Effects of 4-Br-A23187 on Bacillus subtilis cells and unilamellar vesicles reveal it to be a potent copper ionophore.

Ionophores are small molecules or peptides that transport metal ions across biological membranes. Their transport capabilities are typically characterized in vitro using vesicles and single ion species. It is difficult to infer from these data which effects ionophores have on living cells in a complex environment (e.g., culture medium), since net ion movement is influenced by many factors including ion composition of the medium, concentration gradients, pH gradient, and protein-mediated transport processes across the membrane. To gain insights into the antibacterial mechanism of action of the semisynthetic polyether ionophore 4-Br-A23187, known to efficiently transport zinc and manganese in vitro, we investigated its effects on the gram-positive model organism Bacillus subtilis. In addition to monitoring cellular ion concentrations, the physiological impact of treatment was assessed on the proteome level. 4-Br-A23187 treatment resulted in an increase in intracellular copper levels, the extent of which depended on the copper concentration of the medium. Effects of copper accumulation mirrored by the proteomic response included oxidative stress, disturbance of proteostasis, metal and sulfur homeostasis. The antibiotic effect of 4-Br-A23187 is further aggravated by a decrease in intracellular manganese and magnesium. A liposome model confirmed that 4-Br-A23187 acts as copper ionophore in vitro.

Cezomycin Is Activated by CalC to Its Ester Form for Further Biosynthesis Steps in the Production of Calcimycin in Streptomyces chartreusis NRRL 3882.

Calcimycin, N-demethyl calcimycin, and cezomycin are polyether divalent cation ionophore secondary metabolites produced by Streptomyces chartreusis A thorough understanding of the organization of their encoding genes, biosynthetic pathway(s), and cation specificities is vitally important for their efficient future production and therapeutic use. So far, this has been lacking, as has information concerning any biosynthetic relationships that may exist between calcimycin and cezomycin. In this study, we observed that when a Cal- (calB1 mutant) derivative of a calcimycin-producing strain of S. chartreusis (NRRL 3882) was grown on cezomycin, calcimycin production was restored. This suggested that calcimycin synthesis may have resulted from postsynthetic modification of cezomycin rather than from a de novo process through a novel and independent biosynthetic mechanism. Systematic screening of a number of Cal-S. chartreusis mutants lacking the ability to convert cezomycin to calcimycin allowed the identification of a gene, provisionally named calC, which was involved in the conversion step. Molecular cloning and heterologous expression of the CalC protein along with its purification to homogeneity and negative-staining electron microscopy allowed the determination of its apparent molecular weight, oligomeric forms in solution, and activity. These experiments allowed us to confirm that the protein possessed ATP pyrophosphatase activity and was capable of ligating coenzyme A (CoA) with cezomycin but not 3-hydroxyanthranilic acid. The CalC protein's apparent Km and kcat for cezomycin were observed to be 190 µM and 3.98 min-1, respectively, and it possessed the oligomeric form in solution. Our results unequivocally show that cezomycin is postsynthetically modified to calcimycin by the CalC protein through its activation of cezomycin to a CoA ester form.IMPORTANCE Calcimycin is a secondary metabolite divalent cation-ionophore that has been studied in the context of human health. However, detail is lacking with respect to both calcimycin's biosynthesis and its biochemical/biophysical properties as well as information regarding its, and its analogues', divalent cation binding specificities and other activities. Such knowledge would be useful in understanding how calcimycin and related compounds may be effective in modifying the calcium channel ion flux and might be useful in influencing the homeostasis of magnesium and manganese ions for the cure or control of human and bacterial infectious diseases. The results presented here unequivocally show that CalC protein is essential for the production of calcimycin, which is essentially a derivative of cezomycin, and allow us to propose a biosynthetic mechanism for calcimycin's production.

Recycling of Overactivated Acyls by a Type II Thioesterase during Calcimycin Biosynthesis in Streptomyces chartreusis NRRL 3882.

