Ca Homeostasis Research Articles

HIV-1 Tat-Mediated Calcium Dysregulation and Neuronal Dysfunction in Vulnerable Brain Regions.

Despite the success of combined antiretroviral therapy, more than half of HIV-1-infected patients in the USA show HIV-associated neurological and neuropsychiatric deficits. This is accompanied by anatomical and functional alterations in vulnerable brain regions of the mesocorticolimbic and nigrostriatal systems that regulate cognition, mood and motivation-driven behaviors, and could occur at early stages of infection. Neurons are not infected by HIV, but HIV-1 proteins (including but not limited to the HIV-1 trans-activator of transcription, Tat) induce Ca(2+) dysregulation, indicated by abnormal and excessive Ca(2+) influx and increased intracellular Ca(2+) release that consequentially elevate cytosolic free Ca(2+) levels ([Ca(2+)]in). Such alterations in intracellular Ca(2+) homeostasis significantly disturb normal functioning of neurons, and induce dysregulation, injury, and death of neurons or non-neuronal cells, and associated tissue loss in HIV-vulnerable brain regions. This review discusses certain unique mechanisms, particularly the over-activation and/or upregulation of the ligand-gated ionotropic glutamatergic NMDA receptor (NMDAR), the voltage-gated L-type Ca(2+) channel (L-channel) and the transient receptor potential canonical (TRPC) channel (a non-selective cation channel that is also permeable for Ca(2+)), which may underlie the deleterious effects of Tat on intracellular Ca(2+) homeostasis and neuronal hyper-excitation that could ultimately result in excitotoxicity. This review also seeks to provide summarized information for future studies focusing on comprehensive elucidation of molecular mechanisms underlying the pathophysiological effects of Tat (as well as some other HIV-1 proteins and immunoinflammatory molecules) on neuronal function, particularly in HIV-vulnerable brain regions.

3,4-Methylenedioxypyrovalerone (MDPV): in vitro mechanisms of hepatotoxicity under normothermic and hyperthermic conditions.

Synthetic cathinones have emerged in recreational drug markets as legal alternatives for classical amphetamines. Though currently banned in several countries, 3,4-methylenedioxypyrovalerone (MDPV) is one of the most commonly abused cathinone derivatives worldwide. We have recently reported the potential of MDPV to induce hepatocellular damage, but the underlying mechanisms responsible for such toxicity remain to be elucidated. Similar to amphetamines, a prominent toxic effect of acute intoxications by MDPV is hyperthermia. Therefore, the present in vitro study aimed to provide insights into cellular mechanisms involved in MDPV-induced hepatotoxicity and also evaluate the contribution of hyperthermia to the observed toxic effects. Primary cultures of rat hepatocytes were exposed to 0.2-1.6mM MDPV for 48h, at 37 or 40.5°C, simulating the rise in body temperature that follows MDPV intake. Cell viability was measured through the MTT reduction and LDH leakage assays. Oxidative stress endpoints and cell death pathways were evaluated, namely the production of reactive oxygen and nitrogen species (ROS and RNS), intracellular levels of reduced (GSH) and oxidized (GSSG) glutathione, adenosine triphosphate (ATP) and free calcium (Ca(2+)), as well as the activities of caspases 3, 8 and 9, and nuclear morphological changes with Hoechst 33342/PI double staining. At 37°C, MDPV induced a concentration-dependent loss of cell viability that was accompanied by GSH depletion, as one of the first signs of toxicity, observed already at low concentrations of MDPV, with negligible changes on GSSG levels, followed by accumulation of ROS and RNS, depletion of ATP contents and increases in intracellular Ca(2+) concentrations. Additionally, activation of caspases 3, 8, and 9 and apoptotic nuclear morphological changes were found in primary rat hepatocytes exposed to MDPV, indicating that this cathinone derivative activates both intrinsic and extrinsic apoptotic death pathways. The cytotoxic potential of MDPV and all the studied endpoints were markedly aggravated under hyperthermic conditions (40.5°C). In conclusion, these data suggest that MDPV toxicity in primary rat hepatocytes is mediated by oxidative stress, subsequent to GSH depletion and increased ROS and RNS accumulation, mitochondrial dysfunction, and impairment of Ca(2+) homeostasis. Furthermore, the rise in body temperature subsequent to MDPV abuse greatly exacerbates its hepatotoxic potential.

Transient receptor potential channel C5 in cancer chemoresistance.

