ABSTRACT
Cell-to-cell transmission of α-synuclein (α-syn) pathology underlies the spread of neurodegeneration in Parkinson's disease. α-Syn secretion is an important factor in the transmission of α-syn pathology. However, it is unclear how α-syn secretion is therapeutically modulated. Here, we investigated effects of monoamine oxidase (MAO)-B inhibitor selegiline on α-syn secretion. Treatment with selegiline promoted α-syn secretion in mouse primary cortical neuron cultures, and this increase was kept under glial cell-eliminated condition by Ara-C. Selegiline-induced α-syn secretion was blocked by cytosolic Ca2+ chelator BAPTA-AM in primary neurons. Selegiline-induced α-syn secretion was retained in MAOA siRNA knockdown, whereas it was abrogated by ATG5 knockdown in SH-SY5Y cells. Selegiline increased LC3-II generation with a reduction in intracellular p62/SQSTM1 levels in primary neurons. The increase in LC3-II generation was blocked by co-treatment with BAPTA-AM in primary neurons. Additionally, fractionation experiments showed that selegiline-induced α-syn secretion occurred in non-extracellular vesicle fractions of primary neurons and SH-SY5Y cells. Collectively, these findings show that selegiline promotes neuronal autophagy involving secretion of non-exosomal α-syn via a change of cytosolic Ca2+ levels.
Subject(s)
Autophagy , Neurons , Selegiline , alpha-Synuclein , Selegiline/pharmacology , Animals , Autophagy/drug effects , alpha-Synuclein/metabolism , Neurons/drug effects , Neurons/metabolism , Mice , Monoamine Oxidase/metabolism , Humans , Calcium/metabolism , Cells, Cultured , Monoamine Oxidase Inhibitors/pharmacology , Egtazic Acid/analogs & derivatives , Egtazic Acid/pharmacology , Mice, Inbred C57BL , Cell Line, Tumor , Autophagy-Related Protein 5/metabolism , Autophagy-Related Protein 5/geneticsABSTRACT
Extracellular secretion is an essential mechanism for α-synuclein (α-syn) proteostasis. Although it has been reported that neuronal activity affects α-syn secretion, the underlying mechanisms remain unclear. Here, we investigated the autophagic processes that regulate the physiological release of α-syn in mouse primary cortical neurons and SH-SY5Y cells. Stimulating neuronal activity with glutamate or depolarization with high KCl enhanced α-syn secretion. This glutamate-induced α-syn secretion was blocked by a mixture of NMDA receptor antagonist AP5 and AMPA receptor antagonist NBQX, as well as by cytosolic Ca2+ chelator BAPTA-AM. Additionally, mTOR inhibitor rapamycin increased α-syn and p62/SQSTM1 (p62) secretion, and this effect of rapamycin was reduced in primary cortical neurons deficient in the autophagy regulator beclin 1 (derived from BECN1+/- mice). Glutamate-induced α-syn and p62 secretion was suppressed by the knockdown of ATG5, which is required for autophagosome formation. Glutamate increased LC3-II generation and decreased intracellular p62 levels, and the increase in LC3-II levels was blocked by BAPTA-AM. Moreover, glutamate promoted co-localization of α-syn with LC3-positive puncta, but not with LAMP1-positive structures in the neuronal somas. Glutamate-induced α-syn and p62 secretion were also reduced by the knockdown of RAB8A, which is required for autophagosome fusion with the plasma membrane. Collectively, these findings suggest that stimulating neuronal activity mediates autophagic α-syn secretion in a cytosolic Ca2+-dependent manner, and autophagosomes may participate in autophagic secretion by functioning as α-syn carriers.
Subject(s)
Autophagy , Neurons , Sequestosome-1 Protein , alpha-Synuclein , Animals , alpha-Synuclein/metabolism , alpha-Synuclein/genetics , Neurons/metabolism , Mice , Humans , Sequestosome-1 Protein/metabolism , Sequestosome-1 Protein/genetics , Autophagy-Related Protein 5/metabolism , Autophagy-Related Protein 5/genetics , Glutamic Acid/metabolism , Beclin-1/metabolism , Beclin-1/genetics , Calcium/metabolism , Cell Line, Tumor , Egtazic Acid/analogs & derivatives , Egtazic Acid/pharmacology , Sirolimus/pharmacologyABSTRACT
Antisense oligonucleotide (ASO) therapy is a novel therapeutic approach in which ASO specifically binds target mRNA, resulting in mRNA degradation; however, cellular uptake of ASOs remains critically low, warranting improvement. Transient receptor potential canonical (TRPC) channels regulate Ca2+ influx and are activated upon stimulation by phospholipase C-generated diacylglycerol. Herein, we report that a novel TRPC3/C6/C7 activator, L687, can induce cellular ASO uptake. L687-induced ASO uptake was enhanced in a dose- and incubation-time-dependent manner. L687 enhanced the knockdown activity of various ASOs both in vitro and in vivo. Notably, suppression of TRPC3/C6 by specific siRNAs reduced ASO uptake in A549 cells. Application of BAPTA-AM, a Ca2+ chelator, and SKF96365, a TRPC3/C6 inhibitor, suppressed Ca2+ influx via TRPC3/C6, resulting in reduced ASO uptake, thereby suggesting that Ca2+ influx via TRPC3/C6 is critical for L687-mediated increased ASO uptake. L687 also induced dextran uptake, indicating that L687 increased endocytosis. Adding ASO to L687 resulted in endosome accumulation; however, the endosomal membrane disruptor UNC7938 facilitated endosomal escape and enhanced knockdown activity. We discovered a new function for TRPC activators regarding ASO trafficking in target cells. Our findings provide an opportunity to formulate an innovative drug delivery system for the therapeutic development of ASO.