Type II thioesterases typically function as editing enzymes, removing acyl groups that have been misconjugated to acyl carrier proteins during polyketide secondary metabolite biosynthesis as a consequence of biosynthetic errors. Streptomyces chartreusis NRRL 3882 produces the pyrrole polyether ionophoric antibiotic, and we have identified the presence of a putative type II thioesterase-like sequence, calG, within the biosynthetic gene cluster involved in the antibiotic's synthesis. However, targeted gene mutagenesis experiments in which calG was inactivated in the organism did not lead to a decrease in calcimycin production but rather reduced the strain's production of its biosynthetic precursor, cezomycin. Results from in vitro activity assays of purified, recombinant CalG protein indicated that it was involved in the hydrolysis of cezomycin coenzyme A (cezomycin-CoA), as well as other acyl CoAs, but was not active toward 3-S-N-acetylcysteamine (SNAC; the mimic of the polyketide chain-releasing precursor). Further investigation of the enzyme's activity showed that it possessed a cezomycin-CoA hydrolysis Km of 0.67 mM and a kcat of 17.77 min-1 and was significantly inhibited by the presence of Mn2+ and Fe2+ divalent cations. Interestingly, when S. chartreusis NRRL 3882 was cultured in the presence of inorganic nitrite, NaNO2, it was observed that the production of calcimycin rather than cezomycin was promoted. Also, supplementation of S. chartreusis NRRL 3882 growth medium with the divalent cations Ca2+, Mg2+, Mn2+, and Fe2+ had a similar effect. Taken together, these observations suggest that CalG is not responsible for megasynthase polyketide precursor chain release during the synthesis of calcimycin or for retaining the catalytic efficiency of the megasynthase enzyme complex as is supposed to be the function for type II thioesterases. Rather, our results suggest that CalG is a dedicated thioesterase that prevents the accumulation of cezomycin-CoA when intracellular nitrogen is limited, an apparently new and previously unreported function of type II thioesterases.IMPORTANCE Type II thioesterases (TEIIs) are generally regarded as being responsible for removing aberrant acyl groups that block polyketide production, thereby maintaining the efficiency of the megasynthase involved in this class of secondary metabolites' biosynthesis. Specifically, this class of enzyme is believed to be involved in editing misprimed precursors, controlling initial units, providing key intermediates, and releasing final synthetic products in the biosynthesis of this class of secondary metabolites. Our results indicate that the putative TEII CalG present in the calcimycin (A23187)-producing organism Streptomyces chartreusis NRRL 3882 is not important either for the retention of catalytic efficiency of, or for the release of the product compound from, the megasynthase involved in calcimycin biosynthesis. Rather, the enzyme is involved in regulating/controlling the pool size of the calcimycin biosynthetic precursor, cezomycin, by hydrolysis of its CoA derivative. This novel function of CalG suggests a possible additional activity for enzymes belonging to the TEII protein family and promotes better understanding of the overall biosynthetic mechanisms involved in the production of this class of secondary metabolites.

Characterization of Microvesicles Released from Human Red Blood Cells.

BACKGROUND/AIMS: Extracellular vesicles (EVs) are spherical fragments of cell membrane released from various cell types under physiological as well as pathological conditions. Based on their size and origin, EVs are classified as exosome, microvesicles (MVs) and apoptotic bodies. Recently, the release of MVs from human red blood cells (RBCs) under different conditions has been reported. MVs are released by outward budding and fission of the plasma membrane. However, the outward budding process itself, the release of MVs and the physical properties of these MVs have not been well investigated. The aim of this study is to investigate the formation process, isolation and characterization of MVs released from RBCs under conditions of stimulating Ca2+ uptake and activation of protein kinase C. METHODS: Experiments were performed based on single cell fluorescence imaging, fluorescence activated cell sorter/flow cytometer (FACS), scanning electron microscopy (SEM), atomic force microscopy (AFM) and dynamic light scattering (DLS). The released MVs were collected by differential centrifugation and characterized in both their size and zeta potential. RESULTS: Treatment of RBCs with 4-bromo-A23187 (positive control), lysophosphatidic acid (LPA), or phorbol-12 myristate-13 acetate (PMA) in the presence of 2 mM extracellular Ca2+ led to an alteration of cell volume and cell morphology. In stimulated RBCs, exposure of phosphatidylserine (PS) and formation of MVs were observed by using annexin V-FITC. The shedding of MVs was also observed in the case of PMA treatment in the absence of Ca2+, especially under the transmitted bright field illumination. By using SEM, AFM and DLS the morphology and size of stimulated RBCs, MVs were characterized. The sizes of the two populations of MVs were 205.8 ± 51.4 nm and 125.6 ± 31.4 nm, respectively. Adhesion of stimulated RBCs and MVs was observed. The zeta potential of MVs was determined in the range from - 40 mV to - 10 mV depended on the solutions and buffers used. CONCLUSION: An increase of intracellular Ca2+ or an activation of protein kinase C leads to the formation and release of MVs in human RBCs.

