Asante Calcium Green and Asante Calcium Red—Novel Calcium Indicators for Two-Photon Fluorescence Lifetime Imaging

For a comprehensive understanding of cellular processes and potential dysfunctions therein, an analysis of the ubiquitous intracellular second messenger calcium is of particular interest. This study examined the suitability of the novel Ca²⁺-sensitive fluorescent dyes Asante Calcium Red (ACR) and Asante Calcium Green (ACG) for two-photon (2P)-excited time-resolved fluorescence measurements. Both dyes displayed sufficient 2P fluorescence excitation in a range of 720–900 nm. In vitro, ACR and ACG exhibited a biexponential fluorescence decay behavior and the two decay time components in the ns-range could be attributed to the Ca²⁺-free and Ca²⁺-bound dye species. The amplitude-weighted average fluorescence decay time changed in a Ca²⁺-dependent way, unraveling in vitro dissociation constants K _D of 114 nM and 15 nM for ACR and ACG, respectively. In the presence of bovine serum albumin, the absorption and steady-state fluorescence behavior of ACR was altered and its biexponential fluorescence decay showed about 5-times longer decay time components indicating dye-protein interactions. Since no ester derivative of ACG was commercially available, only ACR was evaluated for 2P-excited fluorescence lifetime imaging microscopy (2P-FLIM) in living cells of American cockroach salivary glands. In living cells, ACR also exhibited a biexponential fluorescence decay with clearly resolvable short (0.56 ns) and long (2.44 ns) decay time components attributable to the Ca²⁺-free and Ca²⁺-bound ACR species. From the amplitude-weighted average fluorescence decay times, an in situ K _D of 180 nM was determined. Thus, quantitative [Ca²⁺]_i recordings were realized, unraveling a reversible dopamine-induced [Ca²⁺]_i elevation from 21 nM to 590 nM in salivary duct cells. It was concluded that ACR is a promising new Ca²⁺ indicator dye for 2P-FLIM recordings applicable in diverse biological systems.     

Calcium - how and why?

Calcium is among the most commonly used ions, in a multitude of biological functions, so much so that it is impossible to imagine life without calcium. In this article I have attempted to address the question as to how calcium has achieved this status with a brief mention of the history of calcium research in biology. It appears that during the origin and early evolution of life the Ca²⁺ ion was given a unique opportunity to be used in several biological processes because of its unusual physical and chemical properties.     

Calcium Signalling and Alzheimer’s Disease

New insights into how Ca²⁺ regulates learning and memory have begun to provide clues as to how the amyloid-dependent remodelling of neuronal Ca²⁺ signalling pathways can disrupt the mechanisms of learning and memory in Alzheimer’s disease (AD). The calcium hypothesis of AD proposes that activation of the amyloidogenic pathway remodels the neuronal Ca²⁺ signalling pathways responsible for cognition by enhancing the entry of Ca²⁺ and/or the release of internal Ca²⁺ by ryanodine receptors or InsP₃ receptors. The specific proposal is that Ca²⁺ signalling remodelling results in a persistent elevation in the level of Ca²⁺ that constantly erases newly acquired memories by enhancing the mechanism of long-term depression (LTD). Neurons can still form memories through the process of LTP, but this stored information is rapidly removed by the persistent activation of LTD. Further dysregulation in Ca²⁺ signalling will then go on to induce the neurodegeneration that characterizes the later stages of dementia.     

Structures and Functions of Calcium Channel β Subunits

Calcium channel subunits have profound effects on how ₁ subunits perform. In this article we summarize our present knowledge of the primary structures of subunits as deduced from cDNAs and illustrate their different properties. Upon co-expression with ₁ subunits, the effects of subunits vary somewhat between L-type and non-L-type channels mostly because the two types of channels have different responses to voltage which are affected by subunits, such as long-lasting prepulse facilitation of _1C (absent in _1E) and inhibition by G protein dimer of _1E, absent in _1C. One subunit, a brain 2a splice variant that is palmitoylated, has several effects not seen with any of the others, and these are due to palmitoylation. We also illustrate the finding that functional expression of ₁ in oocytes requires a subunit even if the final channel shows no evidence for its presence. We propose two structural models for Ca²⁺ channels to account for ₁ alone channels seen in cells with limited subunit expression. In one model, dissociates from the mature ₁ after proper folding and membrane insertion. Regulated channels seen upon co-expression of high levels of would then have subunit composition ₁. In the other model, the chaperoning remains associated with the mature channel and ₁ alone channels would in fact be ₁ channels. Upon co-expression of high levels of the regulated channels would have composition [₁].     

