Dependence of cardiac mitochondrial pyruvate dehydrogenase activity on intramitochondrial free Ca2+ concentration.

(1) The free Ca2+ concentration of the matrix of rat heart mitochondria ([Ca2+]m) was determined from the fluorescence of internalized indo-1. The value of the Kd of indo-1-Ca2+ in the mitochondrial matrix was determined to be 95 nM, on the basis of equilibration of [Ca2+]m with the extramitochondrial free Ca2+ ([Ca2+]o) in the presence of rotenone, nigericin, valinomycin and Br-A23187. (2) [Ca2+]m responded to energization/de-energization protocols, the inhibition of Ca2+-uptake by Ruthenium Red and the potentiation of Ca2+-efflux by Na+ in a manner which was consistent with the known kinetic properties of the mitochondrial Ca2+-transport processes. (3) The concentration gradient [Ca2+]m/[Ca2+]o was found to be near unity (0.82 +/- 0.18) when mitochondria were incubated in media containing 10 mM-Na+; the additional presence of 1 mM-Mg2+ reduced the gradient to values below unity (0.26 +/- 0.03). The polyamine spermine increased the Ca2+ concentration gradient in the presence of 1 mM-Mg2+. (4) The fraction of pyruvate dehydrogenase in the active form (PDHA) was found to increase with [Ca2+]m, with a K0.5 for activation of approximately 300 nM-Ca2+. This value of the activation constant was not affected by conditions, e.g. addition of Mg2+, which changed the [Ca2+]m/[Ca2+]o concentration gradient, and the presence of different oxidizable substrates, which changed the [NADH/NAD+]m concentration ratio. Thus pyruvate dehydrogenase interconversion responds directly to changes in [Ca2+]m, as inferred in earlier work.     

Mitochondrial activation directly triggers the exocytosis of insulin in permeabilized pancreatic beta-cells.

In the pancreatic beta-cell, insulin secretion is stimulated by glucose metabolism resulting in membrane potential-dependent elevation of cytosolic Ca2+ ([Ca2+]c). This cascade involves the mitochondrial membrane potential (delta psi[m]) hyperpolarization and elevation of mitochondrial Ca2+ ([Ca2+]m) which activates the Ca(2+)-sensitive NADH-generating dehydrogenases. Metabolism-secretion coupling requires unidentified signals, other than [Ca2+]c, possibly generated by the mitochondria through the rise in [Ca2+]m. To test this paradigm, we have established an alpha-toxin permeabilized cell preparation permitting the simultaneous monitoring of [Ca2+] with mitochondrially targeted aequorin and insulin secretion under conditions of saturating [ATP] (10 mM) and of clamped [Ca2+]c at substimulatory levels (500 nM). The tricarboxylic acid (TCA) cycle intermediate succinate hyperpolarized delta psi(m), raised [Ca2+]m up to 1.5 microM and stimulated insulin secretion 20-fold, without changing [Ca2+]c. Blockade of the uniporter-mediated Ca2+ influx into the mitochondria abolished the secretory response. Moreover, glycerophosphate, which raises [Ca2+]m by hyperpolarizing delta psi(m) without supplying carbons to the TCA cycle, failed to stimulate exocytosis. Activation of the TCA cycle with citrate evoked secretion only when combined with glycerophosphate. Thus, mitochondrially driven insulin secretion at permissive [Ca2+]c requires both a substrate for the TCA cycle and a rise in [Ca2+]m. Therefore, mitochondrial metabolism generates factors distinct from Ca2+ and ATP capable of inducing insulin exocytosis.     

Measurement of the matrix free Ca2+ concentration in heart mitochondria by entrapped fura-2 and quin2.

A method was developed to monitor continuously the matrix free Ca2+ concentration ([Ca2+]m) of heart mitochondria by use of the fluorescent Ca2+ indicators, fura-2 and quin2. The acetoxymethyl esters of fura-2 and quin2 were accumulated in and hydrolysed by isolated mitochondria. An increase of the mitochondrial Ca content from 0.3 nmol/mg of protein to 6 nmol/mg corresponded to a rise of [Ca2+]m from 30 to 1000 nM. The results indicate that physiological fluctuations of the mitochondrial Ca content elicit changes of [Ca2+]m in that range which regulates the matrix dehydrogenases.     

