four groups: control, SiNP (100 µM for 48 h), 5-FU (25 μM for 24 h), and 5-FU + SiNP. SiNP treatment through TRPV1 activation (via capsaicin) stimulated the oxidant and apoptotic actions of 5-FU in the cells, whereas they were diminished in the cells by the TRPV1 antagonist (capsazepine) treatment. The apoptotic and cell death actions of 5-FU were determined by increasing the propidium iodide/Hoechst rate, caspase-3, -8, and -9 activations, mitochondrial membrane depolarization, lipid peroxidation, and ROS, but decreasing the glutathione and glutathione peroxidase. The increase of cytosolic free Ca2+ and Zn2+ into mitochondria via the stimulation of TRPV1 current density increased oxidant and apoptotic properties of 5-FU in the cells. For the therapy of HT-29 tumor cells, I found that the combination of SiNPs and 5-FU was synergistic via TRPV1 activation.

Total alkaloids from the seed embryo of Nelumbo nucifera Gaertn. improve cognitive impairment in APP/PS1 mice and protect Aβ-damaged PC12 cells

ABSTRACT The seed embryo of Nelumbo nucifera Gaertn. is a famous traditional Chinese medicine and food which is considered conducive to the prevention of Alzheimer's disease (AD). In this study, the effect and mechanism of TASENN (total alkaloids from the seed embryo of Nelumbo nucifera Gaertn.) on AD mice and amyloid-β (Aβ) injured PC12 cells were evaluated. HPLC-UV analysis showed that the extracted TASENN (purity = 95.6%) mainly contains Liensinine, Isoliensinine, and Neferine (purity was 23.01, 28.02, and 44.57%, respectively). In vivo, oral treatment with TASENN (50 mg/kg/day for 28 days) improved the learning and memory functions of APP/PS1 transgenic mice, ameliorated the histopathological changes of cortical and hippocampal neurons, and inhibited neuronal apoptosis. We found that TASENN reduced the phosphorylation of Tau and the formation of neurofibrillary tangles (NFTs) in APP/PS1 mouse brain. Moreover, TASENN down-regulated the expression of APP and BACE1, ameliorated Aβ deposition, and inhibited microglial proliferation and aggregation. The elevated protein expression of CaM and p-CaMKII in APP/PS1 mouse brain was also reduced by TASENN. In vitro, TASENN inhibited the apoptosis of PC12 cells injured by Aβ25–35 and increased the cell viability. Aβ25–35-induced increase of cytosolic free Ca2+ level and high expression of CaM, p-CaMKII, and p-Tau were decreased by TASENN. Our findings indicate that TASENN has a potential therapeutic effect on AD mice and a protective effect on PC12 cells. The anti-AD activity of TASENN may be closely related to its negative regulation of the CaM pathway.

Mechanosensitive cation currents through TRPC6 and Piezo1 channels in human pulmonary arterial endothelial cells.

Mechanosensitive cation channels and Ca2+ influx through these channels play an important role in the regulation of endothelial cell functions. Transient receptor potential canonical channel 6 (TRPC6) is a diacylglycerol-sensitive nonselective cation channel that forms receptor-operated Ca2+ channels in a variety of cell types. Piezo1 is a mechanosensitive cation channel activated by membrane stretch and shear stress in lung endothelial cells. In this study, we report that TRPC6 and Piezo1 channels both contribute to membrane stretch-mediated cation currents and Ca2+ influx or increase in cytosolic-free Ca2+ concentration ([Ca2+]cyt) in human pulmonary arterial endothelial cells (PAECs). The membrane stretch-mediated cation currents and increase in [Ca2+]cyt in human PAECs were significantly decreased by GsMTX4, a blocker of Piezo1 channels, and by BI-749327, a selective blocker of TRPC6 channels. Extracellular application of 1-oleoyl-2-acetyl-sn-glycerol (OAG), a membrane permeable analog of diacylglycerol, rapidly induced whole cell cation currents and increased [Ca2+]cyt in human PAECs and human embryonic kidney (HEK)-cells transiently transfected with the human TRPC6 gene. Furthermore, membrane stretch with hypo-osmotic or hypotonic solution enhances the cation currents in TRPC6-transfected HEK cells. In HEK cells transfected with the Piezo1 gene, however, OAG had little effect on the cation currents, but membrane stretch significantly enhanced the cation currents. These data indicate that, while both TRPC6 and Piezo1 are involved in generating mechanosensitive cation currents and increases in [Ca2+]cyt in human PAECs undergoing mechanical stimulation, only TRPC6 (but not Piezo1) is sensitive to the second messenger diacylglycerol. Selective blockers of these channels may help develop novel therapies for mechanotransduction-associated pulmonary vascular remodeling in patients with pulmonary arterial hypertension.

