The dynamics of mitochondrial Ca fluxes

We have investigated the kinetics of mitochondrial Ca²⁺ influx and efflux and their dependence on cytosolic [Ca²⁺] and [Na⁺] using low-Ca²⁺-affinity aequorin. The rate of Ca²⁺ release from mitochondria increased linearly with mitochondrial [Ca²⁺] ([Ca²⁺]_M). Na⁺-dependent Ca²⁺ release was predominant al low [Ca²⁺]_M but saturated at [Ca²⁺]_M around 400 μM, while Na⁺-independent Ca²⁺ release was very slow at [Ca²⁺]_M below 200 μM, and then increased at higher [Ca²⁺]_M, perhaps through the opening of a new pathway. Half-maximal activation of Na⁺-dependent Ca²⁺ release occurred at 5-10 mM [Na⁺], within the physiological range of cytosolic [Na⁺]. Ca²⁺ entry rates were comparable in size to Ca²⁺ exit rates at cytosolic [Ca²⁺] ([Ca²⁺]_c) below 7 μM, but the rate of uptake was dramatically accelerated at higher [Ca²⁺]_c. As a consequence, the presence of [Na⁺] considerably reduced the rate of [Ca²⁺]_M increase at [Ca²⁺]_c below 7 μM, but its effect was hardly appreciable at 10 μM [Ca²⁺]_c. Exit rates were more dependent on the temperature than uptake rates, thus making the [Ca²⁺]_M transients to be much more prolonged at lower temperature. Our kinetic data suggest that mitochondria have little high affinity Ca²⁺ buffering, and comparison of our results with data on total mitochondrial Ca²⁺ fluxes indicate that the mitochondrial Ca²⁺ bound/Ca²⁺ free ratio is around 10- to 100-fold for most of the observed [Ca²⁺]_M range and suggest that massive phosphate precipitation can only occur when [Ca²⁺]_M reaches the millimolar range.     

Mitochondrial regulation of [Ca2+]i oscillations during cell cycle resumption of the second meiosis of oocyte

Oocyte is arrested at metaphase of the second meiosis until fertilization switching on [Ca²⁺]i oscillations. Oocyte activation inefficiency is the most challenging problem for failed fertilization and embryonic development. Mitochondrial function and intracellular [Ca²⁺]i oscillations are two critical factors for the oocyte’s developmental potential. We aimed to understand the possible correlation between mitochondrial function and [Ca²⁺]i oscillations in oocytes. To this end, mitochondrial uncoupler CCCP which damages mitochondrial function and two small molecule mitochondrial agonists, L-carnitine (LC) and BGP-15, were used to examine the regulation of [Ca²⁺]i by mitochondrial functions. With increasing CCCP concentrations, [Ca²⁺]i oscillations were gradually diminished and high concentrations of CCCP led to oocyte death. LC enhanced mitochondrial membrane potential and [Ca²⁺]i oscillations and even improved the damage induced by CCCP, however, BGP-15 had no beneficial effect on oocyte activation. We have found that mitochondrial function plays a vital role in the generation of [Ca²⁺]i oscillations in oocytes, and thus mitochondria may interact with the ER to generate [Ca²⁺]i oscillations during oocyte activation. Improvement of mitochondrial functions with small molecules can be expected to improve oocyte activation and embryonic development in infertile patients without invasive micromanipulation.     