Calcium and connexin-based intercellular communication, a deadly catch?

Ca²⁺ is known as a universal messenger mediating a wide variety of cellular processes, including cell death. In fact, this ion has been proposed as the ‘cell death master’, not only at the intracellular but also at the intercellular level. The most direct form of intercellular spread of cell death is mediated by gap junction channels. These channels have been shown to propagate cell death as well as cell survival signals between the cytoplasm of neighbouring cells, reflecting the dual role of Ca²⁺ signals, i.e. cell death versus survival. Its precursor, the unopposed hemichannel (half of a gap junction channel), has recently joined in as a toxic pore connecting the intracellular with the extracellular environment and allowing the passage of a range of substances. The biochemical nature of the so-called intercellular cell death molecule, transferred through gap junctions or released/taken up via hemichannels, remains elusive but several studies pinpoint Ca²⁺ itself or its messenger inositol trisphosphate as the responsible masters in crime. Although direct evidence is still lacking, indirect data including Ca²⁺ involvement in intercellular communication and cell death, and effects of intercellular communication on intracellular Ca²⁺ homeostasis, support this hypothesis. In addition, hemichannels and their molecular building blocks, connexin or pannexin proteins, may exert their effects on Ca²⁺-dependent cell death at the intracellular level, independently from their channel functions. This review provides a cutting edge overview of the current knowledge and underscores the intimate connection between intercellular communication, Ca²⁺ signalling and cell death.     

Calcium signalling and pancreatic cell death: apoptosis or necrosis?

Secretagogues, such as cholecystokinin and acetylcholine, utilise a variety of second messengers (inositol trisphosphate, cADPR and nicotinic acid adenine dinucleotide phosphate) to induce specific oscillatory patterns of calcium (Ca(2+)) signals in pancreatic acinar cells. These are tightly controlled in a spatiotemporal manner, and are coupled to mitochondrial metabolism necessary to fuel secretion. When Ca(2+) homeostasis is disrupted by known precipitants of acute pancreatitis, for example, hyperstimulation or non-oxidative ethanol metabolites, Ca(2+) stores (endoplasmic reticulum and acidic pool) become depleted and sustained cytosolic [Ca(2+)] elevations replace transient signals, leading to severe consequences. Sustained mitochondrial depolarisation, possibly via opening of the mitochondrial permeability transition pore (MPTP), elicits cellular ATP depletion that paralyses energy-dependent Ca(2+) pumps causing cytosolic Ca(2+) overload, while digestive enzymes are activated prematurely within the cell; Ca(2+)-dependent cellular necrosis ensues. However, when stress to the acinar cell is milder, for example, by application of the oxidant menadione, release of Ca(2+) from stores leads to oscillatory global waves, associated with partial mitochondrial depolarisation and transient MPTP opening; apoptotic cell death is promoted via the intrinsic pathway, when associated with generation of reactive oxygen species. Apoptosis, induced by menadione or bile acids, is potentiated by inhibition of an endogenous detoxifying enzyme NAD(P)H:quinone oxidoreductase 1 (NQO1), suggesting its importance as a defence mechanism that may influence cell fate.     

Protein Kinase C: The “Masters” of Calcium and Lipid

The coordinated and physiological behavior of living cells in an organism critically depends on their ability to interact with surrounding cells and with the extracellular space. For this, cells have to interpret incoming stimuli, correctly process the signals, and produce meaningful responses. A major part of such signaling mechanisms is the translation of incoming stimuli into intracellularly understandable signals, usually represented by second messengers or second-messenger systems. Two key second messengers, namely the calcium ion and signaling lipids, albeit extremely different in nature, play an important and often synergistic role in such signaling cascades. In this report, we will shed some light on an entire family of protein kinases, the protein kinases C, that are perfectly designed to exactly decode these two second messengers in all of their properties and convey the signaling content to downstream processes within the cell.Once generated, second messengers relay their information content in a plethora of properties, including time, quantity (i.e., concentration), space (i.e., subcellular distribution), and interestingly into any combination of these three characteristics. Nevertheless, such information is meaningless for the cell unless it has a toolkit of read-out systems that can actually interpret such second-messenger properties and relate them further downstream into complex signaling networks, or directly to effector systems. An important system is the family of protein kinase Cs (PKCs) that can read-out lipid signals alone, or combine the ability to read-out simultaneous lipid and Ca²⁺ signals. A common denominator of all PKCs is the property to convey signals downstream by phosphorylation of additional signaling partners or effector proteins. We will briefly introduce the PKC subfamilies with particular emphasis on their signaling ability, discuss the important sensing domains, and their properties, before concentrating on sensing details of the subfamily of conventional PKCs and their role in signal integration in greater depth.     

