Dynamics of mitochondrial [Ca2+] measured with the low-Ca2+-affinity dye rhod-5N (original) (raw)
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Simultaneous Measurements of Cytosolic and Mitochondrial Ca2+ Transients in HT29 Cells
Journal of Biological Chemistry, 1998
Loading of HT 29 cells with the Ca 2؉ dye fura-2/AM resulted in an nonhomogeneous intracellular distribution of the dye. Cellular compartments with high fura-2 concentrations were identified by correlation with mitochondrial markers, cellular autofluorescence induced by UV, and dynamic measurement of autofluorescence after inhibition of oxidative phosphorylation. Stimulation with carbachol (10 ؊4 mol/liter) increased cytosolic, nuclear, and mitochondrial Ca 2؉ activity ([Ca 2؉ ] c , [Ca 2؉ ] n , and [Ca 2؉ ] m , respectively) measured by UV confocal and conventional imaging. Similar results were obtained with a prototype two-photon microscope (Zeiss, Jena, Germany) allowing for fura-2 excitation. The increase of [Ca 2؉ ] m lagged behind that of [Ca 2؉ ] c and [Ca 2؉ ] n by 10-20 s, and after removing the agonist, [Ca 2؉ ] m also decreased with a delay. A strong increase of [Ca 2؉ ] m occurred only when a certain threshold of [Ca 2؉ ] c (around 1 mol/liter) was exceeded. In a very similar way, ATP, neurotensin, and thapsigargin increased [Ca 2؉ ] c and [Ca 2؉ ] m. Carbonyl cyanide p-trifluoromethoxyphenylhyrdrazone reversibly reduced the increase of [Ca 2؉ ] m. The source of the mitochondrial Ca 2؉ increase had intra-and extracellular components, as revealed by experiments in low extracellular Ca 2؉. We conclude that agonist-induced Ca 2؉ signals are transduced into mitochondria. 1) Mitochondria could serve as a Ca 2؉ sink, 2) mitochondria could allow the modulation of [Ca 2؉ ] c and [Ca 2؉ ] n signals, and 3) [Ca 2؉ ] m may serve as a stimulatory metabolic signal when a cell is highly stimulated.
Mitochondrial Ca2+ concentrations in live cells: quantification methods and discrepancies
FEBS Letters, 2019
Intracellular Ca2+ signaling controls numerous cellular functions. Mitochondria respond to cytosolic Ca2+ changes by adapting mitochondrial functions and, in some cell types, shaping the spatiotemporal properties of the cytosolic Ca2+ signal. Numerous methods have been developed to specifically and quantitatively measure the mitochondrial‐free Ca2+ concentrations ([Ca2+]m), but there are still significant discrepancies in the calculated absolute values of [Ca2+]m in stimulated live cells. These discrepancies may be due to the distinct properties of the methods used to measure [Ca2+]m, the calcium‐free/bound ratio, and the cell‐type and stimulus‐dependent Ca2+ dynamics. Critical processes happening in the mitochondria, such as ATP generation, ROS homeostasis, and mitochondrial permeability transition opening, depend directly on the [Ca2+]m values. Thus, precise determination of absolute [Ca2+]m values is imperative for understanding Ca2+ signaling. This review summarizes the reported...
Mitochondrial Participation in the Intracellular Ca2+ Network
The Journal of Cell Biology, 1997
Calcium can activate mitochondrial metabolism, and the possibility that mitochondrial Ca2+ uptake and extrusion modulate free cytosolic [Ca2+] (Cac) now has renewed interest. We use whole-cell and perforated patch clamp methods together with rapid local perfusion to introduce probes and inhibitors to rat chromaffin cells, to evoke Ca2+ entry, and to monitor Ca2+-activated currents that report near-surface [Ca2+]. We show that rapid recovery from elevations of Cac requires both the mitochondrial Ca2+ uniporter and the mitochondrial energization that drives Ca2+ uptake through it. Applying imaging and single-cell photometric methods, we find that the probe rhod-2 selectively localizes to mitochondria and uses its responses to quantify mitochondrial free [Ca2+] (Cam). The indicated resting Cam of 100–200 nM is similar to the resting Cac reported by the probes indo-1 and Calcium Green, or its dextran conjugate in the cytoplasm. Simultaneous monitoring of Cam and Cac at high temporal res...