Oscillations of cytosolic free calcium in bombesin-stimulated HIT-T15 cells

The mechanism underlying the generation of cytosolic free Ca²⁺ ([Ca²⁺_i) oscillations by bombesin, a receptor agonist activating phospholipase C, in insulin secreting HIT-T15 cells was investigated. At 25 μM, 61% of cells displayed [Ca²⁺]_i oscillations with variable patterns. The bombesin-induced [Ca²⁺]_i oscillations could last more than 1 h and glucose was required for maintaining these [Ca²⁺ fluctuations. Bombesin-evoked [Ca²⁺]_i oscillations were dependent on extracellular Ca²⁺ entry and were attenuated by membrane hype rpolarization or by L-type Ca²⁺ channel blockers. These [Ca²⁺]_i oscillations were apparently not associated with fluctuations in plasma membrane Ca²⁺ permeability as monitored by the Mn²⁺ quenching technique. 2,5-di-(tert-butyl)-1,4-benzohydroquinone (tBuBHQ) and 4-chloro-m-cresol, which interfere with intracellular Ca²⁺ stores, respectively, by inhibiting Ca²⁺-ATPase of endoplasmic reticulum and by affecting Ca²⁺-induced Ca²⁺ release, disrupted bombesin-induced [Ca²⁺]_i oscillations. 4-chloro-m-resol raised [Ca²⁺]_i by mobilizing an intracellular Ca²⁺ pool, an effect not altered by ryanodine. Caffeine exerted complex actions on [Ca²⁺]_i It raised [Ca²⁺]_i by promoting Ca²⁺ entry while inhibiting bombesin-elicited [Ca²⁺]_i oscillations. Our results suggest that in bombesin-elicited [Ca²⁺]_i oscillations in HIT-T15 cells: (i) the oscillations originate primarily from intracellular Ca²⁺ stores; and (ii) the Ca²⁺ influx required for maintaining the oscillations is in part membrane potential-sensitive and not coordinated with [Ca²⁺]_i oscillations. The interplay between intracellular Ca²⁺ stores and voltage-sensitive and voltage-insensitive extracellular Ca²⁺ entry determines the [Ca²⁺]_i oscillations evoked by bombesin.     

Modulation of calcium signalling by mitochondria

In this review we will attempt to summarise the complex and sometimes contradictory effects that mitochondria have on different forms of calcium signalling. Mitochondria can influence Ca²⁺ signalling indirectly by changing the concentration of ATP, NAD(P)H, pyruvate and reactive oxygen species — which in turn modulate components of the Ca²⁺ signalling machinery i.e. buffering, release from internal stores, influx from the extracellular solution, uptake into cellular organelles and extrusion by plasma membrane Ca²⁺ pumps. Mitochondria can directly influence the calcium concentration in the cytosol of the cell by importing Ca²⁺ via the mitochondrial Ca²⁺ uniporter or transporting Ca²⁺ from the interior of the organelle into the cytosol by means of Na⁺/Ca²⁺ or H⁺/Ca²⁺ exchangers. Considerable progress in understanding the relationship between Ca²⁺ signalling cascades and mitochondrial physiology has been accumulated over the last few years due to the development of more advanced optical techniques and electrophysiological approaches.     

Mitochondrial inhibitors activate influx of external Ca in sea urchin sperm

Sea urchin sperm have a single mitochondrion which, aside from its main ATP generating function, may regulate motility, intracellular Ca²⁺ concentration ([Ca²⁺]_i) and possibly the acrosome reaction (AR). We have found that acute application of agents that inhibit mitochondrial function via differing mechanisms (CCCP, a proton gradient uncoupler, antimycin, a respiratory chain inhibitor, oligomycin, a mitochondrial ATPase inhibitor and CGP37157, a Na⁺/Ca²⁺ exchange inhibitor) increases [Ca²⁺]_i with at least two differing profiles. These increases depend on the presence of extracellular Ca²⁺, which indicates they involve Ca²⁺ uptake and not only mitochondrial Ca²⁺ release. The plasma membrane permeation pathways activated by the mitochondrial inhibitors are permeable to Mn²⁺. Store-operated Ca²⁺ channel (SOC) blockers (Ni²⁺, SKF96365 and Gd²⁺) and internal-store ATPase inhibitors (thapsigargin and bisphenol) antagonize Ca²⁺ influx induced by the mitochondrial inhibitors. The results indicate that the functional status of the sea urchin sperm mitochondrion regulates Ca²⁺ entry through SOCs. As neither CCCP nor dicycloexyl carbodiimide (DCCD), another mitochondrial ATPase inhibitor, eliminate the oligomycin induced increase in [Ca²⁺]_i, apparently oligomycin also has an extra mitochondrial target.     

Calcium signaling in pancreatic β-cells in health and in Type 2 diabetes

Changes in cytosolic free Ca²⁺ concentration ([Ca²⁺]_c) play a crucial role in the control of insulin secretion from the electrically excitable pancreatic β-cell. Secretion is controlled by the finely tuned balance between Ca²⁺ influx (mainly through voltage-dependent Ca²⁺ channels, but also through voltage-independent Ca²⁺ channels like store-operated channels) and efflux pathways. Changes in [Ca²⁺]_c directly affect [Ca²⁺] in various organelles including the endoplasmic reticulum (ER), mitochondria, the Golgi apparatus, secretory granules and lysosomes, as imaged using recombinant targeted probes. Because most of these organelles have specific Ca²⁺ influx and efflux pathways, they mutually influence free [Ca²⁺] in the others. In this article, we review the mechanisms of control of [Ca²⁺] in various compartments and particularly the cytosol, the endoplasmic reticulum ([Ca²⁺]_ER), acidic stores and mitochondrial matrix ([Ca²⁺]_mito), focusing chiefly on the most important physiological stimulus of β-cells, glucose. We also briefly review some alterations of β-cell Ca²⁺ homeostasis in Type 2 diabetes.     