NHERF2 protein mobility rate is determined by a unique C-terminal domain that is also necessary for its regulation of NHE3 protein in OK cells.

Na(+)/H(+) exchanger regulatory factor (NHERF) proteins are a family of PSD-95/Discs-large/ZO-1 (PDZ)-scaffolding proteins, three of which (NHERFs 1-3) are localized to the brush border in kidney and intestinal epithelial cells. All NHERF proteins are involved in anchoring membrane proteins that contain PDZ recognition motifs to form multiprotein signaling complexes. In contrast to their predicted immobility, NHERF1, NHERF2, and NHERF3 were all shown by fluorescence recovery after photobleaching/confocal microscopy to be surprisingly mobile in the microvilli of the renal proximal tubule OK cell line. Their diffusion coefficients, although different among the three, were all of the same magnitude as that of the transmembrane proteins, suggesting they are all anchored in the microvilli but to different extents. NHERF3 moves faster than NHERF1, and NHERF2 moves the slowest. Several chimeras and mutants of NHERF1 and NHERF2 were made to determine which part of NHERF2 confers the slower mobility rate. Surprisingly, the slower mobility rate of NHERF2 was determined by a unique C-terminal domain, which includes a nonconserved region along with the ezrin, radixin, moesin (ERM) binding domain. Also, this C-terminal domain of NHERF2 determined its greater detergent insolubility and was necessary for the formation of larger multiprotein NHERF2 complexes. In addition, this NHERF2 domain was functionally significant in NHE3 regulation, being necessary for stimulation by lysophosphatidic acid of activity and increased mobility of NHE3, as well as necessary for inhibition of NHE3 activity by calcium ionophore 4-Br-A23187. Thus, multiple functions of NHERF2 require involvement of an additional domain in this protein.

Precezomycin, a novel antibiotic biosynthetic precursor of cezomycin, from actinomycete Kitasatospora putterlickiae 10-13.

A novel antibiotic biosynthetic precursor of cezomycin, named precezomycin (1), was isolated from culture broth of actinomycete Kitasatospora putterlickiae 10-13. The planar structure was determined by 1D/2D NMR and HR(ESI)MS data analyses, and the absolute configurations were established by TDDFT calculation of ECD spectra. Precezomycin (1) exhibited moderate antibacterial activity against gram-positive bacteria including Staphylococcus aureus and Bacillus subtilis. The discovery of 1 extends the natural product family of cezomycin and provides a new insight into understanding the biosynthetic process of these polyether ionophore metabolites.

Regulation of phosphatidylserine exposure in red blood cells.

The exposure of phosphatidylserine (PS) on the outer membrane leaflet of red blood cells (RBCs) serves as a signal for eryptosis, a mechanism for the RBC clearance from blood circulation. The process of PS exposure was investigated as function of the intracellular Ca(2+) content and the activation of PKCα in human and sheep RBCs. Cells were treated with lysophosphatidic acid (LPA), 4-bromo-A23187, or phorbol-12 myristate-13 acetate (PMA) and analysed by flow cytometry, single cell fluorescence video imaging, or confocal microscopy. For human RBCs, no clear correlation existed between the number of cells with an elevated Ca(2+) content and PS exposure. Results are explained by three different mechanisms responsible for the PS exposure in human RBCs: (i) Ca(2+)-stimulated scramblase activation (and flippase inhibition) by LPA, 4-bromo-A23187, and PMA; (ii) PKC activation by LPA and PMA; and (iii) enhanced lipid flop caused by LPA. In sheep RBCs, only the latter mechanism occurs suggesting absence of scramblase activity.