A nematode effector inhibits plant immunity by preventing cytosolic free Ca2+ rise.

The Meloidogyne enterolobii effector MeTCTP is a member of the translationally controlled tumour protein (TCTP) family, involved in M. enterolobii parasitism. In this study, we found that MeTCTP forms homodimers and, in this form, binds calcium ions (Ca2+ ). At the same time, Ca2+ could induce homodimerization of MeTCTP. We further identified that MeTCTP inhibits the increase of cytosolic free Ca2+ concentration ([Ca2+ ]cyt ) in plant cells and suppresses plant immune responses. This includes suppression of reactive oxygen species burst and cell necrosis, further promoting M. enterolobii parasitism. Our results have elucidated that the effector MeTCTP can directly target Ca2+ by its homodimeric form and prevent [Ca2+ ]cyt rise in plant roots, revealing a novel mechanism utilized by plant pathogens to suppress plant immunity.

ORAI1 regulates sustained cytosolic free calcium fluctuations during breast cancer cell apoptosis and apoptotic resistance via a STIM1 independent pathway.

Excessive rapid increases in cytosolic free Ca2+ have a clear association with the induction of cancer cell death. Whereas, characterizing the Ca2+ signaling events that occur during the progression of the apoptotic cascade over a period of hours or days, has not yet been possible. Now using genetically encoded Ca2+ indicators complemented with automated epifluorescence microscopy we have shown that staurosporine-induced apoptosis in MDA-MB-231 breast cancer cells was associated with delayed development of cytosolic free Ca2+ fluctuations, which were then maintained for 24 h. These cytosolic free Ca2+ fluctuations were dependent on the Ca2+ channel ORAI1. Silencing of ORAI1, but not its canonical activators STIM1 and STIM2, promoted apoptosis in this model. The pathway for this regulation implicates a mechanism previously associated with the migration of cancer cells involving ORAI1, the chaperone protein SigmaR1, and Ca2+ -activated K+ channels.

Damage Signaling by Extracellular Nucleotides: A Role for Cyclic Nucleotides in Elevating Cytosolic Free Calcium?

Extracellular ATP (eATP) is now held to be a constitutive damage-associated molecular pattern (DAMP) that is released by wounding, herbivory or pathogen attack. The concentration of eATP must be tightly regulated as either depletion or overload leads to cell death. In Arabidopsis thaliana, sensing of eATP is by two plasma membrane legume-like lectin serine–threonine receptor kinases (P2K1 and P2K2), although other receptors are postulated. The transcriptional response to eATP is dominated by wound- and defense-response genes. Wounding and pathogen attack can involve the cyclic nucleotides cyclic AMP (cAMP) and cyclic GMP (cGMP) which, in common with eATP, can increase cytosolic-free Ca2+ as a second messenger. This perspective on DAMP signaling by eATP considers the possibility that the eATP pathway involves production of cyclic nucleotides to promote opening of cyclic nucleotide-gated channels and so elevates cytosolic-free Ca2+. In silico analysis of P2K1 and P2K2 reveals putative adenylyl and guanylyl kinase sequences that are the hallmarks of “moonlighting” receptors capable of cAMP and cGMP production. Further, an Arabidopsis loss of function cngc mutant was found to have an impaired increase in cytosolic-free Ca2+ in response to eATP. A link between eATP, cyclic nucleotides, and Ca2+ signaling therefore appears credible.