Calcium Signaling in Plant Endosymbiotic Organelles： Mechanism and Role in Physiology

Recent studies have demonstrated that chloroplasts and mitochondria evoke specific Ca2＋ signals in response to biotic and abiotic stresses in a stress-dependent manner. The identification of Ca2＋ transporters and Ca2＋signaling mol- ecules in chloroplasts and mitochondria implies that they play roles in controlling not only intra-organellar functions, but also extra-organellar processes such as plant immunity and stress responses. It appears that organellar Ca2＋ signaling might be more important to plant cell functions than previously thought. This review briefly summarizes what is known about the molecular basis of Ca2＋ signaling in plant mitochondria and chloroplasts.     

Calcium ions and polyamines activate the plasma membrane-located 1,3-β-glucan synthase

Sucrose-density-gradient centrifugation and partitioning in a polyethylene glycol/dextran two-phase system were used to isolate plasmamembrane vesicles from microsomal preparations of soybean cell suspension cultures. Both methods resulted in the enrichment of the activity of a 1,3--glucan synthase which forms a polymer consisting of more than 99% of 1,3-linked glucose (callose). Digitonin increases the 1,3--glucan synthase activity in the various membrane fractions to a different degree, supporting the suggestion that this enzyme is vectorially arranged in the plasma membrane. The enzyme is greatly activated either by poly-l-ornithine or synergistically by Ca²⁺ and spermine, indicating that the same enzyme is affected and exhibits the regulatory properties necessary for callose synthesis.Abbreviations GSI glucan synthase I - 1,3--GS 1,3--glucan synthase (EC 2.4.1.34) - IDPase inosine 5-diphosphatase - poly-l-Orn poly-l-ornithine - PEG polyethyleneglycol - SGT sterol-glucosyl-transferase - UDPG uridine 5-diphosphoglucose Dedicated to W. Nultsch on the occasion of his 60th birthday     

Calcium signaling in pancreatic β-cells in health and in Type 2 diabetes

Changes in cytosolic free Ca²⁺ concentration ([Ca²⁺]_c) play a crucial role in the control of insulin secretion from the electrically excitable pancreatic β-cell. Secretion is controlled by the finely tuned balance between Ca²⁺ influx (mainly through voltage-dependent Ca²⁺ channels, but also through voltage-independent Ca²⁺ channels like store-operated channels) and efflux pathways. Changes in [Ca²⁺]_c directly affect [Ca²⁺] in various organelles including the endoplasmic reticulum (ER), mitochondria, the Golgi apparatus, secretory granules and lysosomes, as imaged using recombinant targeted probes. Because most of these organelles have specific Ca²⁺ influx and efflux pathways, they mutually influence free [Ca²⁺] in the others. In this article, we review the mechanisms of control of [Ca²⁺] in various compartments and particularly the cytosol, the endoplasmic reticulum ([Ca²⁺]_ER), acidic stores and mitochondrial matrix ([Ca²⁺]_mito), focusing chiefly on the most important physiological stimulus of β-cells, glucose. We also briefly review some alterations of β-cell Ca²⁺ homeostasis in Type 2 diabetes.     

Are Calcium Ions and Calcium Channels Involved in the Mechanisms of Cu2+ Toxicity in Bean Plants? The Influence of Leaf Age