9-Phenanthrol and flufenamic acid inhibit calcium oscillations in HL-1 mouse cardiomyocytes

It is well established that intracellular calcium ([Ca²⁺]_i) controls the inotropic state of the myocardium, and evidence mounts that a “Ca²⁺ clock” controls the chronotropic state of the heart. Recent findings describe a calcium-activated nonselective cation channel (NSC_Ca) in various cardiac preparations sharing hallmark characteristics of the transient receptor potential melastatin 4 (TRPM4). TRPM4 is functionally expressed throughout the heart and has been implicated as a NSC_Ca that mediates membrane depolarization. However, the functional significance of TRPM4 in regards to Ca²⁺ signaling and its effects on cellular excitability and pacemaker function remains inconclusive. Here, we show by Fura2 Ca-imaging that pharmacological inhibition of TRPM4 in HL-1 mouse cardiac myocytes by 9-phenanthrol (10 μM) and flufenamic acid (10 and 100 μM) decreases Ca²⁺ oscillations followed by an overall increase in [Ca²⁺]_i. The latter occurs also in HL-1 cells in Ca²⁺-free solution and after depletion of sarcoplasmic reticulum Ca²⁺ with thapsigargin (10 μM). These pharmacologic agents also depolarize HL-1 cell mitochondrial membrane potential. Furthermore, by on-cell voltage clamp we show that 9-phenanthrol reversibly inhibits membrane current; by fluorescence immunohistochemistry we demonstrate that HL-1 cells display punctate surface labeling with TRPM4 antibody; and by immunoblotting using this antibody we show these cells express a 130–150 kDa protein, as expected for TRPM4. We conclude that 9-phenanthrol inhibits TRPM4 ion channels in HL-1 cells, which in turn decreases Ca²⁺ oscillations followed by a compensatory increase in [Ca²⁺]_i from an intracellular store other than the sarcoplasmic reticulum. We speculate that the most likely source is the mitochondrion.     

Cold stress increases reactive oxygen species formation via TRPA1 activation in A549 cells

Reactive oxygen species (ROS) are responsible for lung damage during inhalation of cold air. However, the mechanism of the ROS production induced by cold stress in the lung is still unclear. In this work, we measured the changes of ROS and the cytosolic Ca²⁺ concentration ([Ca²⁺]_c) in A549 cell. We observed that cold stress (from 20 to 5 °C) exposure of A549 cell resulted in an increase of ROS and [Ca²⁺]_c, which was completely attenuated by removing Ca²⁺ from medium. Further experiments showed that cold-sensing transient receptor potential subfamily member 1 (TRPA1) agonist (allyl isothiocyanate, AITC) increased the production of ROS and the level of [Ca²⁺]_c in A549 cell. Moreover, HC-030031, a TRPA1 selective antagonist, significantly inhibited the enhanced ROS and [Ca²⁺]_c induced by AITC or cold stimulation, respectively. Taken together, these data demonstrated that TRPA1 activation played an important role in the enhanced production of ROS induced by cold stress in A549 cell.     