The transient receptor potential (TRP) superfamily contains at least 28 homologs in mammalian. These proteins form TRP channels are permeable to monovalent and divalent cations and participate in a variety of physiological functions. Dysregulation of TRP channels is responsible for numerous diseases. This review provides a brief short overview of mammalian TRP channels with a focus on TRPC5 and its role in cancers. Dysregulation of TRPC5 interrupts Ca(2+) homeostasis in cancer cells, which activates signaling pathways that are highly associated with cancer progression, especially cancer chemoresistance. Based on the important role of TRPC5, we also discuss the potential of TRPC5 as a target for therapeutic intervention. Either direct targeting of TRPC5 or indirect interruption of TRPC5-related signaling pathways may effectively overcome cancer chemoresistance.

Sex differences in the mechano-energetic effects of genistein on stunned rat and guinea pig hearts.

Although the phytoestrogen genistein (Gen) is considered protective in cardiovascular diseases, its direct effects on stunned hearts after transient ischemia-reperfusion (I/R) are unknown. This report studied the effects of 20 μmol/L Gen on the mechano-calorimetric behaviour during I/R of rat and guinea pig hearts to evaluate the energetics of Ca(2+) homeostasis. Isolated beating hearts were perfused with control Krebs solution inside a calorimeter with or without perfusion of Gen before a transient period of I/R. Left ventricular pressure development (P) and total heat rate (Ht) were continuously measured. At 37°C, Gen did not change post-ischemic contractile recovery (PICR), but it increased the relaxation rate. However, PICR was reduced in hearts of male rats and guinea pigs at 30°C. Total muscle economy (P/Ht) showed the same behaviour as P at each temperature. Inhibition of phosphatases with orthovanadate during Gen perfusion prevented a decrease in PICR in male rat hearts, suggesting that this effect is due to tyrosine kinase inhibition. Reperfusing ischemic hearts with 10 mmol/L caffeine-36 mmol/L Na(+)-Krebs induced contracture dependent on the sarcoreticular Ca(2+) content. Contracture relaxation depends on mitochondrial Ca(2+) uptake and Gen reduced the relaxation rate. Moreover, Gen prevented the increase in Rhod-2 fluorescence (free [Ca(2+)]m) of rat cardiomyocytes. In guinea pig hearts, Gen maintained ischemic preconditioning, but was reduced by 5-hydroxydecanoate, suggesting the participation of mitochondrial adenosine triphosphate (ATP)-dependent K channels. Results suggest that Gen acts on several mechanisms that regulate myocardial calcium homeostasis and energetics during I/R, which differ in a temperature- and sex-dependent manner.

NRIP is newly identified as a Z-disc protein, activating calmodulin signaling for skeletal muscle contraction and regeneration

Nuclear receptor interaction protein (NRIP, also known as DCAF6 and IQWD1) is a Ca(2+)-dependent calmodulin-binding protein. In this study, we newly identify NRIP as a Z-disc protein in skeletal muscle. NRIP-knockout mice were generated and found to have reduced muscle strength, susceptibility to fatigue and impaired adaptive exercise performance. The mechanisms of NRIP-regulated muscle contraction depend on NRIP being downstream of Ca(2+) signaling, where it stimulates activation of both 'calcineurin-nuclear factor of activated T-cells, cytoplasmic 1' (CaN-NFATc1; also known as NFATC1) and calmodulin-dependent protein kinase II (CaMKII) through interaction with calmodulin (CaM), resulting in the induction of mitochondrial activity and the expression of genes encoding the slow class of myosin, and in the regulation of Ca(2+) homeostasis through the internal Ca(2+) stores of the sarcoplasmic reticulum. Moreover, NRIP-knockout mice have a delayed regenerative capacity. The amount of NRIP can be enhanced after muscle injury and is responsible for muscle regeneration, which is associated with the increased expression of myogenin, desmin and embryonic myosin heavy chain during myogenesis, as well as for myotube formation. In conclusion, NRIP is a novel Z-disc protein that is important for skeletal muscle strength and regenerative capacity.

