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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.

The homeostatic modulation of neurotransmitter release, termed presynaptic homeostatic potentiation (PHP), is a fundamental type of neuromodulation, conserved from Drosophila to humans, that stabilizes information transfer at synaptic connections throughout the nervous system. Here, we demonstrate that α2δ-3, an auxiliary subunit of the presynaptic calcium channel, is required for PHP. The α2δ gene family has been linked to chronic pain, epilepsy, autism, and the action of two psychiatric drugs: gabapentin and pregabalin. We demonstrate that loss of α2δ-3 blocks both the rapid induction and sustained expression of PHP due to a failure to potentiate presynaptic calcium influx and the RIM-dependent readily releasable vesicle pool. These deficits are independent of α2δ-3-mediated regulation of baseline calcium influx and presynaptic action potential waveform. α2δ proteins reside at the extracellular face of presynaptic release sites throughout the nervous system, a site ideal for mediating rapid, transsynaptic homeostatic signaling in health and disease.

Background: Sperm cryopreservation-thawing process has damaging effects on the structure and function of sperm, namely cryoinjury. Calcium overload has been reported as a postulated mechanism for sperm damage during the first steps after thawing. This study was designed to assess the intracellular calcium (Ca2+i) after cryopreservation and to clarify the role of a calcium chelator ethylene glycol-bis (2-aminoethyl ether)-N, N, N′, N′-tetraacetic acid (EGTA) on human sperm quality.Methods: Forty semen samples were obtained from fertile men (March 2017 to 2018). The samples were randomly divided into fresh (F) and cryopreserved-thawed (CT) groups. The F and CT samples were divided into control and 1 mM EGTA-treated groups. Sperm kinematics and membrane integrity were assessed. The reactive oxygen species (ROS) and adenosine triphosphate (ATP) were measured by luminescent methods. Ca2+i, apoptotic rate, and mitochondrial membrane potential (MMP) were evaluated using flow cytometric methods. Data were compared using SPSS software, version 16.0 by ANOVA and Kruskal-Wallis test. PResults: Cryopreservation decreased sperm motility, viability, membrane integrity, Ca2+i, MMP, and induced cell apoptosis and ROS production. EGTA could not protect the cryopreserved sperm from cryoinjury. It was found to have destructive effects on fresh sperm motility and viability (P=0.009) relative to cryopreserved sperm. ATP was reduced (P=0.02) and ROS production (P=0.0001) was increased in the EGTA-treated F and CT sperms.Conclusion: Despite Ca2+i reduction by EGTA, it had no protective effects on fresh or cryopreserved sperm. We concluded that sperm cryoinjury was not dependent on calcium overload, and it was suggested that cryoinjury was mainly related to cell membranes damage.

In this study, the influence of ultrasonication (USN) combined with egtazic acid fragmentation method on sugar industry waste (SWS) sludge was studied for improving biohydrogen production. Initially, USN method was proceeded by varying power and fragmentation time. At an optimal power (115 W) and fragmentation time (30 min), USN method demanded 23,000 kJ/kg TS specific energy for achieving 15.2% complex organics solubilization rate (COR). To further upsurge of SWS sludge solubilization, USN assisted with egtazic acid (USNEG) method was performed by varying fragmentation time and egtazic acid dosage at an optimal power (115 W). USNEG method has achieved more COR (23%) at minimum specific energy of 11,500 kJ/kg TS (fragmentation time: 15 min and EGTA dosage: 0.03 g/g TS). Then, biohydrogen potential (BHP) assay was investigated in which USNEG method has obtained higher biohydrogen production. Also, energy analysis revealed that USNEG method exhibited an energy ratio of 1.78. • USN method demanded 23000 kJ/kg TS of specific energy for attaining 15.2% COR. • USNEG method exhibited a higher COR of 23% at 11500 kJ/kg TS. • A higher intracellular components release was noted for USNEG method. • USNEG method was attained a higher biohydrogen production of 28 mL/g VS. • USNEG method exhibited positive net energy of 2.5 kWh/kg of SWS sludge. [ABSTRACT FROM AUTHOR]

The actin cortex both facilitates and hinders the exocytosis of secretory granules. How cells consolidate these two opposing roles was not well understood. Here we show that antigen activation of mast cells induces oscillations in Ca(2+) and PtdIns(4,5)P(2) lipid levels that in turn drive cyclic recruitment of N-WASP and cortical actin level oscillations. Experimental and computational analysis argues that vesicle fusion correlates with the observed actin and Ca(2+) level oscillations. A vesicle secretion cycle starts with the capture of vesicles by actin when cortical F-actin levels are high, followed by vesicle passage through the cortex when F-actin levels are low, and vesicle fusion with the plasma membrane when Ca(2+) levels subsequently increase. Thus, cells employ oscillating levels of Ca(2+), PtdIns(4,5)P(2) and cortical F-actin to increase secretion efficiency, explaining how the actin cortex can function as a carrier as well as barrier for vesicle secretion.