Spatial and temporal patterns of intracellular calcium in colonic smooth muscle

Summary Intracellular calcium [Ca²⁺] _i measurements in cell suspension of gastrointestinal myocytes have suggested a single [Ca²⁺] _i transient followed by a steady-state increase as the characteristic [Ca²⁺] _i response of these cells. In the present study, we used digital video imaging techniques in freshly dispersed myocytes from the rabbit colon, to characterize the spatiotemporal pattern of the [Ca²⁺] _i signal in single cells. The distribution of [Ca²⁺] _i in resting and stimulated cells was nonhomogeneous, with gradients of high [Ca²⁺] _i present in the subplasmalemmal space and in one cell pole. [Ca²⁺] _i gradients within these regions were not constant but showed temporal changes in the form of [Ca²⁺] _i oscillations and spatial changes in the form of [Ca²⁺] _i waves. [Ca²⁺] _i oscillations in unstimulated cells (n = 60) were independent of extracellular [Ca²⁺] and had a mean frequency of 12.6 +1.1 oscillations per min. The baseline [Ca²⁺], was 171 ± 13 nm and the mean oscillation amplitude was 194 ± 12 nm. Generation of [Ca²⁺] _i waves was also independent of influx of extracellular Ca²⁺. [Ca²⁺] _i waves originated in one cell pole and were visualized as propagation mostly along the subplasmalemmal space or occasionally throughout the cytoplasm. The mean velocity was 23 +3 m per sec (n = 6). Increases of [Ca²⁺] _i induced by different agonists were encoded into changes of baseline [Ca²⁺] _i and the amplitude of oscillations, but not into their frequency. The observed spatiotemporal pattern of [Ca²⁺] _i regulation may be the underlying mechanism for slow wave generation and propagation in this tissue. These findings are consistent with a [Ca²⁺] _i regulation whereby cell regulators modulate the spatiotemporal pattern of intracellularly generated [Ca²⁺] _i oscillations.The authors thank Debbie Anderson for excellent technical assistance with the electron microscopy and Dr. M. Regoli for providing the NK-1 agonist [Sar⁹,Met(O₂)¹¹]-SP. This work was supported by National Institutes of Health Grants DK 40919 and DK 40675 and Veterans Administration Grant SMI.     

Tunicamycin desensitizes store-operated Ca entry to ATP and mitochondrial potential

Tunicamycin effect on thapsigargin-induced store-operated calcium entry was investigated. Ca²⁺ influx was stimulated by 50% upon exposure of Jurkat cells to tunicamycin. Moreover, tunicamycin efficiently prevented the inhibition of store-operated calcium entry caused by dissipation of mitochondrial membrane potential. Protective action of tunicamycin on store-operated Ca²⁺ entry was also partially preserved in Jurkat cells depleted of ATP, while Ca²⁺ entry into ATP-deprived cells grown in tunicamycin-free medium was almost completely inhibited. Tunicamycin-evoked changes in cellular Ca²⁺ fluxes coincided with decreased glycosylation of STIM1 protein. Although the latter observation is correlative and needs additional confirmation it may suggest that deglycosylation of STIM1 protein deprives store-operated calcium entry system of an important regulatory mechanism. This study suggests a novel mechanism of modulation of the activity of store-operated calcium channels in lymphoidal cells.     

Age-dependent changes in diastolic Ca and Na concentrations in dystrophic cardiomyopathy: Role of Ca entry and IP3

Duchenne muscular dystrophy (DMD) is a lethal X-inherited disease caused by dystrophin deficiency. Besides the relatively well characterized skeletal muscle degenerative processes, DMD is also associated with a dilated cardiomyopathy that leads to progressive heart failure at the end of the second decade. The aim of the present study was to characterize the diastolic Ca²⁺ concentration ([Ca²⁺]_d) and diastolic Na⁺ concentration ([Na⁺]_d) abnormalities in cardiomyocytes isolated from 3-, 6-, 9-, and 12-month old mdx mice using ion-selective microelectrodes. In addition, the contributions of gadolinium (Gd³⁺)-sensitive Ca²⁺ entry and inositol triphosphate (IP₃) signaling pathways in abnormal [Ca²⁺]_d and [Na⁺]_d were investigated. Our results showed an age-dependent increase in both [Ca²⁺]_d and [Na⁺]_d in dystrophic cardiomyocytes compared to those isolated from age-matched wt mice. Gd³⁺ treatment significantly reduced both [Ca²⁺]_d and [Na⁺]_d at all ages. In addition, blockade of the IP₃-pathway with either U-73122 or xestospongin C significantly reduced ion concentrations in dystrophic cardiomyocytes. Co-treatment with U-73122 and Gd³⁺ normalized both [Ca²⁺]_d and [Na⁺]_d at all ages in dystrophic cardiomyocytes. These data showed that loss of dystrophin in mdx cardiomyocytes produced an age-dependent intracellular Ca²⁺ and Na⁺ overload mediated at least in part by enhanced Ca²⁺ entry through Gd³⁺ sensitive transient receptor potential channels (TRPC), and by IP₃ receptors.     

