External Ca2 Research Articles

Mitochondria regulate TRPV4-mediated release of ATP.

Ca2+ influx via TRPV4 channels triggers Ca2+ release from the IP3 -sensitive internal store to generate repetitive oscillations. Although mitochondria are acknowledged regulators of IP3 -mediated Ca2+ release, how TRPV4-mediated Ca2+ signals are regulated by mitochondria is unknown. We show that depolarised mitochondria switch TRPV4 signalling from relying on Ca2+ -induced Ca2+ release at IP3 receptors to being independent of Ca2+ influx and instead mediated by ATP release via pannexins. TRPV4-evoked Ca2+ signals were individually examined in hundreds of cells in the endothelium of rat mesenteric resistance arteries using the indicator Cal520. TRPV4 activation with GSK1016790A (GSK) generated repetitive Ca2+ oscillations that required Ca2+ influx. However, when the mitochondrial membrane potential was depolarised, by the uncoupler CCCP or complex I inhibitor rotenone, TRPV4 activation generated large propagating, multicellular, Ca2+ waves in the absence of external Ca2+ . The ATP synthase inhibitor oligomycin did not potentiate TRPV4-mediated Ca2+ signals. GSK-evoked Ca2+ waves, when mitochondria were depolarised, were blocked by the TRPV4 channel blocker HC067047, the SERCA inhibitor cyclopiazonic acid, the PLC blocker U73122 and the inositol trisphosphate receptor blocker caffeine. The Ca2+ waves were also inhibited by the extracellular ATP blockers suramin and apyrase and the pannexin blocker probenecid. These results highlight a previously unknown role of mitochondria in shaping TRPV4-mediated Ca2+ signalling by facilitating ATP release. When mitochondria are depolarised, TRPV4-mediated release of ATP via pannexin channels activates plasma membrane purinergic receptors to trigger IP3 -evoked Ca2+ release.

Promotional effects of defects on Ni/HAP catalyst for carbon resistance and durability during dry reforming of methane

Excellent carbon resistance and stable operations are essential for the industrialization of catalytic dry reforming of methane (DRM). In this study, a series of Ni/HAP and Ni/HAP-D catalysts are used to investigate their DRM reaction performance, including activity, durability, and resistance to carbon deposition. Combined with H2-TPR and XPS results, Ni in the Ni/HAP prefers to replace the external Ca atoms in hydroxyapatite. The inherently “Ca-deficient” HAP-D support facilitates a more robust anchor positioning for the Ni metal, and a significant fraction of the Ni atoms will be embedded in the inner structure of hydroxyapatite in the form of Ni2+[I] and Ni2+[II]. The catalytic activity of the Ni/HAP-D series of catalysts for CH4 and CO2 is significantly higher than that of the Ni/HAP series of catalysts, demonstrating that the Ni morphology, the combined form between Ni and support plays a crucial role in the DRM reaction. The deactivation rate of the Ni0.5/HAP-D catalyst is only 0.008% h−1 and 0.007% h−1 for CH4 and CO2, respectively, in a 200-h durability test. In the spent Ni0.5/HAP catalyst after 118-h reaction, the presence of NiO crystalline phase is detected, indicating that partial Ni is oxidized by CO2 during the reaction, and amorphous carbon and graphitic carbon are also detected. In contrast, no NiO crystalline phase is detected in the spent Ni/HAP-D catalyst after 200-h reaction, and only trace amounts of graphitic carbon are observed, indicating that the Ni presence pattern of Ni0.5/HAP-D contributes to its stability and resistance to carbon deposition. This work provides insights into the design of supported nickel-based catalysts with both excellent stability and surprisingly resistant to carbon deposits for DRM applications.

Differential activation of Ca2+ influx channels modulate stem cell potency, their proliferation/viability and tissue regeneration

Stem cells have indefinite self-renewable capability; however, factors that modulate their pluripotency/function are not fully identified. Here we show that store-dependent Ca2+ entry is essential for modulating the function of bone marrow-derived mesenchymal stem cells (MSCs). Increasing external Ca2+ modulated cell cycle progression that was critical for MSCs survival. Additionally, Ca2+ was critical for stem proliferation, its differentiation, and maintaining stem cell potential. Ca2+ channel characterization, including gene silencing, showed two distinct Ca2+ entry channels (through Orai1/TRPC1 or via Orai3) that differentially regulate the proliferation and viability of MSCs. Importantly, NFκB translocation, but not JNK/ERK into the nucleus, was observed upon store depletion, which was blocked by the addition of Ca2+ channel inhibitors. Radiation lead to a decrease in saliva secretion, decrease in acinar cell number, and enlarged ducts were observed, which were restored by the transplantation of stem cells that were propagated in higher Ca2+. Finally radiation showed a decrese in TRPC1 expression along with a decrese in AQP5, which was again restored upon MSC tranplantation. Together these results suggest that Ca2+ entry is essential for stem cell function that could be critical for regenerative medicine.