Rab3 interacting molecules (RIMs) are evolutionarily conserved scaffolding proteins that are located at presynaptic active zones. In the mammalian nervous system, RIMs have two major activities that contribute to the fidelity of baseline synaptic transmission: they concentrate calcium channels at the active zone and facilitate synaptic vesicle docking/priming. Here we confirm that RIM has an evolutionarily conserved function at the Drosophila neuromuscular junction and then define a novel role for RIM during homeostatic synaptic plasticity. We show that loss of RIM disrupts baseline vesicle release, diminishes presynaptic calcium influx, and diminishes the size of the readily-releasable pool (RRP) of synaptic vesicles, consistent with known activities of RIM. However, loss of RIM also completely blocks the homeostatic enhancement of presynaptic neurotransmitter release that normally occurs after inhibition of postsynaptic glutamate receptors, a process termed synaptic homeostasis. It is established that synaptic homeostasis requires enhanced presynaptic calcium influx as a mechanism to potentiate vesicle release. However, despite a defect in baseline calcium influx in rim mutants, the homeostatic modulation of calcium influx proceeds normally. Synaptic homeostasis is also correlated with an increase in the size of the RRP of synaptic vesicles, although the mechanism remains unknown. Here we demonstrate that the homeostatic modulation of the RRP is blocked in the rim mutant background. Therefore, RIM-dependent modulation of the RRP is a required step during homeostatic plasticity. By extension, homeostatic plasticity appears to require two genetically separable processes, the enhancement of presynaptic calcium influx and a RIM-dependent modulation of the RRP.

We describe the integrated use of pulsed laser microbeam irradiation and microfluidic cell culture methods to examine the dynamics of axonal injury and regeneration in vitro. Microfabrication methods are used to place high purity dissociated central nervous system neurons in specific regions that allow the axons to interact with permissive and inhibitory substrates. Acute injury to neuron bundles is produced via the delivery of single 180 ps duration, lambda = 532 nm laser pulses. Laser pulse energies of 400 nJ and 800 nJ produce partial and complete transection of the axons, respectively, resulting in elliptical lesions 25 mum and 50 mum in size. The dynamics of the resulting degeneration and regrowth of proximal and distal axonal segments are examined for up to 8 h using time-lapse microscopy. We find the proximal and distal dieback distances from the site of laser microbeam irradiation to be roughly equal for both partial and complete transection of the axons. In addition, distinct growth cones emerge from the proximal neurite segments within 1-2 h post-injury, followed by a uniform front of regenerating axons that originate from the proximal segment and traverse the injury site within 8 h. We also examine the use of EGTA to chelate the extracellular calcium and potentially reduce the severity of the axonal degeneration following injury. While we find the addition of EGTA to reduce the severity of the initial dieback, it also hampers neurite repair and interferes with the formation of neuronal growth cones to traverse the injury site. This integrated use of laser microbeam dissection within a micropatterned cell culture system to produce precise zones of neuronal injury shows potential for high-throughput screening of agents to promote neuronal regeneration.

Group IVA phospholipase A(2) (GIVA PLA(2)) catalyzes the release of arachidonic acid (AA) from the sn-2 position of glycerophospholipids. AA is then further metabolized into terminal signaling molecules including numerous prostaglandins. We have now demonstrated the involvement of phosphatidic acid phosphohydrolase 1 (PAP-1) and protein kinase C (PKC) in the Toll-like receptor-4 (TLR-4) activation of GIVA PLA(2). We also studied the effect of PAP-1 and PKC on Ca+2 induced and synergy enhanced GIVA PLA(2) activation. We observed that the AA release induced by exposure of RAW 264.7 macrophages to the TLR-4 specific agonist Kdo(2)-Lipid A is blocked by the PAP-1 inhibitors bromoenol lactone (BEL) and propranolol as well as the PKC inhibitor Ro 31-8220; however these inhibitors did not reduce AA release stimulated by Ca+2 influx induced by the P2X7 purinergic receptor agonist ATP. Additionally, stimulation of cells with diacylglycerol (DAG), the product of PAP-1 mediated hydrolysis, initiated AA release from unstimulated cells as well as restored normal AA release from cells treated with PAP-1 inhibitors. Finally, neither PAP-1 nor PKC inhibition reduced GIVA PLA(2) synergistic activation by stimulation with Kdo(2)-Lipid A and ATP.

Using whole-cell recording in Drosophila S2 cells, we characterized a Ca(2+)-selective current that is activated by depletion of intracellular Ca2+ stores. Passive store depletion with a Ca(2+)-free pipette solution containing 12 mM BAPTA activated an inwardly rectifying Ca2+ current with a reversal potential >60 mV. Inward currents developed with a delay and reached a maximum of 20-50 pA at -110 mV. This current doubled in amplitude upon increasing external Ca2+ from 2 to 20 mM and was not affected by substitution of choline for Na+. A pipette solution containing approximately 300 nM free Ca2+ and 10 mM EGTA prevented spontaneous activation, but Ca2+ current activated promptly upon application of ionomycin or thapsigargin, or during dialysis with IP3. Isotonic substitution of 20 mM Ca2+ by test divalent cations revealed a selectivity sequence of Ba2+ > Sr2+ > Ca2+ >> Mg2+. Ba2+ and Sr2+ currents inactivated within seconds of exposure to zero-Ca2+ solution at a holding potential of 10 mV. Inactivation of Ba2+ and Sr2+ currents showed recovery during strong hyperpolarizing pulses. Noise analysis provided an estimate of unitary conductance values in 20 mM Ca2+ and Ba2+ of 36 and 420 fS, respectively. Upon removal of all external divalent ions, a transient monovalent current exhibited strong selectivity for Na+ over Cs+. The Ca2+ current was completely and reversibly blocked by Gd3+, with an IC50 value of approximately 50 nM, and was also blocked by 20 microM SKF 96365 and by 20 microM 2-APB. At concentrations between 5 and 14 microM, application of 2-APB increased the magnitude of Ca2+ currents. We conclude that S2 cells express store-operated Ca2+ channels with many of the same biophysical characteristics as CRAC channels in mammalian cells.