ATP-Induced Oscillations of Cytosolic Ca2+ Activity in Cultured Astrocytes from Rat Brain are Modulated by Medium Osmolarity Indicating a Control of [Ca2+]i Oscillations by Cell Volume

Oscillations of cytosolic Ca²⁺ activity ([Ca²⁺]_i) induced by stimulation with ATP in rat astrocytes in primary cultures were analysed. Astrocytes, prepared from the brains of newborn rats, loaded with the fluorescent Ca²⁺ indicator fura-2/AM, were continuously stimulated with ATP (10 M). ATP caused a large initial [Ca²⁺ peak, followed by regular [Ca²⁺]_i oscillations (frequencies 1–5/min). Astrocytes were identified by glial fibrillary acidic protein staining of cells after [Ca²⁺]_i recording. The oscillations were reversibly blocked by the P₂ purinoceptor antagonist suramin (30 M). Influx of extracellular Ca²⁺ and mobilization of Ca²⁺ from intracellular stores both contributed to the oscillations. The effects of hypertonic and hypotonic superfusion medium on ATP-induced [Ca²⁺]_i oscillations were examined. Hypertonic medium (430 mOsm) reversibly suppressed the ATP-induced oscillations. Hypotonic medium (250 mOsm), in spite of having heterogeneous effects, most frequently induced a rise in [Ca²⁺]_i, or reversibly increased the frequency of the oscillations. Thus, a change in cell volume might be closely connected with [Ca²⁺]_i oscillations in astrocytes indicating that [Ca²⁺]_i oscillations in glial cells play an important role in regulatory volume regulation in the brain.     

Differential regulation of intracellular calcium oscillations by mitochondria and gap junctions

Fluctuations of intracellular Ca²⁺ ([Ca²⁺]_i) regulate a variety of cellular functions. The classical Ca²⁺ transport pathways in the endoplasmic reticulum (ER) and plasma membrane are essential to [Ca²⁺]_i oscillations. Although mitochondria have recently been shown to absorb and release Ca²⁺ during G protein-coupled receptor (GPCR) activation, the role of mitochondria in [Ca²⁺]_i oscillations remains to be elucidated. Using fluo-3-loaded human teratocarcinoma NT2 cells, we investigated the regulation of [Ca²⁺]_i oscillations by mitochondria. Both the muscarinic GPCR agonist carbachol and the ER Ca²⁺-adenosine triphosphate inhibitor thapsigargin (Tg) induced [Ca²⁺]_i oscillations in NT2 cells. The [Ca²⁺]_i oscillations induced by carbachol were unsynchronized among individual NT2 cells; in contrast, Tg-induced oscillations were synchronized. Inhibition of mitochondrial functions with either mitochondrial blockers or depletion of mitochondrial DNA eliminated carbachol—but not Tg-induced [Ca²⁺]_i oscillations. Furthermore, carbachol-induced [Ca²⁺]_i oscillations were partially restored to mitochondrial DNA-depleted NT2 cells by introduction of exogenous mitochondria. Treatment of NT2 cells with gap junction blockers prevented Tg-induced but not carbachol-induced [Ca²⁺]_i oscillations. These data suggest that the distinct patterns of [Ca²⁺]_i oscillations induced by GPCR and Tg are differentially modulated by mitochondria and gap junctions.     

Salt stress-induced programmed cell death via Ca<Superscript>2+</Superscript>-mediated mitochondrial permeability transition in tobacco protoplasts

The change in cytosolic free concentration of calcium ([Ca²⁺]_cyt) plays a key role in regulating apoptosis in animal cells. In our experiment, we tried to investigate the function of Ca²⁺ in programmed cell death (PCD) in tobacco (Nicotiana tobacum, cultivar BY-2) protoplasts induced by salt stress. An obvious increase in [Ca²⁺]_cyt was observed a few minutes after treatment and the onset of a decrease in mitochondrial membrane potential (ΔΨ_m) was also observed before the appearance of PCD, pre-treatment of protoplasts with EGTA or LaCl₃ effectively retarded the increase in [Ca²⁺]_cyt, which was concomitant with the decrease in the percentage of cell death and higher ΔΨ_m, pre-treatment with cyclosporine A (CsA) also effectively retarded the increase in [Ca²⁺]_cyt, the decrease in ΔΨ_m and the onset of PCD. All these results suggest that Ca²⁺ is a necessary element in regulating PCD and the increase in [Ca²⁺]_cyt and the opening of mitochondrial permeability transition pore (MPTP) could promote each other in regulating PCD in tobacco protoplasts induced by salt stress.Jiusheng Lin and Yuan Wang-These authors contributed equally for this work.     

