Calcium Green-5N Research Articles

Analyzing Mitochondrial Calcium Influx in Isolated Mitochondria.

Mitochondria play a crucial role in Ca2+ signaling and homeostasis and can contribute to shaping the cytosolic Ca2+ landscape as well as regulate a variety of pathways including energy production and cell death. Dysregulation of mitochondrial Ca2+ homeostasis promotes pathologies including neurodegenerative diseases, cardiovascular disorders, and metabolic syndromes. The significance of mitochondria to Ca2+ signaling and regulation underscores the value of methods to assess mitochondrial Ca2+ import. Here we present a plate reader-based method using the Ca2+-sensitive fluorescent probe calcium green-5N to measure mitochondrial Ca2+ import in isolated cardiac mitochondria. This technique can be expanded to measure Ca2+ uptake in mitochondria isolated from other tissue types and from cultured cells.

Ca2+ concentrations in mouse sperm mitochondria fluctuate according to the cytosol.

Mitochondria are important organelles in eukaryotic cells and play an essential role in energy production and cell signaling. However, the importance of mammalian sperm mitochondria as an energy source remains to be elucidated because glycolysis is known to be dominant. In this context, one of the functions of mammalian sperm mitochondria is considered as a calcium ion (Ca2+) homeostasis. Previously, the Ca2+ level within the mitochondria of mouse sperm under resting conditions was reported to be high (in the micromolar range) using the fluorescent Ca2+ indicator Calcium Green-5N (CG-5N). To confirm this fact, we performed the semi-quantitative determination of Ca2+ concentration with several Ca2+ indicators. Although we reproduced the previous report of CG-5N, other Ca2+ indicators do not support the result obtained with CG-5N. The results obtained with Rhod-2, Fluo-3, and Fluo-5N indicate that the free Ca2+ concentration in mitochondria is comparable to that of the cytosol at the resting condition and under the condition stimulated by ATP. Although we still do not understand why CG-5N exhibits a distinct result from other indicators, the regulation of Ca2+ concentration in murine sperm mitochondria is analogous to that observed in somatic cells. Namely, the Ca2+ concentrations within sperm mitochondria fluctuate in response to changes in cytosolic Ca2+ levels. Our results contribute to a revised understanding of the role of mitochondria in Ca2+ homeostasis in mammalian sperm.

Comparative analysis of mitochondrial CRC in permeabilized cells and isolated cell mitochondria

Mitochondria undergo permeability transition (PT) resulting in the opening of the non‐selective PT pores (PTPs) in the inner mitochondrial membrane in response to energy and oxidative stresses associated with Ca2+ overload and ROS accumulation. The mitochondrial PTPs are permeable to ions and solutes with a molecular mass <1.5 kD that increases the colloidal osmotic pressure in the matrix leading to mitochondrial swelling. Calcium retention capacity (CRC) reflects the maximum amount of Ca2+ mitochondria can uptake to provoke the PTP opening. Quantification of CRC is important to study the effects of various pathological stimuli and the efficacy of pharmacological agents on the metabolism and function of mitochondria. Here, we performed a comparative analysis of CRC in mitochondria isolated from H9c2 cardioblasts, and in permeabilized H9c2 cells in situ to highlight the advantages/disadvantages of the fluorescent technique in isolated mitochondria vs. permeabilized cells. The cells were permeabilized using digitonin or saponin, and the CRC was assessed using the Ca2+‐sensitive fluorescence probe Calcium Green‐5N. Results demonstrated the interference of dye‐associated fluorescence signals with saponin and the adverse effects of digitonin on mitochondria at high concentrations. The CRC of saponin‐permeabilized cells was higher than the CRC of digitonin‐permeabilized cells. In addition, the mitochondrial CRC of saponin‐permeabilized cells was higher than isolated mitochondria using the same number of cells. In conclusion, this study demonstrates that the fluorescent technique for CRC analysis in saponin‐permeabilized cells has more advantages than isolated mitochondria.

Analysis of Mitochondrial Calcium Retention Capacity in Cultured Cells: Permeabilized Cells Versus Isolated Mitochondria.