The Protective Effect of Ubiquinone against the Amyloid Peptide in Endothelial Cells Is Isoprenoid Chain Length-Dependent.

Vascular brain pathology constitutes a common feature in neurodegenerative diseases that could underlie their development. Indeed, vascular dysfunction acts synergistically with neurodegenerative changes to exacerbate the cognitive impairment found in Alzheimer’s disease. Different injuries such as hypertension, high glucose, atherosclerosis associated with oxidized low-density lipoprotein or inflammation induce NADPH oxidase activation, overproduction of reactive oxygen species, and apoptosis in endothelial cells. Since it has been shown that pretreatment of cultured endothelial cells with the lipophilic antioxidant coenzyme Q10 (CoQ10) displays a protective effect against the deleterious injuries caused by different agents, this study explores the cytoprotective role of different CoQs homologues against Aβ25–35-induced damage and demonstrates that only pretreatment with CoQ10 protects endothelial brain cells from Aβ25–35-induced damage. Herein, we show that CoQ10 constitutes the most effective ubiquinone in preventing NADPH oxidase activity and reducing both reactive oxygen species generation and the increase in free cytosolic Ca2+ induced by Aβ25–35, ultimately preventing apoptosis and necrosis. The specific cytoprotective effect of CoQ with a side chain of 10 isoprenoid units could be explained by the fact that CoQ10 is the only ubiquinone that significantly reduces the entry of Aβ25–35 into the mitochondria.

Selenium prevents interferon-gamma induced activation of TRPM2 channel and inhibits inflammation, mitochondrial oxidative stress, and apoptosis in microglia.

Microglia as the primary immune cells of brain actprotective effects against injuries and infections in the central nervous system. Inflammation via excessive Ca2+ influx and oxygen radical species (ROS) generation is a known factor in many neurodegenerative disorders. Importantly, the Ca2+ permeable TRPM2 channel is activated by oxidative stress. Thus, TRPM2 could provide the excessive Ca2+ influx in the microglia. Although TRPM2 expressionlevel is high in inflammatory cells, the interplay between mouse microglia and TRPM2 channel during inflammation is not fully identified. Thus, it is important to understand the mechanisms and factors involved in order to enhance neuronal regeneration and repair. The data presented here indicate that TRPM2 channels were activated in microglia cells by interferon-gamma (IFNγ). The IFNγ treatment further increased apoptosis (early and late) and cytokine productions (TNF-α, IL-1β, andIL-6) which were due to increased lipid peroxidation and ROS generations as well as increased activations of caspase -3 (Casp-3) and - 9 (Casp-9). However, selenium treatment diminished activations of TRPM2, cytokine, Casp-3, andCasp-9, and levels of lipid peroxidation andmitochondrial ROS production in the microglia that were treated with IFNγ. Moreover, addition of either PARP1 inhibitors (PJ34 or DPQ) or TRPM2 blockers (2-APB or ACA) potentiated the modulator effects of selenium. These results clearlysuggest that IFNγ leads to TRPM2 activation in microglia cells; whereas, selenium prevents IFNγ-mediated TRPM2 activation and cytokine generation. Together the interplay between IFNγ released from microglia cells is importance in brain inflammation and may affect oxidative cytotoxicity in the microglia. Graphical abstract Summary of pathways involved in IFNγ-induced TRPM2 activation and microglia death through excessive reactive oxygen species (ROS): Modulator role of selenium (Se). The IFNγ causes the microglia activation. Nudix box domain of TRPM2 is sensitive to ROS. The ROS induces DNA damage and ADPR-ribose (ADPR) production in the nucleus via PARP1 enzyme activation. ADPR and ROS-induced TRPM2 activation stimulates excessive Ca2+ influx. ROS are produced in the mitochondria through the increase of free cytosolic Ca2+ (via TRPM2 activation) by the IFNγ treatment, although they are diminished by the TRPM2 channel blocker (ACA and 2-APB) and PARP1 inhibitor treatments. The main mechanism in the cell death and inflammatory effects of IFNγ is mediated by stimulation of ROS-mediated caspase (caspase -3 and - 9) activations and cytokine production (TNF-α, IL-1β, and IL-6) via TRPM2 activation, respectively. The apoptotic, inflammatory, and oxidant actions of IFNγ are modulated through TRPM2 inhibition by the Se treatment.