Characterization of the effects of Ca2+ on the intramitochondrial Ca2+-sensitive enzymes from rat liver and within intact rat liver mitochondria.

The regulatory properties of the Ca2+-sensitive intramitochondrial enzymes (pyruvate dehydrogenase phosphate phosphatase, NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase) in extracts of rat liver mitochondria appeared to be essentially similar to those described previously for other mammalian tissues. In particular, the enzymes were activated severalfold by Ca2+, with half-maximal effects at about 1 microM-Ca2+ (K0.5 value). In intact rat liver mitochondria incubated in a KCl-based medium containing 2-oxoglutarate and malate, the amount of active, non-phosphorylated, pyruvate dehydrogenase could be increased severalfold by increasing extramitochondrial [Ca2+], provided that some degree of inhibition of pyruvate dehydrogenase kinase (e.g. by pyruvate) was achieved. The rates of 14CO2 production from 2-oxo-[1-14C]glutarate at non-saturating, but not at saturating, concentrations of 2-oxoglutarate by the liver mitochondria (incubated without ADP) were similarly enhanced by increasing extramitochondrial [Ca2+]. The rates and extents of NAD(P)H formation in the liver mitochondria induced by non-saturating concentrations of 2-oxoglutarate, glutamate, threo-DS-isocitrate or citrate were also increased in a similar manner by Ca2+ under several different incubation conditions, including an apparent 'State 3.5' respiration condition. Ca2+ had no effect on NAD(P)H formation induced by beta-hydroxybutyrate or malate. In intact, fully coupled, rat liver mitochondria incubated with 10 mM-NaCl and 1 mM-MgCl2, the apparent K0.5 values for extramitochondrial Ca2+ were about 0.5 microM, and the effective concentrations were within the expected physiological range, 0.05-5 microM. In the absence of Na+, Mg2+ or both, the K0.5 values were about 400, 200 and 100 nM respectively. These effects of increasing extramitochondrial [Ca2+] were all inhibited by Ruthenium Red. When extramitochondrial [Ca2+] was increased above the effective ranges for the enzymes, a time-dependent deterioration of mitochondrial function and ATP content was observed. The implications of these results on the role of the Ca2+-transport system of the liver mitochondrial inner membrane are discussed.     

Mitochondrial localization as a determinant of capacitative Ca2+ entry in HeLa cells

Whether different subsets of mitochondria play distinct roles in shaping intracellular Ca2+ signals is presently unresolved. Here, we determine the role of mitochondria located beneath the plasma membrane in controlling (a) Ca2+ release from the endoplasmic reticulum (ER) and (b) capacitative Ca2+ entry. By over-expression of the dynactin subunit dynamitin, and consequent inhibition of the fission factor, dynamin-related protein (Drp-1), mitochondria were relocalised from the plasma membrane towards the nuclear periphery in HeLa cells. The impact of these changes on free calcium concentration in the cytosol ([Ca2+]c), mitochondria ([Ca2+]m) and ER ([Ca2+]ER) was then monitored with specifically-targeted aequorins. Whilst dynamitin over-expression increased the number of close contacts between the ER and mitochondria by >2.5-fold, assessed using organelle-targeted GFP variants, histamine-induced changes in organellar [Ca2+] were unaffected. By contrast, Ca2+ influx elicited significantly smaller increases in [Ca2+]c and [Ca2+]m in dynamitin-expressing than in control cells. These data suggest that the strategic localisation of a subset of mitochondria beneath the plasma membrane is required for normal Ca2+ influx, but that the transfer of Ca2+ ions between the ER and mitochondria is relatively insensitive to gross changes in the spatial relationship between these two organelles.     

Integrating cytosolic calcium signals into mitochondrial metabolic responses.