Subject(s)
Calcium , Oligonucleotides, Antisense , TRPC Cation Channels , Humans , Oligonucleotides, Antisense/pharmacology , Oligonucleotides, Antisense/metabolism , TRPC Cation Channels/metabolism , TRPC Cation Channels/genetics , TRPC Cation Channels/antagonists & inhibitors , Calcium/metabolism , A549 Cells , Animals , Mice , Imidazoles/pharmacology , TRPC6 Cation Channel/metabolism , TRPC6 Cation Channel/genetics , TRPC6 Cation Channel/antagonists & inhibitors , Egtazic Acid/pharmacology , Egtazic Acid/analogs & derivatives , Endosomes/metabolism , Endosomes/drug effects , Cell Line, TumorABSTRACT
Inherited retinal degenerations (IRDs) are a group of untreatable and commonly blinding diseases characterized by progressive photoreceptor loss. IRD pathology has been linked to an excessive activation of cyclic nucleotide-gated channels (CNGC) leading to Na+- and Ca2+-influx, subsequent activation of voltage-gated Ca2+-channels (VGCC), and further Ca2+ influx. However, a connection between excessive Ca2+ influx and photoreceptor loss has yet to be proven.Here, we used whole-retina and single-cell RNA-sequencing to compare gene expression between the rd1 mouse model for IRD and wild-type (wt) mice. Differentially expressed genes indicated links to several Ca2+-signalling related pathways. To explore these, rd1 and wt organotypic retinal explant cultures were treated with the intracellular Ca2+-chelator BAPTA-AM or inhibitors of different Ca2+-permeable channels, including CNGC, L-type VGCC, T-type VGCC, Ca2+-release-activated channel (CRAC), and Na+/Ca2+ exchanger (NCX). Moreover, we employed the novel compound NA-184 to selectively inhibit the Ca2+-dependent protease calpain-2. Effects on the retinal activity of poly(ADP-ribose) polymerase (PARP), sirtuin-type histone-deacetylase, calpains, as well as on activation of calpain-1, and - 2 were monitored, cell death was assessed via the TUNEL assay.While rd1 photoreceptor cell death was reduced by BAPTA-AM, Ca2+-channel blockers had divergent effects: While inhibition of T-type VGCC and NCX promoted survival, blocking CNGCs and CRACs did not. The treatment-related activity patterns of calpains and PARPs corresponded to the extent of cell death. Remarkably, sirtuin activity and calpain-1 activation were linked to photoreceptor protection, while calpain-2 activity was related to degeneration. In support of this finding, the calpain-2 inhibitor NA-184 protected rd1 photoreceptors.These results suggest that Ca2+ overload in rd1 photoreceptors may be triggered by T-type VGCCs and NCX. High Ca2+-levels likely suppress protective activity of calpain-1 and promote retinal degeneration via activation of calpain-2. Overall, our study details the complexity of Ca2+-signalling in photoreceptors and emphasizes the importance of targeting degenerative processes specifically to achieve a therapeutic benefit for IRDs. Video Abstract.