In rat basophilic leukemia (RBL) cells and Jurkat T cells, Ca(2+) release-activated Ca(2+) (CRAC) channels open in response to passive Ca(2+) store depletion. Inwardly rectifying CRAC channels admit monovalent cations when external divalent ions are removed. Removal of internal Mg(2+) exposes an outwardly rectifying current (Mg(2+)-inhibited cation [MIC]) that also admits monovalent cations when external divalent ions are removed. Here we demonstrate that CRAC and MIC currents are separable by ion selectivity and rectification properties: by kinetics of activation and susceptibility to run-down and by pharmacological sensitivity to external Mg(2+), spermine, and SKF-96365. Importantly, selective run-down of MIC current allowed CRAC and MIC current to be characterized under identical ionic conditions with low internal Mg(2+). Removal of internal Mg(2+) induced MIC current despite widely varying Ca(2+) and EGTA levels, suggesting that Ca(2+)-store depletion is not involved in activation of MIC channels. Increasing internal Mg(2+) from submicromolar to millimolar levels decreased MIC currents without affecting rectification but did not alter CRAC current rectification or amplitudes. External Mg(2+) and Cs(+) carried current through MIC but not CRAC channels. SKF-96365 blocked CRAC current reversibly but inhibited MIC current irreversibly. At micromolar concentrations, both spermine and extracellular Mg(2+) blocked monovalent MIC current reversibly but not monovalent CRAC current. The biophysical characteristics of MIC current match well with cloned and expressed TRPM7 channels. Previous results are reevaluated in terms of separate CRAC and MIC channels.

IntroductionFasting has been shown to limit ischemic injury and improve functional activity after global ischemia. Because calcium overload is considered a mechanism of ischemic injury, we hypothesized that fasting would limit the accumulation of intracellular calcium [Ca]i during ischemia, potentially due to reduced accumulation of intracellular sodium [Na]i.MethodsTo address this hypothesis, hearts isolated from rats fed either a normal diet or fasted for 24 hours underwent 20 min of global ischemia at 37 degrees. In addition to functional parameters, [Na]i and [Ca]i were measured using 21Na and 19F spectroscopy using thulium-DOTP-5 and 5F-BAPTA, respectively. In vitro measurement of sarcoplasmic reticulum calcium uptake and release, as well as activity of the sarcolemmal Na-Ca exchanger, was performed in hearts from fed and fasted animals under baseline and ischemic conditions.ResultsHearts from fasted animals showed greater recovery of developed pressure (37+/-9 vs. 11+/-6 cm H2O, p < 0.05) and less contracture (end-diastolic pressure 25+/-2 vs. 47+/-2 cm H2O, p < 0.05) by the end of the reperfusion period. [Na]i was similar in the 2 groups during the first half of the ischemic period, albeit with a higher concentration of [Na]i in hearts from fed compared to fasted animals at reperfusion. Fasting markedly limited calcium accumulation during ischemia, with end-ischemic calcium being 419+/-46 nM in the hearts from fasted animals and 858+/-140 nM in the hearts from fed animals (p < 0.01). There was no significant effect of fasting on calcium uptake or release by the SR, nor on sarcolemmal Na-Ca exchange activity.ConclusionsFasting for 24 hours improves functional recovery and markedly limits [Ca]i accumulation during ischemia and early reperfusion. The mechanism for this phenomenon remains to be elucidated.

Although the crucial role of Ca(2+) influx in lymphocyte activation has been well documented, little is known about the properties or expression levels of Ca(2+) channels in normal human T lymphocytes. The use of Na(+) as the permeant ion in divalent-free solution permitted Ca(2+) release-activated Ca(2+) (CRAC) channel activation, kinetic properties, and functional expression levels to be investigated with single channel resolution in resting and phytohemagglutinin (PHA)-activated human T cells. Passive Ca(2+) store depletion resulted in the opening of 41-pS CRAC channels characterized by high open probabilities, voltage-dependent block by extracellular Ca(2+) in the micromolar range, selective Ca(2+) permeation in the millimolar range, and inactivation that depended upon intracellular Mg(2+) ions. The number of CRAC channels per cell increased greatly from approximately 15 in resting T cells to approximately 140 in activated T cells. Treatment with the phorbol ester PMA also increased CRAC channel expression to approximately 60 channels per cell, whereas the immunosuppressive drug cyclosporin A (1 microM) suppressed the PHA-induced increase in functional channel expression. Capacitative Ca(2+) influx induced by thapsigargin was also significantly enhanced in activated T cells. We conclude that a surprisingly low number of CRAC channels are sufficient to mediate Ca(2+) influx in human resting T cells, and that the expression of CRAC channels increases approximately 10-fold during activation, resulting in enhanced Ca(2+) signaling.