Cardiomyogenesis of embryonic stem cells upon purinergic receptor activation by ADP and ATP

Purinergic signaling may be involved in embryonic development of the heart. In the present study, the effects of purinergic receptor stimulation on cardiomyogenesis of mouse embryonic stem (ES) cells were investigated. ADP or ATP increased the number of cardiac clusters and cardiac cells, as well as beating frequency. Cardiac-specific genes showed enhanced expression of α-MHC, MLC2v, α-actinin, connexin 45 (Cx45), and HCN4, on both gene and protein levels upon ADP/ATP treatment, indicating increased cardiomyogenesis and pacemaker cell differentiation. Real-time RT-PCR analysis of purinergic receptor expression demonstrated presence of P2X1, P2X4, P2X6, P2X7, P2Y1, P2Y2, P2Y4, and P2Y6 on differentiating ES cells. ATP and ADP as well as the P2X agonists β,γ-methylenadenosine 5′-triphosphate (β,γ-MetATP) and 8-bromoadenosine 5′-triphosphate (8-Br-ATP) but not UTP or UDP transiently increased the intracellular calcium concentration ([Ca²⁺]_i) as evaluated by the calcium indicator Fluo-4, whereas no changes in membrane potential were observed. [Ca²⁺]_i transients induced by ADP/ATP were abolished by the phospholipase C-β (PLC-β) inhibitor U-73122, suggesting involvement of metabotropic P2Y receptors. Furthermore, partial inhibition of [Ca²⁺]_i transients was achieved in presence of MRS2179, a selective P2Y1 receptor antagonist, whereas PPADS, a non-selective P2 receptor inhibitor, completely abolished the [Ca²⁺]_i response. Consequently, cardiomyocyte differentiation was decreased upon long term co-incubation of cells with ADP and P2 receptor antagonists. In summary, activation of purinoceptors and the subsequent [Ca²⁺]_i transients enhance the differentiation of ES cells toward cardiomyocytes. Purinergic receptor stimulation may be a promising strategy to drive the fate of pluripotent ES cells into a particular population of cardiomyocytes.

Electronic supplementary material

The online version of this article (doi:10.1007/s11302-015-9468-1) contains supplementary material, which is available to authorized users.     

Mitochondria impairment correlates with increased sensitivity of aging RPE cells to oxidative stress

Impairment of mitochondria function and cellular antioxidant systems are linked to aging and neurodegenerative diseases. In the eye, the retinal pigment epithelium (RPE) is exposed to a highly oxidative environment that contributes to age-related visual dysfunction. Here, we examined changes in mitochondrial function in human RPE cells and sensitivity to oxidative stress with increased chronological age. Primary RPE cells from young (9–20)-, mid-age (48–60)-, and >60 (62–76)-year-old donors were grown to confluency and examined by electron microscopy and flow cytometry using several mitochondrial functional assessment tools. Susceptibility of RPE cells to H₂O₂ toxicity was determined by lactate dehydrogenase and cytochrome c release, as well as propidium iodide staining. Reactive oxygen species, cytoplasmic Ca²⁺ [Ca²⁺]_c, and mitochondrial Ca²⁺ [Ca²⁺]_m levels were measured using 2′,7′-dichlorodihydrofluorescein diacetate, fluo-3/AM, and Rhod-2/AM, respectively, adenosine triphosphate (ATP) levels were measured by a luciferin/luciferase-based assay and mitochondrial membrane potential (ΔΨm) estimated using 5,5′,6,6′-tetrachloro 1,1′3,3′-tetraethylbenzimid azolocarbocyanine iodide. Expression of mitochondrial and antioxidant genes was determined by real-time polymerase chain reaction. RPE cells show greater sensitivity to oxidative stress, reduction in expression of mitochondrial heat shock protein 70, uncoupling protein 2, and superoxide dismutase 3, and greater expression of superoxide dismutase 2 levels with increased chronological age. Changes in mitochondrial number, size, shape, matrix density, cristae architecture, and membrane integrity were more prominent in samples obtained from >60 years old compared to mid-age and younger donors. These mitochondria abnormalities correlated with lower ATP levels, reduced ΔΨm, decreased [Ca²⁺]_c, and increased sequestration of [Ca²⁺]_m in cells with advanced aging. Our study provides evidence for mitochondrial decay, bioenergetic deficiency, weakened antioxidant defenses, and increased sensitivity of RPE cells to oxidative stress with advanced aging. Our findings suggest that with increased severity of mitochondrial decay and oxidative stress, RPE function may be altered in some individuals in a way that makes the retina more susceptible to age-related injury.     