Regulation of cell quiescence-proliferation balance by Ca2+-CaMKK-Akt signaling.

Compared with our extensive understanding of the cell cycle, we have limited knowledge of how the cell quiescence-proliferation decision is regulated. Using a zebrafish epithelial model, we report a novel signaling mechanism governing the cell quiescence-proliferation decision. Zebrafish Ca2+-transporting epithelial cells, or ionocytes, maintain high cytoplasmic Ca2+ concentration ([Ca2+]c) due to the expression of Trpv6. Genetic deletion or pharmacological inhibition of Trpv6, or reduction of external Ca2+ concentration, lowered the [Ca2+]c and reactivated these cells. The ionocyte reactivation was attenuated by chelating intracellular Ca2+ and inhibiting calmodulin (CaM), suggesting involvement of a Ca2+ and CaM-dependent mechanism. Long-term imaging studies showed that after an initial decrease, [Ca2+]c gradually returned to the basal levels. There was a concomitant decease in endoplasmic reticulum (ER) Ca2+ levels. Lowering the ER Ca2+ store content or inhibiting ryanodine receptors impaired ionocyte reactivation. Further analyses suggest that CaM-dependent protein kinase kinase (CaMKK) is a key molecular link between Ca2+ and Akt signaling. Genetic deletion or inhibition of CaMKK abolished cell reactivation, which could be rescued by expression of a constitutively active Akt. These results suggest that the quiescence-proliferation decision in zebrafish ionocytes is regulated by Trpv6-mediated Ca2+ and CaMKK-Akt signaling.

Increasing cytosolic Ca2+ levels restore cell proliferation and stem cell potency in aged MSCs

Aging is an inescapable complex physiological but extendable process, and all cells, including stem cells, are altered over time. Diverse mechanism(s) could modulate stem cell number, their proliferation rate, and promote tissue repair during aging that leads to longevity. However, the factors that could restore aging stem cell potency and would lead to healthy aging are not fully identified. Here we show that maintaining cytosolic Ca2+ levels was essential for modulating stem cells function in aged mesenchymal stem cells (MSCs). Increasing external Ca2+ induced spindle shape stem cell morphology and maintained stem cell surface marker expression in aged bone marrow-derived MSCs. Similarly, stem cell survival and proliferation of aged MSCs was dependent on cytosolic Ca2+ levels. Importantly, Ca2+ entry potentiated cell cycle progression, and stem cell potential was increased in cells incubated with higher external Ca2+. Moreover, blocking Ca2+ entry using SKF 96365, decreased stem cell survival and its proliferation but, treatment with 2-APB did not significantly affected cell proliferation, rather only modulated cell viability. Evaluation of Ca2+ entry channels, showed that TRPC1/Orai1/Orai3 and their regulator STIM1 was essential for MSCs proliferation/viability as gene silencing of Orai1/Orai3/TRPC1/STIM1 significantly inhibited stem cell viability. Finally, MSCs isolated from aged mice that were subjected to higher Ca2+ levels, were able to rescue age-induced loss of MSCs function. Together these results suggest that Ca2+ entry is essential for preventing the loss of aged stem cell function and supplementing Ca2+ not only restored their proliferative potential but, allowed them to develop into younger stem cell lineages that could be critical for regenerative medicine.

Coordinated glucose-induced Ca2+ and pH responses in yeast Saccharomyces cerevisiae

Ca2+ and pH homeostasis are closely intertwined and this interrelationship is crucial in the cells' ability to adapt to varying environmental conditions. To further understand this Ca2+-pH link, cytosolic Ca2+ was monitored using the aequorin-based bioluminescent assay in parallel with fluorescence reporter-based assays to monitor plasma membrane potentials and intracellular (cytosolic and vacuolar) pH in yeast Saccharomyces cerevisiae. At external pH 5, starved yeast cells displayed depolarized membrane potentials and responded to glucose re-addition with small Ca2+ transients accompanied by cytosolic alkalinization and profound vacuolar acidification. In contrast, starved cells at external pH 7 were hyperpolarized and glucose re-addition induced large Ca2+ transients and vacuolar alkalinization. In external Ca2+-free medium, glucose-induced pH responses were not affected but Ca2+ transients were abolished, indicating that the intracellular [Ca2+] increase was not prerequisite for activation of the two primary proton pumps, being Pma1 at the plasma membrane and the vacuolar and Golgi localized V-ATPases. A reduction in Pma1 expression resulted in membrane depolarization and reduced Ca2+ transients, indicating that the membrane hyperpolarization generated by Pma1 activation governed the Ca2+ influx that is associated with glucose-induced Ca2+ transients. Loss of V-ATPase activity through concanamycin A inhibition did not alter glucose-induced cytosolic pH responses but affected vacuolar pH changes and Ca2+ transients, indicating that the V-ATPase established vacuolar proton gradient is substantial for organelle H+/Ca2+ exchange. Finally, a systematic analysis of yeast deletion strains allowed us to reveal an essential role for both the vacuolar H+/Ca2+ exchanger Vcx1 and the Golgi exchanger Gdt1 in the dissipation of intracellular Ca2+.

