8 research outputs found
Ca2+ homeostasis in the endoplasmic reticulum measured with a new low-Ca2+-affinity targeted aequorin
Producción CientíficaWe use here a new very low-Ca2+-affinity targeted aequorin to measure the [Ca2+] in the endoplasmic
reticulum ([Ca2+]ER). The new aequorin chimera has the right Ca2+-affinity to make long-lasting measurements
of [Ca2+]ER in the millimolar range. Moreover, previous Ca2+-depletion of the ER is no longer
required. The steady-state [Ca2+]ER obtained is 1–2 mM, higher than previously reported. In addition,
we find evidence that there is significant heterogeneity in [Ca2+]ER among different regions of the ER.
About half of the ER had a [Ca2+]ER of 1 mM or below, and the rest had [Ca2+]ER values above 1 mM and
in some parts even above 2 mM. About 5% of the ER was also found to have high [Ca2+]ER levels but to be
thapsigargin-insensitive and inositol trisphosphate insensitive. The rate of refilling with Ca2+ of the ER
was almost linearly dependent on the extracellular [Ca2+] between 0.1 and 3 mM, and was only partially
affected by mitochondrial membrane depolarization. Instead, it was significantly reduced by loading cells
with chelators, and the fast chelator BAPTA was much more effective than the slow chelator EGTA. This
suggests that local [Ca2+] microdomains connecting the store operated Ca2+ channels with the ER Ca2+
pumps may be important during refilling
Mitochondrial Ca2+ dynamics in MCU knockout C. elegans worms
Producción CientíficaMitochondrial [Ca2+] plays an important role in the regulation of mitochondrial function, controlling ATP production and apoptosis triggered by mitochondrial Ca2+ overload. This regulation depends on Ca2+ entry into the mitochondria during cell activation processes, which is thought to occur through the mitochondrial Ca2+ uniporter (MCU). Here, we have studied the mitochondrial Ca2+ dynamics in control and MCU-defective C. elegans worms in vivo, by using worms expressing mitochondrially-targeted YC3.60 yellow cameleon in pharynx muscle. Our data show that the small mitochondrial Ca2+ oscillations that occur during normal physiological activity of the pharynx were very similar in both control and MCU-defective worms, except for some kinetic differences that could mostly be explained by changes in neuronal stimulation of the pharynx. However, direct pharynx muscle stimulation with carbachol triggered a large and prolonged increase in mitochondrial [Ca2+] that was much larger in control worms than in MCU-defective worms. This suggests that MCU is necessary for the fast mitochondrial Ca2+ uptake induced by large cell stimulations. However, low-amplitude mitochondrial Ca2+ oscillations occurring under more physiological conditions are independent of the MCU and use a different Ca2+ pathway.Ministerio de Economía y Competitividad - (Proyecto BFU2017-83509-R)Fondo Europeo de Desarrollo Regional (FEDER) y Junta de Castilla y León - (Projecto VA011G18
The role of Ca2+ signaling in aging and neurodegeneration: Insights from caenorhabditis elegans models
Producción CientíficaCa2+ is a ubiquitous second messenger that plays an essential role in physiological processes such as muscle contraction, neuronal secretion, and cell proliferation or differentiation. There is ample evidence that the dysregulation of Ca2+ signaling is one of the key events in the development of neurodegenerative processes, an idea called the “calcium hypothesis” of neurodegeneration. Caenorhabditis elegans (C. elegans) is a very good model for the study of aging and neurodegeneration. In fact, many of the signaling pathways involved in longevity were first discovered in this nematode, and many models of neurodegenerative diseases have also been developed therein, either through mutations in the worm genome or by expressing human proteins involved in neurodegeneration (β-amyloid, α-synuclein, polyglutamine, or others) in defined worm tissues. The worm is completely transparent throughout its whole life, which makes it possible to carry out Ca2+ dynamics studies in vivo at any time, by expressing Ca2+ fluorescent probes in defined worm tissues, and even in specific organelles such as mitochondria. This review will summarize the evidence obtained using this model organism to understand the role of Ca2+ signaling in aging and neurodegeneration