Calcium Homeostasis Modulator (CALHM1/2) and Pulmonary Arterial Hypertension

RATIONALEConcentric pulmonary vascular remodeling is a major cause for the elevated pulmonary vascular resistance (PVR) and pulmonary arterial pressure (PAP) in patients with pulmonary arterial hypertension (PAH). Excessive PASMC proliferation and inhibited PASMC apoptosis have been implicated in the development and progression of pulmonary vascular wall thickening in PAH. Transition from the contractile phenotype to the synthetic or proliferative phenotype of PASMC is thus an important pathogenic process that promotes vascular remodeling. An increase in cytosolic free Ca2+ concentration ([Ca2+]cyt) in PASMC is not only a trigger for PASMC contraction (and pulmonary vasoconstriction), but also an important stimulus for PASMC proliferation. Calcium homeostasis modulators (CALHM), CALHM1/2, are membrane Ca2+ channels that are allosterically regulated by voltage and extracellular Ca2+. CALHM1/2 are closed at the resting membrane potential but can be opened by strong membrane depolarization. Reduction of extracellular [Ca2+] increases CALHM open probability, which allows the channel to be activated at negative potential. We aimed to study whether CALHM1/2 are involved in the phenotypical transition of PASMC and whether CALHM1/2 are upregulated in highly proliferative PASMC from patients with PAH.METHODSFreshly isolated pulmonary artery with the endothelium removed (as contractile PASMC) and primary cultured PASMC (as synthetic or proliferative PASMC) from rats were used for this study. Western blot analysis was used to compare protein expression levels of CALHM1 and CALHM2 in fresh PA tissue and cultured PASMC derived from the same rats. In addition, we compared protein expression levels of CALHM1/2 in PASMC from normal subjects and PAH patients.RESULTSThe protein expression level of CALHM2 in proliferative PASMC (the primary cultured PASMC in growth factor‐containing medium) was significantly higher than in contractile PASMC (the freshly isolated PA tissue). The increased CALHM2 in proliferative PASMC was indeed associated with a significantly increased expression of PCNA, a cell proliferation marker, and a significantly decreased expression of MHC and TGLN4, markers for contractile phenotype of SMC. Furthermore, we also observed that mRNA expression level of CALHM2 was significantly higher in PASMC isolated from PAH patients than in PASMC from normal subjects.CONCLUSIONUpregulation of CALHM2 may play an important role in the phenotypical transition of PASMC from the contractile phenotype to the synthetic or proliferative phenotype. Ca2+ influx through voltage‐ and extracellular Ca2+‐regulated CALHM2 in the plasma membrane of PASMC is an important trigger for PASMC proliferation and may play an important role in stimulating PASMC proliferation in patients with PAH.Support or Funding InformationNIH

Tetrahydrobenzimidazole TMQ0153 triggers apoptosis, autophagy and necroptosis crosstalk in chronic myeloid leukemia