The Depolarization-Evoked, Ca2+-Dependent Release of Exosomes From Mouse Cortical Nerve Endings: New Insights Into Synaptic Transmission.

Whether exosomes can be actively released from presynaptic nerve terminals is a matter of debate. To address the point, mouse cortical synaptosomes were incubated under basal and depolarizing (25 mM KCl-enriched medium) conditions, and extracellular vesicles were isolated from the synaptosomal supernatants to be characterized by dynamic light scattering, transmission electron microscopy, Western blot, and flow cytometry analyses. The structural and biochemical analysis unveiled that supernatants contain vesicles that have the size and the shape of exosomes, which were immunopositive for the exosomal markers TSG101, flotillin-1, CD63, and CD9. The marker content increased upon the exposure of nerve terminals to the high-KCl stimulus, consistent with an active release of the exosomes from the depolarized synaptosomes. High KCl-induced depolarization elicits the Ca2+-dependent exocytosis of glutamate. Interestingly, the depolarization-evoked release of exosomes from cortical synaptosomes also occurred in a Ca2+-dependent fashion, since the TSG101, CD63, and CD9 contents in the exosomal fraction isolated from supernatants of depolarized synaptosomes were significantly reduced when omitting external Ca2+ ions. Differently, (±)-baclofen (10 µM), which significantly reduced the glutamate exocytosis, did not affect the amount of exosomal markers, suggesting that the GABAB-mediated mechanism does not control the exosome release. Our findings suggest that the exposure of synaptosomes to a depolarizing stimulus elicits a presynaptic release of exosomes that occurs in a Ca2+-dependent fashion. The insensitivity to the presynaptic GABAB receptors, however, leaves open the question on whether the release of exosomes could be a druggable target for new therapeutic intervention for the cure of synaptopathies.

Machine Learning Establishes Single-Cell Calcium Dynamics as an Early Indicator of Antibiotic Response.

Changes in bacterial physiology necessarily precede cell death in response to antibiotics. Herein we investigate the early disruption of Ca2+ homeostasis as a marker for antibiotic response. Using a machine learning framework, we quantify the temporal information encoded in single-cell Ca2+ dynamics. We find Ca2+ dynamics distinguish kanamycin sensitive and resistant cells before changes in gross cell phenotypes such as cell growth or protein stability. The onset time (pharmacokinetics) and probability (pharmacodynamics) of these aberrant Ca2+ dynamics are dose and time-dependent, even at the resolution of single-cells. Of the compounds profiled, we find Ca2+ dynamics are also an indicator of Polymyxin B activity. In Polymyxin B treated cells, we find aberrant Ca2+ dynamics precedes the entry of propidium iodide marking membrane permeabilization. Additionally, we find modifying membrane voltage and external Ca2+ concentration alters the time between these aberrant dynamics and membrane breakdown suggesting a previously unappreciated role of Ca2+ in the membrane destabilization during Polymyxin B treatment. In conclusion, leveraging live, single-cell, Ca2+ imaging coupled with machine learning, we have demonstrated the discriminative capacity of Ca2+ dynamics in identifying antibiotic-resistant bacteria.

Low-voltage-activated inward current in murine antral smooth muscle cells is an artifact

Two types of voltage-dependent inward currents were evoked by depolarization in murine antral smooth muscle cells (SMCs) bathed in Ca2+-containing physiological solution: high-voltage-activated (HVA) and low-voltage-activated (LVA) inward currents. We examined whether the LVA current was due to: 1) T-type Ca2+ channels, 2) Ca2+-activated Cl−channels, 3) nonselective cation channels (NSCC), or 4) voltage-dependent K+ channels. Replacement of external Ca2+ (2 mM) with equimolar Ba2+ increased the amplitude of the HVA current but blocked the LVA current. Nicardipine blocked the HVA current, and in the presence of nicardipine, T-type Ca2+ blockers failed to block LVA current. A Cl− channel antagonist had little effect on LVA current. Cation-free external solution completely abolished both HVA and LVA currents. Addition of Ca2+ to the solution restored only HVA currents. Addition of K+ (5 mM) to otherwise cation-free solution induced LVA current that reversed at −20 mV. These data suggest that LVA current is not due to T-type Ca2+ channels, Ca2+-activated Cl− channels, or NSCC. A-type K+ (KA) currents and delayed rectifying K+ (KDR) currents can be resolved in antral SMCs dialyzed with a solution containing 140 mM K+. When cells were exposed to high K+ external solution and dialyzed with Cs+-rich solution in the presence of nicardipine, LVA current was evoked and reversed at positive potentials. LVA currents were blocked by K+ channel blockers, 4-aminopyridine, and tetraethylammonium. In conclusion, LVA inward currents can be generated by K+ influx via KA channels in murine antral SMCs when cells were dialyzed with Cs+-rich solution.

