-
[show abstract]
[hide abstract]
ABSTRACT: We use here a new very low-Ca(2+)-affinity targeted aequorin to measure the [Ca(2+)] in the endoplasmic reticulum ([Ca(2+)]ER). The new aequorin chimera has the right Ca(2+)-affinity to make long-lasting measurements of [Ca(2+)]ER in the millimolar range. Moreover, previous Ca(2+)-depletion of the ER is no longer required. The steady-state [Ca(2+)]ER obtained is 1-2mM, higher than previously reported. In addition, we find evidence that there is significant heterogeneity in [Ca(2+)]ER among different regions of the ER. About half of the ER had a [Ca(2+)]ER of 1mM or below, and the rest had [Ca(2+)]ER values above 1mM and in some parts even above 2mM. About 5% of the ER was also found to have high [Ca(2+)]ER levels but to be thapsigargin-insensitive and inositol trisphosphate insensitive. The rate of refilling with Ca(2+) of the ER was almost linearly dependent on the extracellular [Ca(2+)] between 0.1 and 3mM, 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 [Ca(2+)] microdomains connecting the store operated Ca(2+) channels with the ER Ca(2+) pumps may be important during refilling.
Cell calcium 05/2013; · 4.29 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Mitochondria have a very large capacity to accumulate Ca(2+) during cell stimulation driven by the mitochondrial membrane potential. Under these conditions, [Ca(2+)](M) (mitochondrial [Ca(2+)]) may well reach millimolar levels in a few seconds. Measuring the dynamics of [Ca(2+)](M) during prolonged stimulation has been previously precluded by the high Ca(2+) affinity of the probes available. We have now developed a mitochondrially targeted double-mutated form of the photoprotein aequorin which is able to measure [Ca(2+)] in the millimolar range for long periods of time without problems derived from aequorin consumption. We show in the present study that addition of Ca(2+) to permeabilized HeLa cells triggers an increase in [Ca(2+)](M) up to an steady state of approximately 2-3 mM in the absence of phosphate and 0.5-1 mM in the presence of phosphate, suggesting buffering or precipitation of calcium phosphate when the free [Ca(2+)] reaches 0.5-1 mM. Mitochondrial pH acidification partially re-dissolved these complexes. These millimolar [Ca(2+)](M) levels were stable for long periods of time provided the mitochondrial membrane potential was not collapsed. Silencing of the mitochondrial Ca(2+) uniporter largely reduced the rate of [Ca(2+)](M) increase, but the final steady-state [Ca(2+)](M) reached was similar. In intact cells, the new probe allows monitoring of agonist-induced increases of [Ca(2+)](M) without problems derived from aequorin consumption.
Biochemical Journal 06/2012; 445(3):371-6. · 4.90 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Available methods to measure mitochondrial [Ca(2+)] ([Ca(2+)](M)) include both targeted proteins and fluorescent dyes. Targeted proteins usually report much higher [Ca(2+)](M) values than fluorescent dyes, up to two orders of magnitude. However, we show here that the low-Ca(2+)-affinity dye rhod-5N provides [Ca(2+)](M) values similar to those reported by targeted aequorin, suggesting that the discrepancies are mainly due to the higher Ca(2+)-affinity of the fluorescent dyes used. We find rhod-5N has an apparent in situ intramitochondrial Kd around 0.5mM. Addition of Ca(2+) buffers containing between 4.5 and 10μM [Ca(2+)] to permeabilized cells loaded with rhod-5N induced increases in calibrated [Ca(2+)](M) up to the 100μM-1mM range, which were dependent on mitochondrial membrane potential. Ca(2+) release from mitochondria was largely dependent on [Na(+)]. We have then used rhod-5N loaded cells to investigate the [Ca(2+)](M) response to agonist stimulation at the single-cell and subcellular level. The [Ca(2+)](M) peaks induced by histamine varied by nearly 10-fold among different cells, with a mean about 25μM. In the presence of the Ca(2+) uniporter stimulator kaempferol, the [Ca(2+)](M) peaks induced by histamine were also highly variable, and the mean [Ca(2+)](M) peak was 3-fold higher. Simultaneous measurement of cytosolic and mitochondrial [Ca(2+)] peaks showed little correlation among the heights of the peaks in both compartments. Studying the [Ca(2+)](M) peaks at the subcellular level, we found significant heterogeneities among regions in the same cell. In particular, the [Ca(2+)](M) increase in mitochondrial regions close to the nucleus was more than double that of mitochondrial regions far from the nucleus.