Presynaptic ryanodine receptor-activated calmodulin kinase II increases vesicle mobility and potentiates neuropeptide release.

Although it has been postulated that vesicle mobility is increased to enhance release of transmitters and neuropeptides, the mechanism responsible for increasing vesicle motion in nerve terminals and the effect of perturbing this mobilization on synaptic plasticity are unknown. Here, green fluorescent protein-tagged dense-core vesicles (DCVs) are imaged in Drosophila motor neuron terminals, where DCV mobility is increased for minutes after seconds of activity. Ca2+-induced Ca2+ release from presynaptic endoplasmic reticulum (ER) is shown to be necessary and sufficient for sustained DCV mobilization. However, this ryanodine receptor (RyR)-mediated effect is short-lived and only initiates signaling. Calmodulin kinase II (CaMKII), which is not activated directly by external Ca2+ influx, then acts as a downstream effector of released ER Ca2+. RyR and CaMKII are essential for post-tetanic potentiation of neuropeptide secretion. Therefore, the presynaptic signaling pathway for increasing DCV mobility is identified and shown to be required for synaptic plasticity.

ATP2C1 is specifically localized in the basal layer of normal epidermis and its depletion triggers keratinocyte differentiation.

BACKGROUND: ATP2C1 is a calcium/manganese-ATPase localized in the Golgi apparatus and known as responsible gene for Hailey-Hailey disease. But its localization and roles in the epidermis are not fully elucidated. OBJECTIVE: To explore the localization and biological role of ATP2C1 in normal epidermis in terms of differentiation states. METHODS: We examined the immunohistochemical distribution of ATP2C1 in normal epidermis and measured the expression of ATP2C1 in cultured keratinocytes following forced detachment from culture dish or following treatment with high concentrations of calcium. Furthermore, we knockdown ATP2C1 expression in cultured keratinocytes by using RNA interference procedure to abrogate cation accumulation in cell organelles. RESULTS: ATP2C1 is specifically localized at the basal cell layer in normal epidermis. Neither detachment of keratinocyte from culture dish nor treatment with high concentrations of calcium suppressed ATP2C1 expression, while both procedures induced differentiation markers, K10 keratin and involucrin. In contrast, knockdown of ATP2C1 induced these differentiation markers of cultured keratinocytes. Furthermore, treatment of keratinocytes with a calcium ionophore, A23187, did not up-regulate differentiation markers of keratinocytes, while a more manganese selective ionophore Br-A23187 up-regulated these differentiation markers. CONCLUSION: Our results suggest that ATP2C1 plays an essential role for basal keratinocytes to keep in the undifferentiated state and that its reduction evokes differentiation and up-localization to suprabasal layers most likely via the manganese starvation in the Golgi apparatus of keratinocytes.

Tubulovascular nitric oxide crosstalk: buffering of angiotensin II-induced medullary vasoconstriction.

Studies were designed to determine the source of NO responsible for buffering of the angiotensin II (Ang II)-mediated decrease of blood flow in the renal medulla. Intracellular Ca2+ concentration ([Ca2+]i) and NO production ([NO]i) of pericytes and endothelium of the vasa recta were independently measured with the use of fura 2-AM and 4,5-diaminofluorescein diacetate (DAF-2DA), respectively, in microtissue strips of the vascular bundles of the outer medullary vasa recta. Disruption of the endothelium of the vasa recta by perfusion with latex microspheres enabled imaging of the pericytes. Ang II (1 micromol/L) produced an increase of [NO]i of 19+/-6 U in pericytes of the vasa recta when the vessels were adjacent to medullary thick ascending limbs (mTALs). Pericytes of isolated vasa recta without surrounding mTALs showed a rapid peak increase in [Ca2+]i of 248+/-107 nmol/L, with a sustained elevation of 107+/-75 nmol/L, but did not show an increase in [NO]i to either Ang II (1 micromol/L) or the Ca2+ ionophore 4-bromo-A23187 (5 micromol/L). These observations indicated the lack of Ang II and Ca2+-sensitive NO production in pericytes of the vasa recta. In isolated vasa recta with intact endothelium, Ang II reduced [Ca2+]i from 128+/-28 to 62+/-13 nmol/L and failed to increase [NO]i. However, the Ca2+ ionophore did increase [NO]i in the endothelium (47+/-8 U), indicating the presence of Ca2+-sensitive NO production. Significant increases of [NO]i were observed in single isolated mTALs in response to both Ang II (33+/-6 U) and the Ca2+ ionophore (51+/-18 U). We conclude that Ang II increases [Ca2+]i in pericytes of the descending vasa recta as part of its constrictor action and that this vasoconstriction is buffered by the NO from the surrounding tubular elements, such as mTALs.