Subject(s)
Egtazic Acid/analogs & derivatives , Retinal Degeneration , Sirtuins , Mice , Animals , Retinal Degeneration/metabolism , Calpain/metabolism , Sodium-Calcium Exchanger , Photoreceptor Cells/metabolism , Photoreceptor Cells/pathology , Cell Death , Sirtuins/metabolismABSTRACT
Calcium overload, a notable instigator of acute pancreatitis (AP), induces oxidative stress and an inflammatory cascade, subsequently activating both endogenous and exogenous apoptotic pathways. However, there is currently lack of available pharmaceutical interventions to alleviate AP by addressing calcium overload. In the present study, the potential clinical application of liposome nanoparticles (LNs) loaded with 1,2bis(2aminophenoxy)ethaneN,N,N',N'tetraacetic acid tetrakis (acetoxymethyl ester) (BAPTAAM), a cellpermeant calcium chelator, was investigated as a therapeutic approach for the management of AP. To establish the experimental models in vitro, AR42J cells were exposed to high glucose/sodium oleate (HGO) to induce necrosis, and in vivo, intraductal taurocholate (TC) infusion was used to induce AP. The findings of the present study indicated that the use of BAPTAAMloaded LN (BLN) effectively and rapidly eliminated excessive Ca2+ and reactive oxygen species, suppressed mononuclear macrophage activation and the release of inflammatory cytokines, and mitigated pancreatic acinar cell apoptosis and necrosis induced by HGO. Furthermore, the systemic administration of BLN demonstrated promising therapeutic potential in the rat model of AP. Notably, BLN significantly enhanced the survival rates of rats subjected to the TC challenge, increasing from 37.5 to 75%. This improvement was attributed to the restoration of pancreatic function, as indicated by improved blood biochemistry indices and alleviation of pancreatic lesions. The potential therapeutic efficacy of BLN in rescuing patients with AP is likely attributed to its capacity to inhibit oxidative stress, prevent premature activation of zymogens and downregulate the expression of TNFα, IL6 and cathepsin B. Thus, BLN demonstrated promising value as a novel therapeutic approach for promptly alleviating the burden of intracellular Ca2+ overload in patients with AP.
Subject(s)
Egtazic Acid/analogs & derivatives , Pancreatitis , Humans , Rats , Animals , Pancreatitis/metabolism , Liposomes/metabolism , Calcium/metabolism , Acute Disease , Acinar Cells/pathology , Necrosis/metabolismABSTRACT
PLEKHG4B is a Cdc42-targeting guanine-nucleotide exchange factor implicated in forming epithelial cell-cell junctions. Here we explored the mechanism regulating PLEKHG4B localization. PLEKHG4B localized to the basal membrane in normal Ca2+ medium but accumulated at cell-cell junctions upon ionomycin treatment. Ionomycin-induced junctional localization of PLEKHG4B was suppressed upon disrupting its annexin-A2 (ANXA2)-binding ability. Thus, Ca2+ influx and ANXA2 binding are crucial for PLEKHG4B localization to cell-cell junctions. Treatments with low Ca2+ or BAPTA-AM (an intracellular Ca2+ chelator) suppressed PLEKHG4B localization to the basal membrane. Mutations of the phosphoinositide-binding motif in the pleckstrin homology (PH) domain of PLEKHG4B or masking of membrane phosphatidylinositol-4,5-biphosphate [PI(4,5)P2] suppressed PLEKHG4B localization to the basal membrane, indicating that basal membrane localization of PLEKHG4B requires suitable intracellular Ca2+ levels and PI(4,5)P2 binding of the PH domain. Activation of mechanosensitive ion channels (MSCs) promoted PLEKHG4B localization to cell-cell junctions, and their inhibition suppressed it. Moreover, similar to the PLEKHG4B knockdown phenotypes, inhibition of MSCs or treatment with BAPTA-AM disturbed the integrity of actin filaments at cell-cell junctions. Taken together, our results suggest that Ca2+ influx plays crucial roles in PLEKHG4B localization to cell-cell junctions and the integrity of junctional actin organization, with MSCs contributing to this process.
Subject(s)
Calcium , Egtazic Acid/analogs & derivatives , Intercellular Junctions , Calcium/metabolism , Ionomycin , Intercellular Junctions/metabolism , Actin Cytoskeleton/metabolismABSTRACT
Male sex is one of the most important risk factors of atrial fibrillation (AF), with the incidence in men being almost double that in women. However, the reasons for this sex difference are unknown. Accordingly, in this study, we sought to determine whether there are sex differences in intracellular Ca2+ homeostasis in mouse atrial myocytes that might help explain male predisposition to AF. AF susceptibility was assessed in male (M) and female (F) mice (4-5 months old) using programmed electrical stimulation (EPS) protocols. Males were 50% more likely to develop AF. The Ca2+ transient amplitude was 28% higher in male atrial myocytes. Spontaneous systolic and diastolic Ca2+ releases, which are known sources of triggered activity, were significantly more frequent in males than females. The time to 90% decay of Ca2+ transient was faster in males. Males had 54% higher Na+-Ca2+ exchanger (NCX1) current density, and its expression was also more abundant. L-type Ca2+ current (ICaL) was recorded with and without BAPTA, a Ca2+ chelator. ICaL density was lower in males only in the absence of BAPTA, suggesting stronger Ca2+-dependent inactivation in males. CaV1.2 expression was similar between sexes. This study reports major sex differences in Ca2+ homeostasis in mouse atria, with larger Ca2+ transients and enhanced NCX1 function and expression in males resulting in more spontaneous Ca2+ releases. These sex differences may contribute to male susceptibility to AF by promoting triggered activity.