Throughout the body, epithelial tissues contain curved features (e.g. cysts, ducts and crypts) that influence cell behaviors. These structures have varied curvature, with flat structures having zero curvature and structures such as crypts having large curvature. In the ovary, cortical inclusion cysts (CICs) of varying curvatures are found, and fallopian tube epithelial (FTE) cells have been found trapped within these cysts. FTE are the precursor for ovarian cancer, and the CIC niche has been proposed to play a role in ovarian cancer progression. We hypothesized that variations in ovarian CIC curvature that occur during cyst resolution impact the ability of trapped FTE cells to invade into the surrounding stroma. Using a lumen model in collagen gels, we determined that increased curvature resulted in more invasions of mouse FTE cells. To isolate curvature as a system parameter, we developed a novel technique to pattern concave curvatures into collagen gels. When FTE cells were seeded to confluency on curved substrates, increases in curvature increased the number of invading FTE cells and the invasion distance. FTE invasion into collagen substrates with higher curvature depended on matrix metalloproteinases (MMPs), but expression of collagen I degrading Mmps was not different on curved and flat regions. A finite-element model predicted that contractility and cell–cell connections were essential for increased invasion on substrates with higher curvature, while cell–substrate interactions had minimal effect. Experiments supported these predictions, with invasion decreased by blebbistatin, ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) or N-cadherin-blocking antibody, but with no effect from a focal adhesion kinase inhibitor. Finally, experimental evidence supports that cell invasion on curved substrates occurs in two phases—a cell–cell-dependent initiation phase where individual cells break away from the monolayer and an MMP-dependent phase as cells migrate further into the collagen matrix. [ABSTRACT FROM AUTHOR]

BackgroundPhotolabile chelators that release Ca2+ upon illumination have been used extensively to dissect the role of this important second messenger in cellular processes such as muscle contraction and synaptic transmission. The caged calcium chelators that are presently available are often limited by their inadequate changes in Ca2+ affinity, selectivity for Ca2+ over Mg2+ and sensitivity to light. As these chelators are all based on nitrobenzyl photochemistry, we explored the use of other photosensitive moieties to generate a new caged calcium with improved properties.ResultsAzid-1 is a novel caged calcium in which a fluorescent Ca2+ indicator, fura-2, has been modified with an azide substituent on the benzofuran 3-position. Azid-1 binds Ca2+ with a dissociation constant (Kd) of approximately 230 nM, which changes to 120 microM after photolysis with ultraviolet light (330-380 nm). Mg2+ binding is weak (8-9 mM Kd) before or after photolysis. Azid-1 photolyzes with unit quantum efficiency, making it 40-170-fold more sensitive to light than caged calciums used previously. The photolysis of azid-1 probably releases N2 to form a nitrenium ion that adds water to yield an amidoxime cation; the electron-withdrawing ability of the amidoxime cation reduces the chelator's Ca2+ affinity within at most 2 ms following a light flash. The ability of azid-1 to function as a caged calcium in living cells was demonstrated in cerebellar Purkinje cells, in which Ca2+ photolytically released from azid-1 could replace the normal depolarization-induced Ca2+ transient in triggering synaptic plasticity.ConclusionsAzid-1 promises to be a useful tool for generating highly controlled spatial and temporal increases of Ca2+ in studies of the many Ca2+-dependent biological processes. Unlike other caged calciums, azid-1 has a substantial cross section or shows a high susceptibility for two-photon photolysis, the only technique that confines the photochemistry to a focal spot that is localized in three dimensions. Azide photolysis could be a useful and more photosensitive alternative to nitrobenzyl photochemistry.

Activity-induced short-term synaptic enhancement (STE) is a common property of neurons, one that can endow neural circuits with the capacity for rapid and flexible information processing. Evidence from a variety of systems indicates that the expression of STE depends largely on the action of residual Ca2+, which enters the presynaptic terminal during activity. We have shown previously that a Ca2+-dependent STE in the inhibitory synapse between interneurons L30 and L29 in the abdominal ganglion of Aplysia californica has a functional role in regulating the gain of the siphon withdrawal circuit through facilitated recurrent inhibition onto the L29s. In the present paper, we further explore the role of Ca2+ in L30 STE by examining two basic issues: 1) What is the role of residual presynaptic Ca2+ in the maintenance of L30 STE? We examine this question by first inducing STE in the L30s then rapidly buffering presynaptic free calcium through the use of the photoactivated Ca2+ chelator diazo-4, which was preloaded into the L30 neurons. Three forms of STE in the L30s were examined: frequency facilitation (FF), augmentation (AUG), and posttetanic potentiation (PTP). In each case, the activation-induced enhancement of the L30 to L29 synapse was reduced to preactivation levels at the first test pulse following photolysis of diazo-4. 2) What is the role of postsynaptic Ca2+ in the induction of L30 STE? We examine whether there is a postsynaptic requirement of elevated Ca2+ for the induction of L30 STE by first injecting the calcium chelator bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA) into the postsynaptic cell L29 (at levels sufficient to block transmitter release from the L29s), to prevent any increase in postsynaptic intracellular Ca2+ that may occur during L30 (presynaptic) activation. We found that BAPTA injection did not effect either the induction or the time course of FF, AUG, or PTP in the L30s. Taken collectively, our data indicate that all forms of STE in the L30s depend on presynaptic free cytosolic Ca2+ for their maintenance but do not require the elevation of postsynaptic Ca2+ for their induction.