Cell calcium 11/2011; 51(1):65-71. · 4.29 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Secretory 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 GH(3) 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.
Journal of Structural Biology 12/2010; 172(3):261-9. · 3.41 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We have investigated the dynamics of the free [Ca(2+)] inside the secretory granules of neurosecretory PC12 and INS1 cells using a low-Ca(2+)-affinity aequorin chimera fused to synaptobrevin-2. The steady-state secretory granule [Ca(2+)] ([Ca(2+)](SG)] was around 20-40 μM in both cell types, about half the values previously found in chromaffin cells. Inhibition of SERCA-type Ca(2+) pumps with thapsigargin largely blocked Ca(2+) uptake by the granules in Ca(2+)-depleted permeabilized cells, and the same effect was obtained when the perfusion medium lacked ATP. Consistently, the SERCA-type Ca(2+) pump inhibitor benzohydroquinone induced a rapid release of Ca(2+) from the granules both in intact and permeabilized cells, suggesting that the continuous activity of SERCA-type Ca(2+) pumps is essential to maintain the steady-state [Ca(2+)](SG). Both inositol 1,4,5-trisphosphate (InsP(3)) and caffeine produced a rapid Ca(2+) release from the granules, suggesting the presence of InsP(3) and ryanodine receptors in the granules. The response to high-K(+) depolarization was different in both cell types, a decrease in [Ca(2+)](SG) in PC12 cells and an increase in [Ca(2+)](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 [Ca(2+)](SG) in INS1 cells. In conclusion, our data show that the secretory granules of PC12 and INS1 cells take up Ca(2+) through SERCA-type Ca(2+) pumps and can release it through InsP(3) and ryanodine receptors, supporting the hypothesis that secretory granule Ca(2+) may be released during cell stimulation and contribute to secretion.
Cellular and Molecular Neurobiology 11/2010; 30(8):1267-74. · 1.97 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We have investigated the kinetics of mitochondrial Ca(2+) influx and efflux and their dependence on cytosolic [Ca(2+)] and [Na(+)] using low-Ca(2+)-affinity aequorin. The rate of Ca(2+) release from mitochondria increased linearly with mitochondrial [Ca(2+)] ([Ca(2+)](M)). Na(+)-dependent Ca(2+) release was predominant al low [Ca(2+)](M) but saturated at [Ca(2+)](M) around 400muM, while Na(+)-independent Ca(2+) release was very slow at [Ca(2+)](M) below 200muM, and then increased at higher [Ca(2+)](M), perhaps through the opening of a new pathway. Half-maximal activation of Na(+)-dependent Ca(2+) release occurred at 5-10mM [Na(+)], within the physiological range of cytosolic [Na(+)]. Ca(2+) entry rates were comparable in size to Ca(2+) exit rates at cytosolic [Ca(2+)] ([Ca(2+)](c)) below 7muM, but the rate of uptake was dramatically accelerated at higher [Ca(2+)](c). As a consequence, the presence of [Na(+)] considerably reduced the rate of [Ca(2+)](M) increase at [Ca(2+)](c) below 7muM, but its effect was hardly appreciable at 10muM [Ca(2+)](c). Exit rates were more dependent on the temperature than uptake rates, thus making the [Ca(2+)](M) transients to be much more prolonged at lower temperature. Our kinetic data suggest that mitochondria have little high affinity Ca(2+) buffering, and comparison of our results with data on total mitochondrial Ca(2+) fluxes indicate that the mitochondrial Ca(2+) bound/Ca(2+) free ratio is around 10- to 100-fold for most of the observed [Ca(2+)](M) range and suggest that massive phosphate precipitation can only occur when [Ca(2+)](M) reaches the millimolar range.