Involvement of mitogen-activated protein kinases and reactive oxygen species in the inotropic action of ouabain on cardiac myocytes. A potential role for mitochondrial KATP channels

Binding of ouabain to Na⁺/K⁺-ATPase activated multiple signal transduction pathways including stimulation of Src, Ras, p42/44 MAPKs and production of reactive oxygen species (ROS) in rat cardiac myocytes. Inhibition of either Src or Ras ablated ouabain-induced increase in both [Ca²⁺]_i and contractility. While PD98059 abolished the effects of ouabain on [Ca²⁺]_i, it only caused a partial inhibition of ouabain-induced increases in contractility. On the other hand, pre-incubation of myocytes with N-acetyl cysteine (NAC) reduced the effects of ouabain on contractility, but not [Ca²⁺]_i. Furthermore, 5-hydroxydecanoate (5-HD) blocked ouabain-induced ROS production and partially inhibited ouabain-induced increases in contractility in cardiac myocytes. Pre-incubation of myocytes with both 5-HD and PD98059 completely blocked ouabain's effect on contractility. Finally, we found that opening of mitochondrial K_ATP channel by diazoxide increased intracellular ROS and significantly raised contractility in cardiac myocytes. These new findings indicate that ouabain regulates cardiac contractility via both [Ca²⁺]_i and ROS. While activation of MAPKs leads to increases in [Ca²⁺]_i, opening of mitochondrial K_ATP channel relays the ouabain signal to increased ROS production in cardiac myocytes.     

The Na(+)/Ca(2+)-exchanger: an essential component in the mechanism governing cardiac steroid-induced slow Ca(2+) oscillations

Plasma membrane (PM) Na⁺, K⁺-ATPase, plays crucial roles in numerous physiological processes. Cardiac steroids (CS), such as ouabain and bufalin, specifically bind to the Na⁺, K⁺-ATPase and affect ionic homeostasis, signal transduction, and endocytosed membrane traffic. CS-like compounds, synthesized in and released from the adrenal gland, are considered a new family of steroid hormones. Previous studies showed that ouabain induces slow Ca²⁺ oscillations in COS-7 cells by enhancing the interactions between Na⁺, K⁺-ATPase, inositol 1,4,5-trisphosphate receptor (IP₃R) and Ankyrin B (Ank-B) to form a Ca²⁺ signaling micro-domain. The activation of this micro-domain, however, is independent of InsP3 generation. Thus, the mechanism underlying the induction of these slow Ca²⁺ oscillations remained largely unclear. We now show that other CS, such as bufalin, can also induce Ca²⁺ oscillations. These oscillations depend on extracellular Ca²⁺ concentrations [Ca²⁺]_out and are inhibited by Ni²⁺. Furthermore, we found that these slow oscillations are Na⁺_out dependent, abolished by Na⁺/Ca²⁺ exchanger1 (NCX1)-specific inhibitors and markedly attenuated by NCX1 siRNA knockdown. Based on these results, a model is presented for the CS-induced slow Ca²⁺ oscillations in COS-7 cells.     

Extracellular ATP induces spikes in cytosolic free Ca but not in NADPH oxidase activity in neutrophils

In order to establish whether non-mitochondrial oxidase activity in human neutrophils is tightly related to cytosolic Ca²⁺ concentration, we simultaneously measured Ca²⁺ oscillations induced by ATP and oxidant production in single adherent neutrophils using confocal microscopy. ATP induced fast damped Ca²⁺ spikes with a period of 15 s and slower irregular spikes with a period greater than 50 s. Spikes in Ca²⁺ occurred in the absence of Ca²⁺ influx, but the amplitude was damped by inhibition of Ca²⁺ influx. Using the oxidation of hydroethidine as a cytosolic marker of oxidant production, we show that the generation of reactive oxygen species by neutrophils adherent to glass was accelerated by ATP. The step-up in NADPH oxidase activity followed the first elevation of cytosolic Ca²⁺ but, despite subsequent spikes in Ca²⁺ concentration, no oscillations in oxidase activity could be detected. ATP induced spikes in Ca²⁺ in a very reproducible way and we propose that the Ca²⁺ signal is an on-switch for oxidase activity, but the activity is apparently not directly correlated with spiking activity in cytosolic Ca²⁺.