Store-operated Ca2+ Entry Mediated by Orai1 and TRPC1 Participates to Insulin Secretion in Rat β-Cells

Store-operated Ca(2+) channels (SOCs) are voltage-independent Ca(2+) channels activated upon depletion of the endoplasmic reticulum Ca(2+) stores. Early studies suggest the contribution of such channels to Ca(2+) homeostasis in insulin-secreting pancreatic β-cells. However, their composition and contribution to glucose-stimulated insulin secretion (GSIS) remains unclear. In this study, endoplasmic reticulum Ca(2+) depletion triggered by acetylcholine (ACh) or thapsigargin stimulated the formation of a ternary complex composed of Orai1, TRPC1, and STIM1, the key proteins involved in the formation of SOCs. Ca(2+) imaging further revealed that Orai1 and TRPC1 are required to form functional SOCs and that these channels are activated by STIM1 in response to thapsigargin or ACh. Pharmacological SOCs inhibition or dominant negative blockade of Orai1 or TRPC1 using the specific pore mutants Orai1-E106D and TRPC1-F562A impaired GSIS in rat β-cells and fully blocked the potentiating effect of ACh on secretion. In contrast, pharmacological or dominant negative blockade of TRPC3 had no effect on extracellular Ca(2+) entry and GSIS. Finally, we observed that prolonged exposure to supraphysiological glucose concentration impaired SOCs function without altering the expression levels of STIM1, Orai1, and TRPC1. We conclude that Orai1 and TRPC1, which form SOCs regulated by STIM1, play a key role in the effect of ACh on GSIS, a process that may be impaired in type 2 diabetes.

Proteomic analysis of physiological versus pathological cardiac remodeling in animal models expressing mutations in myosin essential light chains.

In this study we aimed to provide an in-depth proteomic analysis of differentially expressed proteins in the hearts of transgenic mouse models of pathological and physiological cardiac hypertrophy using tandem mass tag labeling and liquid chromatography tandem mass spectrometry. The Δ43 mouse model, expressing the 43-amino-acid N-terminally truncated myosin essential light chain (ELC) served as a tool to study the mechanisms of physiological cardiac remodeling, while the pathological hypertrophy was investigated in A57G (Alanine 57→Glycine) ELC mice. The results showed that 30 proteins were differentially expressed in Δ43 versus A57G hearts as determined by multiple pair comparisons of the mutant versus wild-type (WT) samples with P<0.05. The A57G hearts showed differential expression of nine mitochondrial proteins involved in metabolic processes compared to four proteins for ∆43 hearts when both mutants were compared to WT hearts. Comparisons between ∆43 and A57G hearts showed an upregulation of three metabolically important mitochondrial proteins but downregulation of nine proteins in ∆43 hearts. The physiological model of cardiac hypertrophy (∆43) showed no changes in the levels of Ca(2+)-binding proteins relative to WT, while the pathologic model (A57G) showed the upregulation of three Ca(2+)-binding proteins, including sarcalumenin. Unique differences in chaperone and fatty acid metabolism proteins were also observed in Δ43 versus A57G hearts. The proteomics data support the results from functional studies performed previously on both animal models of cardiac hypertrophy and suggest that the A57G- and not ∆43- mediated alterations in fatty acid metabolism and Ca(2+) homeostasis may contribute to pathological cardiac remodeling in A57G hearts.

The role of continuous versus fractionated physical training on muscle oxidative stress parameters and calcium-handling proteins in aged rats.

Age-associated decline in skeletal muscle mass and strength is associated with oxidative stress and Ca(2+) homeostasis disturbance. Exercise should be considered a viable modality to combat aging of skeletal muscle. This study aimed to investigate whether continuous and fractionated training could be useful tools to attenuate oxidative damage and retain calcium-handling proteins. We conducted the study using 24-month-old male Wistar rats, divided into control, continuous, and fractionated groups. Animals ran at 13mmin(-1) for five consecutive days (except weekends) for 6weeks, for a total period of 42days. Each session comprised 45min of exercise, either continuous or divided into three daily sessions of 15min each. Metabolic and oxidative stress markers, protein levels of mitochondrial transcription factors, and calcium-handling proteins were analyzed. Continuous exercise resulted in reduced ROS production as well as showed a decrease in TBARS levels and carbonyl content. On the other hand, fractionated training increased the antioxidant enzyme activities. The ryanodine receptor and phospholamban protein were regulated by continuous training while sodium calcium exchange protein was increased by the fractionated training. These data suggest that intracellular Ca(2+) can be modulated by various training stimuli. In addition, the modulation of oxidative stress by continuous and fractionated training may play an important regulatory role in the muscular contraction mechanism of aged rats, due to changes in calcium metabolism.

Endoplasmic reticulum stress and disturbed calcium homeostasis are involved in copper-induced alteration in hepatic lipid metabolism in yellow catfish Pelteobagrus fulvidraco.