Stimulation of hepatocytes with vasopressin evokes increases in cytosolic free Ca2+ ([Ca2+]c) that are relayed into the mitochondria, where the resulting mitochondrial Ca2+ ([Ca2+]m) increase regulates intramitochondrial Ca2+-sensitive targets. To understand how mitochondria integrate the [Ca2+]c signals into a final metabolic response, we stimulated hepatocytes with high vasopressin doses that generate a sustained increase in [Ca2+]c. This elicited a synchronous, single spike of [Ca2+]m and consequent NAD(P)H formation, which could be related to changes in the activity state of pyruvate dehydrogenase (PDH) measured in parallel. The vasopressin-induced [Ca2+]m spike evoked a transient increase in NAD(P)H that persisted longer than the [Ca2+]m increase. In contrast, PDH activity increased biphasically, with an initial rapid phase accompanying the rise in [Ca2+]m, followed by a sustained secondary activation phase associated with a decline in cellular ATP. The decline of NAD(P)H in the face of elevated PDH activity occurred as a result of respiratory chain activation, which was also manifest in a calcium-dependent increase in the membrane potential and pH gradient components of the proton motive force (PMF). This is the first direct demonstration that Ca2+-mobilizing hormones increase the PMF in intact cells. Thus, Ca2+ plays an important role in signal transduction from cytosol to mitochondria, with a single [Ca2+]m spike evoking a complex series of changes to activate mitochondrial oxidative metabolism.     

Increased [Mg2+]o reduces Ca2+ influx and disruption of mitochondrial membrane potential during reoxygenation

Increase in extracellular Mg2+ concentration ([Mg2+]o) reduces Ca2+ accumulation during reoxygenation of hypoxic cardiomyocytes and exerts protective effects. The aims of the present study were to investigate the effect of increased [Mg(2+)](o) on Ca2+ influx and efflux, free cytosolic Ca2+ ([Ca2+]i) and Mg2+ concentrations ([Mg2+]i), Ca2+ accumulation in the presence of inhibitors of mitochondrial or sarcoplasmatic reticulum Ca2+ transport, and finally mitochondrial membrane potential (Delta(psi)m). Isolated adult rat cardiomyocytes were exposed to 1 h of hypoxia and subsequent reoxygenation. Cell Ca2+ was determined by 45Ca2+ uptake, and the levels of [Mg2+]i and [Ca2+]i were determined by flow cytometry as the fluorescence of magnesium green and fluo 3, respectively. Ca2+ influx rate was significantly reduced by approximately 40%, whereas Ca2+ efflux was not affected by increased [Mg2+]o (5 mM) during reoxygenation. [Ca2+]i and [Mg2+]i were increased at the end of hypoxia, fell after reoxygenation, and were unaffected by increased [Mg2+]o. Clonazepam, a selective mitochondrial Na+/Ca2+ exchange inhibitor (100 microM), significantly reduced Ca2+ accumulation by 70% and in combination with increased [Mg2+]o by 90%. Increased [Mg2+]o, clonazepam, and the combination of both attenuated the hypoxia-reoxygenation-induced reduction in Delta(psi)m, determined with the cationic dye JC-1 by flow cytometry. A significant inverse correlation was observed between Delta(psi)m and cell Ca2+ in reoxygenated cells treated with increased [Mg2+]o and clonazepam. In conclusion, increased [Mg2+]o (5 mM) inhibits Ca2+ accumulation by reducing Ca2+ influx and preserves Delta(psi)m without affecting [Ca2+]i and [Mg2+]i during reoxygenation. Preservation of mitochondria may be an important effect whereby increased [Mg2+]o protects the postischemic heart.     

Control of cytosolic free calcium by intracellular organelles in bovine adrenal glomerulosa cells. Effects of sodium and inositol 1,4,5-trisphosphate

