8 research outputs found
Mitochondrial free [Ca2+] levels and the permeability transition
Producción CientíficaMitochondrial Ca2+ activates many processes, from mitochondrial metabolism to opening of the permeability
transition pore (PTP) and apoptosis. However, there is considerable controversy regarding the
free mitochondrial [Ca2+] ([Ca2+]M) levels that can be attained during cell activation or even in mitochondrial
preparations. Studies using fluorescent dyes (rhod-2 or similar), have reported that phosphate
precipitation precludes [Ca2+]M from increasing above 2–3 M. Instead, using low-Ca2+-affinity aequorin
probes, we have measured [Ca2+]M values more than two orders of magnitude higher. We confirm here
these values by making a direct in situ calibration of mitochondrial aequorin, and we show that a prolonged
increase in [Ca2+]M to levels of 0.5–1mM was actually observed at any phosphate concentration
(0–10mM) during continuous perfusion of 3.5–100 MCa2+-buffers. In spite of this high and maintained
(>10 min) [Ca2+]M, mitochondria retained functionality and the [Ca2+]M drop induced by a protonophore
was fully reversible. In addition, this high [Ca2+]M did not induce PTP opening unless additional activators
(phenyl arsine oxide, PAO) were present. PAO induced a rapid, concentration-dependent and irreversible
drop in [Ca2+]M. In conclusion [Ca2+]M levels of 0.5–1mM can be reached and maintained for prolonged
periods (>10 min) in phosphate-containing medium, and massive opening of PTP requires additional pore
activators
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
The mitochondrial Na+/Ca2+ exchanger plays a key role in the control of cytosolic Ca2+ oscillations
Producción CientíficaThere is increasing evidence that mitochondria play an important role in the control of cytosolic Ca2+ signaling. We show here that the
main mitochondrial Ca2+-exit pathway, the mitochondrial Na+/Ca2+ exchanger, controls the pattern of cytosolic Ca2+ oscillations in nonexcitable
cells. In HeLa cells, the inhibitor of the mitochondrial Na+/Ca2+ exchanger CGP37157 changed the pattern of the oscillations
induced by histamine from a high-frequency irregular one to a lower frequency baseline spike type, surprisingly with little changes in the
average Ca2+ values of a large cell population. In human fibroblasts, CGP37157 increased the frequency of the baseline oscillations in cells
having spontaneous activity and induced the generation of oscillations in cells without spontaneous activity. This effect was dose-dependent,
disappeared when the inhibitor was washed out and was not mimicked by mitochondrial depolarization. CGP37157 increased mitochondrial
[Ca2+] and ATP production in histamine-stimulated HeLa cells, but the effect on ATP production was only transient. CGP37157 also activated
histamine-induced Ca2+ release from the endoplasmic reticulum and increased the size of the cytosolic Ca2+ peak induced by histamine
in HeLa cells. Our results suggest that the mitochondrial Na+/Ca2+ exchanger directly modulates inositol 1,4,5-trisphosphate-induced Ca2+
release and in that way controls cytosolic Ca2+ oscillations
Leptolide improves insulin resistance in diet-induced obese mice
Producción CientíficaType 2 diabetes (T2DM) is a complex disease linked to pancreatic beta-cell failure and insulin resistance. Current antidiabetic treatment regimens for T2DM include insulin sensitizers and insulin secretagogues. We have previously demonstrated that leptolide, a member of the furanocembranolides family, promotes pancreatic beta-cell proliferation in mice. Considering the beneficial effects of leptolide in diabetic mice, in this study, we aimed to address the capability of leptolide to improve insulin resistance associated with the pathology of obesity. To this end, we tested the hypothesis that leptolide should protect against fatty acid-induced insulin resistance in hepatocytes. In a time-dependent manner, leptolide (0.1 µM) augmented insulin-stimulated phosphorylation of protein kinase B (PKB) by two-fold above vehicle-treated HepG2 cells. In addition, leptolide (0.1 µM) counteracted palmitate-induced insulin resistance by augmenting by four-fold insulin-stimulated phosphorylation of PKB in HepG2 cells. In vivo, acute intraperitoneal administration of leptolide (0.1 mg/kg and 1 mg/kg) improved glucose tolerance and insulin sensitivity in lean mice. Likewise, prolonged leptolide treatment (0.1 mg/kg) in diet-induced obese mice improved insulin sensitivity. These effects were paralleled with an ~50% increased of insulin-stimulated phosphorylation of PKB in liver and skeletal muscle and reduced circulating pro-inflammatory cytokines in obese mice. We concluded that leptolide significantly improves insulin sensitivity in vitro and in obese mice, suggesting that leptolide may be another potential treatment for T2DM.This research has been funded by Sociedad Española de Diabetes (Ayudas Investigación Básica 2014), Salud Castilla y León (BIO/VA40/15)Ministerio de Economía y Competitividad, (SAF2014-58702-C2-1-R),(SAF2014-58702-C2-2-R
Calcium dynamics in catecholamine-containing secretory vesicles
Producción CientíficaWe have used an aequorin chimera targeted to the membrane of the secretory granules to monitor the free [Ca2+] inside them in neurosecretory
PC12 cells. More than 95% of the probe was located in a compartment with an homogeneous [Ca2+] around 40 M. Cell stimulation with
either ATP, caffeine or high-K+ depolarization increased cytosolic [Ca2+] and decreased secretory granule [Ca2+] ([Ca2+]SG). Inositol-(1,4,5)-
trisphosphate, cyclic ADP ribose and nicotinic acid adenine dinucleotide phosphate were all ineffective to release Ca2+ from the granules.
Changes in cytosolic [Na+] (0–140 mM) or [Ca2+] (0–10 M) did not modify either ([Ca2+]SG). Instead, [Ca2+]SG was highly sensitive to
changes in the pH gradient between the cytosol and the granules. Both carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) and
nigericin, as well as cytosolic acidification, reversibly decreased [Ca2+]SG, while cytosolic alcalinization reversibly increased [Ca2+]SG. These
results are consistent with the operation of a H+/Ca2+ antiporter in the vesicular membrane. This antiporter could also mediate the effects
of ATP, caffeine and high-K+ on [Ca2+]SG, because all of them induced a transient cytosolic acidification. The FCCP-induced decrease in
[Ca2+]SG was reversible in 10–15 min even in the absence of cytosolic Ca2+ or ATP, suggesting that most of the calcium content of the vesicles
is bound to a slowly exchanging Ca2+ buffer. This large store buffers [Ca2+]SG changes in the long-term but allows highly dynamic free [Ca2+]SG
changes to occur in seconds or minutes
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