In response to various pathological stimuli, such as oxidative and energy stress accompanied by high Ca2+, mitochondria undergo permeability transition (PT) leading to the opening of the non-selective PT pores (PTP) in the inner mitochondrial membrane. Opening of the pores at high conductance allows the passage of ions and solutes <1.5 kD across the membrane, that increases colloid osmotic pressure in the matrix leading to excessive mitochondrial swelling. Calcium retention capacity (CRC) reflects maximum Ca2+ overload of mitochondria that occurs just before PTP opening. Quantification of CRC is important for elucidating the effects of different pathological stimuli and the efficacy of pharmacological agents on the mitochondria. Here, we performed a comparative analysis of CRC in mitochondria isolated from H9c2 cardioblasts, and in permeabilized H9c2 cells in situ to highlight the strengths and weaknesses of the CRC technique in isolated cell mitochondria vs. permeabilized cells. The cells were permeabilized by digitonin or saponin, and the Ca2+-sensitive fluorescence probe Calcium Green-5N was used in both preparations. Results demonstrated the interference of dye-associated fluorescence signals with saponin and the adverse effects of digitonin on mitochondria at high concentrations. Analysis of the CRC in permeabilized cells revealed a higher CRC in the saponin-permeabilized cells in comparison with the digitonin-permeabilized cells. In addition, the mitochondrial CRC in saponin-permeabilized cells was higher than in isolated mitochondria. Altogether, these data demonstrate that the quantification of the mitochondrial CRC in cultured cells permeabilized by saponin has more advantages compared to the isolated mitochondria.

Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells.

Ca2+ handling by mitochondria is a critical function regulating both physiological and pathophysiological processes in a broad spectrum of cells. The ability to accurately measure the influx and efflux of Ca2+ from mitochondria is important for determining the role of mitochondrial Ca2+ handling in these processes. In this report, we present two methods for the measurement of mitochondrial Ca2+ handling in both isolated mitochondria and cultured cells. We first detail a plate reader-based platform for measuring mitochondrial Ca2+ uptake using the Ca2+ sensitive dye calcium green-5N. The plate reader-based format circumvents the need for specialized equipment, and the calcium green-5N dye is ideally suited for measuring Ca2+ from isolated tissue mitochondria. For our application, we describe the measurement of mitochondrial Ca2+ uptake in mitochondria isolated from mouse heart tissue; however, this procedure can be applied to measure mitochondrial Ca2+ uptake in mitochondria isolated from other tissues such as liver, skeletal muscle, and brain. Secondly, we describe a confocal microscopy-based assay for measurement of mitochondrial Ca2+ in permeabilized cells using the Ca2+ sensitive dye Rhod-2/AM and imaging using 2-dimensional laser-scanning microscopy. This permeabilization protocol eliminates cytosolic dye contamination, allowing for specific recording of changes in mitochondrial Ca2+. Moreover, laser-scanning microscopy allows for high frame rates to capture rapid changes in mitochondrial Ca2+ in response to various drugs or reagents applied in the external solution. This protocol can be applied to measure mitochondrial Ca2+ uptake in many cell types including primary cells such as cardiac myocytes and neurons, and immortalized cell lines.

In situ fiber‐optical monitoring of cytosolic calcium in tissue explant cultures

We present a fluorescence‐lifetime based method for monitoring cell and tissue activity in situ, during cell culturing and in the presence of a strong autofluorescence background. The miniature fiber‐optic probes are easily incorporated in the tight space of a cell culture chamber or in an endoscope. As a first application we monitored the cytosolic calcium levels in porcine tracheal explant cultures using the Calcium Green‐5N (CG5N) indicator. Despite the simplicity of the optical setup we are able to detect changes of calcium concentration as small as 2.5 nM, with a monitoring time resolution of less than 1 s. (© 2013 WILEY‐VCH Verlag GmbH &amp;Co. KGaA, Weinheim)

Effects of all-trans-retinoic acid on the permeability transition and bioenergetic functions of rat liver mitochondria in combination with endoxifen