By comparing imatinib-sensitive and -resistant chronic myeloid leukemia (CML) cell models, we investigated the molecular mechanisms by which tetrahydrobenzimidazole derivative TMQ0153 triggered caspase-dependent apoptosis at low concentrations accompanied by loss of mitochondrial membrane potential (MMP) and increase of cytosolic free Ca2+ levels. Interestingly, at higher concentrations, TMQ0153 induced necroptotic cell death with accumulation of ROS, both preventable by N-acetyl-L-cysteine (NAC) pretreatment. At necroptosis-inducing concentrations, we observed increased ROS and decreased ATP and GSH levels, concomitant with protective autophagy induction. Inhibitors such as bafilomycin A1 (baf-A1) and siRNA against beclin 1 abrogated autophagy, sensitized CML cells against TMQ0153 and enhanced necroptotic cell death. Importantly, TMQ153-induced necrosis led to cell surface exposure of calreticulin (CRT) and ERp57 as well as the release of extracellular ATP and high mobility group box (HMGB1) demonstrating the capacity of this compound to release immunogenic cell death (ICD) markers. We validated the anti-cancer potential of TMQ0153 by in vivo inhibition of K562 microtumor formation in zebrafish. Taken together, our findings provide evidence that cellular stress and redox modulation by TMQ0153 concentration-dependently leads to different cell death modalities including controlled necrosis in CML cell models.

IgGs from patients with amyotrophic lateral sclerosis and diabetes target CaVα2δ1 subunits impairing islet cell function and survival

Patients with amyotrophic lateral sclerosis (ALS) often show hallmarks of type 2 diabetes mellitus (T2DM). However, the causal link between ALS and T2DM has remained a mystery. We now demonstrate that 60% of ALS patients with T2DM (ALS-T2DM) have sera that exaggerated K+-induced increases in cytosolic free Ca2+ concentration ([Ca2+]i) in mouse islet cells. The effect was attributed to the presence of pathogenic immunoglobulin Gs (IgGs) in ALS-T2DM sera. The pathogenic IgGs immunocaptured the voltage-dependent Ca2+ (CaV) channel subunit CaVα2δ1 in the plasma membrane enhancing CaV1 channel-mediated Ca2+ influx and [Ca2+]i, resulting in impaired mitochondrial function. Consequently, impairments in [Ca2+]i dynamics, insulin secretion, and cell viability occurred. These data reveal that patients with ALS-T2DM carry cytotoxic ALS-T2DM-IgG autoantibodies that serve as a causal link between ALS and T2DM by immunoattacking CaVα2δ1 subunits. Our findings may lay the foundation for a pharmacological treatment strategy for patients suffering from a combination of these diseases.

NaCl- and cold-induced stress activate different Ca2+-permeable channels in Arabidopsis thaliana

Salinity (NaCl) and low temperatures significantly affect plant growth, development, and agricultural productivity. Both these stresses trigger an increase in free cytosolic Ca2+ concentration ([Ca2+]i) via Ca2+ influx across the plasma membrane. However, the interaction between salinity- and low temperature-induced [Ca2+]i increases are still poorly understood. We used aequorin to investigate the NaCl- and cold-induced [Ca2+]i signals in Arabidopsis thaliana. We found that the combination of NaCl and cold stimuli induced a greater increase in [Ca2+]i than that by either NaCl or cold separately. Therefore, we showed that these factors had an additive effect on [Ca2+]i increase. Following a pre-treatment of either NaCl or cold, the increase in [Ca2+]i significantly declined in response to a second NaCl treatment or cold exposure. After pre-treatment with NaCl, a subsequent cold stimulus suppressed [Ca2+]i increase to a lesser degree than that by a second NaCl treatment. A similar response was observed after pre-treatment with cold and subsequent treatment with NaCl, but the peak [Ca2+]i differed between them. We propose that NaCl-induced Ca2+ channels differ from those induced by cold and that a feedback mechanism may exist between NaCl- and cold-evoked [Ca2+]i. The Ca2+-permeable channels activated by NaCl were similar to those induced by cold stress; however, they may be expressed in different cells and activated by different signaling pathways. Our results indicated that there is a cross-adaptation in [Ca2+]i changes in response to NaCl- and cold-induced stresses.

Both NaCl and H2O2 Long-Term Stresses Affect Basal Cytosolic Ca2+ Levels but Only NaCl Alters Cytosolic Ca2+ Signatures in Arabidopsis.