The regulation of cytosolic free Ca2+ concentration ([Ca2+]c) by intracellular organelles was studied in permeabilized bovine adrenal glomerulosa cells. Two compartments, with distinct characteristics, were able to pump Ca2+. A first pool, sensitive to ruthenium red and presumably mitochondrial, required respiratory chain substrates to maintain [Ca2+]c around 700 nM. Ca2+ efflux from this compartment was activated by Na+ (ED50 = 5 mM). Inositol 1,4,5-trisphosphate (IP3) had no effect on this pool. A second nonmitochondrial pool required ATP to lower [Ca2+]c to about 200 nM and released Ca2+ transiently upon addition of IP3. When the two systems were allowed to work simultaneously, the nonmitochondrial pool regulated [Ca2+]c and IP3 released Ca2+ in a concentration-dependent manner (EC50 = 0.6 microM). Under these conditions the mitochondria seemed Ca2+ depleted. Upon repeated stimulations with IP3, a marked attenuation of the response was observed. This phenomenon was due to Ca2+ sequestration by a nonmitochondrial IP3-insensitive pool. Neither dantrolene (200 microM) nor 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (10 microM) were able to abolish IP3-induced Ca2+ release, though both compounds efficiently inhibited aldosterone production in intact cells stimulated with angiotensin II (10 nM) or K+ (12 mM). These results suggest that in permeabilized adrenal glomerulosa cells: the nonmitochondrial pool is responsible for buffering [Ca2+]c and for releasing Ca2+ in response to IP3; at resting [Ca2+]c levels, the mitochondria appear Ca2+ depleted; when [Ca2+]c rises above their set point, the mitochondria accumulate Ca2+ as a function of [Na+]c; 4) the mitochondria are not involved in the desensitization mechanism of the response to IP3.     

Mouse mast cell secretory granules can function as intracellular ionic oscillators

Fluorescent Ca2+ probes and digital photo-sectioning techniques were used to directly study the dynamics of Ca2+ in isolated mast cell granules of normal (CB/J) and beige (Bg(j)/Bg(j)) mice. The resting intraluminal free Ca2+ concentration ([Ca2+]L) is 25 +/- 4.2 microM (mean +/- SD, n = 68). Exposure to 3 microM inositol 1,4,5-trisphosphate (InsP3) induced periodic oscillations of luminal Ca2+ ([Ca2+]L) of approximately 10 microM amplitude and a period around 8-10 s. The [Ca2+]L oscillations were accompanied by a corresponding oscillatory release of [Ca2+]L to the extraluminal space. Control experiments using ruthenium red (2 microM) and thapsigargin (100 nM) ruled out artifacts derived from the eventual presence of mitochondria or endoplasmic reticulum in the isolated granule preparation. Oscillations of [Ca2+]L and Ca2+ release result from a Ca2+/K+ exchange process whereby bound Ca is displaced from the heparin polyanionic matrix by inflow of K+ into the granular lumen via an apamin-sensitive Ca2+-sensitive K+ channel (ASK(Ca)), whereas Ca2+ release takes place via an InsP3-receptor-Ca2+ (InsP3-R) channel. These results are consistent with previous observations of [Ca2+]L oscillations and release in/from the endoplasmic reticulum and mucin granules, and suggest that a highly conserved common mechanism might be responsible for [Ca2+]L oscillations and quantal periodic Ca2+ release in/from intracellular Ca2+ storage compartments.     

Mitochondria exert a negative feedback on the propagation of intracellular Ca2+ waves in rat cortical astrocytes.

We have used digital fluorescence imaging techniques to explore the interplay between mitochondrial Ca2+ uptake and physiological Ca2+ signaling in rat cortical astrocytes. A rise in cytosolic Ca2+ ([Ca2+]cyt), resulting from mobilization of ER Ca2+ stores was followed by a rise in mitochondrial Ca2+ ([Ca2+]m, monitored using rhod-2). Whereas [Ca2+]cyt recovered within approximately 1 min, the time to recovery for [Ca2+]m was approximately 30 min. Dissipating the mitochondrial membrane potential (Deltapsim, using the mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxy-phenyl-hydrazone [FCCP] with oligomycin) prevented mitochondrial Ca2+ uptake and slowed the rate of decay of [Ca2+]cyt transients, suggesting that mitochondrial Ca2+ uptake plays a significant role in the clearance of physiological [Ca2+]cyt loads in astrocytes. Ca2+ signals in these cells initiated either by receptor-mediated ER Ca2+ release or mechanical stimulation often consisted of propagating waves (measured using fluo-3). In response to either stimulus, the wave traveled at a mean speed of 22.9 +/- 11.2 micrometer/s (n = 262). This was followed by a wave of mitochondrial depolarization (measured using tetramethylrhodamine ethyl ester [TMRE]), consistent with Ca2+ uptake into mitochondria as the Ca2+ wave traveled across the cell. Collapse of Deltapsim to prevent mitochondrial Ca2+ uptake significantly increased the rate of propagation of the Ca2+ waves by 50%. Taken together, these data suggest that cytosolic Ca2+ buffering by mitochondria provides a potent mechanism to regulate the localized spread of astrocytic Ca2+ signals.     