AimsThe clinical utilization of the combinations of all-trans-retinoic acid (RA) with antiestrogens, which present synergism of action in breast cancer, has been limited by RA adverse effects, including hepatotoxicity, which may be related with mitochondrial damage. This work evaluated the effects of RA alone and in combination with the antiestrogen endoxifen (EDX) on liver mitochondria. Main methodsMitochondrial permeability transition (MPT) was assessed by using Calcium Green-5N fluorescence and a tetraphenylphosphonium selective electrode. Oxidative stress was evaluated by oxygen consumption and thiobarbituric acid method. Mitochondrial bioenergetic was monitored by measuring oxygen consumption and mitochondrial membrane potential (ΔΨ). Osmotic volume changes of mitochondria were followed at 540nm. Key findingsEDX prevents the MPT induced by RA, allowing mitochondria pre-incubated with RA to accumulate Ca2+ and inhibiting the depolarization of ΔΨ. RA above 10nmol/mg protein depresses the phosphorylation capacity of mitochondria, as shown by the increase in the time required for ADP phosphorylation as well as by the decrease in state 3 respiration. At 20nmol/mg protein, RA decreases the ΔΨ and increases the state 4 respiration, suggesting that high concentrations of RA permeabilize the membrane to protons, possibly due to a proton leak through the Fo fraction of complex V. Moreover, the effects of RA on mitochondrial bioenergetics are not changed by EDX. SignificanceRA-induced hepatotoxicity may be related with induction of MPT and alterations in bioenergetic parameters; the combination with EDX, which reduces mitochondrial dysfunction and synergistically potentiates the anticancer activity, may provide a safer therapeutic strategy.

Regulation of Mitochondrial Ca2+ Dynamics by Inorganic Phosphate

The mitochondrial Ca2+ uniporter (mCU) is the main influx pathway for the uptake of mitochondria Ca2+ and plays an important role in regulating energy production as well as cell life and death. In our previous study, we reported two modes of the mCU (mode1 and mode 2) that have different properties with respect to Ca2+ affinity (mode1 > mode 2) and Ru360 sensitivity (mode 1 < mode 2). This study further investigates the role of concomitant inorganic phosphate (Pi) transport on mitochondrial uptake of Ca2+ mediated by the mCU in isolated cardiac mitochondria loaded with mitochondrial and extra-mitochondrial Ca2+ fluorescent dyes (Fura-FF and Calcium Green-5N). In Pi-depleted mitochondria, the maximal Ca2+ uptake rate is shown here to be limited by extramitochondrial Pi. The mitochondrial Ca2+ uptake rate was accelerated in a concentration-dependent manner by phosphate concentrations ranging from 0.01 mM to a maximum at ∼0.1 mM. The effect of Pi on mode 2 Ca2+ uptake was largely inhibited in the presence of the mitochondrial phosphate carrier inhibitor N-ethylmaleimide; however, mode 1 uptake was still observed, i.e., bulk Ca2+ uptake through mCU mode 2 was more Pi- dependent than mode 1. These experiments demonstrate another distinction between mCU modes 1 and 2 and contribute to an understanding of their possible physiological roles in mitochondrial function as either a signal for regulating energetics or as a Ca2+ sink.

AMPK Activation Stimulates Autophagy and Ameliorates Muscular Dystrophy in the mdx Mouse Diaphragm

Duchenne muscular dystrophy (DMD) is characterized by myofiber death from apoptosis or necrosis, leading in many patients to fatal respiratory muscle weakness. Among other pathological features, DMD muscles show severely deranged metabolic gene regulation and mitochondrial dysfunction. Defective mitochondria not only cause energetic deficiency, but also play roles in promoting myofiber atrophy and injury via opening of the mitochondrial permeability transition pore. Autophagy is a bulk degradative mechanism that serves to augment energy production and eliminate defective mitochondria (mitophagy). We hypothesized that pharmacological activation of AMP-activated protein kinase (AMPK), a master metabolic sensor in cells and on-switch for the autophagy-mitophagy pathway, would be beneficial in the mdx mouse model of DMD. Treatment of mdx mice for 4 weeks with an established AMPK agonist, AICAR (5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside), potently triggered autophagy in the mdx diaphragm without inducing muscle fiber atrophy. In AICAR-treated mdx mice, the exaggerated sensitivity of mdx diaphragm mitochondria to calcium-induced permeability transition pore opening was restored to normal levels. There were associated improvements in mdx diaphragm histopathology and in maximal force-generating capacity, which were not linked to increased mitochondrial biogenesis or up-regulated utrophin expression. These findings suggest that agonists of AMPK and other inducers of the autophagy-mitophagy pathway can help to promote the elimination of defective mitochondria and may thus serve as useful therapeutic agents in DMD.