Ca(2+) currents activated by depletion of Ca(2+) stores in Xenopus oocytes were studied with a two-electrode voltage clamp. Buffering of cytosolic Ca(2+) with EGTA and MeBAPTA abolished I(Cl(Ca)) and unmasked a current in oocytes that was activated by InsP(3) or ionomycin in minutes and by thapsigargin or the chelators themselves over hours. At -60 mV in 10 mM extracellular CaCl(2), the current was typically around -90 or -160 nA in oocytes loaded with EGTA or MeBAPTA, respectively. This current was judged to be a Ca(2+)-selective current for the following reasons: (a) it was inwardly rectifying and reversed at membrane potentials usually more positive than +40 mV; (b) it was dependent on extracellular [CaCl(2)] with K(m) = 11.5 mM; (c) it was highly selective for Ca(2+) against monovalent cations Na(+) and K(+), because replacing Na(+) and K(+) by N-methyl-d-glucammonium did not reduce the amplitude or voltage dependence of the current significantly; and (d) Ca(2+), Sr(2+), and Ba(2+) currents had similar instantaneous conductances, but Sr(2+) and Ba(2+) currents appeared to inactivate more strongly than Ca(2+). This Ca(2+) current was blocked by metal ions with the following potency sequence: Mg(2+) << Ni(2+) approximately Co(2+) approximately Mn(2+) < Cd(2+) << Zn(2+) << La(3+). It was also inhibited by niflumic acid, which is commonly used to block I(Cl(Ca)). PMA partially inhibited the Ca(2+) current, and this effect was mostly abolished by calphostin C, indicating that the Ca(2+) current is sensitive to protein kinase C. These results are the first detailed electrophysiological characterization of depletion-activated Ca(2+) current in nondialyzed cells. Because exogenous molecules and channels are easy to introduce into oocytes and the distortions in measuring I(Cl(Ca)) can now be bypassed, oocytes are now a superior system in which to analyze the activation mechanisms of capacitative Ca(2+) influx.

Phagosome-lysosome membrane fusion is a highly regulated event that is essential for intracellular killing of microorganisms. Functionally, it represents a form of polarized regulated secretion, which is classically dependent on increases in intracellular ionized calcium ([Ca2+]i). Indeed, increases in [Ca2+]i are essential for phagosome-granule (lysosome) fusion in neutrophils and for lysosomal fusion events that mediate host cell invasion by Trypanosoma cruzi trypomastigotes. Since several intracellular pathogens survive in macrophage phagosomes that do not fuse with lysosomes, we examined the regulation of phagosome-lysosome fusion in macrophages. Macrophages (M phi) were treated with 12.5 microM bis-(2-amino-S-methylphenoxy) ethane-N,N,N',N',-tetraacetic acid tetraacetoxymethyl ester (MAPT/AM), a cell-permeant calcium chelator which reduced resting cytoplasmic [Ca2+]; from 80 nM to < or = 20 nM and completely blocked increases in [Ca2+]i in response to multiple stimuli, even in the presence of extracellular calcium. Subsequently, M phi phagocytosed serum-opsonized zymosan, staphylococci, or Mycobacterium bovis. Microbes were enumerated by 4',6-diamidino-2-phenylindole, dihydrochloride (DAPI) staining, and phagosome-lysosome fusion was scored using both lysosome-associated membrane protein (LAMP-1) as a membrane marker and rhodamine dextran as a content marker for lysosomes. Confirmation of phagosome-lysosome fusion by electron microscopy validated the fluorescence microscopy findings. We found that phagosome-lysosome fusion in M phi occurs noramlly at very low [Ca2+]i (< or = 20 nM). Kinetic analysis showed that in M phi none of the steps leading from particle binding to eventual phagosome-lysosome fusion are regulated by [Ca2+]i in a rate-limiting way. Furthermore, confocal microscopy revealed no difference in the intensity of LAMP-1 immunofluorescence in phagolysosome membranes in calcium-buffered vs. control macrophages. We conclude that neither membrane recognition nor fusion events in the phagosomal pathway in macrophages are dependent on or regulated by calcium.