A confocal study on the visualization of chromaffin cell secretory vesicles with fluorescent targeted probes and acidic dyes
Producción CientíficaSecretory vesicles have low pH and have been classically identified as those labelled by a series of acidic
fluorescent dyes such as acridine orange or neutral red, which accumulate into the vesicles according to
the pH gradient. More recently, several fusion proteins containing enhanced green fluorescent protein
(EGFP) and targeted to the secretory vesicles have been engineered. Both targeted fluorescent proteins
and acidic dyes have been used, separately or combined, to monitor the dynamics of secretory vesicle
movements and their fusion with the plasma membrane. We have now investigated in detail the degree
of colocalization of both types of probes using several fusion proteins targeted to the vesicles (synaptobrevin2-
EGFP, Cromogranin A-EGFP and neuropeptide Y-EGFP) and several acidic dyes (acridine orange,
neutral red and lysotracker red) in chromaffin cells, PC12 cells and GH3 cells. We find that all the acidic
dyes labelled the same population of vesicles. However, that population was largely different from the
one labelled by the targeted proteins, with very little colocalization among them, in all the cell types
studied. Our data show that the vesicles containing the proteins more characteristic of the secretory vesicles
are not labelled by the acidic dyes, and vice versa. Peptide glycyl-L-phenylalanine 2-naphthylamide
(GPN) produced a rapid and selective disruption of the vesicles labelled by acidic dyes, suggesting that
they could be mainly lysosomes. Therefore, these labelling techniques distinguish two clearly different
sets of acidic vesicles in neuroendocrine cells. This finding should be taken into account whenever vesicle
dynamics is studied using these techniques
Ca2+ Dynamics in the Secretory Vesicles of Neurosecretory PC12 and INS1 Cells
Producción CientíficaWe have investigated the dynamics of the free
[Ca2+] inside the secretory granules of neurosecretory PC12
and INS1 cells using a low-Ca2+-affinity aequorin chimera
fused to synaptobrevin-2. The steady-state secretory granule
[Ca2+] ([Ca2+]SG] was around 20–40 lM in both cell types,
about half the values previously found in chromaffin cells.
Inhibition of SERCA-type Ca2+ pumps with thapsigargin
largely blocked Ca2+ uptake by the granules in
Ca2+-depleted permeabilized cells, and the same effect was
obtained when the perfusion medium lacked ATP. Consistently,
the SERCA-type Ca2+ pump inhibitor benzohydroquinone
induced a rapid release of Ca2+ from the granules
both in intact and permeabilized cells, suggesting that the
continuous activity of SERCA-type Ca2+ pumps is essential
to maintain the steady-state [Ca2+]SG. Both inositol 1,4,
5-trisphosphate (InsP3) and caffeine produced a rapid Ca2+
release from the granules, suggesting the presence of InsP3
and ryanodine receptors in the granules. The response to
high-K+ depolarization was different in both cell types, a
decrease in [Ca2+]SG in PC12 cells and an increase in
[Ca2+]SG in INS1 cells. The difference may rely on the
heterogeneous response of different vesicle populations in
each cell type. Finally, increasing the glucose concentration
triggered a decrease in [Ca2+]SG in INS1 cells. In conclusion,
our data show that the secretory granules of PC12 and INS1
cells take up Ca2+ through SERCA-type Ca2+ pumps and
can release it through InsP3 and ryanodine receptors, supporting
the hypothesis that secretory granule Ca2+ may be
released during cell stimulation and contribute to secretion
Monitoring mitochondrial [Ca2+] dynamics with rhod-2, ratiometric pericam and aequorin
Producción CientíficaThe dynamics of mitochondrial [Ca2+] ([Ca2+]M) plays a key role in a variety of cellular processes. The
most important methods available to monitor [Ca2+]M are fluorescent dyes such as rhod-2 and specifically
targeted proteins such as aequorin and pericam. However, significant discrepancies, both quantitative