Subject(s)
Atrial Fibrillation , Sodium-Calcium Exchanger/metabolism , Animals , Atrial Fibrillation/metabolism , Calcium/metabolism , Chelating Agents/metabolism , Egtazic Acid/analogs & derivatives , Female , Heart Atria/metabolism , Humans , Male , Mice , Myocytes, Cardiac/metabolism , Patch-Clamp Techniques , Sarcoplasmic Reticulum/metabolism , Sex CharacteristicsABSTRACT
We have developed a convenient synthesis of a series of ß-fluoramides in 65% yield. The process involved a tandem fluorination/Ritter reaction to synthesize ß-fluoramides using α-diazo 2H-benzopyran-4-one compounds. Selectfluor was used as the electrophilic fluoride source in acetonitrile to build the ß-fluorinated quaternary carbon center and amide derivatives of 2H-benzopyran-4-one in one step. The products N-(2-(2-fluoro-2,3-dihydro-3-oxobenzofuran-2-yl)propan-2-yl)acetamides were a series of bifunctional compounds with a 2-fluoro-2,3-dihydro-3-oxobenzofuran motif and amide groups.
Subject(s)
Acetamides , Benzopyrans , Diazomethane/analogs & derivatives , Egtazic Acid/analogs & derivatives , Molecular StructureABSTRACT
N-(3-oxododecanoyl) homoserine lactone (3oc) is a Pseudomonas aeruginosa secreted quorum-sensing signal molecule playing a crucial role in regulating quorum-sensing (QS) dependent biofilm formation and secretion of virulence factors. In addition to regulating quorum sensing, 3oc also plays an immunomodulatory role in the host by triggering regulated cell death in immune cells. The molecular mechanisms of 3oc in modulating macrophage pathologies are still unclear. In this study, we hypothesized the novel 3oc mediated crosstalk between autophagy and apoptosis at the interphase of calcium signaling in human macrophages. The study showed that 3oc induces mitochondrial dysfunction and apoptosis in macrophages through elevating cytosolic Ca+2 ([Ca+2]cyt) levels. Pre-treatment with the calcium-specific chelator BAPTA-AM effectively abrogated 3oc-induced apoptotic events, like mitochondrial ROS generation (mROS), mitochondrial membrane potential (MMP) drop, and phosphatidylserine (PS) exposure. The study also showed that 3oc induces autophagy, as assessed by the accumulation of autophagic vacuoles, induction of lysosomal biogenesis, upregulation of autophagy genes (LC3, BECLIN 1, STX17, PINK1, and TFEB), autophagosomes formation, and LC3 lipidation. Mechanistically, our study proved that 3oc-induced autophagy was [Ca+2]cyt dependent as BAPTA-AM pre-treatment reduced autophagosome formation. Furthermore, inhibiting autophagy with chloroquine attenuated 3oc-induced apoptosis, while autophagy induction with rapamycin aggravated cell death, suggesting autophagy plays a role in cell death in 3oc-treated macrophages. In conclusion, our findings indicate that 3oc activates a multifaceted death signaling by activating autophagy and apoptosis through Ca+2 signaling, and we propose pharmacological modulation of Ca+2 signaling may act as a combinatorial therapeutic intervention in patients with Pseudomonas aeruginosa-associated infections.
Subject(s)
Pseudomonas Infections , Quorum Sensing , 4-Butyrolactone/analogs & derivatives , 4-Butyrolactone/pharmacology , Apoptosis , Autophagy , Beclin-1/metabolism , Calcium/metabolism , Calcium Signaling , Chelating Agents/metabolism , Chelating Agents/pharmacology , Chloroquine/pharmacology , Egtazic Acid/analogs & derivatives , Homoserine , Humans , Macrophages/metabolism , Phosphatidylserines/metabolism , Protein Kinases/metabolism , Pseudomonas aeruginosa , Reactive Oxygen Species/metabolism , Sirolimus/pharmacology , Virulence Factors/metabolism , Virulence Factors/pharmacologyABSTRACT
Astrocytes are glial cells that exhibit calcium signaling-mediated activity. Here, we present a protocol to monitor and manipulate astrocyte calcium activity from mouse amygdala slices. In the first part of this protocol, we describe the procedure of astrocyte calcium imaging. In the second part, we detail how to disrupt astrocyte calcium activity by patch-clamp-mediated loading of BAPTA. These two approaches are presented separately but they can also be used simultaneously to monitor the effects of disruption on an astrocyte network. For complete details on the use and execution of this protocol, please refer to Wahis et al. (2021).
