Modulation of an early step in the secretory machinery in hippocampal nerve terminals

In hippocampal neurons, neurotransmitter release can be regulated by protein kinase A (PKA) through a direct action on the secretory machinery. To identify the site of PKA modulation, we have taken advantage of the ability of the neurotoxin Botulinum A to cleave the synaptic protein SNAP-25. Cleavage of this protein decreases the Ca2+ responsiveness of the secretory machinery by partially uncoupling Ca2+-sensing from fusion per se. This is expressed as a shift toward higher Ca2+ levels of the Ca2+ to neurotransmitter release relationship and as a perturbation of synaptic delay under conditions where secretion induced by the Ca2+-independent secretagogue ruthenium red is unimpaired. We find that SNAP-25 cleavage also perturbs PKA-dependent modulation of secretion; facilitation of ruthenium red-evoked neurotransmitter release by the adenylyl cyclase activator forskolin is blocked completely after Botulinum toxin A action. Together with our observation that forskolin modifies the Ca2+ to neurotransmitter release relationship, our results suggest that SNAP-25 acts as a functional linker between Ca2+ detection and fusion and that PKA modulates an early step in the secretory machinery related to calcium sensing to facilitate synaptic transmission.

Mitochondrial Ca2+-induced Ca2+ Release Mediated by the Ca2+ Uniporter

We have reported that a population of chromaffin cell mitochondria takes up large amounts of Ca2+ during cell stimulation. The present study focuses on the pathways for mitochondrial Ca2+ efflux. Treatment with protonophores before cell stimulation abolished mitochondrial Ca2+ uptake and increased the cytosolic [Ca2+] ([Ca2+]c) peak induced by the stimulus. Instead, when protonophores were added after cell stimulation, they did not modify [Ca2+]c kinetics and inhibited Ca2+ release from Ca2+-loaded mitochondria. This effect was due to inhibition of mitochondrial Na+/Ca2+ exchange, because blocking this system with CGP37157 produced no further effect. Increasing extramitochondrial [Ca2+]c triggered fast Ca2+ release from these depolarized Ca2+-loaded mitochondria, both in intact or permeabilized cells. These effects of protonophores were mimicked by valinomycin, but not by nigericin. The observed mitochondrial Ca2+-induced Ca2+ release response was insensitive to cyclosporin A and CGP37157 but fully blocked by ruthenium red, suggesting that it may be mediated by reversal of the Ca2+ uniporter. This novel kind of mitochondrial Ca2+-induced Ca2+ release might contribute to Ca2+ clearance from mitochondria that become depolarized during Ca2+ overload.

Direct Measurement of Calcium Transport across Chloroplast Inner-Envelope Vesicles1

The initial rate of Ca2+ movement across the inner-envelope membrane of pea (Pisum sativum L.) chloroplasts was directly measured by stopped-flow spectrofluorometry using membrane vesicles loaded with the Ca2+-sensitive fluorophore fura-2. Calibration of fura-2 fluorescence was achieved by combining a ratiometric method with Ca2+-selective minielectrodes to determine pCa values. The initial rate of Ca2+ influx in predominantly right-side-out inner-envelope membrane vesicles was greater than that in largely inside-out vesicles. Ca2+ movement was stimulated by an inwardly directed electrochemical proton gradient across the membrane vesicles, an effect that was diminished by the addition of valinomycin in the presence of K+. In addition, Ca2+ was shown to move across the membrane vesicles in the presence of a K+ diffusion potential gradient. The potential-stimulated rate of Ca2+ transport was slightly inhibited by diltiazem and greatly inhibited by ruthenium red. Other pharmacological agents such as LaCl3, verapamil, and nifedipine had little or no effect. These results indicate that Ca2+ transport across the chloroplast inner envelope can occur by a potential-stimulated uniport mechanism.