Salinity is one of the formidable environmental factors that affect plant growth and development and constrain agricultural productivity. Experimentally imposed short-term NaCl treatment triggers a transient increase in cytosolic free Ca2+ concentration ([Ca2+]i) via Ca2+ influx across the plasma membrane. Salinity stress, as well as other stresses, induces the production of reactive oxygen species (ROS), such as H2O2. It is well established that short-term H2O2 treatment also triggers a transient increase in [Ca2+]i. However, whether and how long-term NaCl and H2O2 treatments affect the basal levels of [Ca2+]i as well as plant responses to additional NaCl and H2O2 stresses remain poorly understood. Using an aequorin-based Ca2+ imaging assay, we found that the long-term treatment of Arabidopsis seedlings with both moderate NaCl and H2O2 in the growth media reduced the basal [Ca2+]i levels. Interestingly, we found that the long-term treatment with NaCl, but not H2O2, affected the responses of plants to additional NaCl stress, and remarkably the roots displayed enhanced responses while the leaves showed reduced responses. These findings suggest that plants adapt to the long-term NaCl stress, while H2O2 might be an integrator of many stresses.

STIM2 (Stromal Interaction Molecule 2)-Mediated Increase in Resting Cytosolic Free Ca2+ Concentration Stimulates PASMC Proliferation in Pulmonary Arterial Hypertension.

An increase in cytosolic free Ca2+ concentration ([Ca2+]cyt) in pulmonary artery smooth muscle cells (PASMCs) triggers pulmonary vasoconstriction and stimulates PASMC proliferation leading to vascular wall thickening. Here, we report that STIM2 (stromal interaction molecule 2), a Ca2+ sensor in the sarcoplasmic reticulum membrane, is required for raising the resting [Ca2+]cyt in PASMCs from patients with pulmonary arterial hypertension (PAH) and activating signaling cascades that stimulate PASMC proliferation and inhibit PASMC apoptosis. Downregulation of STIM2 in PAH-PASMCs reduces the resting [Ca2+]cyt, whereas overexpression of STIM2 in normal PASMCs increases the resting [Ca2+]cyt The increased resting [Ca2+]cyt in PAH-PASMCs is associated with enhanced phosphorylation (p) of CREB (cAMP response element-binding protein), STAT3 (signal transducer and activator of transcription 3), and AKT, increased NFAT (nuclear factor of activated T-cell) nuclear translocation, and elevated level of Ki67 (a marker of cell proliferation). Furthermore, the STIM2-associated increase in the resting [Ca2+]cyt also upregulates the antiapoptotic protein Bcl-2 in PAH-PASMCs. Downregulation of STIM2 in PAH-PASMCs with siRNA (1) decreases the level of pCREB, pSTAT3, and pAKT and inhibits NFAT nuclear translocation, thereby attenuating proliferation, and (2) decreases Bcl-2, which leads to an increase of apoptosis. In summary, these data indicate that upregulated STIM2 in PAH-PASMCs, by raising the resting [Ca2+]cyt, contributes to enhancing PASMC proliferation by activating the CREB, STAT3, AKT, and NFAT signaling pathways and stimulating PASMC proliferation. The STIM2-associated increase in the resting [Ca2+]cyt is also involved in upregulating Bcl-2 that makes PAH-PASMCs resistant to apoptosis, and thus plays an important role in sustained pulmonary vasoconstriction and excessive pulmonary vascular remodeling in patients with PAH.

