Regulation of Ca2+ efflux in rat liver mitochondria. Role of membrane potential

The paper analyzes the relationship between membrane potential (delta psi), steady state pCao (-log [Ca2+] in the outer aqueous phase) and rate of ruthenium-red-induced Ca2+ efflux in liver mitochondria. Energized liver mitochondria maintain a pCao of about 6.0 in the presence of 1.5 mM Mg2+ and 0.5 mM Pi. A slight depression of delta psi results in net Ca2+ uptake leading to an increased steady state pCao. On the other hand, a more marked depression of delta psi results in net Ca2+ efflux, leading to a decreased steady-state pCao. These results reflect a biphasic relationship between delta psi and pCao, in that pCao increases with the increase of delta psi up to a value of about 130 mV, whereas a further increase of delta psi above 130 mV results in a decrease of pCao. The phenomenon of Ca2+ uptake following a depression of delta psi is independent of the tool used to affect delta psi whether by inward K+ current via valinomycin, or by inward H+ current through protonophores or through F1-ATP synthase, or by restriction of e- flow. The pathway for Ca2+ efflux is considerably activated by stretching of the inner membrane in hypotonic media. This activation is accompanied by a decreased pCao at steady state and by an increased rate of ruthenium-red-induced Ca2+ efflux. By restricting the rate of e- flow in hypotonically treated mitochondria, a marked dependence of the rate of ruthenium-red-induced Ca2+ efflux on the value of delta psi is observed, in that the rate of Ca2+ efflux increases with the value of delta psi. The pCao is linearly related to the rate of Ca2+ efflux. Activation of oxidative phosphorylation via addition of hexokinase + glucose to ATP-supplemented mitochondria, is followed by a phase of Ca2+ uptake, which is reversed by atractyloside. These findings support the view that Ca2+ efflux in steady state mitochondria occurs through an independent, delta psi-controlled pathway and that changes of delta psi during oxidative phosphorylation can effectively modulate mitochondrial Ca2+ distribution by inhibiting or activating the delta psi-controlled Ca2+ efflux pathway.     

Na+-dependent regulation of extramitochondrial Ca2+ by rat-liver mitochondria

The presence and significance of Na+-induced Ca2+ release from rat liver mitochondria was investigated by the arsenazo technique. Under the experimental conditions used, the mitochondria, as expected, avidly extracted Ca2+ from the medium. However, when the uptake pathway was blocked with ruthenium red, only a small rate of 'basal' release of Ca2+ was seen (0.3 nmol Ca2+ X min-1 X mg-1), in marked contrast to earlier reports on a rapid loss of sequestered Ca2+ from rat liver mitochondria. The addition of Na+ in 'cytosolic' levels (20 mM) led to an increase in the release rate by about 1 nmol Ca2+ X min-1 X mg-1. This effect was specific for Na+. The significance of this Na+-induced Ca2+ release, in relation to the Ca2+ uptake mechanism, was investigated (in the absence of uptake inhibitors) by following the change in the extramitochondrial Ca2+ steady-state level (set point) induced by Na+. A five-fold increase in this level, from less than 0.2 microM to more than 1 microM, was induced by less than 20 mM Na+. The presence of K+ increased the sensitivity of the Ca2+ homeostat to Na+. The effect of Na+ on the extramitochondrial level was equally well observed in an K+/organic-anion buffer as in a sucrose buffer. Liver mitochondria incubated under these circumstances actively counteracted a Ca2+ or EGTA challenge by taking up or releasing Ca2+, so that the initial level, as well as the Na+-controlled level, was regained. It was concluded that liver mitochondria should be considered Na+-sensitive, that the capacity of the Na+-induced efflux pathway was of sufficient magnitude to enable it to influence the extramitochondrial Ca2+ level biochemically and probably also physiologically, and that the mitochondria have the potential to act as active, Na+-dependent regulators of extramitochondrial ('cytosolic') Ca2+. It is suggested that changes of cytosolic Na+ could be a mediator between certain hormonal signals (notably alpha 1-adrenergic) and changes in this extramitochondrial ('cytosolic') Ca2+ steady state level.     

The regulation of extramitochondrial steady state free Ca2+ concentration by rat insulinoma mitochondria

For the study of Ca2+ handling by mitochondria of an insulin secretory tissue, a method for the isolation of functionally intact insulinoma mitochondria is described. The mitochondria had a respiratory control ratio of 6.3 +/- 0.3 with succinate as a substrate. The regulation of extramitochondrial [Ca2+]o concentration by suspensions of insulinoma mitochondria was studied using Ca2+-selective minielectrodes. The mitochondria were found to maintain an ambient free Ca2+ concentration of about 0.3 and 0.9 microM in the absence or presence of Mg2+ (1 mM), respectively. The addition of Na+ resulted in a dose-dependent (half-maximal 4 mM Na+) increase in steady state [Ca2+]o. Na+ accelerated the ruthenium red-induced Ca2+ efflux, suggesting the existence of a Ca2+/2Na+ antiporter, as described in mitochondria of excitable tissues. Experiments were performed to study the effects of various agents on the steady state extramitochondrial free Ca2+. cAMP, 3-isobutyl-1-methylxanthine, and NADH were found to have no effect, whereas phosphoenolpyruvate induced a net Ca2+ efflux, the kinetic of which suggests deleterious effects on mitochondrial functions. A small decrease in pH (0.1 unit) of the incubation buffer resulted in an increase of the extramitochondrial Ca2+ steady state that was reversible upon restoration of the pH to its initial value. In conclusion, insulinoma mitochondria were able to maintain an extramitochondrial [Ca2+]o steady state in the submicromolar range that was markedly influenced by the ionic composition of the incubation medium. Thus, mitochondria may play a role in the regulation of cellular calcium homeostasis and insulin release.     