In situ characterization of nonmitochondrial Ca2+ stores in individual pancreatic beta-cells.

Free Ca2+ was measured in intracellular stores of individual mouse pancreatic beta-cells using dual-wavelength microfluorometry and the low-affinity Ca2+ indicator furaptra. Controlled permeabilization of the plasma membrane with 4 micromol/l digitonin revealed that 22% of the furaptra was trapped in intracellular nonnuclear compartments. When 3 mmol/l ATP and 200 nmol/l Ca2+ were simultaneously present, this cation rapidly accumulated in the organelle pool, reaching an average concentration of 200-500 micromol/l. Whereas agents affecting the mitochondrial function (5 mmol/l succinate, 2 micromol/l ruthenium red, or 10 micromol/l antimycin A + 2 microg/ml oligomycin) had little effects, the Ca2+-ATPase inhibitor thapsigargin released 92% of the Ca2+ mobilizable with the ionophore Br-A23187. Digital imaging revealed regional differences in the organelle Ca2+. The regions with the highest Ca2+ concentration were particularly responsive to inositol 1,4,5-trisphosphate (IP3). IP3 mobilized Ca2+ in a dose-dependent way with half-maximal and maximal effects at about 1 and 5 micromol/l, respectively. High concentrations of IP3 released about half of the thapsigargin-sensitive Ca2+, but there were no responses to agents known to activate ryanodine receptors, such as 10 mmol/l caffeine, 0.1-1 micromol/l ryanodine, or 1-5 micromol/l cyclic ADP ribose. The results reinforce the concept that mobilization of intracellular Ca2+ in the pancreatic beta-cell is mediated by IP3 receptors rather than ryanodine receptors.

A Novel Allyl Transfer Coupled with a Grob Fragmentation.

A novel acid-promoted rearrangement is disclosed. In the previously unknown transformation, an allyl group migrated to an in situ formed carbocation stabilized by an electron-rich aryl or heteroaryl group, resulting in a stereoselective intramolecular Grob fragmentation. The outcome of the rearrangement observed with an array of substrates can be satisfactorily rationalized using a working hypothesis with the aid of a six-membered transition state similar to those proposed for the anionic oxy-Cope or oxonia-Cope rearrangements, but involving only one instead of two double bonds.

Characteristics of intracellular calcium changes required for augmentation of phorbol ester-stimulated pancreatic enzyme secretion.

Previous studies demonstrated that Ca2+ ionophores augment the pancreatic enzyme secretion caused by phorbol esters. The present study was performed to determine the nature of the cellular Ca2+ effects responsible for the augmentation. Relatively low concentrations (0.3-1.0 microM) of the nonfluorescent Ca2+ ionophore, 4-bromo-A23187 (Br-A23187), did not measurably increase free cytosolic Ca2+ ([Ca2+]i) and caused little or no enzyme release from guinea pig pancreatic acini. However, these concentrations of Br-A23187 augmented the amylase release caused by the phorbol ester, 4 beta-phorbol 12-myristate 13-acetate (PMA). This augmentation occurred in the absence of extracellular Ca2+ as long as the intracellular agonist-sensitive pool contained Ca2+. Greater concentrations of Br-A23187 (3-10 microM) alone caused transient increases in [Ca2+]i and transient increases in amylase release. Although not resulting in an increase in [Ca2+]i, the low concentrations of Br-A23187 caused release of Ca2+ from the intracellular agonist-sensitive pool. These results suggest that Ca2+ mediates enzyme release by two distinct mechanisms in the pancreatic acinar cell. First, an increase in [Ca2+]i alone mediates enzyme release. Second, Ca2+ release from the agonist-sensitive pool not resulting in a measurable increase in [Ca2+]i augments enzyme release stimulated by a phorbol ester. The second effect of Ca2+ may be due to a small localized change in cell Ca2+ or an induction of cytosolic Ca2+ oscillations.