Quasi-synaptic calcium signal transmission between endoplasmic reticulum and mitochondria.

Transmission of cytosolic [Ca2+] ([Ca2+]c) oscillations into the mitochondrial matrix is thought to be supported by local calcium control between IP3 receptor Ca2+ channels (IP3R) and mitochondria, but study of the coupling mechanisms has been difficult. We established a permeabilized cell model in which the Ca2+ coupling between endoplasmic reticulum (ER) and mitochondria is retained, and mitochondrial [Ca2+] ([Ca2+]m) can be monitored by fluorescence imaging. We demonstrate that maximal activation of mitochondrial Ca2+ uptake is evoked by IP3-induced perimitochondrial [Ca2+] elevations, which appear to reach values >20-fold higher than the global increases of [Ca2+]c. Incremental doses of IP3 elicited [Ca2+]m elevations that followed the quantal pattern of Ca2+ mobilization, even at the level of individual mitochondria. In contrast, gradual increases of IP3 evoked relatively small [Ca2+]m responses despite eliciting similar [Ca2+]c increases. We conclude that each mitochondrial Ca2+ uptake site faces multiple IP3R, a concurrent activation of which is required for optimal activation of mitochondrial Ca2+ uptake. This architecture explains why calcium oscillations evoked by synchronized periodic activation of IP3R are particularly effective in establishing dynamic control over mitochondrial metabolism. Furthermore, our data reveal fundamental functional similarities between ER-mitochondrial Ca2+ coupling and synaptic transmission.     

Characterization of the effects of Ca2+ on the intramitochondrial Ca2+-sensitive dehydrogenases within intact rat-kidney mitochondria

The regulatory properties of the Ca2+-sensitive intramitochondrial enzymes (pyruvate dehydrogenase phosphate phosphatase, NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase) in extracts of rat kidney mitochondria were found to be essentially similar to those described previously for other mammalian tissues; in particular each enzyme could be activated severalfold by Ca2+ with half-maximal effects (K0.5 values) of about 1 microM and effective ranges of approx. 0.1-10 microM Ca2+. In intact mitochondria prepared from whole rat kidneys incubated in a KCl-based medium containing respiratory substrates, the amount of active, nonphosphorylated pyruvate dehydrogenase could be increased severalfold by increases in extramitochondrial [Ca2+]; these effects could be blocked by ruthenium red. Similarly, Ca2+-dependent activations of NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase could be demonstrated in intact, fully coupled, rat kidney mitochondria by either following O2 uptake (in the presence of ADP) and NAD(P)H reduction (in the absence of ADP) on presentation of non-saturating concentrations of either threo-Ds-isocitrate or 2-oxoglutarate, respectively, under appropriate conditions, or for the latter enzyme only, also by following 14CO2 production from 2-oxo[1-14C]glutarate (in the absence or presence of ADP). Effects of Na+ (as a promoter of egress) and Mg2+ (as an inhibitor of uptake) on Ca2+-transport by rat kidney mitochondria could be readily demonstrated by assaying for the Ca2+-sensitive properties of the intramitochondrial Ca2+-sensitive dehydrogenases within intact rat kidney mitochondria. In the presence of physiological concentrations of Na+ (10 mM) and Mg2+ (2 mM), activation of the enzymes was achieved by increases in extramitochondrial [Ca2+] within the expected physiological range (0.05-5 microM) and with apparent K0.5 values in the approximate range of 300-500 nM. The implications of these results on the role of the Ca2+-transport system of kidney mitochondria are discussed.     

Parallel measurement of oxoglutarate dehydrogenase activity and matrix free Ca2+ in fura-2-loaded heart mitochondria

The entrapment of the Ca2+-sensitive fluorescence indicators fura-2 or quin2 in the matrix space of isolated heart mitochondria renders possible the direct monitoring of the matrix free Ca2+ [( Ca2+]m) [(1987) Biochem J. 248, 609-613]. In this paper the correlation between the [Ca2+]m and the in situ activity of oxoglutarate dehydrogenase (OGDH) in fura-2-loaded mitochondria is shown. At the initial value of [Ca2+]m, 64 nM, which corresponded to 0.36 nmol/mg mitochondrial Ca content, the OGDH activity was 12% of the maximal. Half-maximal and maximal activation were attained at 0.8 and 1.6 microM [Ca2+]m, respectively. The results indicate that an increase of the mitochondrial Ca content in the physiological range enhances the OGDH activity by means of elevation of [Ca2+]m.     