Nitric oxide protects the heart from ischemia-induced apoptosis and mitochondrial damage via protein kinase G mediated blockage of permeability transition and cytochrome c release

BackgroundHeart ischemia can rapidly induce apoptosis and mitochondrial dysfunction via mitochondrial permeability transition-induced cytochrome c release. We tested whether nitric oxide (NO) can block this damage in isolated rat heart, and, if so, by what mechanisms.MethodsHearts were perfused with 50 μM DETA/NO (NO donor), then subjected to 30 min stop-flow ischemia or ischemia/reperfusion. Isolated heart mitochondria were used to measure the rate of mitochondrial oxygen consumption and membrane potential using oxygen and tetraphenylphosphonium-selective electrodes. Mitochondrial and cytosolic cytochrome c levels were measured spectrophotometrically and by ELISA. The calcium retention capacity of isolated mitochondria was measured using the fluorescent dye Calcium Green-5N. Apoptosis and necrosis were evaluated by measuring the activity of caspase-3 in cytosolic extracts and the activity of lactate dehydrogenase in perfusate, respectively.Results30 min ischemia caused release of mitochondrial cytochrome c to the cytoplasm, inhibition of the mitochondrial respiratory chain, and stimulation of mitochondrial proton permeability. 3 min perfusion with 50 μM DETA/NO of hearts prior to ischemia decreased this mitochondrial damage. The DETA/NO-induced blockage of mitochondrial cytochrome c release was reversed by a protein kinase G (PKG) inhibitor KT5823, or soluble guanylate cyclase inhibitor ODQ or protein kinase C inhibitors (Ro 32-0432 and Ro 31-8220). Ischemia also stimulated caspase-3-like activity, and this was substantially reduced by pre-perfusion with DETA/NO. Reperfusion after 30 min of ischemia caused no further caspase activation, but was accompanied by necrosis, which was completely prevented by DETA/NO, and this protection was blocked by the PKG inhibitor. Incubation of isolated heart mitochondria with activated PKG blocked calcium-induced mitochondrial permeability transition and cytochrome c release. Perfusion of non-ischemic heart with DETA/NO also made the subsequently isolated mitochondria resistant to calcium-induced permeabilisation, and this protection was blocked by the PKG inhibitor.ConclusionThe results indicate that NO rapidly protects the ischemic heart from apoptosis and mitochondrial dysfunction via PKG-mediated blockage of mitochondrial permeability transition and cytochrome c release.

Phosphate Is Essential for Inhibition of the Mitochondrial Permeability Transition Pore by Cyclosporin A and by Cyclophilin D Ablation

Energized mouse liver mitochondria displayed the same calcium retention capacity (a sensitive measure of the propensity of the permeability transition pore (PTP) to open) irrespective of whether phosphate, arsenate, or vanadate was the permeating anion. Unexpectedly, however, phosphate was specifically required for PTP desensitization by cyclosporin A (CsA) or by genetic inactivation of cyclophilin D (CyP-D). Indeed, when phosphate was replaced by arsenate, vanadate, or bicarbonate, the inhibitory effects of CsA and of CyP-D ablation on the PTP disappeared. After loading with the same amount of Ca(2+) in the presence of arsenate or vanadate but in the absence of phosphate, the sensitivity of the PTP to a variety of inducers was identical in mitochondria from wild-type mice, CyP-D-null mice, and wild-type mice treated with CsA. These findings call for a reassessment of conclusions on the role of the PTP in cell death that are based on the effects of CsA or of CyP-D ablation.