Ryanodine receptor-mediated Ca2+ release and atlastin-2 GTPase activity contribute to IP3-induced dendritic Ca2+ signals in primary hippocampal neurons

Neuronal Ca2+ signals are fundamental for synaptic transmission and activity-dependent changes in gene expression. Voltage-gated Ca2+ channels and N-methyl-d-aspartate receptors play major roles in mediating external Ca2+ entry during action potential firing and glutamatergic activity. Additionally, the inositol-1,4,5-trisphosphate receptor (IP3R) and the ryanodine receptor (RyR) channels expressed in the endoplasmic reticulum (ER) also contribute to the generation of Ca2+ signals in response to neuronal activity. The ER forms a network that pervades the entire neuronal volume, allowing intracellular Ca2+ release in dendrites, soma and presynaptic boutons. Despite its unique morphological features, the contributions of ER structure and of ER-shaping proteins such as atlastin - an ER enriched GTPase that mediates homotypic ER tubule fusion - to the generation of Ca2+ signals in dendrites remains unreported. Here, we investigated the contribution of RyR-mediated Ca2+ release to IP3-generated Ca2+ signals in dendrites of cultured hippocampal neurons. We also employed GTPase activity-deficient atlastin-2 (ATL2) mutants to evaluate the potential role of atlastin on Ca2+ signaling and ER-resident Ca2+ channel distribution. We found that pharmacological suppression of RyR channel activity increased the rising time and reduced the magnitude and propagation of IP3-induced Ca2+ signals. Additionally, ATL2 mutants induced specific ER morphological alterations, delayed the onset and increased the rising time of IP3-evoked Ca2+ signals, and caused RyR2 and IP3R1 aggregation and RyR2 redistribution. These results indicate that both RyR and ATL2 activity regulate IP3-induced Ca2+ signal dynamics through RyR-mediated Ca2+-induced Ca2+ release, ER shaping and RyR2 distribution.

Facile Synthesis of In Situ Formable Alginate Composite Hydrogels with Ca2+-Induced Healing Ability.

Dental caries have plagued humans for many years. At present, photocrosslinking resin is commonly used in clinics to repair narrow tooth defects, but the ultraviolet light used in this process has unavoidable cytotoxicity. In situ hydrogels with a similar structure to that of the natural extracellular matrix have gradually attracted attention in the field of hard tissue repair engineering. The injectable molding properties of hydrogel also give it the potential to fill irregularly shaped or fine tissue defects. Through a rapid and facile Michael addition reaction, we prepared maleic chitosan (CS-maleic anhydride [MA]) and thiolated alginate (sodium alginate [SA]-SH) to form a CS-MA/SA-SH hydrogel. To endue its mineralize ability, β-glycerophosphate calcium phosphate and calcium carbonate as the precursor of hydroxyapatite (HAp) were premixed in the hydrogel at certain ratios. This kind of hydrogel can quickly form into different shapes within 10 min. It is worth noting that external Ca2+ can react with the residual carboxyl groups of SA and provide the hydrogel with a self-healing ability. At the same time, we creatively propose a method that uses alkaline phosphatase to promote the mineralization of HAp in hydrogels, to achieve the purpose of regenerating hard tissue in situ. By examining the properties of hydrogels at different concentrations of calcium and phosphorus salts, we find that the CS-MA/SA-SH hydrogel with 50% (wt.%) inorganic matter presented the best self-healing properties, excellent mineralization of highly crystallized Hap, and has ideal cell compatibility. The potential application of the CS-MA/SA-SH hydrogel in repairing exposed dentin tubules in decayed teeth was explored through preliminary in vitro dental restoration experiments. Obviously, the penetration depth through dentin tubules was better than that of commercial dental sensitizers. In addition, the HAp morphology was affected by the local environment. We believe that this hydrogel can utilize tissues for dental regeneration and mineralization, and the healing ability provides the hydrogel flexibility for further application in hard tissue regeneration. Impact statement In this article, we report a simple, gentle, and rapid method for the synthesis of new, in situ-formable polysaccharide-based hydrogels that are capable of healing through the Michael addition reaction, which does not require any crosslinking agents. The rapid healing ability of the hydrogel can be obtained within 1 min by introducing calcium ions, giving the hydrogel the possibility of self-healing in the cap-enriched state. Second, we conferred in vitro mineralization of hydrogels and used a simple novel "enzyme-promoted" approach. Simulated mineralization using human body-enriched alkaline phosphatase as an inducer, allowing hydrogels rich in calcium glycerophosphate and calcium chloride to be highly similar to natural hydroxyphospholipids in a simulated body fluid environment at 37°C, stone-structured, and well-shaped thin-section mineralized products. Finally, we use the self-healing hydrogel with in vitro mineralization ability to carry out simple repair and mineralization regeneration of enamel and dentin, and this result is related to the tooth desensitizing agent commonly used in Chinese dental clinics. Comparison of commercial hydrogels further proves that this polysaccharide-based hydrogel is not only convenient for clinical operation in oral prosthesis but also has obvious advantages in the sealing of enamel by dental tubules and the enhancement of tooth hardness.