The influence of calcium channel blockers and ionophore on Cu₂₊-induced changes of the photosynthetic activity of runner bean plants (Phaseolus coccineus L.) was investigated. Excess Cu₂₊ was applied to leaves by injection or via the roots to examine a short/local or a long time/systemic effect of this metal, respectively. The changes in fluorescence parameters indicated that the mechanism of toxic action of Cu₂₊ ions on the photosynthetic apparatus was only partially connected with Ca₂₊ or Ca₂₊ channels. In young plants Ca₂₊ diminished especially photochemical and nonphotochemical dissipative processes induced by short- and long-term influence of excess Cu₂₊. Blocking of Ca₂₊ channels did not change direct Cu₂₊ action on the photosynthetic activity, however, their opening distinctly intensified the inhibitory effect of the metal. After a longer accumulation peri od the effect of Cu₂₊ ions did not change significantly due to modified Ca₂₊ penetration through membranes (except that caused by La³⁺). Copper directly introduced into older leaves diminished only at its highest concentration the activity both of the donor and acceptor sides of photosystem 2 (PS2) connected with Rfd decrease and increase of LNU. A similar effect was observed also after a long-term Cu₂₊ action, but disturbances on the acceptor side of PS2 were observed only at a higher Ca₂₊ content in the nutrient solution. Ca₂₊ ions, particularly after openning of channels, intensified direct inhibitory Cu₂₊ action on the photosynthetic activity expressed by decreased values of F_v/F₀ and Rfd. Lanthanum and verapamil, at a lower Ca₂₊ content in the medium, decreased the photosynthetic activity of Cu₂₊-treated plants. This effect was also seen after additional Ca₂₊ supply to the leaves. This revised version was published online in September 2006 with corrections to the Cover Date.     

Calcium ion as cellular messenger

<正>The prominent role Ca2+ion plays as a major small biological messenger is fascinating.There are many physiologically important ions,such as Na+,K+,H+,Cl?,Ca2+,Mg2+,Fe3+,and PO43?that participate in cell signaling.Among them,monovalent ions primarily contribute to rapid electrical signaling,while multivalent ions generally act as a co-factor for chemical reactions by associating with the host molecule through electrostatic or covalent interactions.Ca2+plays both roles.Its transmembrane influx supports the plateau phase of cardiac action potential and helps set the speed of pacemaker potential.Inside the cell,Ca2+switches     

Aminopyridines Potentiate Synaptic and Neuromuscular Transmission by Targeting the Voltage-activated Calcium Channel �� Subunit

Aminopyridines such as 4-aminopyridine (4-AP) are widely used as voltage-activated K⁺ (Kv) channel blockers and can improve neuromuscular function in patients with spinal cord injury, myasthenia gravis, or multiple sclerosis. Here, we present novel evidence that 4-AP and several of its analogs directly stimulate high voltage-activated Ca²⁺ channels (HVACCs) in acutely dissociated neurons. 4-AP, 4-(aminomethyl)pyridine, 4-(methylamino)pyridine, and 4-di(methylamino)pyridine profoundly increased HVACC, but not T-type, currents in dissociated neurons from the rat dorsal root ganglion, superior cervical ganglion, and hippocampus. The widely used Kv channel blockers, including tetraethylammonium, α-dendrotoxin, phrixotoxin-2, and BDS-I, did not mimic or alter the effect of 4-AP on HVACCs. In HEK293 cells expressing various combinations of N-type (Cav2.2) channel subunits, 4-AP potentiated Ca²⁺ currents primarily through the intracellular β₃ subunit. In contrast, 4-AP had no effect on Cav3.2 channels expressed in HEK293 cells. Furthermore, blocking Kv channels did not mimic or change the potentiating effects of 4-AP on neurotransmitter release from sensory and motor nerve terminals. Thus, our findings challenge the conventional view that 4-AP facilitates synaptic and neuromuscular transmission by blocking Kv channels. Aminopyridines can directly target presynaptic HVACCs to potentiate neurotransmitter release independent of Kv channels.     

The pH Dependence and the Binding Equilibria of the Calcium Indicator — Arsenazo III

The underlying principles of binding equilibria of arsenazo III with Ca²⁺ and Mg²⁺ are presented. Ca²⁺ and Mg²⁺ can bind arsenazo III in several different protonated forms depending on pH. The binding affinities of these different protonated forms of arsenazo III with Ca²⁺ increase in the order of H₄A_4- <H₃A^5- >H₂A^6- and with Mg²⁺, H₄A^4- > H₃A^5- > H₂A^6-. Arsenazo III is not membrane bound. The sensitivity ratio of arsenazo III with Ca²⁺ to arsenazo III with Mg²⁺ is close to two orders of magnitude. Arsenazo III and its complexes are extremely sensitive to pH changes. With 5 μM arsenazo III, the minimum detectable amount of Ca²⁺ can be as low as 0.08 μM. Contrary to current belief, we found that Mg²⁺ can bind to arsenazo III in a slightly acidic medium. Potential applications of arsenazo III to the study of membrane Ca²⁺ transport are also discussed.     