Biochimica et Biophysica Acta 10/2010; 1797(10):1727-35. · 4.66 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The dynamics of mitochondrial [Ca(2+)] ([Ca(2+)](M)) plays a key role in a variety of cellular processes. The most important methods available to monitor [Ca(2+)](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 Ca(2+)-sensitive proteins, aequorin and pericam. Our results show that measurements obtained with aequorin and pericam are consistent in terms of dynamic Ca(2+) changes. Instead, fluorescent dyes failed to follow Ca(2+) changes adequately, especially during repetitive stimulation. In particular, measures obtained with rhod-2 or rhod-FF evidenced the previously reported Ca(2+)-dependent inhibition of mitochondrial Ca(2+) 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 microM), 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 [Ca(2+)](M) data obtained with these dyes should be taken with care.
Cell calcium 07/2010; 48(1):61-9. · 4.29 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The 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 microM 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.
European Journal of Neuroscience 11/2008; 28(7):1265-74. · 3.63 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The endoplasmic reticulum (ER) has been posited as a potential anticancer target. The synthetic antitumor alkyl-lysophospholipid analogue edelfosine accumulates in the ER of solid tumor cells. This ER accumulation of the drug leads to the inhibition of phosphatidylcholine and protein synthesis, G(2)-M arrest, depletion of ER-stored Ca(2+), Bax up-regulation and activation, transcriptional factor growth arrest and DNA damage-inducible gene 153 up-regulation, caspase-4 and caspase-8 activation, and eventually to apoptosis. Edelfosine prompted ER stress apoptotic signaling, but not the survival unfolded protein response. Edelfosine also induced persistent c-Jun NH(2)-terminal kinase (JNK) activation. Gene transfer-mediated overexpression of apoptosis signal-regulating kinase 1, which plays a crucial role in ER stress, enhanced edelfosine-induced JNK activation and apoptosis. Inhibition of JNK, caspase-4, or caspase-8 activation diminished edelfosine-induced apoptosis. Edelfosine treatment led to the generation of the p20 caspase-8 cleavage fragment of BAP31, directing proapoptotic signals between the ER and the mitochondria. bax(-/-)bak(-/-) double-knockout cells fail to undergo edelfosine-induced ER-stored Ca(2+) release and apoptosis. Wild-type and bax(-/-)bak(-/-) cells showed similar patterns of phosphatidylcholine and protein synthesis inhibition, despite their differences in drug sensitivity. Thus, edelfosine-induced apoptosis is dependent on Bax/Bak-mediated ER-stored Ca(2+) release, but phosphatidylcholine and protein synthesis inhibition is not critical. Transfection-enforced expression of Bcl-X(L), which localizes specifically in mitochondria, prevented apoptosis without inhibiting ER-stored Ca(2+) release. These data reveal that edelfosine induces an ER stress response in solid tumor cells, providing novel insights into the edelfosine-mediated antitumor activity. Our data also indicate that mitochondria are indispensable for this edelfosine-induced cell death initiated by ER stress.