A Transverse Tubule NADPH Oxidase Activity Stimulates Calcium Release from Isolated Triads via Ryanodine Receptor Type 1 S -Glutathionylation

We report here the presence of an NADPH oxidase (NOX) activity both in intact and in isolated transverse tubules and in triads isolated from mammalian skeletal muscle, as established by immunochemical, enzymatic, and pharmacological criteria. Immunohistochemical determinations with NOX antibodies showed that the gp91(phox) membrane subunit and the cytoplasmic regulatory p47(phox) subunit co-localized in transverse tubules of adult mice fibers with the alpha1s subunit of dihydropyridine receptors. Western blot analysis revealed that isolated triads contained the integral membrane subunits gp91(phox) and p22(phox), which were markedly enriched in isolated transverse tubules but absent from junctional sarcoplasmic reticulum vesicles. Isolated triads and transverse tubules, but not junctional sarcoplasmic reticulum, also contained varying amounts of the cytoplasmic NOX regulatory subunits p47(phox) and p67(phox). NADPH or NADH elicited superoxide anion and hydrogen peroxide generation by isolated triads; both activities were inhibited by NOX inhibitors but not by rotenone. NADH diminished the total thiol content of triads by one-third; catalase or apocynin, a NOX inhibitor, prevented this effect. NADPH enhanced the activity of ryanodine receptor type 1 (RyR1) in triads, measured through [3H]ryanodine binding and calcium release kinetics, and increased significantly RyR1 S-glutathionylation over basal levels. Preincubation with reducing agents or NOX inhibitors abolished the enhancement of RyR1 activity produced by NADPH and prevented NADPH-induced RyR1 S-glutathionylation. We propose that reactive oxygen species generated by the transverse tubule NOX activate via redox modification the neighboring RyR1 Ca2+ release channels. Possible implications of this putative mechanism for skeletal muscle function are discussed.

Finding of Optimal Calcium Ion Probes for Fluorescence Lifetime Measurement

We have investigated the fluorescence lifetime properties of 8 calcium ion probes, calcium-green-1, calcium green-2, calcium green-5N, calcium orange, oregon green 488 BAPTA-6F, fluo-3, fluo-4, and fluo-5N. We found that the decay time of calcium green-5N varied more sensitively with calcium concentration than calcium green-1 which was known to be a highly sensitive probe. We have also found that the center of observable range of calcium concentration by fluorescence lifetime measurement is lower than that by fluorescence intensity measurement.

Properties of the Permeability Transition Pore in Mitochondria Devoid of Cyclophilin D

We have studied the properties of the permeability transition pore (PTP) in mitochondria from the liver of mice where the Ppif gene encoding for mitochondrial Cyclophilin D (CyP-D) had been inactivated. Mitochondria from Ppif-/- mice had no CyP-D and displayed a striking desensitization of the PTP to Ca2+, in that pore opening required about twice the Ca2+ load necessary to open the pore in strain-matched, wild-type mitochondria. Mitochondria lacking CyP-D were insensitive to Cyclosporin A (CsA), which increased the Ca2+ retention capacity only in mitochondria from wild-type mice. The PTP response to ubiquinone 0, depolarization, pH, adenine nucleotides, and thiol oxidants was similar in mitochondria from wild-type and Ppif-/- mice. These experiments demonstrate that (i) the PTP can form and open in the absence of CyP-D, (ii) that CyP-D represents the target for PTP inhibition by CsA, and (iii) that CyP-D modulates the sensitivity of the PTP to Ca2+ but not its regulation by the proton electrochemical gradient, adenine nucleotides, and oxidative stress. These results have major implications for our current understanding of the PTP and its modulation in vitro and in vivo.

The acrosomal vesicle of mouse sperm is a calcium store.