In the P388D1 macrophage-like cell line, phospholipase A2 activity and prostaglandin production are stimulated by platelet-activating factor (PAF) and bacterial lipopolysaccharide (LPS). We have investigated the role of Ins(1,4,5)P3 and Ca2+ in signal transduction of PAF-induced prostaglandin E2 (PGE2) formation in these cells. The role of Ca2+ in the activation mechanism was studied by fluorescence imaging of intracellular Ca2+ in individual adherent cells and by determining the PGE2 production in the same population of cells. This new approach enabled us to correlate directly events on the single-cell level with a physiologically relevant response of the cell population. Priming the cells with LPS was required for PAF to stimulate PGE2 formation, yet LPS affected neither the intracellular free Ca2+ concentration ([Ca2+]i) nor the PAF-induced rise in [Ca2+]i. In addition, basal and PAF-stimulated Ins(1,4,5)P3 levels were not affected by LPS priming. However, the Ca2+ transient, the release of Ins(1,4,5)P3 and the formation of PGE2 induced by PAF were inhibited in cells pretreated with pertussis toxin. Buffering the [Ca2+]i with intracellular BAPTA [bis-(o-aminophenoxy)ethane-NNN'N'-tetra-acetic acid] blocked the PAF-stimulated rise in [Ca2+]i and PGE2 formation. Removal of extracellular Ca2+ during PAF stimulation prevented the influx of Ca2+, but did not affect the initial [Ca2+]i transient, nor did it inhibit PGE2 formation. Under the same conditions, ionomycin stimulated an identical [Ca2+]i transient, but, in contrast with PAF stimulation, no PGE2 formation was observed. PGE2 production could be rescued by prompt subsequent addition of PAF, which caused no further [Ca2+]i change on its own. These results show that the transient initial rise in [Ca2+]i, produced either by PAF via the formation of Ins(1,4,5)P3 or directly by ionomycin, is necessary, but not sufficient for the formation of PGE2 in LPS-primed P388D1 cells. Furthermore, we have demonstrated for the first time that PAF triggers a second signal that is not mediated by a change in [Ca2+]i. However, both signals are required to induce PGE2 formation.

A sustained high voltage-activated (HVA), nifedipine- and cadmium-sensitive calcium current and a sustained calcium action potential (AP) were recorded from horizontal cells isolated from catfish retina. pH indicator dyes showed that superfusion with NH4Cl alkalinized these cells and that washout of NH4Cl or superfusion with Na-acetate acidified them. HVA current was slightly enhanced during superfusion of NH4Cl but was suppressed upon NH4Cl washout or application of Na-acetate. When 25 mM HEPES was added to the patch pipette to increase intracellular pH buffering, the effects of NH4Cl and Na-acetate on HVA current were reduced. These results indicated that intracellular acidification reduces HVA calcium current and alkalinization increases it. Sustained APs, recorded with high resistance, small diameter microelectrodes, were blocked by cobalt and cadmium and their magnitude varied with extracellular calcium concentration. These results provide confirmatory evidence that the HVA current is a major component of the AP and indicate that the AP can be used as a measure of how the HVA current can be modified in intact, undialyzed cells. The duration of APs was increased by superfusion with NH4Cl and reduced by washout of NH4Cl or superfusion with Na-acetate. The Na-acetate and NH4Cl washout-dependent shortening of the APs was observed in the presence of intracellular BAPTA, a calcium chelator, IBMX, a phosphodiesterase inhibitor, and in Na-free or TEA-enriched saline. These findings provide supportive evidence that intracellular acidification may directly suppress the HVA calcium current in intact cells. Intracellular pH changes would thereby be expected to modulate not only the resting membrane potential of these cells in darkness, but calcium-dependent release of neurotransmitter from these cells as well. Furthermore, this acidification-dependent suppression of calcium current could serve a protective role by reducing calcium entry during retinal ischemia, which is usually thought to be accompanied by intracellular acidosis.

Intrathecal administration of Ca2+ has been shown to produce antinociception which is thought to be partially mediated by the release of adenosine. In the present study we have examined directly the effects of varying Ca2+ concentrations on the release of endogenous adenosine, measured by HPLC with fluorescence detection, from rat spinal cord synaptosomes. Although increasing the concentration of extracellular Ca2+ reduces the total amount of adenosine detected extrasynaptosomally, the component derived from the release of adenosine per se is actually augmented. This release of adenosine occurs from dorsal but not ventral spinal cord synaptosomes and appears to originate from capsaicin-sensitive nerve terminals. The Ca2+ ionophore A23187 also releases adenosine, but this release is due to the extrasynaptosomal conversion of released nucleotide(s) to adenosine, as it is markedly reduced by ecto-5'-nucleotidase inhibitors. Release of adenosine by A23187 occurs from both the dorsal and ventral spinal cord, and is not capsaicin-sensitive. Ethanol, used as a vehicle for the ionophore, releases adenosine which is a mixture of adenosine and nucleotide from both dorsal and ventral spinal cord synaptosomes. These observations provide direct support for behavioural studies which demonstrate that methylxanthines block antinociception produced by intrathecal administration of Ca2+.

Intracellular movement of vesiculated pigment granules in angelfish melanophores is regulated by a signalling pathway that triggers kinesin and dyneinlike microtubule motor proteins. We have tested the relative importance of intracellular Ca2+ ([Ca2+]i) vs cAMP ([cAMP]i) in the control of such motility by adrenergic agonists, using fluorescence ratio imaging and many ways to artificially stimulate or suppress signals in these pathways. Fura-2 imaging reported a [Ca2+]i elevation accompanying pigment aggregation, but this increase was not essential since movement was not induced with the calcium ionophore, ionomycin, nor was movement blocked when the increases were suppressed by withdrawal of extracellular Ca2+ or loading of intracellular BAPTA. The phosphatase inhibitor, okadaic acid, blocked aggregation and induced dispersion at concentrations that suggested that the protein phosphatase PP-1 or PP-2A was continuously turning phosphate over during intracellular motility. cAMP was monitored dynamically in single living cells by microinjecting cAMP-dependent kinase in which the catalytic and regulatory subunits were labeled with fluorescein and rhodamine respectively (Adams et al., 1991. Nature (Lond.). 349:694-697). Ratio imaging of F1CRhR showed that the alpha 2-adrenergic receptor-mediated aggregation was accompanied by a dose-dependent decrease in [cAMP]i. The decrease in [cAMP]i was both necessary and sufficient for aggregation, since cAMP analogs or microinjected free catalytic subunit of A kinase-blocked aggregation or caused dispersal, whereas the cAMP antagonist RpcAMPs or the microinjection of the specific kinase inhibitor PKI5-24 amide induced aggregation. Our conclusion that cAMP, not calcium, controls bidirectional microtubule dependent motility in melanophores might be relevant to other instances of non-muscle cell motility.