Subject(s)
Astrocytes , Calcium , Amygdala/diagnostic imaging , Animals , Astrocytes/metabolism , Calcium/metabolism , Calcium Signaling , Calcium, Dietary , Egtazic Acid/analogs & derivatives , MiceABSTRACT
Capsaicin and zinc have recently been highlighted as potential treatments for glucose metabolism disorders; however, the effect of these two natural compounds on signalling pathways involved in glucose metabolism is still uncertain. In this study, we assessed the capsaicin- or zinc- induced activation of signalling molecules including calcium/calmodulin-dependent protein kinase 2 (CAMKK2), cAMP-response element-binding protein (CREB), and target of rapamycin kinase complex 1 (TORC1). Moreover, the expression status of genes associated with the control of glucose metabolism was measured in treated cells. The activation of cell signalling proteins was then evaluated in capsaicin- or zinc treated cells in the presence or absence of cell-permeant calcium chelator (BAPTA-AM) and the CAMKK inhibitor (STO-609). Finally, capsaicin- and zinc-induced glucose uptake was measured in the cells pre-treated with or without BAPTA-AM. Our results indicate that calcium flux induced by capsaicin or zinc led to activation of calcium signalling molecules and promoting glucose uptake in skeletal muscle cells. Pharmacological inhibition of CAMKK diminished activation of signalling molecules. Moreover, we observed an increase in intracellular cAMP levels in the cells after treatment with capsaicin and zinc. Our data show that capsaicin and zinc mediate glucose uptake in C2C12 skeletal muscle cells through the activation of calcium signalling.
Subject(s)
Calcium Signaling/drug effects , Calcium/metabolism , Capsaicin/pharmacology , Glucose/metabolism , Muscle Fibers, Skeletal/drug effects , Zinc/pharmacology , AMP-Activated Protein Kinases/metabolism , Animals , Benzimidazoles/pharmacology , Calcium-Calmodulin-Dependent Protein Kinase Kinase/metabolism , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Carbohydrate Metabolism/drug effects , Cell Line , Egtazic Acid/analogs & derivatives , Egtazic Acid/pharmacology , Muscle Fibers, Skeletal/metabolism , Naphthalimides/pharmacology , Phosphorylation/drug effectsABSTRACT
Rationale: Disruption of intracellular calcium (Ca2+) homeostasis is implicated in inflammatory responses. Here we investigated endoplasmic reticulum (ER) Ca2+ efflux through the Inositol 1,4,5-trisphosphate receptor (IP3R) as a potential mechanism of inflammatory pathophysiology in a ventilator-induced lung injury (VILI) mouse model. Methods: C57BL/6 mice were exposed to mechanical ventilation using high tidal volume (HTV). Mice were pretreated with the IP3R agonist carbachol, IP3R inhibitor 2-aminoethoxydiphenyl borate (2-APB) or the Ca2+ chelator BAPTA-AM. Lung tissues and bronchoalveolar lavage fluid (BALF) were collected to measure Ca2+ concentrations, inflammatory responses and mRNA/protein expression associated with ER stress, NLRP3 inflammasome activation and inflammation. Analyses were conducted in concert with cultured murine lung cell lines. Results: Lungs from mice subjected to HTV displayed upregulated IP3R expression in ER and mitochondrial-associated-membranes (MAMs), with enhanced formation of MAMs. Moreover, HTV disrupted Ca2+ homeostasis, with increased flux from the ER to the cytoplasm and mitochondria. Administration of carbachol aggravated HTV-induced lung injury and inflammation while pretreatment with 2-APB or BAPTA-AM largely prevented these effects. HTV activated the IRE1α and PERK arms of the ER stress signaling response and induced mitochondrial dysfunction-NLRP3 inflammasome activation in an IP3R-dependent manner. Similarly, disruption of IP3R/Ca2+ in MLE12 and RAW264.7 cells using carbachol lead to inflammatory responses, and stimulated ER stress and mitochondrial dysfunction. Conclusion: Increase in IP3R-mediated Ca2+ release is involved in the inflammatory pathophysiology of VILI via ER stress and mitochondrial dysfunction. Antagonizing IP3R/Ca2+ and/or maintaining Ca2+ homeostasis in lung tissue represents a prospective treatment approach for VILI.