Cytosolic and mitochondrial [Ca2+] in whole hearts using indo-1 acetoxymethyl ester: effects of high extracellular Ca2+.

Assessment of free cytosolic [Ca2+] ([Ca2+]c) using the acetoxymethyl ester (AM) form of indo-1 may be compromised by loading of indo-1 into noncytosolic compartments, primarily mitochondria. To determine the fraction of noncytosolic fluorescence in whole hearts loaded with indo-1 AM, Mn2+ was used to quench cytosolic fluorescence. Residual (i.e., noncytosolic) fluorescence was subtracted from the total fluorescence before calculating [Ca2+]c. Noncytosolic fluorescence was used to estimate mitochondrial [Ca2+]. In hearts paced at 5 Hz (N = 17), noncytosolic fluorescence was 0.61 +/- 0.06 and 0.56 +/- 0.07 of total fluorescence at lambda 385 and lambda 456, respectively. After taking into account noncytosolic fluorescence, systolic and diastolic [Ca2+]c was 673 +/- 72 and 132 +/- 9 nM, respectively, noncytosolic [Ca2+] was 183 +/- 36 nM and increased to 272 +/- 12 when extracellular Ca2+ was increased from 2 to 6 mM. This increase in noncytosolic [Ca2+] was inhibited by ruthenium red, a blocker of Ca2+ uptake by mitochondria. We conclude that cytosolic and mitochondrial [Ca2+] can be determined in whole hearts loaded with indo-1 AM by using Mn2+ to quench cytosolic fluorescence.     

Mitochondria suppress local feedback activation of inositol 1,4, 5-trisphosphate receptors by Ca2+.

The concerted action of inositol 1,4,5-trisphosphate (IP3) and Ca2+ on the IP3 receptor Ca2+ release channel (IP3R) is a fundamental step in the generation of cytosolic Ca2+ oscillations and waves, which underlie Ca2+ signaling in many cells. Mitochondria appear in close association with regions of endoplasmic reticulum (ER) enriched in IP3R and are particularly responsive to IP3-induced increases of cytosolic Ca2+ ([Ca2+]c). To determine whether feedback regulation of the IP3R by released Ca2+ is modulated by mitochondrial Ca2+ uptake, the interactions between ER and mitochondrial Ca2+ pools were examined by fluorescence imaging of compartmentalized Ca2+ indicators in permeabilized hepatocytes. IP3 decreased luminal ER Ca2+ ([Ca2+]ER), and this was paralleled by an increase in mitochondrial matrix Ca2+ ([Ca2+]m) and activation of Ca2+-sensitive mitochondrial metabolism. Remarkably, the decrease in [Ca2+]ER evoked by submaximal IP3 was enhanced when mitochondrial Ca2+ uptake was blocked with ruthenium red or uncoupler. Moreover, subcellular regions that were relatively deficient in mitochondria demonstrated greater sensitivity to IP3 than regions of the cell with a high density of mitochondria. These data demonstrate that Ca2+ uptake by the mitochondria suppresses the local positive feedback effects of Ca2+ on the IP3R, giving rise to subcellular heterogeneity in IP3 sensitivity and IP3R excitability. Thus, mitochondria can play an important role in setting the threshold for activation and establishing the subcellular pattern of IP3-dependent [Ca2+]c signaling.     