Putrescine transport in Neurospora is saturable and concentrative in dilute buffers, but in the growth medium putrescine simply equilibrates across the cell membrane. We describe a mutant, puu-1, that can concentrate putrescine from the growth medium because the polyamine transport system has lost its normal sensitivity to Ca2+. The wild type closely resembles the mutant if it is washed with citrate and ethylene glycol bis(beta-aminoethyl ether)N,N'-tetraacetic acid. The mutant phenotype also appears in the wild type after treatment with cycloheximide. The results suggest that putrescine uptake is normally regulated by an unstable Ca(2+)-binding protein that restricts polyamine uptake. This protein is evidently distinct from the polyamine-binding function for uptake, which is normal in mutant and in cycloheximide-treated wild type cells. The puu-1 mutation, stripping of Ca2+, and cycloheximide treatment all cause an impairment of amino acid transport, indicating that other membrane transport functions rely upon the product of the puu-1+ gene. Preliminary evidence suggests that the putrescine carrier is not the Ca(2+)-sensitive, low-affinity K(+)-transport system, but K+ efflux does accompany putrescine uptake.

Global Ca2+ transients have been observed to precede nuclear envelope breakdown and the onset of anaphase in Swiss 3T3 fibroblasts in 8% (vol/vol) FBS. The occurrence of these Ca2+ transients was dependent on intracellular stores. These Ca2+ transients could be (a) abolished by serum removal without halting mitosis, and (b) eliminated by increasing intracellular Ca2+ buffering capacity through loading the cells with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) buffer, via the tetra(acetoxymethyl) ester, without hindering the transition into anaphase. Microinjection of sufficient concentrations of BAPTA buffer could block nuclear envelope breakdown. Pulses of Ca2+ generated by flash photolysis of intracellularly trapped nitr-5, a "caged" Ca2+, could precipitate precocious nuclear envelope breakdown in prophase cells. In metaphase cells, photochemically generated Ca2+ pulses could cause changes in the appearance of the chromosomes, but the length of time required for cells to make the transition from metaphase to anaphase remained essentially unchanged regardless of whether a Ca2+ pulse was photoreleased during metaphase. The results from these photorelease experiments were not dependent on the presence of serum in the medium. Discharging intracellular Ca2+ stores with ionomycin in the presence of 1.8 mM extracellular Ca2+ doubled the time for cells to pass from late metaphase into anaphase, whereas severe Ca2+ deprivation by treatment with ionomycin in EGTA-containing medium halted mitosis. Our results collectively indicate that Ca2+ is actively involved in nuclear envelope breakdown, but Ca2+ signals are likely unnecessary for the metaphase-anaphase transition in Swiss 3T3 fibroblasts. Additional studies of intracellular Ca2+ concentrations in mitotic REF52 and PtK1 cells revealed that Ca2+ transients are not observed at all mitotic stages in all cells. The absence of observable global Ca2+ transients, where calcium buffers can block and pulses of Ca2+ can advance mitotic stages, may imply that the relevant Ca2+ movements are too local to be detected.

Intracellular Ca 2+ signals control several physiological and pathophysiological processes. The main tool to chelate intracellular Ca 2+ is intracellular BAPTA (BAPTA i ), usually introduced into cells as a membrane-permeant acetoxymethyl ester (BAPTA-AM). Previously, we demonstrated that BAPTA i enhanced apoptosis induced by venetoclax, a BCL-2 antagonist, in diffuse large B-cell lymphoma (DLBCL). This finding implied a novel interplay between intracellular Ca 2+ signaling and anti-apoptotic BCL-2 function. Hence, we set out to identify the underlying mechanisms by which BAPTA i enhances cell death in B-cell cancers. In this study, we discovered that BAPTA i alone induced apoptosis in hematological cancer cell lines that were highly sensitive to S63845, an MCL-1 antagonist. BAPTA i provoked a rapid decline in MCL-1-protein levels by inhibiting mTORC1-driven Mcl-1 translation. These events were not a consequence of cell death, as BAX/BAK-deficient cancer cells exhibited similar downregulation of mTORC1 activity and MCL-1-protein levels. Next, we investigated how BAPTA i diminished mTORC1 activity and identified its ability to impair glycolysis by directly inhibiting 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) activity, a previously unknown effect of BAPTA i . Notably, these effects were also induced by a BAPTA i analog with low affinity for Ca 2+ . Consequently, our findings uncover PFKFB3 inhibition as an Ca 2+ -independent mechanism through which BAPTA i impairs cellular metabolism and ultimately compromises the survival of MCL-1-dependent cancer cells. These findings hold two important implications. Firstly, the direct inhibition of PFKFB3 emerges as a key regulator of mTORC1 activity and a promising target in MCL-1-dependent cancers. Secondly, cellular effects caused by BAPTA i are not necessarily related to Ca 2+ signaling. Our data support the need for a reassessment of the role of Ca 2+ in cellular processes when findings were based on the use of BAPTA i ., (© 2023. The Author(s).)