ATP-evoked intracellular Ca2+ transients shape the ionic permeability of human microglia from epileptic temporal cortex

BackgroundIntracellular Ca2+ modulates several microglial activities, such as proliferation, migration, phagocytosis, and inflammatory mediator secretion. Extracellular ATP, the levels of which significantly change during epileptic seizures, activates specific receptors leading to an increase of intracellular free Ca2+ concentration ([Ca2+]i). Here, we aimed to functionally characterize human microglia obtained from cortices of subjects with temporal lobe epilepsy, focusing on the Ca2+-mediated response triggered by purinergic signaling.MethodsFura-2 based fluorescence microscopy was used to measure [Ca2+]i in primary cultures of human microglial cells obtained from surgical specimens. The perforated patch-clamp technique, which preserves the cytoplasmic milieu, was used to measure ATP-evoked Ca2+-dependent whole-cell currents.ResultsIn human microglia extracellular ATP evoked [Ca2+]i increases depend on Ca2+ entry from the extracellular space and on Ca2+ mobilization from intracellular compartments. Extracellular ATP also induced a transient fivefold potentiation of the total transmembrane current, which was completely abolished when [Ca2+]i increases were prevented by removing external Ca2+ and using an intracellular Ca2+ chelator. TRAM-34, a selective KCa3.1 blocker, significantly reduced the ATP-induced current potentiation but did not abolish it. The removal of external Cl− in the presence of TRAM-34 further lowered the ATP-evoked effect. A direct comparison between the ATP-evoked mean current potentiation and mean Ca2+ transient amplitude revealed a linear correlation. Treatment of microglial cells with LPS for 48 h did not prevent the ATP-induced Ca2+ mobilization but completely abolished the ATP-mediated current potentiation. The absence of the Ca2+-evoked K+ current led to a less sustained ATP-evoked Ca2+ entry, as shown by the faster Ca2+ transient kinetics observed in LPS-treated microglia.ConclusionsOur study confirms a functional role for KCa3.1 channels in human microglia, linking ATP-evoked Ca2+ transients to changes in membrane conductance, with an inflammation-dependent mechanism, and suggests that during brain inflammation the KCa3.1-mediated microglial response to purinergic signaling may be reduced.

Non-Selective Cation Currents Mediated by Cx43 Hemichannel-P2X4 Receptor Signaling Pathway in Rat Atrial Myocytes under Shear Stress

Cardiac myocytes are exposed to shear stress under physiological conditions and hemodynamic disturbances. Shear stress has been thought to trigger two distinct global Ca2+ waves in atrial myocytes through autocrine activation of P2X4 receptor (P2X4R) and P2Y1-receptor (P2Y1R) via connexin 43 (Cx43)-mediated ATP release. In this study, we examined whether such shear stress-triggered Cx43-ATP-P2 receptor signaling generates depolarizing currents in atrial myocytes. Whole-cell patch clamp and dye-flux assays were used with genetic and pharmacological interventions in rat atrial myocytes and HL-1 cells. We found that shear stress activated a non-selective inward cation (Cs+) current (IS,Cs) at resting potential which was eliminated by Cx43 blockade using siRNA, antibodies, carbenoxolone or La3+. IS,Cs was enhanced by removal of external Ca2+, whereas internal Ca2+ buffering suppressed IS,Cs. Hemichannel-mediated dye (calcein red-orange) flux was induced by shear stress. This response was augmented by removing external Ca2+ but was not affected by pannexin blocker (probenecid). The shear-activated current was significantly reduced when Cs+ was replaced with NMDG+, and this was enhanced by quinine and suppressed by La3+. P2X4R antagonists (5-BDBD and PSB-12054), P2XR-blocker (iso-PPADS) and anti-P2X4R antibodies each blocked IS,Cs by approximately 50%. About 25% of IS,Cs was suppressed by P2Y1R inhibitor (MRS2179) and/or transient receptor potential melastatin 4 blocker (9-phenanthrol). P2X4R, but not P2X5R, was co-localized with Cx43 at the cell ends. P2X4R-dependent IS,Cs was augmented in atrial myocytes from hypertrophied rat hearts by short-term aortic constriction. Our data suggest that shear stress triggers Cx43 hemichannels to gate P2X4Rs in their vicinity. This shear stress signaling may play an important role in membrane depolarization at negative potentials and in atrial remodeling under increased afterload.