The present study was conducted to investigate the effect of Cu exposure on ER stress and Ca(2+) homeostasis, and explore the underlying mechanism of the ER stress and disturbed Ca(2+) homeostasis in the regulation of hepatic lipid metabolism in yellow catfish Pelteobagrus fulvidraco. To this end, three experiments were conducted. In experiment 1, P. fulvidraco were exposed to three waterborne Cu concentrations for 56 days. Waterborne Cu exposure evoked ER stress and SREBP-1c activation and resulted in dysregulation of hepatic lipid metabolism in liver of P. fulvidraco in a time-dependent manner. In experiment 2, specific inhibitors 2-APB (IP3 receptor inhibitor) and dantrolene (RyR receptor inhibitor) were used to explore whether Ca(2+) release from ER was involved in the Cu-induced ER stress change. Dantrolene and 2-APB prevented Cu-induced intracellular Ca(2+) elevation, demonstrating that the release of Ca(2+) from the ER, mediated by both RyR and IP3R, contributed to dysregulation of lipid metabolism. In experiment 3, a chemical chaperone (PBA) was used to demonstrate whether Cu-induced alteration in lipid metabolism was suppressed through the attenuation of ER stress. PBA attenuated the Cu-induced elevation of mRNA expression of SREBP-1c, SCAP, ACC, FAS, GRP78/BiP, GRP94, CRT, eIF2α and XBP-1, and alleviated the Cu-induced downregulation of Insig-1. Based on these observations, these results reveal a link between ER stress and the change of lipid metabolism induced by Cu, which will help to understand the Cu-induced toxicity on cellular and molecular level, and provide some novel insights into the regulation of lipid metabolism in fish.

Activation of SK2 channels preserves ER Ca²⁺ homeostasis and protects against ER stress-induced cell death.

Alteration of endoplasmic reticulum (ER) Ca(2+) homeostasis leads to excessive cytosolic Ca(2+) accumulation and delayed neuronal cell death in acute and chronic neurodegenerative disorders. While our recent studies established a protective role for SK channels against excessive intracellular Ca(2+) accumulation, their functional role in the ER has not been elucidated yet. We show here that SK2 channels are present in ER membranes of neuronal HT-22 cells, and that positive pharmacological modulation of SK2 channels with CyPPA protects against cell death induced by the ER stressors brefeldin A and tunicamycin. Calcium imaging of HT-22 neurons revealed that elevated cytosolic Ca(2+) levels and decreased ER Ca(2+) load during sustained ER stress could be largely prevented by SK2 channel activation. Interestingly, SK2 channel activation reduced the amount of the unfolded protein response transcription factor ATF4, but further enhanced the induction of CHOP. Using siRNA approaches we confirmed a detrimental role for ATF4 in ER stress, whereas CHOP regulation was dispensable for both, brefeldin A toxicity and CyPPA-mediated protection. Cell death induced by blocking Ca(2+) influx into the ER with the SERCA inhibitor thapsigargin was not prevented by CyPPA. Blocking the K(+) efflux via K(+)/H(+) exchangers with quinine inhibited CyPPA-mediated neuroprotection, suggesting an essential role of proton uptake and K(+) release in the SK channel-mediated neuroprotection. Our data demonstrate that ER SK2 channel activation preserves ER Ca(2+) uptake and retention which determines cell survival in conditions where sustained ER stress contributes to progressive neuronal death.

Abstract 11016: Bag3 Regulates Contractility and Ca Homeostasis in Adult Mouse Ventricular Myocytes