Subject(s)
Boron Compounds/pharmacology , Calcium Chelating Agents/pharmacology , Calcium Signaling/drug effects , Calcium/metabolism , Egtazic Acid/analogs & derivatives , Endoplasmic Reticulum/drug effects , Inositol 1,4,5-Trisphosphate Receptors/antagonists & inhibitors , Lung/drug effects , Mitochondria/drug effects , Ventilator-Induced Lung Injury/prevention & control , Animals , Apoptosis/drug effects , Carbachol/toxicity , Disease Models, Animal , Egtazic Acid/pharmacology , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum/pathology , Endoplasmic Reticulum Stress/drug effects , Inflammasomes/metabolism , Inflammation Mediators/metabolism , Inositol 1,4,5-Trisphosphate Receptors/metabolism , Lung/metabolism , Lung/pathology , Macrophages/drug effects , Macrophages/metabolism , Macrophages/pathology , Male , Mice , Mice, Inbred C57BL , Mitochondria/metabolism , Mitochondria/pathology , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , RAW 264.7 Cells , Ventilator-Induced Lung Injury/metabolism , Ventilator-Induced Lung Injury/pathologyABSTRACT
Osteoarthritis (OA) is a common joint disease that is characterized by cartilage degradation. Iron deposition in the joints is common during the pathogenic progression of OA and recent studies have indicated that iron overload is an important contributor to OA progression. Calcium chelators have been reported to inhibit iron influx via modulating transferrin receptor protein 1 internalization, and they have been identified as a potential approach to the treatment of iron overloadinduced diseases. The aim of the present study was to investigate the effect of calcium chelators on the progression of iron overloadinduced OA. Primary chondrocytes were treated with various concentrations of ferric ammonium citrate (FAC) to mimic iron overload in vitro, followed by cotreatment with the calcium chelator BAPTA acetoxymethyl ester (BAPTAAM). Subsequently, intracellular iron levels, cell viability, reactive oxygen species (ROS) levels, mitochondrial function and morphological changes, as well as MMP levels, were detected using commercial kits. It was demonstrated that FAC treatment significantly promoted chondrocyte apoptosis and the expression of MMPs, and these effects were reversed by cotreatment with BAPTAAM. Moreover, BAPTAAM suppressed iron influx into chondrocytes and inhibited iron overloadinduced ROS production and mitochondrial dysfunction. These results indicated that calcium chelators may be of value in the treatment of iron metabolismrelated diseases and iron overloadinduced OA progression.
Subject(s)
Calcium Chelating Agents/pharmacology , Cartilage Diseases/drug therapy , Chondrocytes/drug effects , Egtazic Acid/analogs & derivatives , Iron Overload/complications , Mitochondrial Diseases/drug therapy , Protective Agents/pharmacology , Animals , Apoptosis/drug effects , Cartilage Diseases/metabolism , Cartilage, Articular/drug effects , Cartilage, Articular/metabolism , Cell Survival/drug effects , Cells, Cultured , Chondrocytes/metabolism , Egtazic Acid/pharmacology , Ferric Compounds/pharmacology , Iron Overload/metabolism , Male , Mice , Mice, Inbred C57BL , Mitochondria/drug effects , Mitochondria/metabolism , Mitochondrial Diseases/chemically induced , Mitochondrial Diseases/metabolism , Osteoarthritis/drug therapy , Osteoarthritis/metabolism , Quaternary Ammonium Compounds/pharmacology , Reactive Oxygen Species/metabolismABSTRACT
In 1980, Roger Tsien published a paper, in this journal [Tsien, R. Y. (1980) Biochemistry, 19 (11), 2396], titled "New calcium indicators and buffers with high selectivity against magnesium and protons: design, synthesis, and properties of prototype structures". These new buffers included 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, or BAPTA, which is still widely used today. And so, the world was set alight with new ways in which to visualize Ca2+. The ability to watch fluctuations in intracellular Ca2+ revolutionized the life sciences, although the fluorescent indicators used today, particularly in neurobiology, no longer rely exclusively on BAPTA but on genetically encoded fluorescent Ca2+ indicators. In this Perspective, we reflect on the origins of Ca2+ imaging with a special focus on the contributions made by Roger Tsien, from the early concept of selective Ca2+ binding described in Biochemistry to optical Ca2+ indicators based on chemically synthesized fluorophores to genetically encoded fluorescent Ca2+ indicators.