TRPM7 Permeant Divalent Metal Cytotoxicity During Magnesium Depletion and Loading

TRPM7 channels, highly expressed in immune cells, are believed to conduct divalent metal cations such as Mg2+, Zn2+, Cd2+ and Mn2+. Normally, TRPM7 channels are inhibited by cytosolic Mg2+, polyamines and protons, resulting in very small basal current magnitudes. In patch-clamp, these channels are traditionally activated by removal of intracellular Mg2+. Here, we have investigated the consequences of long-term reductions in extracellular [Mg2+] on TRPM7 channel activity. Jurkat T lymphocytes were maintained at external Mg2+ concentrations of 400 nM to 1.4 mM [Mg2+]o for 1-2 days. Incubating cells in 400 nM or 8 uM [Mg2+] resulted in almost complete current activation at break-in (t=0). Conversely, 1.4 mM external [Mg2+] was sufficient to eliminate basal currents. These findings suggested that long-term culture in low [Mg2+] results in cytosolic Mg2+ depletion and TRPM7 current activation. Presence or absence of external Ca2+, did not influence Mg2+ depletion or loading. Next, we investigated if entry and cytotoxicity of divalent metals is influenced by TRPM7 channel activity. We exposed the cells to 0.4 mM Zn2+, Cd2+, Mn2+, Co2+, Ni2+, Ba2+, Sr2+ for 1-2 days and quantified cell viability by trypan blue exclusion. In cells grown in 8 uM and 0.4 mM [Mg2+] the cytotoxicity sequence was: Cd2+ > Zn2+ > Co2+ > Ni2+ >Mn2+ with Ba2+ and Sr2+ virtually nontoxic. Maintaining cells in 0.4 or 1.4 mM Mg2+ had a ≤20% protective effect over 8 uM Mg2+ for the tested metal ions except Cd2+. Blockade of TRPM7 channels with NS8593 or La3+ also had a modest protective effect. In TRPM7 CRISPR knockdown HAP1 cells, metal cytotoxicity persisted and was modestly reduced for Co2+, Ni2+, Mn2+ but not Cd2+, Zn2+. Our experiments suggest that only a fraction of divalent metal cation entry depends on TRPM7 channels.

Responses of sweet pepper (Capsicum annum L.) cultivated in a closed hydroponic system to variable calcium concentrations in the nutrient solution.

The use of water containing calcium bicarbonate (Ca(HCO3 )2 ) at excessively high concentrations in closed hydroponic crops can cause calcium ion (Ca2+ ) accumulation in the recycled nutrient solution (NS) and concomitantly negatively affect yield and product quality. The aim of the study was to determine maximum Ca2+ concentrations that do not harm the crop and to simulate the pattern of Ca2+ accumulation when the Ca2+ concentration in the irrigation water, and concomitantly in the replenishment nutrient solution (RNS), is excessive. In the current study, irrigation water containing 1.5, 3.0, 4.5 and 6.0 mmol L-1 Ca2+ was used to prepare the RNS supplied to pepper cultivated in a closed hydroponic system. At 1.5 mmol L-1 Ca2+ , no Ca2+ accumulation was observed in the recirculating NS. However, at 3.0, 4.5 and 6.0 mmol L-1 in the irrigation water, the Ca2+ concentration in the recirculating NS, increased by the latter cropping stages to 17, 28 and 37 mmol L-1 , corresponding to 6.4, 9.0 and 10.8 dS m-1 . The accumulation of Ca2+ in the recirculating NS affected both tissue nutrient concentrations and uptake concentrations of Ca2+ , sulphate ion (SO4 2- ) and magnesium ion (Mg2+ ), but this was not the case for nitrogen (N) or potassium ion (K+ ). Growth, yield and plant water uptake were restricted at moderate (3.0 and 4.5 mmol L-1 ) and high (6.0 mmol L-1 ) external Ca2+ levels. In soilless pepper crops with zero discharge of fertigation effluents, the Ca2+ concentration in the irrigation water and the RNS should be lower than 3.0 mmol L-1 to avoid yield restrictions due to salinity. © 2021 Society of Chemical Industry.