Bcl2-associated athanogene 3 (BAG3) is a 575 amino acid anti-apoptotic protein that is constitutively expressed in the heart. BAG3 mutations are associated with familial dilated cardiomyopathy and reduced BAG3 expression was observed in murine transverse aortic constriction and porcine myocardial infarction models. BAG3 is expressed in the sarcolemma and t-tubules in adult myocytes. To simulate reduced BAG3 expression in disease states, we downregulated BAG3 in adult myocytes by shRNA (shBAG3). Despite ~50% reduction in BAG3 by shRNA, expression of α-subunit of L-type Ca channel, SR Ca-ATPase, Na/Ca exchanger, α1- and α2-subunits of Na-K-ATPase, and ryanodine receptor was unchanged. At baseline, BAG3 downregulation had no effect on myocyte contraction and [Ca]i dynamics. After isoproterenol, BAG3 downregulation resulted in reduced myocyte contraction amplitudes, shortening and re-lengthening velocities, lower systolic [Ca]i and [Ca]i transient amplitudes, and prolongation in both APD50 and APD90. L-type Ca current (ICa) and sarcoplasmic reticulum (SR) Ca content but not Na/Ca exchange current or SR Ca uptake were reduced in isoproterenol-treated shBAG3 myocytes. Forskolin or dibutyrl cAMP restored ICa amplitude in shBAG3 myocytes to that observed in WT myocytes. On the other hand, BAG3 overexpression resulted in enhanced myocyte contractility after isoproterenol. In addition, BAG3 overexpression rescued the depressed contraction amplitudes in myocytes overexpressing the HIV protein Tat, and improved myocardial contractility in Tg26 (HIV-1 transgenic) mice. We conclude: (1) BAG3 modulates myocyte contraction, Ca dynamics and action potential duration by regulating ion channels and provides a novel paradigm for contractile dysfunction and increased arrhythmia risks in familial dilated cardiomyopathy; (2) BAG3 is a therapeutic target for cardiomyopathy including HIV-associated cardiomyopathy.

Ca(2+) -regulated and diurnal rhythm-regulated Na(+) /Ca(2+) exchanger AtNCL affects flowering time and auxin signalling in Arabidopsis.

Calcium (Ca(2+) ) is vital for plant growth, development, hormone response and adaptation to environmental stresses, yet the mechanisms regulating plant cytosolic Ca(2+) homeostasis are not fully understood. Here, we characterize an Arabidopsis Ca(2+) -regulated Na(+) /Ca(2+) exchanger AtNCL that regulates Ca(2+) and multiple physiological processes. AtNCL was localized to the tonoplast in yeast and plant cells. AtNCL appeared to mediate sodium (Na(+) ) vacuolar sequestration and meanwhile Ca(2+) release. The EF-hand domains within AtNCL regulated Ca(2+) binding and transport of Ca(2+) and Na(+) . Plants with diminished AtNCL expression were more tolerant to high CaCl2 but more sensitive to both NaCl and auxin; heightened expression of AtNCL rendered plants more sensitive to CaCl2 but tolerant to NaCl. AtNCL expression appeared to be regulated by the diurnal rhythm and suppressed by auxin. DR5::GUS expression and root responses to auxin were altered in AtNCL mutants. The auxin-induced suppression of AtNCL was attenuated in SLR/IAA14 and ARF6/8 mutants. The mutants with altered AtNCL expression also altered flowering time and FT and CO expression; FT may mediate AtNCL-regulated flowering time change. Therefore, AtNCL is a vacuolar Ca(2+) -regulated Na(+) /Ca(2+) exchanger that regulates auxin responses and flowering time.

The RyR2-P2328S mutation downregulates Nav1.5 producing arrhythmic substrate in murine ventricles

Catecholaminergic polymorphic ventricular tachycardia (CPVT) predisposes to ventricular arrhythmia due to altered Ca2+ homeostasis and can arise from ryanodine receptor (RyR2) mutations including RyR2-P2328S. Previous reports established that homozygotic murine RyR2-P2328S (RyR2S/S) hearts show an atrial arrhythmic phenotype associated with reduced action potential (AP) conduction velocity and sodium channel (Nav1.5) expression. We now relate ventricular arrhythmogenicity and slowed AP conduction in RyR2S/S hearts to connexin-43 (Cx43) and Nav1.5 expression and Na+ current (INa). Stimulation protocols applying extrasystolic S2 stimulation following 8 Hz S1 pacing at progressively decremented S1S2 intervals confirmed an arrhythmic tendency despite unchanged ventricular effective refractory periods (VERPs) in Langendorff-perfused RyR2S/S hearts. Dynamic pacing imposing S1 stimuli then demonstrated that progressive reductions of basic cycle lengths (BCLs) produced greater reductions in conduction velocity at equivalent BCLs and diastolic intervals in RyR2S/S than WT, but comparable changes in AP durations (APD90) and their alternans. Western blot analyses demonstrated that Cx43 protein expression in whole ventricles was similar, but Nav1.5 expression in both whole tissue and membrane fractions were significantly reduced in RyR2S/S compared to wild-type (WT). Loose patch-clamp studies similarly demonstrated reduced INa in RyR2S/S ventricles. We thus attribute arrhythmogenesis in RyR2S/S ventricles resulting from arrhythmic substrate produced by reduced conduction velocity to downregulated Nav1.5 reducing INa, despite normal determinants of repolarization and passive conduction. The measured changes were quantitatively compatible with earlier predictions of linear relationships between conduction velocity and the peak INa of the AP but nonlinear relationships between peak INa and maximum Na+ permeability.

