Exocytotic release of ATP from cultured astrocytes.

Astrocytes appear to communicate with each other as well as with neurons via ATP. However, the mechanisms of ATP release are controversial. To explore whether stimuli that increase [Ca(2+)](i) also trigger vesicular ATP release from astrocytes, we labeled ATP-containing vesicles with the fluorescent dye quinacrine, which exhibited a significant co-localization with atrial natriuretic peptide. The confocal microscopy study revealed that quinacrine-loaded vesicles displayed mainly non-directional spontaneous mobility with relatively short track lengths and small maximal displacements, whereas 4% of vesicles exhibited directional mobility. After ionomycin stimulation only non-directional vesicle mobility could be observed, indicating that an increase in [Ca(2+)](i) attenuated vesicle mobility. Total internal reflection fluorescence (TIRF) imaging in combination with epifluorescence showed that a high percentage of fluorescently labeled vesicles underwent fusion with the plasma membrane after stimulation with glutamate or ionomycin and that this event was Ca(2+)-dependent. This was confirmed by patch-clamp studies on HEK-293T cells transfected with P2X(3) receptor, used as sniffers for ATP release from astrocytes. Glutamate stimulation of astrocytes was followed by an increase in the incidence of small transient inward currents in sniffers, reminiscent of postsynaptic quantal events observed at synapses. Their incidence was highly dependent on extracellular Ca(2+). Collectively, these findings indicate that glutamate-stimulated ATP release from astrocytes was most likely exocytotic and that after stimulation the fraction of quinacrine-loaded vesicles, spontaneously exhibiting directional mobility, disappeared

Peptide hormone release monitored from single vesicles in 'membrane lawns' of differentiated male pituitary cells: SNAREs and fusion pore widening

In this study we used live-cell immunocytochemistry and confocal microscopy to study the release from a single vesicle in a simplified system called membrane lawns. The lawns were prepared by exposing differentiated pituitary prolactin (PRL)-secreting cells to a hypoosmotic shear stress. The density of the immunolabeled ternary soluble N-ethylmaleimide-sensitive factor-attachment protein receptor (SNARE) complexes that bind complexin was approximately 10 times lower than the PRL-positive, lawn-resident vesicles; this indicates that some but not all vesicles are associated with ternary SNARE complexes. However, lawn-resident PRL vesicles colocalized relatively well with particular SNARE proteins: synaptobrevin 2 (35%), syntaxin 1 (22%), and 25-kDa synaptosome associated protein (6%). To study vesicle discharge, we prepared lawn-resident vesicles, derived from atrial natriuretic peptide tagged with emerald fluorescent protein (ANP.emd)-transfected cells, which label vesicles. These maintained the structural passage to the exterior because approximately 40% of ANP.emd-loaded vesicles were labeled by extracellular PRL antibodies. Cargo release from the lawn-resident vesicles, monitored by the decline in the ANP.emd fluorescence intensity, was similar to that in intact cells. It is likely that SNARE proteins are required for calcium-dependent release from these vesicles. This is because the expression of the dominant-negative SNARE peptide, which interferes with SNARE complex formation, reduced the number of PRL-positive spots per cell (PRL antibodies placed extracellularly) significantly, from 58 ± 9 to 4 ± 2. In dominant-negative SNARE-treated cells, the PRL-positive area was reduced from 0.259 ± 0.013 to 0.123 ± 0.014 μm(2), which is consistent with a hindered vesicle luminal access for extracellular PRL antibodies. These results indicate that vesicle discharge is regulated by SNARE-mediated fusion pore widening.We thank Mrs Sonja Grilc for initial assistance with the immunocytochemical experiments on fixed cells.. This work was supported by Grants P3 310 381 and BI-PT-10-11 from the Slovenian Research Agency (ARRS) and by grant Proc° 441.00 ESLOVENIA from the Portuguese Foundation for Science and Technology.publishe

Immunoglobulins G from patients with sporadic amyotrophic lateral sclerosis affects cytosolic Ca2+ homeostasis in cultured rat astrocytes

Astrocytes are considered essential in the etiopathogenesis of amyotrophic lateral sclerosis (ALS). We have demonstrated previously that immunoglobulins G (IgG) isolated from patients with ALS enhance the mobility of acidic vesicles in cultured astrocytes in a Ca2+-dependent manner. Here we directly examined the impact of purified sporadic ALS IgG on cytosolic [Ca2+] ([Ca2+](i)) in astrocytes. Confocal time-lapse images were acquired and fluorescence of a non-ratiometric Ca2+ indicator was recorded before and after the application of IgG. ALS IgG (0.1 mg/ml) from 7 patients evoked transient increases in [Ca2+](i) in similar to 50% of tested astrocytes. The probability of observing a response was independent of extracellular Ca2+. The peak increase in [Ca2+](i) developed similar to 3 times faster and the time integral of evoked transients was similar to 2-fold larger; the peak amplitude itself was not affected by extracellular Ca2+. Application of pharmacological inhibitors revealed that activation of inositol-1,4,5-triphosphate receptors is necessary and sufficient to initiate transients in [Ca2+](i) the Ca2+ influx through store-operated calcium entry prolongs the transient increase in [Ca2+](i). Thus, ALS IgG acutely affect [Ca2+](i) by mobilizing both, intra- and extracellular Ca2+ into the cytosol of cultured astrocytes. (C) 2013 Elsevier Ltd. All rights reserved

Life and death in aluminium-exposed cultures of rat lactotrophs studied by flow cytometry

Prolonged exposure to aluminium may impact health. Aluminium's deleterious effects are mostly attributed to its selective accumulation in particular organs and cell types. Occupational exposure to aluminium is allied with a reduced level of serum prolactin, a stress peptide hormone mainly synthesised and secreted by the anterior pituitary lactotrophs. Our aim was to study the effect of aluminium on the viability of rat lactotrophs in primary suspension cultures where multicellular aggregates tend to form, comprising approximately two thirds of the total cell population as confirmed by confocal microscopy. Flow cytometric light scattering of calcein acetoxymethyl ester and ethidium homodimer-1 labelled cells was used to define subpopulations of live and dead cells in heterogeneous suspensions comprised of single cells and multicellular aggregates of distinct size. Concentration-dependent effects of AlCl(3) were observed on aggregate size and cell survival. After 24-h exposure to 3 mM AlCl(3), viability of single cells declined from 5% to 3%, while in multicellular aggregates, viability declined from 23% to 20%. The proportion of single cells increased from 30% to 42% within the same concentration range, while in large aggregates, the proportion remained approximately constant representing 35% of the cell suspension. In large aggregates, cell viability (75%) remained unaltered after exposure to AlCl(3) concentrations up to 300 mu M, while in single cells, viability was halved at 30 mu M. In conclusion, our finding indicates that prolonged exposure to aluminium may lead to significant loss of pituitary cells.Ministry of Higher Education, Sciences and Technology of the Republic of SloveniaP3 310 381Z3 7476 1683BI-PT/06-07-002FCTSFRH/BD/41217/2007SFRH/BD27467/2006SFRH/BPD/14677/2003GRICES - MCTES (4.1.1 Eslovénia