Sodium Influx and Potassium Efflux Currents in Sunflower Root Cells Under High Salinity.

Helianthus annuus L. is an important oilseed crop, which exhibits moderate salt tolerance and can be cultivated in areas affected by salinity. Using patch-clamp electrophysiology, we have characterized Na+ influx and K+ efflux conductances in protoplasts of salt-tolerant H. annuus L. hybrid KBSH-53 under high salinity. This work demonstrates that the plasma membrane of sunflower root cells has a classic set of ionic conductances dominated by K+ outwardly rectifying channels (KORs) and non-selective cation channels (NSCCs). KORs in sunflower show extreme Na+ sensitivity at high extracellular [Ca2+] that can potentially have a positive adaptive effect under salt stress (decreasing K+ loss). Na+ influx currents in sunflower roots demonstrate voltage-independent activation, lack time-dependent component, and are sensitive to Gd3+. Sunflower Na+-permeable NSCCs mediate a much weaker Na+ influx currents on the background of physiological levels of Ca2+ as compared to other species. This suggests that sunflower NSCCs have greater Ca2+ sensitivity. The responses of Na+ influx to Ca2+ correlates well with protection of sunflower growth by external Ca2+ in seedlings treated with NaCl. It can be, thus, hypothesized that NaCl tolerance in sunflower seedling roots is programmed at the ion channel level via their sensitivity to Ca2+ and Na+.

Phase-Locking Requires Efficient Ca2+ Extrusion at the Auditory Hair Cell Ribbon Synapse.

Proper perception of sounds in the environment requires auditory signals to be encoded with extraordinary temporal precision up to tens of microseconds, but how it originates from the hearing organs in the periphery is poorly understood. In particular, sound-evoked spikes in auditory afferent fibers in vivo are phase-locked to sound frequencies up to 5 kHz, but it is not clear how hair cells can handle intracellular Ca2+ changes with such high speed and efficiency. In this study, we combined patch-clamp recording and two-photon Ca2+ imaging to examine Ca2+ dynamics in hair cell ribbon synapses in the bullfrog amphibian papilla of both sexes. We found that Ca2+ clearance from single synaptic ribbons followed a double exponential function, and the weight of the fast component, but not the two time constants, was significantly reduced for prolonged stimulation, and during inhibition of the plasma membrane Ca2+ ATPase (PMCA), the mitochondrial Ca2+ uptake (MCU), or the sarcolemma/endoplasmic reticulum Ca2+ ATPase (SERCA), but not the Na+/Ca2+ exchanger (NCX). Furthermore, we found that both the basal Ca2+ level and the Ca2+ rise during sinusoidal stimulation were significantly increased by inhibition of PMCA, MCU, or SERCA. Consistently, phase-locking of synaptic vesicle releases from hair cells was also significantly reduced by blocking PMCA, MCU, or SERCA, but not NCX. We conclude that, in addition to fast diffusion mediated by mobile Ca2+ buffer, multiple Ca2+ extrusion pumps are required for phase-locking at the auditory hair cell ribbon synapse.SIGNIFICANCE STATEMENT Hair cell synapses can transmit sound-driven signals precisely in the kHz range. However, previous studies of Ca2+ handling in auditory hair cells have often been conducted in immature hair cells, with elevated extracellular Ca2+ concentration, or through steady-state stimulation that may not be physiologically relevant. Here we examine Ca2+ clearance from hair cell synaptic ribbons in a fully mature preparation at physiological concentration of external Ca2+ and at physiological temperature. By stimulating hair cells with sinusoidal voltage commands that mimic pure sound tones, we recapitulated the phase-locking of hair cell exocytosis with an in vitro approach. This allowed us to reveal the Ca2+ extrusion mechanisms that are required for phase-locking at auditory hair cell ribbon synapses.

Long-Term Exercise Reduces Formation of Tubular Aggregates and Promotes Maintenance of Ca2+ Entry Units in Aged Muscle.