Subject(s)
Calcium/metabolism , Fluorescent Dyes/chemistry , Intravital Microscopy/methods , Optical Imaging/methods , Calcium/chemistry , Egtazic Acid/analogs & derivatives , Egtazic Acid/chemistry , History, 20th Century , Intravital Microscopy/history , Optical Imaging/historyABSTRACT
BACKGROUND: Phagocytosis is an essential process that maintains cellular homeostasis. In the epidermis, the phagocytosis of melanosomes into keratinocytes is important to protect their DNA against damage from ultraviolet B (UVB) radiation. Furthermore, it is considered that UVB activates the phagocytosis by keratinocytes but the detailed mechanism involved is not fully understood. OBJECTIVE: To clarify the mechanism of UVB-enhanced phagocytosis in keratinocytes, we investigated the relationship between the phagocytic ability of keratinocytes and the cell cycle stage of keratinocytes. METHODS: The phagocytic ability of keratinocytes was evaluated using the incorporation of fluorescent beads after exposure to UVB or oxidative stress. S-phase was evaluated by BrdU incorporation and immunostaining of cyclin D1. Intracellular calcium levels of keratinocytes were measured using the probe Fluo-4AM. RESULTS: The phagocytosis of fluorescent beads into keratinocytes was enhanced by UVB and also by oxidative stress. We found that keratinocytes exposed to UVB or oxidative stress were at S-phase of the cell cycle. Furthermore, keratinocytes synchronized to S-phase showed a higher phagocytic ability according to the increased intracellular ROS level. The UVB-enhanced phagocytosis and entrance into S-phase of keratinocytes was abolished by ascorbic acid, a typical antioxidant. Keratinocytes synchronized to S-phase and exposed to UVB or oxidative stress had increased levels of intracellular calcium and their enhanced phagocytic abilities were diminished by the calcium ion chelator BAPTA-AM. CONCLUSION: Taken together, intracellular oxidative stress induced by intracellular calcium influx mediates the UVB-enhanced phagocytic ability of keratinocytes accumulating at S-phase of the cell cycle.
Subject(s)
Calcium/metabolism , Keratinocytes/radiation effects , Phagocytosis/radiation effects , S Phase Cell Cycle Checkpoints/radiation effects , Ultraviolet Rays/adverse effects , Antioxidants/pharmacology , Ascorbic Acid/pharmacology , Cell Line , Chelating Agents/pharmacology , DNA Damage/drug effects , DNA Damage/radiation effects , Egtazic Acid/analogs & derivatives , Egtazic Acid/pharmacology , Humans , Keratinocytes/cytology , Keratinocytes/metabolism , Melanins/biosynthesis , Melanosomes/metabolism , Oxidative Stress/drug effects , Oxidative Stress/genetics , Oxidative Stress/radiation effects , Phagocytosis/drug effects , Phagocytosis/genetics , Reactive Oxygen Species/metabolismABSTRACT
In 15% of cases, autosomal dominant polycystic kidney disease arises from defects in polycystin-2 (PC2). PC2 is a member of the polycystin transient receptor potential subfamily of cation-conducting channels and is expressed in the endoplasmic reticulum and primary cilium of renal epithelial cells. PC2 opposes a procystogenic influence of the cilium, and it has been proposed that this beneficial effect is mediated in part by a flow of Ca2+ through PC2 channels into the primary cilium. However, previous efforts to determine the permeability of PC2 channels to Ca2+ have yielded widely varying results. Here, we report the mean macroscopic Ca2+ influx through native PC2 channels in the primary cilia of mIMCD-3 cells, which are derived from the murine inner medullary collecting duct. Under conditions designed to isolate inward Ca2+ currents, a small inward Ca2+ current was detected in cilia with active PC2 channels but not in cilia lacking those channels. The current was activated by the addition of 10 µM internal Ca2+, which is known to activate ciliary PC2 channels. It was blocked by 10 µM isosakuranetin, which blocks the same channels. On average, the current amplitude was -1.8 pA at -190 mV; its conductance from -50 to -200 mV averaged 20 pS. Thus, native PC2 channels of renal primary cilia are able to conduct a small but detectable Ca2+ influx under the conditions tested. The possible consequences of this influx are discussed.NEW & NOTEWORTHY In autosomal dominant polycystic kidney disease, it is proposed that Ca2+ entering the primary cilium through polycystin-2 (PC2) channels may limit the formation of cysts. Recent studies predict that any macroscopic Ca2+ influx through these channels should be small. We report that the native PC2 channels in primary cilia of cultured renal epithelial cells can allow a small macroscopic calcium influx. This may allow a significant accumulation of Ca2+ in the cilium in vivo.