Spatial gradients of cytosolic calcium concentration in neurones during paradoxical activation by calcium.

4-Br-A23187 caused a calcium influx into chick sensory neurones and raised cytosolic calcium from a rest level of 97 +/- 7 nM to a peak of 296 +/- 30 nM. Despite the continued presence of ionophore, however, cytosolic calcium concentrations then fell. After 30 min in ionophore, cytosolic calcium concentration had returned to 105 +/- 5 nM, not significantly different from the value before ionophore addition. The permeability of the plasmalemma to divalent cations, as estimated by the manganese quench technique, was no lower at 30 min than at the peak of the cytosolic calcium transient. Thus the fall of calcium from its peak was not due to a slowing of calcium influx, but was due to an upregulation of mechanisms that remove calcium from the cytosol- an upregulation that persists even though cytosolic calcium has apparently returned to pre-stimulus levels. We used a novel fixed slit confocal microscope to examine the calcium concentration profile close to the plasmalemma. We found that after 25-30 min ionophore treatment, calcium concentration was elevated only in the cytoplasm within 1 micron of the plasmalemma. A maintained, elevated calcium under the plasmalemma can help explain the phenomenon of paradoxical activation seen in this and other cell types.

Biochemical, biophysical and haemorheological effects of dimethylsulphoxide on human erythrocyte calcium loading.

The studies using dimethylsulphoxide (DMSO) and/or the 4-bromo-calcium ionophore A23187 (Br-A23187) often neglect the precise knowledge of some of their biochemical, biophysical and haemorheological effects. The aim of the present study was to evaluate these effects on erythrocytes after whole blood incubations with DMSO or Br-A23187 dissolved in DMSO. There were no significant differences between the different aliquots in the values of P(50), pH, erythrocyte deformability, erythrocyte membrane fluidity, haemoglobin and intracellular Ca(2+) concentrations ([Ca(2+)](i)). Aliquots with DMSO (independently of the presence of Br-A23187 or added Ca(2+)) had lower erythrocyte aggregation indexes and higher plasma concentrations of K(+)], Na(+)] and Ca(2+) than the aliquots without DMSO (independently of the presence of added Ca(2+)). Aliquots with added calcium (without the presence of Br-A23187 in DMSO) had a significantly higher erythrocyte acetylcholinesterase activity. Our data shows that calcium loading, the usual objective of Br-A23187 incubations, cannot be fulfilled with the studied experimental conditions. The coherence between our results and those obtained by other authors with different biological systems and different modulators of the rise on [Ca(2+)](i) suggests a non-specific effect of DMSO, disabling the action of the modulator. It can be reasoned that the decreased erythrocyte aggregation (without significant changes on the deformability or membrane fluidity) can result either from the decrease of the hydrogen bonding contribution to erythrocyte aggregation or the increased ionic strength influence on the erythrocyte membrane surface.

Calcium buffering and protection from excitotoxic cell death by exogenous calbindin-D28k in HEK 293 cells.