Orai1, 2, 3 and STIM1 promote store-operated calcium entry in pulmonary arterial smooth muscle cells

Previous studies have demonstrated that besides the classic canonical transient receptor potential channel family, Orai family and stromal interaction molecule 1 (STIM1) might also be involved in the regulation of store-operated calcium channels (SOCCs). An increase in cytosolic free Ca2+ concentration promoted by store-operated Ca2+ entry (SOCE) in pulmonary arterial smooth muscle cells (PASMCs) is a major trigger for pulmonary vasoconstriction and proliferation and migration of PASMCs. In this study, our data revealed the following: (1) in both rat distal pulmonary arteries and PASMCs, chronic hypoxia exposure upregulated the expression of Orai1 and Orai2, without affecting Orai3 and STIM1; (2) either heterozygous knockout of HIF-1α in mice or knockdown of HIF-1α in PASMCs abolished the hypoxic upregulation of Orai2, but not Orai1, suggesting the hypoxic upregulation of Orai2 depends on HIF-1α; and (3) using small interference RNA knockdown strategies, Orai1, 2, 3 and STIM1 were all shown to mediate SOCE in hypoxic PASMCs. Together, these results suggested that the components of SOCCs, including Orai1, 2, 3 and STIM1, may lead to novel therapeutic targets for the treatment of chronic hypoxia-induced pulmonary hypertension.

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

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. 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. 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. 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.

Biotic and Abiotic Stresses Activate Different Ca2+ Permeable Channels in Arabidopsis.

To survive, plants must respond rapidly and effectively to various stress factors, including biotic and abiotic stresses. Salinity stress triggers the increase of cytosolic free Ca2+ concentration ([Ca2+]i) via Ca2+ influx across the plasma membrane, as well as bacterial flg22 and plant endogenous peptide Pep1. However, the interaction between abiotic stress-induced [Ca2+]i increases and biotic stress-induced [Ca2+]i increases is still not clear. Employing an aequorin-based Ca2+ imaging assay, in this work, we investigated the [Ca2+]i changes in response to flg22, Pep1, and NaCl treatments in Arabidopsis thaliana. We observed an additive effect on the [Ca2+]i increase which induced by flg22, Pep1, and NaCl. Our results indicate that biotic and abiotic stresses may activate different Ca2+ permeable channels. Further, calcium signal induced by biotic and abiotic stresses was independent in terms of spatial and temporal patterning.

Spontaneous Exit from Diplotene Arrest Associates with the Increase of Calcium and Reactive Oxygen Species in Rat Oocytes Cultured In Vitro

Several signal molecules modulate the physiology of mammalian oocytes. Changes in the level of various signal molecules and their downstream impact on maturation promoting factor (MPF) during the achievement of meiotic competency remain ill understood. Therefore, the present study aimed to analyze levels of various signal molecules and MPF during the achievement of meiotic competency in rat oocytes. For this purpose, cumulus oocyte complexes (COCs) were collected from animals treated with 20 IU pregnant mare's serum gonadotropin (PMSG) for 48 h, followed by 20 IU human chorionic gonadotropin (hCG) for 14 h. The morphological changes, meiotic status and oocyte competency, cytosolic free calcium (Ca2+), total reactive oxygen species (ROS), inducible nitric oxide synthase (iNOS), adenosine 3′,5′-cyclic monophosphate (cAMP), guanosine 3′,5′-cyclic monophosphate (cGMP), cell division cycle 25B (Cdc25B), calcium-calmodulin-dependent protein kinase II (CaMKII), Wee1, specific phosphorylation status of cyclin-dependent kinase 1 (Cdk1), and cyclin B1 expression levels were analyzed. Our data suggest that the culture of denuded diplotene-arrested oocytes resulted in spontaneous exit from diplotene arrest (EDA) in a time-dependent manner. The supplementation of hCG (2 IU) did not show any additive effect over spontaneous EDA in vitro. The hCG (20 IU) surge induced achievement of meiotic competency as most of the ovulated oocytes showed metaphase-II (M-II) arrest with extrusion of first polar body (PB-I). The spontaneous EDA was associated with a moderate increase of cytosolic free Ca2+ as well as total ROS levels. Furthermore, iNOS, cAMP, cGMP, Cdc25B, Thr161 phosphorylated Cdk1, as well as cyclin B1 levels were significantly decreased, while CaMKII, Wee1, and Thr14/Tyr15 phosphorylated Cdk1 levels were increased significantly during spontaneous EDA. These changes were reversed in ovulated oocytes that showed M-II arrest. The above results suggest that the oscillation of signal molecules modulates MPF destabilization during the achievement of meiotic competency in rat oocytes cultured in vitro.