Tubular aggregates (TAs) in skeletal muscle fibers are unusual accumulation of sarcoplasmic reticulum (SR) tubes that are found in different disorders including TA myopathy (TAM). TAM is a muscular disease characterized by muscle pain, cramping, and weakness that has been recently linked to mutations in STIM1 and ORAI1. STIM1 and ORAI1 are the two main proteins mediating store-operated Ca2+ entry (SOCE), a mechanism activated by depletion of intracellular Ca2+ stores (e.g., SR) that allows recovery of Ca2+ from the extracellular space during repetitive muscle activity. We have recently shown that exercise triggers the formation of unique intracellular junctions between SR and transverse tubules named Ca2+ entry units (CEUs). CEUs promote colocalization of STIM1 with ORAI1 and improve muscle function in presence of external Ca2+. TAs virtually identical to those of TAM patients are also found in fast-twitch fibers of aging male mice. Here, we used a combination of electron and confocal microscopy, Western blotting, and ex vivo stimulation protocols (in presence or absence of external Ca2+) to evaluate the presence of TAs, STIM1-ORAI1 localization and expression and fatigue resistance of intact extensor digitorum longus (EDL) muscles in wild-type male adult (4-month-old) and aged (24-month-old) mice and in mice trained in wheel cages for 15 months (from 9 to 24 months of age). The results collected indicate that (i) aging causes STIM1 and ORAI1 to accumulate in TAs and (ii) long-term exercise significantly reduced formation of TAs. In addition, (iii) EDL muscles from aged mice exhibited a faster decay of contractile force than adult muscles, likely caused by their inability to refill intracellular Ca2+ stores, and (iv) exercise in wheel cages restored the capability of aged EDL muscles to use external Ca2+ by promoting maintenance of CEUs. In conclusion, exercise prevented improper accumulation of STIM1 and ORAI1 in TAs during aging, maintaining the capability of aged muscle to refill intracellular Ca2+ stores via SOCE.

Impact of Maternal Vitamin D Receptor (VDR) Gene Polymorphisms on Spontaneous Preterm Birth (Egyptian Case-Control Study)

Diverse effects of Ba^(2+) and Co^(2+) on fura-2 fluorescence ratio signals (FR, F350/F380) were examined in cultured bovine aortic endothelial cells (BAECs). ATP (3 μM) induced FR oscillations in normal Ca^(2+)-containing solutions. Co^(2+), but not Ba^(2+), inhibited ATP-induced FR oscillations. In Ca^(2+)-free solutions, 3 μM ATP only elicited a single FR transient. When Ba^(2+) replaced Ca^(2+) in the perfusate, 3 μM ATP provoked a distinct FR transient. The maximal rate of rise of the signal was greater than seen without Ba^(2+) and the FR increased and maintained to a high steady level. When external Ca^(2+) was replaced by Co^(2+), ATP-induced FR transient oscillated with a long recovery time constant. In a survey of the single fluorescence at excitation wavelength 350 nm (F350), Co^(2+) decreased the intensity in the presence of ATP and Ca^(2+). Ba^(2+), however, increased F350 and FR as well, especially in Ca^(2+)-free solutions. The results suggest that ATP-induced Ca^(2+) fluctuation is balanced by Ca^(2+) release from internal stores and influx from external Ca^(2+). Ba2+ seems to be permeable through Ca^(2+) channels and competes with Ca^(2+) for the dye. Co^(2+) blocks Ca^(2+) channels but does not inhibit ATP-mediated Ca^(2+) release in cells.

Green tea catechins and intracellular calcium dynamics in prostate cancer cells

Perturbations of internal Ca²⁺ ([Ca²⁺]_i) homeostasis play a key role in several pathologies and in neoplastic transformation, where deregulated cell proliferation, together with the suppression of apoptosis, provides the condition for abnormal tissue growth and invasion. Green tea catechins have been shown to affect cancer development by interference with basic cellular functions, most of which are mediated by [Ca²⁺]_i. Prostate cancer (PCa) is one of the most common malignancy in men in Western countries and the androgen-independent carcinoma is a lethal form for which there is still no effective therapy. Different evidences suggested that consumption of green tea may have beneficial effects against PCa. We have previously described how the main green tea flavonoid, (−)-epigallocatechin-3-gallate (EGCG), inhibited proliferation and induced dose-dependent peaks of [Ca²⁺]_i in metastatic androgen-insensitive DU145 and PC3 PCa cells, by a mechanism that combined Ca²⁺ entry and Ca²⁺-induced Ca²⁺ release. In the present study, we studied the effect of green tea extract (GTE) on the same cell lines. Proliferation, measured by MTT assay, was inhibited by GTE with IC₅₀ close to 60 µg/ml, a value that is higher than that expected by EGCG effect alone. [Ca²⁺]_i, measured in real time by the fluorescent dye Fura-2, was transiently increased by GTE by a mechanism that resembled that described for EGCG, but was largely independent of external Ca²⁺. These observations suggested that other components, acting in synergy with EGCG, were involved in GTE effect, and confirmed the view that the alleged health benefits of green tea for PCa prevention may be related to [Ca²⁺]_i deregulation in malignant cells. These results may be significant to understand the functional mechanisms by which flavonoids exert their beneficial or toxic actions.