A Calcium-Dependent Protein Kinase Interactswith and Activates A Calcium Channel toRequlate Pollen Tube Growth

ABSTRACT Calcium, as a ubiquitous second messenger, plays essential roles in tip-growing cells, such as animal neu-rons, plant pollen tubes, and root hairs. However, little is known concerning the regulatory mechanisms that code anddecode Ca2＋ signals in plants. The evidence presented here indicates that a calcium-dependent protein kinase, CPK32,controls polar growth of pollen tubes. Overexpression of CPK32 disrupted the polar growth along with excessive Ca2＋accumulation in the tip. A search of downstream effector molecules for CPK32 led to identification of a cyclic nucleotide-gated channel, CNGC18, as an interacting partner for CPK32. Co-expression of CPK32 and CNGC18 resulted in activationof CNGC18 in Xenopus oocytes where expression of CNGC18 alone did not exhibit significant calcium channel activity.Overexpression of CNGC18 produced a growth arrest phenotype coupled with accumulation of calcium in the tip, simi-lar to that induced by CPK32 overexpression. Co-expression of CPK32 and CNGC18 had a synergistic effect leading tomore severe depolarization of pollen tube growth. These results provide a potential feed-forward mechanism in whichcalcium-activated CPK32 activates CNGC18, further promoting calcium entry during the elevation phase of Ca2＋ oscilla-tions in the polar growth of pollen tubes.     

Calcium Extrusion Pump PMCA4: A New Player in Renal Calcium Handling?

Calcium (Ca²⁺) is vital for multiple processes in the body, and maintenance of the electrolyte concentration is required for everyday physiological function. In the kidney, and more specifically, in the late distal convoluted tubule and connecting tubule, the fine-tuning of Ca²⁺ reabsorption from the pro-urine takes place. Here, Ca²⁺ enters the epithelial cell via the transient receptor potential vanilloid receptor type 5 (TRPV5) channel, diffuses to the basolateral side bound to calbindin-D_28k and is extruded to the blood compartment via the Na⁺/Ca²⁺ exchanger 1 (NCX1) and the plasma membrane Ca²⁺ ATPase (PMCA). Traditionally, PMCA1 was considered to be the primary Ca²⁺ pump in this process. However, in recent studies TRPV5-expressing tubules were shown to highly express PMCA4. Therefore, PMCA4 may have a predominant role in renal Ca²⁺ handling. This study aimed to elucidate the role of PMCA4 in Ca²⁺ homeostasis by characterizing the Ca²⁺ balance, and renal and duodenal Ca²⁺-related gene expression in PMCA4 knockout mice. The daily water intake of PMCA4 knockout mice was significantly lower compared to wild type littermates. There was no significant difference in serum Ca²⁺ level or urinary Ca²⁺ excretion between groups. In addition, renal and duodenal mRNA expression levels of Ca²⁺-related genes, including TRPV5, TRPV6, calbindin-D_28k, calbindin-D_9k, NCX1 and PMCA1 were similar in wild type and knockout mice. Serum FGF23 levels were significantly increased in PMCA4 knockout mice. In conclusion, PMCA4 has no discernible role in normal renal Ca²⁺ handling as no urinary Ca²⁺ wasting was observed. Further investigation of the exact role of PMCA4 in the distal convoluted tubule and connecting tubule is required.     

Effects of Intracellular Calcium Chelation and Pifithrin-α on Deoxynucleotide Metabolism in Human Lymphocytes

Previously, we have found that activation of deoxycytidine kinase elicited by various DNA-damaging chemical agents could be prevented by BAPTA-AM, a cell-permeable calcium chelator or by pifithrin-α, a pharmacological inhibitor of p53. Here, we show that stimulation of deoxycytidine kinase by UV-light also is calcium-dependent and pifithrin-α-sensitive in tonsillar lymphocytes, while thymidine kinase 1 activity is stabilised in the presence of BAPTA-AM. Importantly, both UV-irradiation and calcium chelation decreased the incorporation of labelled deoxycytidine and thymidine into DNA. Pifithrin-alpha dramatically reduced the labelling of both the nucleotide and DNA fractions, possibly due to inhibition of transmembrane nucleoside transport.