The prion protein constitutively controls neuronal store-operated Ca2+ entry through Fyn kinase

The prion protein (PrPC) is a cell surface glycoprotein mainly expressed in neurons, whose misfolded isoforms generate the prion responsible for incurable neurodegenerative disorders. Whereas PrPC involvement in prion propagation is well established, PrPC physiological function is still enigmatic despite suggestions that it could act in cell signal transduction by modulating phosphorylation cascades and Ca2+ homeostasis. Because PrPC binds neurotoxic protein aggregates with high-affinity, it has also been proposed that PrPC acts as receptor for amyloid-β (Aβ) oligomers associated with Alzheimer’s disease (AD), and that PrPC-Aβ binding mediates AD-related synaptic dysfunctions following activation of the tyrosine kinase Fyn. Here, use of gene-encoded Ca2+ probes targeting different cell domains in primary cerebellar granule neurons (CGN) expressing, or not, PrPC, allowed us to investigate whether PrPC regulates store-operated Ca2+ entry (SOCE) and the implication of Fyn in this control. Our findings show that PrPC attenuates SOCE, and Ca2+ accumulation in the cytosol and mitochondria, by constitutively restraining Fyn activation and tyrosine phosphorylation of STIM1, a key molecular component of SOCE. This data establishes the existence of a PrPC-Fyn-SOCE triad in neurons. We also demonstrate that treating cerebellar granule and cortical neurons with soluble Aβ(1–42) oligomers abrogates the control of PrPC over Fyn and SOCE, suggesting a PrPC-dependent mechanizm for Aβ-induced neuronal Ca2+ dyshomeostasis.

Esculetin, a natural coumarin compound, evokes Ca(2+) movement and activation of Ca(2+)-associated mitochondrial apoptotic pathways that involved cell cycle arrest in ZR-75-1 human breast cancer cells.

Esculetin (6,7-dihydroxycoumarin), a derivative of coumarin compound, is found in traditional medicinal herbs. It has been shown that esculetin triggers diverse cellular signal transduction pathways leading to regulation of physiology in different models. However, whether esculetin affects Ca(2+) homeostasis in breast cancer cells has not been explored. This study examined the underlying mechanism of cytotoxicity induced by esculetin and established the relationship between Ca(2+) signaling and cytotoxicity in human breast cancer cells. The results showed that esculetin induced concentration-dependent rises in the intracellular Ca(2+) concentration ([Ca(2+)]i) in ZR-75-1 (but not in MCF-7 and MDA-MB-231) human breast cancer cells. In ZR-75-1 cells, this Ca(2+) signal response was reduced by removing extracellular Ca(2+) and was inhibited by the store-operated Ca(2+) channel blocker 2-aminoethoxydiphenyl borate (2-APB). In Ca(2+)-free medium, pre-treatment with the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin (TG) abolished esculetin-induced [Ca(2+)]i rises. Conversely, incubation with esculetin abolished TG-induced [Ca(2+)]i rises. Esculetin induced cytotoxicity that involved apoptosis, as supported by the reduction of mitochondrial membrane potential and the release of cytochrome c and the proteolytic activation of caspase-9/caspase-3, which were partially reversed by pre-chelating cytosolic Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM). Moreover, esculetin increased the percentage of cells in G2/M phase and regulated the expressions of p53, p21, CDK1, and cyclin B1. Together, in ZR-75-1 cells, esculetin induced [Ca(2+)]i rises by releasing Ca(2+) from the ER and causing Ca(2+) influx through 2-APB-sensitive store-operated Ca(2+) entry. Furthermore, esculetin activated Ca(2+)-associated mitochondrial apoptotic pathways that involved G2/M cell cycle arrest. Graphical abstract The summary of esculetin-evoked [Ca(2+)]i rises and -activated Ca(2+)-associated mitochondrial apoptotic pathways that involved cell cycle arrest. The natural coumarin derivative esculetin caused Ca(2+) influx via 2-APB-sensitive store-operated Ca(2+) entry and induced Ca(2+) release from the endoplasmic reticulum. Moreover, esculetin activated the mitochondrial pathway of apoptosis in a Ca(2+)-associated manner that involved G2/M arrest.