Journal of Biological Chemistry, 2003
Mitochondria have been found to sequester and release Ca 2؉ during cell stimulation with inositol 1,4,5triphosphate-generating agonists, thereby generating subplasmalemmal microdomains of low Ca 2؉ that sustain activity of capacitative Ca 2؉ entry (CCE). Procedures that prevent mitochondrial Ca 2؉ uptake inhibit local Ca 2؉ buffering and CCE, but it is not clear whether Ca 2؉ has to transit through or remains trapped in the mitochondria. Thus, we analyzed the contribution of mitochondrial Ca 2؉ efflux on the ability of mitochondria to buffer subplasmalemmal Ca 2؉ , to maintain CCE, and to facilitate endoplasmic reticulum (ER) refilling in endothelial cells. Upon the addition of histamine, the initial mitochondrial Ca 2؉ transient, monitored with ratiometric-pericam-mitochondria, was largely independent of extracellular Ca 2؉. However, subsequent removal of extracellular Ca 2؉ produced a reversible decrease in [Ca 2؉ ] mito , indicating that Ca 2؉ was continuously taken up and released by mitochondria, although [Ca 2؉ ] mito had returned to basal levels. Accordingly, inhibition of the mitochondrial Na ؉ /Ca 2؉ exchanger with CGP 37157 increased [Ca 2؉ ] mito and abolished the ability of mitochondria to buffer subplasmalemmal Ca 2؉ , resulting in an increased activity of BK Ca channels and a decrease in CCE. Hence, CGP 37157 also reversibly inhibited ER refilling during cell stimulation. These effects of CGP 37157 were mimicked if mitochondrial Ca 2؉ uptake was prevented with oligomycin/antimycin A. Thus, during cell stimulation a continuous Ca 2؉ flux through mitochondria underlies the ability of mitochondria to generate subplasmalemmal microdomains of low Ca 2؉ , to facilitate CCE, and to relay Ca 2؉ from the plasma membrane to the ER.
Mitochondria and Ca2+ in cell physiology and pathophysiology
Cell Calcium, 2000
There is now a consensus that mitochondria take up and accumulate Ca 2; during physiological [Ca 2; ] c signalling. This contribution will consider some of the functional consequences of mitochondrial Ca 2; uptake for cell physiology and pathophysiology. The ability to remove Ca 2; from local cytosol enables mitochondria to regulate the [Ca 2; ] in microdomains close to IP3-sensitive Ca 2; -release channels. The [Ca 2; ] sensitivity of these channels means that, by regulating local [Ca 2; ] c , mitochondrial Ca 2; uptake modulates the rate and extent of propagation of [Ca 2; ] c waves in a variety of cell types. The coincidence of mitochondrial Ca 2; uptake with oxidative stress may open the mitochondrial permeability transition pore (mPTP). This is a catastrophic event for the cell that will initiate pathways to cell death either by necrotic or apoptotic pathways. A model is presented in which illumination of an intramitochondrial fluorophore is used to generate oxygen radical species within mitochondria. This causes mitochondrial Ca 2; loading from SR and triggers mPTP opening. In cardiomyocytes, mPTP opening leads to ATP consumption by the mitochondrial ATPase and so results in ATP depletion, rigor and necrotic cell death. In central mammalian neurons exposed to glutamate, a cellular Ca 2; overload coincident with NO production also causes loss of mitochondrial potential and cell death, but mPTP involvement has proven more difficult to demonstrate unequivocally. A cartoon to illustrate the features of the mitochondrial permeability transition pore and its pharmacology. The mPTP seems to consist of an association between the adenine nucleotide translocase (ANT), the voltage dependent anion channel (VDAC) of the outer mitochondrial membrane and cyclophilin D (Cyp D). Opening of the pore is modulated primarily by [Ca 2; ] m , ROS, NO, many sulphydryl reagents and by bongrekate or atractyloside, amongst many agents and conditions (from Duchen, 2000, with permission).