Synaptotagmin‐1 (Syt1) is the primary calcium sensor (Ca2+) that mediates neurotransmitter release at the synapse. The tandem C2 domains (C2A and C2B) of Syt1 exhibit functionally critical, Ca2+‐dependent interactions with the plasma membrane. With the surface forces apparatus, we directly measure the binding energy of membrane‐anchored Syt1 to an anionic membrane and find that Syt1 binds with ~6 kBT in EGTA, ~10 kBT in Mg2+ and ~18 kBT in Ca2+. Molecular rearrangements measured during confinement are more prevalent in Ca2+ and Mg2+ and suggest that Syt1 initially binds through C2B, then reorients the C2 domains into the preferred binding configuration. These results provide energetic and mechanistic details of the Syt1 Ca2+‐activation process in synaptic transmission. [ABSTRACT FROM AUTHOR]

Synaptotagmin‐1 (Syt1) is the primary calcium sensor (Ca2+) that mediates neurotransmitter release at the synapse. The tandem C2 domains (C2A and C2B) of Syt1 exhibit functionally critical, Ca2+‐dependent interactions with the plasma membrane. With the surface forces apparatus, we directly measure the binding energy of membrane‐anchored Syt1 to an anionic membrane and find that Syt1 binds with ~6 kBT in EGTA, ~10 kBT in Mg2+ and ~18 kBT in Ca2+. Molecular rearrangements measured during confinement are more prevalent in Ca2+ and Mg2+ and suggest that Syt1 initially binds through C2B, then reorients the C2 domains into the preferred binding configuration. These results provide energetic and mechanistic details of the Syt1 Ca2+‐activation process in synaptic transmission. [ABSTRACT FROM AUTHOR]

The timing and probability of synaptic vesicle fusion from presynaptic terminals is governed by the distance between voltage-gated Ca2+ channels (VGCCs) and Ca2+ sensors for exocytosis. This VGCC-sensor coupling distance can be determined from the fractional block of vesicular release by exogenous Ca2+ chelators, which depends on biophysical factors that have not been thoroughly explored. Using numerical simulations of Ca2+ reaction and diffusion, as well as vesicular release, we examined the contributions of conductance, density, and open duration of VGCCs, and the influence of endogenous Ca2+ buffers on the inhibition of exocytosis by EGTA. We found that estimates of coupling distance are critically influenced by the duration and amplitude of Ca2+ influx at active zones, but relatively insensitive to variations of mobile endogenous buffer. High concentrations of EGTA strongly inhibit vesicular release in close proximity (20-30 nm) to VGCCs if the flux duration is brief, but have little influence for longer flux durations that saturate the Ca2+ sensor. Therefore, the diversity in presynaptic action potential duration is sufficient to alter EGTA inhibition, resulting in errors potentially as large as 300% if Ca2+ entry durations are not considered when estimating VGCC-sensor coupling distances. [ABSTRACT FROM AUTHOR]

The bone-targeting mechanism of clinic bisphosphonate-type drugs, such as alendronate, risedronate, and ibandronate, relies on chelated calcium ions on the surface of the bone mineralized matrix for the treatment of osteoporosis. EGTA with aminocarboxyl chelating ligands can specifically chelate calcium ions. Inspired by the bone-targeting mechanism of bisphosphonates, we hypothesize that EGTA-derived carbon dots (EGTA-CDs) hold bone-targeting ability. For the target-oriented synthesis of EGTA-CDs and to endow CDs with bone targeting, we designed calcium ion chelating agents as precursors, including aminocarboxyl chelating agents (EGTA and EDTA) and bisphosphonate agents (ALN and HEDP) for the target-oriented synthesis of aminocarboxyl-derived CDs (EGTA-CDs and EDTA-CDs) and bisphosphonate-derived CDs (ALN-CDs and HEDP-CDs) with high synthetic yield. The synthetic yield of EGTA-CDs reached 87.6%. Aminocarboxyl-derived CDs and bisphosphonate-derived CDs retain the chelation ability of calcium ions and can specifically bind calcium ions. The chemical environment bone-targeting value coordination constant K and chelation sites of EGTA-CDs were 6.48 × 10 4 M -1 and 4.12, respectively. A novel method was established to demonstrate the bone-targeting capability of chelate-functionalized carbon dots using fluorescence quenching in a simulated bone trauma microenvironment. EGTA-CDs exhibit superior bone-targeting ability compared with other aminocarboxyl-derived CDs and bisphosphonate-derived CDs. EGTA-CDs display exceptional specificity toward calcium ions and better bone affinity than ALN-CDs, suggesting their potential as novel bone-targeting drugs. EGTA-CDs with strong calcium ion chelating ability have calcium ion affinity in simulated body fluid and bone-targeting ability in a simulated bone trauma microenvironment. These findings offer new avenues for the development of advanced bone-targeting strategies.