Paradoxical lower sensitivity of Locus Coeruleus than Substantia Nigra pars compacta neurons to acute actions of rotenone

Parkinson's disease (PD) is not only associated with degeneration of dopaminergic (DAergic) neurons in the Substantia Nigra, but also with profound loss of noradrenergic neurons in the Locus Coeruleus (LC). Remarkably, LC degeneration may exceed, or even precede the loss of nigral DAergic neurons, suggesting that LC neurons may be more susceptible to damage by various insults. Using a combination of electrophysiology, fluorescence imaging and electrochemistry, we directly compared the responses of LC, nigral DAergic and nigral non-dopaminergic (non-DAergic) neurons in rat brain slices to acute application of rotenone, a mitochondrial toxin used to create animal and in vitro models of PD. Rotenone (0.01–5.0μM) dose-dependently inhibited the firing of all three groups of neurons, primarily by activating KATP channels. The toxin also depolarised mitochondrial potential (Ψm) and released reactive oxygen species (H2O2). When KATP channels were blocked, rotenone (1μM) increased the firing of LC neurons by activating an inward current associated with dose-dependent increase of cytosolic free Ca2+ ([Ca2+]i). This effect was attenuated by blocking oxidative stress-sensitive TRPM2 channels, and by pre-treatment of slices with anti-oxidants. These results demonstrate that rotenone inhibits the activity of LC neurons mainly by activating KATP channels, and increases [Ca2+]ivia TRPM2 channels. Since the responses of LC neurons were smaller than those of nigral DAergic neurons, our study shows that LC neurons are paradoxically less sensitive to acute effects of this parkinsonian toxin.

Pathogenic role of calcium-sensing receptors in the development and progression of pulmonary hypertension

An increase in cytosolic free Ca(2+) concentration ([Ca(2+)]cyt) in pulmonary arterial smooth muscle cells (PASMC) is a major trigger for pulmonary vasoconstriction and a critical stimulation for PASMC proliferation and migration. Previously, we demonstrated that expression and function of calcium sensing receptors (CaSR) in PASMC from patients with idiopathic pulmonary arterial hypertension (IPAH) and animals with experimental pulmonary hypertension (PH) were greater than in PASMC from normal subjects and control animals. However, the mechanisms by which CaSR triggers Ca(2+) influx in PASMC and the implication of CaSR in the development of PH remain elusive. Here, we report that CaSR functionally interacts with TRPC6 to regulate [Ca(2+)]cyt in PASMC. Downregulation of CaSR or TRPC6 with siRNA inhibited Ca(2+)-induced [Ca(2+)]cyt increase in IPAH-PASMC (in which CaSR is upregulated), whereas overexpression of CaSR or TRPC6 enhanced Ca(2+)-induced [Ca(2+)]cyt increase in normal PASMC (in which CaSR expression level is low). The upregulated CaSR in IPAH-PASMC was also associated with enhanced Akt phosphorylation, whereas blockade of CaSR in IPAH-PASMC attenuated cell proliferation. In in vivo experiments, deletion of the CaSR gene in mice (casr(-/-)) significantly inhibited the development and progression of experimental PH and markedly attenuated acute hypoxia-induced pulmonary vasoconstriction. These data indicate that functional interaction of upregulated CaSR and upregulated TRPC6 in PASMC from IPAH patients and animals with experimental PH may play an important role in the development and progression of sustained pulmonary vasoconstriction and pulmonary vascular remodeling. Blockade or downregulation of CaSR and/or TRPC6 with siRNA or miRNA may be a novel therapeutic strategy to develop new drugs for patients with pulmonary arterial hypertension.