and qualitative, exist in the literature between the results obtained with different methods. We have
made here a systematic comparison of the response of several fluorescent dyes, rhod-2 and rhod-FF,
and two Ca2+-sensitive proteins, aequorin and pericam. Our results show that measurements obtained
with aequorin and pericam are consistent in terms of dynamic Ca2+ changes. Instead, fluorescent dyes
failed to follow Ca2+ changes adequately, especially during repetitive stimulation. In particular, measures
obtained with rhod-2 or rhod-FF evidenced the previously reported Ca2+-dependent inhibition of
mitochondrial Ca2+ uptake, but data obtained with aequorin or pericam under the same conditions did
not. The reason for the loss of response of fluorescent dyes is unclear. Loading with these dyes produced
changes in mitochondrial morphology and membrane potential, which were small and reversible at low
concentrations (1–2 M), but produced large and prolonged damage at higher concentrations. In addition,
cells loaded with low concentrations of rhod-2 suffered large changes in mitochondrial morphology
after light excitation. Our results suggest that [Ca2+]M data obtained with these dyes should be taken with
care
Calcium dynamics in bovine adrenal medulla chromaffin cell secretory granules
Producción CientíficaThe secretory granules constitute one of the less well-known compartments in terms of Ca2+ dynamics. They contain large amounts
of total Ca2+, but the free intragranular [Ca2+] ([Ca2+]SG), the mechanisms for Ca2+ uptake and release from the granules and their
physiological significance regarding exocytosis are still matters of debate. We used in the present work an aequorin chimera targeted
to the granules to investigate [Ca2+]SG homeostasis in bovine adrenal chromaffin cells. We found that most of the intracellular
aequorin chimera is present in a compartment with 50–100 lm Ca2+. Ca2+ accumulation into this compartment takes place mainly
through an ATP-dependent mechanism, namely, a thapsigargin-sensitive Ca2+-ATPase. In addition, fast Ca2+ release was observed
in permeabilized cells after addition of inositol 1,4,5-trisphosphate (InsP3) or caffeine, suggesting the presence of InsP3 and
ryanodine receptors in the vesicular membrane. Stimulation of intact cells with the InsP3-producing agonist histamine or with caffeine
also induced Ca2+ release from the vesicles, whereas acetylcholine or high-[K+] depolarization induced biphasic changes in vesicular
[Ca2+], suggesting heterogeneous responses of different vesicle populations, some of them releasing and some taking up Ca2+
during stimulation. In conclusion, our data show that chromaffin cell secretory granules have the machinery required for rapid uptake
and release of Ca2+, and this strongly supports the hypothesis that granular Ca2+ may contribute to its own secretion.2015-09-1
Evolution of neutrophil apoptosis in septic shock survivors and nonsurvivors
Producción CientíficaThe aims were to analyze the temporal evolution of neutrophil apoptosis, to determine the differences in neutrophil apoptosis among 28-day survivors and nonsurvivors, and to evaluate the use of neutrophil apoptosis as a predictor of mortality in patients with septic shock. [Materials and Methods]: Prospective multicenter observational study carried out between July 2006 and June 2009. The staining solution study included 80 patients with septic shock and 25 healthy volunteers. Neutrophil apoptosis was assessed by fluorescein isothiocyanate (FITC)-conjugated annexin V and aminoactinomycin D staining. [Results]: The percentage of neutrophil apoptosis was significantly decreased at 24 hours, 5 days, and 12 days after the diagnosis of septic shock (14.8% ± 13.4%, 13.4% ± 8.4%, and 15.4% ± 12.8%, respectively; P .05). The mortality rate at 28 days was 53.7%. The crude hazard ratio for mortality in patients with septic shock did not differ according to the percentage of apoptosis (hazard ratio, 1.006; 95% confidence interval, 0.98-1.03; P = .60). [Conclusions]: During the first 12 days of septic shock development, the level of neutrophil apoptosis decreases and does not recover normal values. No differences were observed between surviving and nonsurviving patients