Strontium-Induced Repetitive Calcium Spikes in a Unicellular Green Alga1

The divalent cation Sr2+ induced repetitive transient spikes of the cytosolic Ca2+ activity [Ca2+]cy and parallel repetitive transient hyperpolarizations of the plasma membrane in the unicellular green alga Eremosphaera viridis. [Ca2+]cy measurements, membrane potential measurements, and cation analysis of the cells were used to elucidate the mechanism of Sr2+-induced [Ca2+]cy oscillations. Sr2+ was effectively and rapidly compartmentalized within the cell, probably into the vacuole. The [Ca2+]cy oscillations cause membrane potential oscillations, and not the reverse. The endoplasmic reticulum (ER) Ca2+-ATPase blockers 2,5-di-tert-butylhydroquinone and cyclopiazonic acid inhibited Sr2+-induced repetitive [Ca2+]cy spikes, whereas the compartmentalization of Sr2+ was not influenced. A repetitive Ca2+ release and Ca2+ re-uptake by the ER probably generated repetitive [Ca2+]cy spikes in E. viridis in the presence of Sr2+. The inhibitory effect of ruthenium red and ryanodine indicated that the Sr2+-induced Ca2+ release from the ER was mediated by a ryanodine/cyclic ADP-ribose type of Ca2+ channel. The blockage of Sr2+-induced repetitive [Ca2+]cy spikes by La3+ or Gd3+ indicated the necessity of a certain influx of divalent cations for sustained [Ca2+]cy oscillations. Based on these data we present a mathematical model that describes the baseline spiking [Ca2+]cy oscillations in E. viridis.

Redox transitions between oxygen intermediates in cytochrome-c oxidase.

Some intermediates in the reduction of O2 to water by cytochrome-c oxidase have been characterized by optical, Raman, and magnetic circular dichroism spectroscopy. The so-called "peroxy" (P) and "ferryl" (F) forms of the enzyme, which have been considered to be intermediates of the oxygen reaction, can be generated when the oxidized enzyme reacts with H2O2, or when the two-electron reduced ("CO mixed-valence") enzyme reacts with O2. The structures as well as the overall redox states of P and F have recently been controversial. We show here, using tris(2,2'-bipyridyl)ruthenium(II) as a photoinducible reductant, that one-electron reduction of P yields F, and that one-electron reduction of F yields the oxidized enzyme. This confirms that the overall redox states of P and F differ from the oxidized enzyme by two and one electron equivalents, respectively. The structures of the P and F states are discussed.

Photochemical energy conversion in a helical oligoproline assembly.

A general method is described for constructing a helical oligoproline assembly having a spatially ordered array of functional sites protruding from a proline-II helix. Three different redox-active carboxylic acids were coupled to the side chain of cis-4-amino-L-proline. These redox modules were incorporated through solid-phase peptide synthesis into a 13-residue helical oligoproline assembly bearing in linear array a phenothiazine electron donor, a tris(bipyridine)ruthenium(II) chromophore, and an anthraquinone electron acceptor. Upon transient 460-nm irradiation in acetonitrile, this peptide triad formed with 53% efficiency an excited state containing a phenothiazine radical cation and an anthraquinone radical anion. This light-induced redox-separated state had a lifetime of 175 ns and stored 1.65 eV of energy.

Photo-induced inactivation of viruses: adsorption of methylene blue, thionine, and thiopyronine on Qbeta bacteriophage.

The adsorption of cationic organic dyes (methylene blue, thionine, and thiopyronine) on Qbeta bacteriophage was studied by UV-visible and fluorescence spectroscopy. The dyes have shown a strong affinity to the virus and some have been used as sensitizers for photo-induced inactivation of virus. In the methylene blue concentration range of 0.1-5 microM and at high ratios of dye to virus (greater than 1000 dye molecules per virion), the dyes bind as aggregates on the virus. Aggregation lowers the efficiency of photoinactivation because of self-quenching of the dye. At lower ratios of dye to virus (lower than 500 dye molecules per virion), the dye binds to the virus as a monomer. Fluorescence polarization and time-resolved studies of the fluorescence support the conclusions based on fluorescence quenching. Increasing the ionic strength (adding NaCl) dissociates bound dye aggregates on the virus and releases monomeric dye into the bulk solution.

Gadolinium(III) texaphyrin: a tumor selective radiation sensitizer that is detectable by MRI.