Molecular Cell, 2010
Although it is widely accepted that mitochondria in living cells can efficiently uptake Ca 2+ during stimulation because of their vicinity to microdomains of high [Ca 2+ ], the direct proof of Ca 2+ hot spots' existence is still lacking. Thanks to a GFP-based Ca 2+ probe localized on the cytosolic surface of the outer mitochondrial membrane, we demonstrate that, upon Ca 2+ mobilization, the [Ca 2+ ] in small regions of the mitochondrial surface reaches levels 5-to 10-fold higher than in the bulk cytosol. We also show that the [Ca 2+ ] to which mitochondria are exposed during capacitative Ca 2+ influx is similar between near plasma membrane mitochondria and organelles deeply located in the cytoplasm, whereas it is 2-to 3-fold higher in subplasma membrane mitochondria upon activation of voltage-gated Ca 2+ channels. These results demonstrate that mitochondria are exposed to Ca 2+ hot spots close to the ER but are excluded from the regions where capacitative Ca 2+ influx occurs.
Mitochondrial Ca2+ homeostasis: mechanism, role, and tissue specificities
Pflügers Archiv - European Journal of Physiology, 2012
Mitochondria from every tissue are quite similar in their capability to accumulate Ca 2+ in a process that depends on the electrical potential across the inner membrane; it is catalyzed by a gated channel (named mitochondrial Ca 2+ uniporter), the molecular identity of which has only recently been unraveled. The release of accumulated Ca 2+ in mitochondria from different tissues is, on the contrary, quite variable, both in terms of speed and mechanism: a Na +-dependent efflux in excitable cells (catalyzed by NCLX) and a H + /Ca 2+ exchanger in other cells. The efficacy of mitochondrial Ca 2+ uptake in living cells is strictly dependent on the topological arrangement of the organelles with respect to the source of Ca 2+ flowing into the cytoplasm, i.e., plasma membrane or intracellular channels. In turn, the structural and functional relationships between mitochondria and other cellular membranes are dictated by the specific architecture of different cells. Mitochondria not only modulate the amplitude and the kinetics of local and bulk cytoplasmic Ca 2+ changes but also depend on the Ca 2+ signal for their own functionality, in particular for their capacity to produce ATP. In this review, we summarize the processes involved in mitochondrial Ca 2+ handling and its integration in cell physiology, highlighting the main common characteristics as well as key differences, in different tissues. Keywords Mitochondria. Ca 2+. MCU. NCLX This article is published as part of the special issue on "Cell-specific roles of mitochondrial Ca 2+ handling."
Nature Cell Biology, 1999
Activation of calcium-ion (Ca 2+ ) channels on the plasma membrane and on intracellular Ca 2+ stores, such as the endoplasmic reticulum, generates local transient increases in the cytosolic Ca 2+ concentration that induce Ca 2+ uptake by neighbouring mitochondria. Here, by using mitochondrially targeted aequorin proteins with different Ca 2+ affinities, we show that half of the chromaffin-cell mitochondria exhibit surprisingly rapid millimolar Ca 2+ transients upon stimulation of cells with acetylcholine, caffeine or high concentrations of potassium ions. Our results show a tight functional coupling of voltage-dependent Ca 2+ channels on the plasma membrane, ryanodine receptors on the endoplasmic reticulum, and mitochondria. Cell stimulation generates localized Ca 2+ transients, with Ca 2+ concentrations above 20-40 µM, at these functional units. Protonophores abolish mitochondrial Ca 2+ uptake and increase stimulated secretion of catecholamines by three-to fivefold. These results indicate that mitochondria modulate secretion by controlling the availability of Ca 2+ for exocytosis.
Measurements of mitochondrial calcium in vivo
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2009
Mitochondria play a pivotal role in intracellular Ca(2+) signalling by taking up and releasing the ion upon specific conditions. In order to do so, mitochondria depend on a number of factors, such as the mitochondrial membrane potential and spatio-temporal constraints. Whereas most of the basic principles underlying mitochondrial Ca(2+) handling have been successfully deciphered over the last 50 years using assays based on in vitro preparations of mitochondria or cultured cells, we have only just started to understand the actual physiological relevance of these processes in the whole animal. Recent advancements in imaging and genetically encoded sensor technologies have allowed us to visualise mitochondrial Ca(2+) transients in live mice. These studies used either two-photon microscopy or bioluminescence imaging of cameleon or aequorin-GFP Ca(2+) sensors, respectively. Both methods revealed a consistent picture of Ca(2+) uptake into mitochondria under physiological conditions even during very short-lasting elevations of cytosolic Ca(2+) levels. The big future challenge is to understand the functional impact of such Ca(2+) signals on the physiology of the observed tissue as well as of the whole organism. To that end, the development of multiparametric in vivo approaches will be mandatory.