Calbindin-D28k (CaBP) is a calcium-binding protein found in specific neuronal populations in the mammalian brain that, as a result of its proposed calcium-buffering action, may protect neurons against potentially harmful increases in intracellular calcium. We have stably transfected HEK 293 cells with recombinant human CaBP in order to determine the influence of this protein upon transient increases in intracellular ionic calcium concentration ([Ca(2+)](i)) induced either by transient transfection of the NR1 and NR2A subunits of the N-methyl-D-aspartate (NMDA) receptor and brief exposure to glutamate, photolysis of the caged calcium compound NP-EGTA, or exposure to the Ca(2+)]-ionophore 4-Br-A23187. The presence of CaBP did not significantly reduce the peak [Ca(2+)](i)stimulated by glutamate activation of NMDA receptors but significantly prolonged the recovery to baseline values. Flash photolysis of NP-EGTA in control cells resulted in an almost instantaneous increase in [Ca(2+)](i)followed by a bi-exponential recovery to baseline values. In cells stably expressing CaBP, the peak [Ca(2+)](i)levels were not statistically different from the controls, however, there was a significant prolongation of the initial portion of the slow recovery phase. In cells exposed to 4-Br-A23187, the presence of CaBP significantly reduced the rate of rise of [Ca(2+)](i), reduced the peak response, slowed the rate of recovery, and reduced the depolarization of mitochondria. In studies of delayed, Ca(2+)]-dependent cell death, CaBP transfected cells exhibited enhanced survival 24h after a 1-h exposure to 200 microM NMDA. However, necrotic cell death observed after the first 6h was not prevented by the presence of CaBP. These results provide direct evidence for a Ca(2+)-buffering effect of CaBP which serves to limit Ca(2+)entry and the depolarization of mitochondria, thereby protecting cells from death mediated most likely by apoptosis.

Use of co-loaded Fluo-3 and Fura Red fluorescent indicators for studying the cytosolic Ca(2+)concentrations distribution in living plant tissue.

A method for visualisation of cytosolic [Ca(2+)] distribution was applied to living plant tissue. A mixture of the fluorescent probes Fluo-3 and Fura Red was used. The emitted fluorescence was scanned simultaneously in two channels with a laser-scanning confocal microscope and rationing was performed. The homogeneity of the Fluo-3/Fura Red concentration ratio throughout the tissue after AM-ester loading was proven. In vitro calibration permitted conversion of Fluo-3/Fura Red fluorescence ratios to [Ca(2+)] values. Apparent K(D)of 286 nM, R(min)of 0.43 and R(max)of 18 were calculated. The in vivo determination of extreme ratio values was performed by permeabilizing the plasmalemma for Ca(2+)with a ionophore and manipulating the extracellular [Ca(2+)]. The resultant R(minv)of 1.33 and R(maxv)of 2.69 for vegetative apices, and R(mini)of 1.26 and R(maxi)of 3.45 for apices induced to flowering, suggested incomplete equalization of extra- and intracellular Ca(2+)levels in these experiments. In Chenopodium rubrum, the cytosolic [Ca(2+)] patterns of apical tissue obtained using Fluo-3 and Fura Red were significantly different between vegetative apices and apices after photoperiodic flower induction. This methodological approach may also be helpful for studying cytosolic [Ca(2+)] distribution in other living plant tissues.

Monitoring neuronal calcium signalling using a new method for ratiometric confocal calcium imaging.

Ca2+ signalling influences many processes in the adult and developing nervous system like exocytosis, synaptic plasticity, and growth cone motility. Optical techniques in combination with fluorescent Ca2+ indicators are the most frequently used methods to measure Ca2+ signalling in cells. In the present study, a new method for ratiometric confocal Ca2+ imaging was developed, and the usefulness of the system was tested with two different neuronal preparations. Developing Manduca sexta antennal lobe neurons were loaded with the Ca2+-sensitive dye Fura Red-AM, and the ratio of fluorescence excited at 457 and 488nm was measured with a confocal laser scanning microscope. During pupal stages 4-12, the antennal lobe neuropil is restructured which includes the ingrowth of olfactory receptor axons, dendritic outgrowth of antennal lobe neurons, and synaptogenesis. In antennal lobe neurons, application of the AChR agonist carbachol induced Ca2+ oscillations the amplitude and frequency of which changed during stages 4-9, while at the end of synaptogenesis, at stages 11 and 12, only single Ca2+ transients were elicited. The Ca2+ oscillations were blocked by D-tubocurarine and Cd2+, indicating that they were due to Ca2+ influx through voltage-gated Ca2+ channels, activated by nAChR-mediated membrane depolarization. To test whether single action potentials can induce Ca2+ transients detectable by Fura Red, individual leech Retzius neurons were injected iontophoretically with the Ca2+ indicator, and the membrane potential was recorded during Ca2+ imaging. Single action potentials induced transient increases in the Fura Red ratio measured in the axon, while trains of action potentials elicited Ca2+ transients that could also be recorded in the cell body and the nucleus. The results show that Fura Red can be used as a ratiometric Ca2+ indicator for confocal imaging.