Electrochemical potential of the inner mitochondrial membrane and Ca(2+) homeostasis of myometrium cells

We demonstrated using Ca(2+)-sensitive fluorescent probe, mitochondria binding dyes, and confocal laser scanning microscopy, that elimination of electrochemical potential of uterus myocytes' inner mitochondrial membrane by aprotonophore carbonyl cyanide m-chlorophenyl hydrazone (10 μM), and by a respiratory chain complex IV inhibitor sodium azide (1 mM) is associated with substantial increase of Ca2+ concentration in myoplasm in the case of the protonophore effect only, but not in the case of the azide effect. In particular, with the use of nonyl acridine orange, a mitochondria-specific dye, and 9-aminoacridine, an agent that binds to membrane compartments in the presence of proton gradient, we showed that both the protonophore and the respiratory chain inhibitor cause the proton gradient on mitochondrial inner membrane to dissipate when introduced into incubation medium. We also proved with the help of 3,3'-dihexyloxacarbocyanine, a potential-sensitive carbocyanine-derived fluorescent probe, that the application of these substances results in dissipation of the membrane's electrical potential. The elimination of mitochondrial electrochemical potential by carbonyl cyanide m-chlorophenyl hydrazone causes substantial increase in fluorescence of Ca(2+)-sensitive Fluo-4 AM dye in myoplasm of smooth muscle cells. The results obtained were qualitatively confirmed with flow cytometry of mitochondria isolated through differential centrifugation and loaded with Fluo-4 AM. Particularly, Ca2+ matrix influx induced by addition of the exogenous cation is totally inhibited by carbonyl cyanide m-chlorophenyl hydrazone. Therefore, using two independent fluorometric methods, namely confocal laser scanning microscopy and flow cytometry, with Ca(2+)-sensitive Fluo-4 AM fluorescent probe, we proved on the models of freshly isolated myocytes and uterus smooth muscle mitochondria isolated by differential centrifugation sedimentation that the electrochemical gradient of inner membrane is an important component of mechanisms that regulate Ca2+ homeostasis in myometrium cells.

BAX inhibitor-1 is a Ca(2+) channel critically important for immune cell function and survival.

The endoplasmic reticulum (ER) serves as the major intracellular Ca(2+) store and has a role in the synthesis and folding of proteins. BAX (BCL2-associated X protein) inhibitor-1 (BI-1) is a Ca(2+) leak channel also implicated in the response against protein misfolding, thereby connecting the Ca(2+) store and protein-folding functions of the ER. We found that BI-1-deficient mice suffer from leukopenia and erythrocytosis, have an increased number of splenic marginal zone B cells and higher abundance and nuclear translocation of NF-κB (nuclear factor-κ light-chain enhancer of activated B cells) proteins, correlating with increased cytosolic and ER Ca(2+) levels. When put into culture, purified knockout T cells and even more so B cells die spontaneously. This is preceded by increased activity of the mitochondrial initiator caspase-9 and correlated with a significant surge in mitochondrial Ca(2+) levels, suggesting an exhausted mitochondrial Ca(2+) buffer capacity as the underlying cause for cell death in vitro. In vivo, T-cell-dependent experimental autoimmune encephalomyelitis and B-cell-dependent antibody production are attenuated, corroborating the ex vivo results. These results suggest that BI-1 has a major role in the functioning of the adaptive immune system by regulating intracellular Ca(2+) homeostasis in lymphocytes.

Diseases caused by mutations in ORAI1 and STIM1.

Ca(2+) release-activated Ca(2+) (CRAC) channels mediate a specific form of Ca(2+) influx called store-operated Ca(2+) entry (SOCE) that contributes to the function of many cell types. CRAC channels are composed of ORAI1 proteins located in the plasma membrane, which form its ion-conducting pore. ORAI1 channels are activated by stromal interaction molecule (STIM) 1 and STIM2 located in the endoplasmic reticulum. Loss- and gain-of-function gene mutations in ORAI1 and STIM1 in human patients cause distinct disease syndromes. CRAC channelopathy is caused by loss-of-function mutations in ORAI1 and STIM1 that abolish CRAC channel function and SOCE; it is characterized by severe combined immunodeficiency (SCID)-like disease, autoimmunity, muscular hypotonia, and ectodermal dysplasia, with defects in sweat gland function and dental enamel formation. The latter defect emphasizes an important role of CRAC channels in tooth development. By contrast, autosomal dominant gain-of-function mutations in ORAI1 and STIM1 result in constitutive CRAC channel activation, SOCE, and increased intracellular Ca(2+) levels that are associated with an overlapping spectrum of diseases, including nonsyndromic tubular aggregate myopathy (TAM) and York platelet and Stormorken syndromes. The latter two syndromes are defined, besides myopathy, by thrombocytopenia, thrombopathy, and bleeding diathesis. The fact that myopathy results from both loss- and gain-of-function mutations in ORAI1 and STIM1 highlights the importance of CRAC channels for Ca(2+) homeostasis in skeletal muscle function. The cellular dysfunction and clinical disease spectrum observed in mutant patients provide important information about the molecular regulation of ORAI1 and STIM1 proteins and the role of CRAC channels in human physiology.