Cancer Research 12/2007; 67(21):10368-78. · 7.86 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Treatment of GH(3) pituitary cells with p-chloromercurybenzenesulfonate (PCMBS) increased the cytosolic Ca(2+) concentration ([Ca(2+)](i)). This effect was reversed by dithiothreitol and blocked by L-type Ca(2+) channel antagonists or Na(+) removal. PCMBS increased membrane conductance and depolarized the plasma membrane. Apart from minor effects on K(+) and Ca(2+) channels, PCMBS increased (6 times at -80 mV) an inward Na(+) current whose properties were similar to those of a background Na(+) conductance (BNC) described previously, necessary for generation of spontaneous electrical activity. In rat lactotropes and somatotropes in primary culture, PCMBS also produced a Na(+)-dependent [Ca(2+)](i) increase, whereas little or no effect was observed in thyrotropes, corticotropes, and gonadotropes. The Na(+) conductance elicited by PCMBS in somatotropes seemed to be the same as that stimulated by the hypothalamic growth hormone (GH)-releasing hormone, which regulates membrane excitability and GH secretion. The BNC studied here could play a physiological role, regulating excitability and spontaneous activity, and explains satisfactorily the [Ca(2+)](i)-increasing actions of the mercurials reported previously in several excitable tissues.
AJP Cell Physiology 05/2002; 282(4):C864-72. · 3.54 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Antitumor ether lipid 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3; edelfosine) induces apoptosis in cancer cells, sparing normal cells. We have found that the apoptotic action of ET-18-OCH3 required drug uptake and Fas in the target cell. Failure to accomplish one of these requirements prevents cell killing by the ether lipid. In human lymphoid leukemic cells, ET-18-OCH3 does not promote Fas or FasL expression and ET-18-OCH3-induced apoptosis is not inhibited by pre-incubation with an anti-Fas blocking antibody that abrogates cell killing mediated by Fas/FasL interactions. ET-18-OCH3-resistant normal human Fas-positive fibroblasts do not incorporate ET-18-OCH3, but undergo apoptosis upon ET-18-OCH3 microinjection. Murine fibroblasts L929 and L929-Fas, stably transfected with human Fas cDNA, do not incorporate ET-18-OCH3 and are resistant to its action when added exogenously. Microinjection of ET-18-OCH3 induces apoptosis in L929-Fas cells, but not in wild-type L929 cells. Confocal laser scanning microscopy shows that ET-18-OCH3 induces Fas clustering and capping during triggering of ET-18-OCH3-induced apoptosis. Microinjection-induced apoptosis and Fas clustering are specific for the molecular structure of ET-18-OCH3. Our data indicate that ET-18-OCH3 induces apoptosis via Fas after the ether lipid is inside the cell, and this Fas activation is independent of the interaction of Fas with its natural ligand FasL. This explains the selective action of ET-18-OCH3 on tumors since only cancer cells incorporate sufficient amounts of the drug. Int. J. Cancer 85:674–682, 2000. © 2000 Wiley-Liss, Inc.
International Journal of Cancer 02/2000; 85(5):674 - 682. · 5.44 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We have studied the effects of cytochrome P450 inhibitors on the entry of Ca2 and Mn2, used here as a Ca2 surrogate for Ca2 channels, in fura-2-loaded GB3 pituitary cells and bovine chromaffin cells depola- rized with high-K solutions. Imidazole antimycotics were potent inhibitors (econazole > miconazole > clotrimazole > ketoconazole). ot-Naphtoflavone and isosafrole, but not metyrapone, also inhibited the entry of Ca2 and Mn2 induced by depolarization. This inhibi- tory profile most resembles that reported for IA-type cytochrome P450. However, carbon monoxide (CO), a well-known cytochrome P450 antagonist, had no effect on Ca2 (Mn2) entry. Given the high selectivity of the imida- zole antimycotics for the heme moiety, our results suggest that a hemoprotein closely related to cytochrome P450 (but insensitive to CO) might be involved in the regula- tion of voltage-gated Ca2 channels. The inhibitory pat- tern was also similar to that previously reported for agonist-induced Ca2 (Mn2) influx in neutrophils and platelets, although CO was an efficient inhibitor in this case. These results pose the question of whether similari- ties in the sensitivity to cytochrome P450 inhibitors ex- hibited by receptor-operated and voltage-gated channels reflect unknown similarities either in structural features or regulation mechanisms.-Villalobos, C.; Fonteriz, R.