Gadolinium(III) texaphyrin (Gd-tex2+) is representative of a new class of radiation sensitizers detectable by magnetic resonance imaging (MRI). This porphyrin-like complex has a high electron affinity [E1/2 (red.) approximately = -0.08 V versus normal hydrogen electrode] and forms a long-lived pi-radical cation upon exposure to hydrated electrons, reducing ketyl radicals, or superoxide ions. Consistent with these chemical findings, Gd-tex2+ was found to be an efficient radiation sensitizer in studies carried out with HT29 cells in in vitro as well as in in vivo single and multifraction irradiation studies with a murine mammary carcinoma model. Selective localization of Gd-tex2+ in tumors was confirmed by MRI scanning.

Detection of exocytosis at individual pancreatic beta cells by amperometry at a chemically modified microelectrode.

Amperometry at a carbon fiber microelectrode modified with a composite of ruthenium oxide and cyanoruthenate was used to monitor chemical secretions of single pancreatic beta cells from rats and humans. When the insulin secretagogues glucose, tolbutamide, and K+ were applied to the cell, a series of randomly occurring current spikes was observed. The current spikes were shown to be due to the detection of chemical substances secreted from the cell. Chromatography showed that the primary secreted substance detected by the electrode was insulin. The current spikes were strongly dependent on external Ca2+, had an average area that was independent of the stimulation method, and had an area distribution which corresponded to the distribution of vesicle sizes in beta cells. It was concluded that the spikes were due to the detection of concentration pulses of insulin secreted by exocytosis.

Effects of mechanical signaling on plant cell cytosolic calcium.

Mechanical signals are important influences on the development and morphology of higher plants. Using tobacco transformed with the Ca(2+)-sensitive luminescent protein aequorin, we recently reported the effects of mechanical signals of touch and wind on the luminescence and thus intracellular calcium of young seedlings. When mesophyll protoplasts are isolated from these transgenic tobacco plants and mechanically stimulated by swirling them in solution, cytoplasmic Ca2+ increases immediately and transiently up to 10 microM, and these transients are unaffected by an excess of EGTA in the medium. The size of the transient effect is related to the strength of swirling. Epidermal strips isolated from transgenic tobacco leaves and containing only viable guard cells and trichomes also respond to the strength of swirling in solution and can increase their cytoplasmic Ca2+ transiently up to 10 microM. Finally, the moss Physcomitrella patens containing recombinant aequorin exhibits transient increases in cytoplasmic Ca2+ up to 5 microM when swirled in solution. This effect is strongly inhibited by ruthenium red. Our data indicate that the effect of mechanical stimulation can be found in a number of different cell types and in a lower plant as well as tobacco and suggest that mechanoperception and the resulting increase in cytoplasmic Ca2+ may be widespread.

Activation of Ca2+ signaling in neutrophils by the mast cell-released immunophilin FKBP12.

The immunophilins of the FK506-binding protein (FKBP) family are intracellular proteins that bind the immunosuppresants FK506 and rapamycin. In this study we show that HMC-1 mast cells sensitized with IgE release FKBP12 upon stimulation with anti-IgE. The release is rapid and not affected by actinomycin D or cycloheximide, suggesting that it is due to exocytosis from a storage compartment. FKBP12 from HMC-1 mast cells exhibits biological activity. When applied extracellularly to human neutrophils, it induces transient changes in the intracellular Ca2+ concentration ([Ca2+]i) due to Ca2+ release from intracellular stores. Inhibition of [Ca2+]i changes by ruthenium red and ryanodine indicates that ryanodine receptor/Ca2+ release channels are involved in FKBP12-induced Ca2+ signaling. Neutrophil activation by mast cell-derived FKBP12 is prevented by complexing FKBP12 with FK506 or rapamycin. These results demonstrate that extracellular FKBP12 functions as a cytokine in cell-to-cell communication. They further suggest a pathophysiological role for FKBP12 as a mediator in immediate or type I hypersensitivity and may have implications for novel therapeutic strategies in the treatment of allergic disorders with FK506 and rapamycin.