Subsequent to binding to the zona pellucida, mammalian sperm undergo a regulated sequence of events that ultimately lead to acrosomal exocytosis. Like most regulated exocytotic processes, a rise in intracellular calcium is sufficient to trigger this event although the precise mechanism of how this is achieved is still unclear. Numerous studies on mouse sperm have indicated that a voltage-operated Ca2+ channel plays some immediate role following sperm binding to the zona pellucida glycoprotein ZP3. However, there is also evidence that the mammalian sperm acrosome contains a high density of IP3 receptors, suggesting that the exocytotic event involves the release of Ca2+ from the acrosome. The release of Ca2+ from the acrosome may directly trigger exocytosis or may activate store-operated Ca2+ channels in the plasma membrane. To test the hypothesis that the acrosome is an intracellular store we loaded mammalian sperm with the membrane permeant forms of three Ca2+-sensitive fluorescent indicator dyes: fura-2, indo-1, and Calcium Green-5N. Fluorescence microscopy revealed that the sperm were labeled in all intracellular compartments. When fura-2 labeled sperm were treated with 150 microM MnCl2 to quench all fluorescence in the cytosol, or when the sperm were labeled with the low affinity dye Calcium Green-5N, there was a large Ca2+ signal in the acrosome. Consistent with the acrosome serving as an intracellular Ca2+ reservoir, the addition of 20 microM thapsigargin, a potent inhibitor of the smooth endoplasmic reticular Ca2+-ATPase (SERCA), to populations of capacitated sperm resulted in nearly 100% acrosomal exocytosis within 60 min (tau1/2 approximately 10 min), in the absence of extracellular Ca2+. Additionally, treatment of sperm with 100 microM thimerosal, an IP3 receptor agonist, also resulted in acrosomal exocytosis. Taken together, these data suggest that the mouse sperm acrosome is a Ca2+ store that regulates its own exocytosis through an IP3 Ca2+ mobilization pathway.

Vanadate inhibits the calcium extrusion in rat pancreatic acinar cells

Our objective was to evaluate the role of vanadate on calcium extrusion in Fura-2-loaded rat pancreatic acinar cells by digital microscopic fluorimetry and spectrofluorimetry. In the absence of extracellular calcium, perfusion of pancreatic acinar cells with 1 nM CCK-8 and 1 mM vanadate did not significantly affect the typical transient calcium spike induced by CCK-8, but the plateau phase of calcium in response to CCK-8 remained elevated. In addition, vanadate was able to inhibit calcium efflux evoked by CCK-8 when we determined directly calcium transport across plasma membrane using Calcium Green-5N hexapotassium salt (cell impermeant form) in cell populations. The effect of vanadate on calcium extrusion was strongly blocked by the sulfhydryl-reducing agent dithiothreitol (DTT). The present results demonstrate that vanadate is able to irreversibly inhibit the calcium extrusion. This effect of vanadate can be blocked using DTT, indicating that its action is probably mediated by oxidation of sulfhydryl groups of Ca 2+-ATPases.

The phosphoinositide signaling cascade is involved in photoreception in the leech Hirudo medicinalis.

The effects of BAPTA, heparin, and neomycin on electrical light responses were studied in the photoreceptors of Hirudo medicinalis. Light activation produces a fast increase in intracellular Ca2+ concentration (Cai) as detected with the fluorescent Ca2+ indicator calcium green-5N. Chelating intracellular calcium by injections of 10 mmol(-1) BAPTA suppresses spontaneous quantum bumps, reduces light sensitivity by more than 2 log(10) units, and substantially increases the latent period of light responses. BAPTA strongly inhibits the plateau phase of responses to long steps of light. Injections of 45-100 mg ml(-1) of heparin act in a similar manner to BAPTA, affecting the latency of the light responses even more. De-N-sulfated heparin, an inactive analog, is almost ineffective at the same concentration compared with heparin. Heparin diminishes the light-induced Cai elevation significantly, whereas de-N-sulfated heparin does not. Intracellular injections of 50-100 mmol l(-1) of the aminoglycoside neomycin, which inhibits phospholipase-C-mediated inositol 1,4,5-trisphosphate formation, acts similar to BAPTA and heparin. Pressure injections of the hydrolysis resistant analog of inositol 1,4,5-trisphosphate, inositol 2,4,5-trisphosphate, strongly depolarize leech photoreceptors and mimic an effect of light adaptation. These results suggest a close similarity between phototransduction mechanisms in leech photoreceptors and existing models for visual transduction in other invertebrate microvillar photoreceptors.