Plasticity of mitochondrial calcium signaling

Evidence is emerging that a quasisynaptic local communication facilitates the calcium signaling between endoplasmic reticulum and mitochondria. However, it remains elusive whether the machinery of mitochondrial calcium signaling displays plasticity similar to the synaptic transmission. Here we studied the relationship between inositol 1,4,5-trisphosphate (IP3)-linked cytosolic [Ca2+] ([Ca2+]c) oscillations and the associated rise in mitochondrial matrix [Ca2+] ([Ca2+]m) in RBL-2H3 mast cells. We observed that the second [Ca2+]c spike is often associated with a larger rise in the [Ca2+]m than the first. It would appear that this phenomenon was not due to a change in the driving force for Ca2+ uptake and therefore must be due to an enhanced Ca2+ permeability of the mitochondrial Ca2+ uptake sites (uniporter). To investigate the activation and deactivation kinetics of the uniporter during IP3 receptor-mediated Ca2+ mobilization, we established novel methods. Using these approaches, we demonstrated that the IP3-induced increase in the permeability of the uniporter lasted longer than the Ca2+ signal. The sustained increase in Ca2+ permeability was bidirectional. Furthermore, the addition of Ca2+ during the decay of the IP3 effect evoked a large further increase in the uniporter permeability. Calmodulin inhibitors did not interfere with the IP3-induced initial activation of the uniporter but inhibited the sustained phase. These results suggest that the uniporter displays a calmodulin-mediated facilitation. This plasticity may allow cooperation among sequential IP3 receptor-mediated [Ca2+] transients in the control of calcium signal propagation to the mitochondria.     

Ca2+ influx pathways mediated by swelling or stores depletion in mouse thymocytes

We used fura-2 video imaging to characterize two Ca2+ influx pathways in mouse thymocytes. Most thymocytes (77%) superfused with hypoosmotic media (60% of isoosmotic) exhibited a sharp, transient rise in the concentration of intracellular free Ca2+ ([Ca2+]i). After a delay of approximately 70 s, these swelling-activated [Ca2+]i (SWAC) transients reached approximately 650 nM from resting levels of approximately 100 nM and declined from a time constant of 20 s. Peak [Ca2+]i during transients correlated with maximum volume during swelling. Regulatory volume decrease (RVD) was enhanced in thymocytes exhibiting SWAC transients. Three lines of evidence indicate that Ca2+ influx, and not the release of Ca2+ from intracellular stores, underlies SWAC transients in thymocytes. First, thymocytes swollen in Ca2+-free media failed to respond. Second, Gd3+ and La3+ inhibited SWAC influx with Kd's of 3.8 and 2.4 microM, respectively. Finally, the depletion of Ca2+ stores with thapsigargin (TG) before swelling did not inhibit the generation, nor decrease the amplitude, of SWAC transients. Cell phenotyping demonstrated that SWAC transients are primarily associated with immature CD4-CD8- and CD4+CD8+ thymocytes. Mature peripheral lymphocytes (mouse or human) did not exhibit SWAC transients. SWAC influx could be distinguished from the calcium release-activated Ca2+ (CRAC) influx pathway stimulated by store depletion with TG. In TG- treated thymocytes, [Ca2+]i rose steadily for approximately 100 s, peaked at approximately 900 nM, and then declined slowly. Simultaneous activation of both pathways produced an additive [Ca2+]i profile. Gd3+ and La3+ blocked Ca2+ entry during CRAC activation more potently (Kd's of 28 and 58 nM, respectively) than Ca2+ influx during SWAC transients. SWAC transients could be elicited in the presence of 1 microM Gd3+, after the complete inhibition of CRAC influx. Finally, whereas SWAC transients were principally restricted to immature thymocytes. TG stimulated the CRAC influx pathway in all four thymic CD4/CD8 subsets and in mature T cells. We conclude that SWAC and CRAC represent separate pathways for Ca2+ entry in thymocytes.     

Dynamic regulation of [Ca2+]i by plasma membrane Ca(2+)-ATPase and Na+/Ca2+ exchange during capacitative Ca2+ entry in bovine vascular endothelial cells.