Mobilization of Ca2+ stores in individual pancreatic beta-cells permeabilized or not with digitonin or alpha-toxin.

The concentration of free Ca2+ in the cytoplasm and organelles of individual mouse pancreatic beta-cells was estimated with dual wavelength microfluorometry and the indicators Fura-2 and furaptra. Measuring the increase of cytoplasmic Ca2+ resulting from intracellular mobilization of the ion in ob/ob mouse beta-cells, most organelle calcium (92%) was found in acidic compartments released when combining the Ca2+ ionophore Br-A23187 with a protonophore. Only 3-4% of organelle calcium was recovered from a pool sensitive to the Ca(2+)-ATPase inhibitor thapsigargin. Organelle Ca2+ was also measured directly in furaptra-loaded beta-cells after controlled plasma membrane permeabilization. The permeabilizing agent alpha-toxin was superior to digitonin in preserving the integrity of intracellular membranes, but digitonin provided more reproducible access to intracellular sites. After permeabilization, the thapsigargin-sensitive fraction of Ca2+ detected by furaptra was as high as 90%, suggesting that the indicator essentially measures Ca2+ in endoplasmic reticulum (ER). Both alpha-toxin- and digitonin-permeabilized cells exhibited ATP-dependent uptake of Ca2+ into thapsigargin-sensitive stores with half-maximal and maximal filling at 6-11 microM and 1 mM ATP respectively. Most of the thapsigargin-sensitive Ca2+ was mobilized by inositol 1,4,5-trisphosphate (IP3), whereas caffeine, ryanodine, cyclic ADP ribose and nicotinic acid adenine dinucleotide phosphate lacked effects both in beta-cells from ob/ob mice and normal NMRI mice. Mobilization of organelle Ca2+ by 4-chloro-3-methylphenol was attributed to interference with the integrity of the ER rather than to activation of ryanodine receptors. The observations emphasize the importance of IP3 for Ca2+ mobilization in pancreatic beta-cells, but question a role for ryanodine receptor agonists.

Actions of ionomycin, 4-BrA23187 and a novel electrogenic Ca2+ ionophore on mitochondria in intact cells.

We have used fluorescence digital imaging techniques to explore the actions of two groups of Ca(2+) ionophores: (i). ferutinin, an electrogenic naturally occurring ionophore, and (ii). the neutral ionophores 4-BrA23187 and ionomycin, on cytosolic [Ca(2+)] ([Ca(2+)](c)), mitochondrial [Ca(2+)] ([Ca(2+)](m)) and mitochondrial membrane potential (deltapsi(m)) in HepG2 cells and primary hippocampal neurones in culture. 4-BrA23187 and ionomycin promoted the equilibration of [Ca(2+)] gradients between cellular compartments, including ER, mitochondria and cytosol. Thus, [Ca(2+)](c) and [Ca(2+)](m) increased together and then recovered in parallel on removal of the ionophore. In contrast, following a rise in [Ca(2+)](c) in response to ferutinin, [Ca(2+)](m) remained elevated for prolonged periods after the recovery of [Ca(2+)](c) levels despite washout of the compound. Both groups of Ca(2+) ionophores caused some mitochondrial depolarisation, although this was highly variable in degree. Mitochondrial depolarisation induced by ionomycin and 4-BrA23187 was often modest, independent of cyclosporin A (CsA), was suppressed in the absence of extracellular Ca(2+) and was enhanced by pre-incubation of cells with the inhibitor of the mitochondrial Ca(2+)/2Na(+)-exchanger, CGP37157, suggesting that the change in potential reflects the prior state of mitochondrial calcium loading. The mitochondrial depolarisation induced by ferutinin was not influenced by CGP37157 but was completely blocked by CsA, suggesting that it reflects opening of the mitochondrial permeability transition pore (mPTP). We suggest that ferutinin may provide a very valuable tool to promote mitochondrial calcium overload experimentally and to promote calcium-dependent opening of the mPTP.