Subject(s)
Calcium Channels/physiology , Calcium/metabolism , Cilia/physiology , Electrophysiological Phenomena , TRPP Cation Channels/metabolism , Animals , Cell Line , Chelating Agents/pharmacology , Egtazic Acid/analogs & derivatives , Egtazic Acid/pharmacology , Epithelial Cells , Kidney Tubules, Collecting/cytology , MiceABSTRACT
AIM: Despite animal evidence of a role of calcium in the pathogenesis of spinal cord injury, several studies conducted in the past found calcium blockade ineffective. However, those studies involved oral or parenteral administration of Ca++ antagonists. We hypothesized that Ca++ blockade might be effective with local/immediate application (LIA) at the time of neural injury. METHODS: In this study, we assessed the effects of LIA of BAPTA (1,2-bis (o-aminophenoxy) ethane-N, N, N', N'-tetraacetic acid), a cell-permeable highly selective Ca++ chelator, after spinal cord transection (SCT) in mice over 4 weeks. Effects of BAPTA were assessed behaviorally and with immunohistochemistry. Concurrently, BAPTA was submitted for the first time to multimodality assessment in an in vitro model of neural damage as a possible spinal neuroprotectant. RESULTS: We demonstrate that BAPTA alleviates neuronal apoptosis caused by physical damage by inhibition of neuronal apoptosis and reactive oxygen species (ROS) generation. This translates to enhanced preservation of electrophysiological function and superior behavioral recovery. CONCLUSION: This study shows for the first time that local/immediate application of Ca++ chelator BAPTA is strongly neuroprotective after severe spinal cord injury.
Subject(s)
Calcium Chelating Agents/therapeutic use , Egtazic Acid/analogs & derivatives , Neuroprotective Agents/therapeutic use , Recovery of Function/drug effects , Spinal Cord Injuries/drug therapy , Thoracic Vertebrae/injuries , Animals , Calcium Chelating Agents/pharmacology , Cells, Cultured , Egtazic Acid/pharmacology , Egtazic Acid/therapeutic use , Female , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Neurons/drug effects , Neurons/physiology , Neuroprotection/drug effects , Neuroprotection/physiology , Neuroprotective Agents/pharmacology , Reactive Oxygen Species/metabolism , Recovery of Function/physiology , Spinal Cord Injuries/metabolism , Spinal Cord Injuries/pathologyABSTRACT
Fetuin-A is an inhibitor of ectopic calcification that is expressed mainly in hepatocytes and is secreted into the circulation after posttranslational processing, including glycosylation and phosphorylation. The molecular weight (MW) of fully modified fetuin-A (FM-fetuin-A) is approximately 60 kDa in an immunoblot, which is much higher than the estimated MW by amino acid sequence. Under conditions of calcification stress such as advanced stage chronic kidney disease, fetuin-A prevents calcification by forming colloidal complexes, which are referred to as calciprotein particles (CPP). Since the significance of CPP in this process is unclear, we investigated the effect of synthetic secondary CPP on the level of FM-fetuin-A in HepG2 cells. Secondary CPP increased the level of FM-fetuin-A in dose- and time-dependent manners, but did not affect expression of mRNA for fetuin-A. Treatment with O- and/or N-glycosidase caused a shift of the 60 kDa band of FM-fetuin-A to a lower MW. Preincubation with brefeldin A, an inhibitor of transport of newly synthesized proteins from the endoplasmic reticulum to the Golgi apparatus, completely blocked the secondary CPP-induced increase in FM-fetuin-A. Treatment with BAPTA-AM, an intracellular calcium chelating agent, also inhibited the CPP-induced increase in the FM-fetuin-A level. Secondary CPP accelerate posttranslational processing of fetuin-A in HepG2 cells.
Subject(s)
Calcinosis/metabolism , Protein Processing, Post-Translational , alpha-2-HS-Glycoprotein/metabolism , Brefeldin A/pharmacology , Calcium/metabolism , Egtazic Acid/analogs & derivatives , Egtazic Acid/metabolism , Glycoside Hydrolases/metabolism , Glycosylation , Hep G2 Cells , Humans , Phosphorylation , Phosphoserine/metabolism , Protein Transport , RNA, Messenger/genetics , RNA, Messenger/metabolism , alpha-2-HS-Glycoprotein/geneticsABSTRACT
We present the first direct nuclear magnetic resonance (NMR) evidence of enhanced entry of Ca2+ ions into human erythrocytes (red blood cells; RBCs), when these cells are mechanically distorted. For this we loaded the RBCs with the fluorinated Ca2+ chelator, 1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N',N'-tetraacetic acid (5FBAPTA), and recorded 19F NMR spectra. The RBCs were suspended in gelatin gel in a special stretching/compression apparatus. The 5FBAPTA was loaded into the cells as the tetraacetoxymethyl ester; and 13C NMR spectroscopy with [1,6-13C]D-glucose as substrate showed active glycolysis albeit at a reduced rate in cell suspensions and gels. The enhancement of Ca2+ influx is concluded to be via the mechanosensitive cation channel Piezo1. The increased rate of influx brought about by the activator of Piezo1, 2-[5-[[(2,6-dichlorophenyl)methyl]thio]-1,3,4-thiadiazol-2-yl]-pyrazine (Yoda1) supported this conclusion; while the specificity of the cation-sensing by 5FBAPTA was confirmed by using the Ca2+ ionophore, A23187.