The dynamic regulation of Ca2+ extrusion by the plasma membrane Ca(2+)-ATPase (PMCA) and Na+/Ca2+ exchange (NCX) was investigated in single cultured calf pulmonary artery endothelial (CPAE) cells using indo-1 microfluorimetry to measure cytoplasmic Ca2+ concentration ([Ca2+]i). The quantitative analysis of the recovery from an increase of [Ca2+]i elicited by activation of capacitative Ca2+ entry (CCE) served to characterize kinetic parameters of these Ca2+ extrusion systems in the intact cell. In CPAE cells the PMCA is activated in a Ca(2+)- and time-dependent manner. Full activation of the pump occurs only after [Ca2+]i has been elevated for at least 1 min which results in an increase of the affinity of the pump for Ca2+ and an increase in the apparent maximal extrusion rate (Vmax). Application of calmodulin antagonists W-7 and calmidazolium chloride (compound R 24571) revealed that calmodulin is a major regulator of PMCA activity in vivo. Sequential and simultaneous inhibition of PMCA and NCX suggested that both contribute to Ca2+ extrusion in a non-additive fashion. The activity of one system is dynamically adjusted to compensate for changes in the extrusion rate by the alternative transporter. It was concluded that in vascular endothelial cells, the PMCA functions as a calmodulin-regulated, high-affinity Ca2+ removal system. The contribution by the low-affinity NCX to Ca2+ clearance became apparent at [Ca2+]i > approximately 150 nM under conditions of submaximal activation of the PMCA.     

Permeability transition pore regulates both mitochondrial membrane potential and agonist-evoked Ca2+ signals in oligodendrocyte progenitors

In this study, we investigated the importance of mitochondrial permeability transition pore (PTP) in agonist-evoked cytosolic Ca2+ ([Ca2+]c) signals in oligodendrocyte progenitor cells (OP cells). We measured transmembrane potential across the mitochondrial inner membrane (delta psi m) and [Ca2+]c in the immediate vicinity simultaneously using tetramethylrhodamine ethyl ester (TMRE) and calcium green respectively. Stimulation of OP cells with methacholine evoked robust [Ca2+]c signals in approximately 80% of cells which were either oscillatory or showed a peak followed by a plateau. Elevations in [Ca2+]c induced by supramaximal concentrations of the agonist (> 200 microM) were accompanied by changes in delta psi m in 33-42% of the total mitochondria investigated. The mitochondria that responded either depolarized (26-29%), hyperpolarized (7-13%) or showed no change (58-67%). Thus, of the responsive mitochondria, most (70%) depolarized during agonist-evoked [Ca2+]c signals. Blockade of PTP with cyclosporin A (CSA) reduced the number of mitochondria that depolarized with a corresponding increase in the number that hyperpolarized. In addition, CSA or its analogue methyl valine-4- CSA (MeVal-CSA), reduced the frequency of agonist-evoked global [Ca2+]c oscillations. In resting cells, CSA (63%) and MeVal-CSA (77%) hyperpolarized a majority of the mitochondria suggesting that PTP is constitutively active and may show flickering openings. Such hyperpolarizations were not mimicked by either cyclosporine H or verapamil and were inhibited by Ru360, which blocks the mitochondrial uniporter. This observation suggested that in resting cells, Ca2+ ions might redistribute between cytosol and mitochondrial matrix through the uniporter and the PTP. Taken together, these data suggest that PTP may play an important role in regulating delta psi m and local [Ca2+]c signals during agonist stimulation in OP cells.     

Visualizing Changes in Cytosolic-Free Ca2+ during the Response of Stomatal Guard Cells to Abscisic Acid

In this paper, we report the results of a detailed investigation into abscisic acid (ABA)[mdash]stimulated elevations of guard cell cytosolic-free Ca2+ ([Ca2+]cyt). Fluorescence ratio photometry and ratio imaging techniques were used to investigate this phenomenon. Guard cells of open and closed (opened to 10 to 12 [mu]m before treatment with ABA) stomata were microinjected with the fluorescent Ca2+ indicator Indo-1. Resting [Ca2+]cyt ranged from 50 to 350 nM. ABA (100 nM) stimulated an increase in [Ca2+]cyt in 68 and 81% of guard cells microinjected in the open and closed configuration, respectively. All stomata were observed to close in response to ABA. Increases ranged from 100 to 750 nM above the resting concentration and were arbitrarily grouped into five "classes." ABA-stimulated increases in [Ca2+]cyt were not uniformly distributed across the cytosol of guard cells. Rapid transient increases in [Ca2+]cyt were also observed in the guard cells of stomata microinjected in the closed configuration. We concluded that the ABA-induced turgor loss in guard cells is a Ca2+-dependent process.