Ca(2+) dynamics in synaptosomes isolated from the squid optic lobe.

Synaptosomes from the optic lobes of squid (Loligo forbesi) were prepared by homogenization and allowed to settle onto glass coverslips. Synaptosomes were loaded with Ca(2+) sensitive dyes (Fura-2 AM, Calcium Green-1 AM and Calcium Green-5N AM), visualized by light microscopy and Ca(2+) sensitive fluorescence signals recorded and analyzed. With Fura-2, resting Ca(2+) was found to be 80 nM (n = 10, SEM 5.7). Addition of K(+) (30 mM), caffeine (3 mM) and thapsigargin (10 microM) evoked transient increases in cytoplasmic Ca(2+). Addition of BAPTA-AM (20 microM) decreased intrasynaptosomal free Ca(2+). Similar results were obtained with Calcium Green-1 AM but not with Calcium Green-5N AM. We conclude that synaptosomes from the squid optic lobe posses intact membranes and mechanisms to regulate intrasynaptosomal free [Ca(2+)], as well as caffeine sensitive Ca(2+) stores. The results of this study are discussed with respect to the role of Ca(2+) in presynaptic protein synthesis.

Effects of calcium buffering on glucose-induced insulin release in mouse pancreatic islets: an approximation to the calcium sensor.

1. The properties of the calcium sensor for glucose-induced insulin secretion have been studied using cell-permeant Ca2+ buffers with distinct kinetics and affinities. In addition, submembrane cytosolic Ca2+ distribution has been modelled after trains of glucose-induced action potential-like depolarizations. 2. Slow Ca2+ buffers (around 1 mmol l-1 intracellular concentration) with different affinities (EGTA and Calcium Orange-5N) did not significantly affect glucose-induced insulin release. Modelling showed no effect on cytosolic Ca2+ concentrations at the outermost shell (0.05 microm), their effects being observed in the innermost shells dependent on Ca2+ affinity. 3. In contrast, fast Ca2+ buffers (around 1 mmol l-1 intracellular concentration) with different affinities (BAPTA and Calcium Green-5N) caused a 50 % inhibition of early insulin response and completely blocked the late phase of glucose-induced insulin response, their simulations showing a decrease of [Ca2+]i at both the inner and outermost shells. 4. These data are consistent with the existence in pancreatic beta-cells of a higher affinity Ca2+ sensor than that proposed for neurons. Moreover, these data are consistent with the proposed existence of two distinct pools of granules: (i) 'primed' vesicles, colocalized with Ca2+ channels and responsible of the first phase of insulin release; and (ii) 'reserved pool' vesicles, not colocalized and responsible for the second phase.

Ca increase in secretory granules of stimulated mast cells

The elemental content of rat peritoneal mast-cell secretory granules has been measured by X-ray microanalysis. Two distinct categories of granules were analyzed: intact granules, seen in control samples, and spumous granules, corresponding to exocytosed granule matrices. The average Ca content of intact granules was found to be approximately equal to cytosolic concentration, and to increase up to 40-fold in spumous granules. A significant increase was also observed for Na and Cl. These changes were not observed (for Ca) or weaker (for Na and Cl) if the cells had been challenged in the absence of nominal extracellular Ca; in this case, there was also a significant decrease in the sulphur content, suggesting a partial dispersion of the organic matrix components.In exocytosed granule matrices, in the presence but not in the absence of extracellular Ca, a slow and long-lasting increase of intragranular free Ca was monitored by changes in the fluorescence of the Ca-sensitive probes Fluo-3 and Calcium Green-5N, accumulated within rat mast-cell secretory granules. These findings are discussed along two lines:• It is proposed that the calcium uptake by the exocytosed mast-cell granule matrices can have a physiological relevance for the surrounding tissue.• Mast-cell granules do not disperse after exocytosis. The major uptake of Ca which is seen after opening of the exocytotic pore could be responsible for the exceptional stability of the externalized matrices.