Deregulation of mitochondrial functions provoked by long-chain fatty acid accumulating in long-chain 3-hydroxyacyl-CoA dehydrogenase and mitochondrial permeability transition deficiencies in rat heart--mitochondrial permeability transition pore opening as a potential contributing pathomechanism of cardiac alterations in these disorders.

Mitochondrial trifunctional protein and long-chain 3-hydroxyacyl-CoA dehydrogenase deficiencies are fatty acid oxidation disorders biochemically characterized by tissue accumulation of long-chain fatty acids and derivatives, including the monocarboxylic long-chain 3-hydroxy fatty acids (LCHFAs) 3-hydroxytetradecanoic acid (3HTA) and 3-hydroxypalmitic acid (3HPA). Patients commonly present severe cardiomyopathy for which the pathogenesis is still poorly established. We investigated the effects of 3HTA and 3HPA, the major metabolites accumulating in these disorders, on important parameters of mitochondrial homeostasis in Ca(2+) -loaded heart mitochondria. 3HTA and 3HPA significantly decreased mitochondrial membrane potential, the matrix NAD(P)H pool and Ca(2+) retention capacity, and also induced mitochondrial swelling. These fatty acids also provoked a marked decrease of ATP production reflecting severe energy dysfunction. Furthermore, 3HTA-induced mitochondrial alterations were completely prevented by the classical mitochondrial permeability transition (mPT) inhibitors cyclosporin A and ADP, as well as by ruthenium red, a Ca(2+) uptake blocker, indicating that LCHFAs induced Ca(2+)-dependent mPT pore opening. Milder effects only achieved at higher doses of LCHFAs were observed in brain mitochondria, implying a higher vulnerability of heart to these fatty acids. By contrast, 3HTA and docosanoic acids did not change mitochondrial homeostasis, indicating selective effects for monocarboxylic LCHFAs. The present data indicate that the major LCHFAs accumulating in mitochondrial trifunctional protein and long-chain 3-hydroxyacyl-CoA dehydrogenase deficiencies induce mPT pore opening, compromising Ca(2+) homeostasis and oxidative phosphorylation more intensely in the heart. It is proposed that these pathomechanisms may contribute at least in part to the severe cardiac alterations characteristic of patients affected by these diseases.

Regulation of cytoskeletal dynamics by redox signaling and oxidative stress: implications for neuronal development and trafficking.

A proper balance between chemical reduction and oxidation (known as redox balance) is essential for normal cellular physiology. Deregulation in the production of oxidative species leads to DNA damage, lipid peroxidation and aberrant post-translational modification of proteins, which in most cases induces injury, cell death and disease. However, physiological concentrations of oxidative species are necessary to support important cell functions, such as chemotaxis, hormone synthesis, immune response, cytoskeletal remodeling, Ca2+ homeostasis and others. Recent evidence suggests that redox balance regulates actin and microtubule dynamics in both physiological and pathological contexts. Microtubules and actin microfilaments contain certain amino acid residues that are susceptible to oxidation, which reduces the ability of microtubules to polymerize and causes severing of actin microfilaments in neuronal and non-neuronal cells. In contrast, inhibited production of reactive oxygen species (ROS; e.g., due to NOXs) leads to aberrant actin polymerization, decreases neurite outgrowth and affects the normal development and polarization of neurons. In this review, we summarize emerging evidence suggesting that both general and specific enzymatic sources of redox species exert diverse effects on cytoskeletal dynamics. Considering the intimate relationship between cytoskeletal dynamics and trafficking, we also discuss the potential effects of redox balance on intracellular transport via regulation of the components of the microtubule and actin cytoskeleton as well as cytoskeleton-associated proteins, which may directly impact localization of proteins and vesicles across the soma, dendrites and axon of neurons.