Effect of temperature on the reversal of the calcium ion pump in sarcoplasmic reticulum.

Sarcoplasmic-reticulum vesicles were actively loaded with Ca2+ in the presence of phosphate, and the ADP-induced Ca2+ efflux and ATP synthesis were measured as a function of temperature. Arrhenius plots show break points for both processes at about 18 and 37 degrees C. Between 18 and 37 degrees C, Ca2+ efflux and ATP synthesis occur with an activation energy of 67.2-71.4 kJ/mol, whereas it is about 189-210 kJ/mol for temperatures below 18 degrees C. Above 37 degrees C, the rates of ADP-induced Ca2+ release and of ATP synthesis sharply decline until the temperature reaches about 42 degrees C. Above this temperature, the Ca2+ efflux increases again even in absence of ADP, although the synthesis of ATP is inhibited, which reflects leakiness of the vesicles. The results show that the transition temperatures for ADP-induced Ca2+ efflux and for ATP synthesis resemble those for active Ca2+ uptake, which indicates that the same coupling mechanism is involved during the inward and outward Ca2+ translocations across the membrane.     

Studies on mitochondrial Ca2+-transport and matrix Ca2+ using fura-2-loaded rat heart mitochondria

Rat heart mitochondria were incubated for 5 min at 30 degrees C and at approx. 40 mg protein.ml-1 and in the presence of 10 microM fura-2/AM. This allowed the entrapment of free fura-2 within the mitochondrial matrix and its use as a probe for Ca2+, but without affecting the apparent viability of the mitochondria. Parallel measurements of the activities of the intramitochondrial Ca2+-sensitive enzymes, pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase, allowed an assessment of their sensitivity to measured free Ca2+ within intact mitochondria incubated under different conditions; the enzymes responded to matrix Ca2+ over the approximate range 0.02-2 microM with half-maximal effects at about 0.3-0.6 microM Ca2+. Effectors of Ca2+-transport across the inner membrane (e.g., Na+, Mg2+, Ruthenium red, spermine) did not appear to affect these ranges, but did bring about expected changes in Ca2+ distribution across this membrane. Significantly, when mitochondria were incubated in the presence of physiological concentrations of both Na+ and Mg2+, and at low extramitochondrial Ca2+ (less than 400 nM), there was a gradient of Ca2+ (in:out) of less than unity; at higher extramitochondrial [Ca2+] (but still within the physiological range) the gradient was greater than unity indicating a highly cooperative nature of transmission of the Ca2+ signal into the matrix under such conditions.     

The isolation of lymphocyte mitochondria and their regulation of extramitochondrial free Ca2+ concentration.

1. A method for the isolation of functionally intact mitochondria from lymphocytes is described. It involves digitonin breakage of the plasma membrane, followed by differential centrifugation. The yield was 36 mg of mitochondrial protein/200 g of pig mesenteric lymph node (6 mg of mitochondrial protein/10(9) lymphocytes). The mitochondrial had a respiratory-control ratio of 2--3.5 with succinate as substrate. 2. Ca2+ transport by these mitochondria was investigated. They were able to regulate the extramitochondrial free [Ca2+] very precisely, by buffering any displacements from the steady-state. The exact extramitochondrial free [Ca2+] of this steady-state depended on the conditions of incubation. In a medium designed to resemble the cytoplasmic environment, with added Ca2+, lymphocyte mitochondria maintained a steady-state free [Ca2+] of 0.63 microM (pCa of 6.2). The rates of Ca2+ uptake and efflux under these conditions, with both lymphocyte and liver mitochondria, were very much lower than those in a less complex medium. 3. Lymphocyte mitochondria were shown to possess an Na+-independent Ruthenium Red-insensitive efflux pathway similar to that of liver mitochondria. Ruthenium Red totally inhibited the electrophoretic uniporter. Although Na+ had no effect on the steady-state maintained by lymphocyte mitochondria, they were shown to possess an Na+/H+ antiporter.     

Ca2+ ions, an allosteric activator of calcium uptake in rat liver mitochondria

In a previous investigation, I have shown that the kinetics of the Ca uniporter change fundamentally when mitochondria have transitorily lost their membrane potential. The sigmoidal kinetics, usually observed in liver mitochondria, became almost hyperbolic. This means an increase in the affinity for calcium, and hence a considerable acceleration of Ca uptake in the range of low, e.g., physiological calcium concentration. In this investigation I show that extramitochondrial calcium released from the deenergized mitochondria causes the allosteric activation of the Ca uniporter. The dependence of the allosterical activation on the extramitochondrial Ca2+ concentration and on time is described. It is also reported that it is possible to activate allosterically the Ca uniporter of energized mitochondria by a short-term elevation of the extramitochondrial Ca2+ concentration. The process of activation is reversible. It is quickly reversed by the addition of chelators for Ca2+, and it is slowly reversed when the activating Ca2+ has to be removed by the mitochondrial Ca uniporter, though the bulk of extramitochondrial calcium is taken up by it very quickly. Several kinetics of the Ca uniporter are described. The implications of continually changing kinetics of the Ca uniporter are considered for carbon tetrachloride intoxication and the action of alpha 1-adrenergic agonists in liver cells.     

Interactions between spermine and Mg2+ on mitochondrial Ca2+ transport

The effects of the polyamine spermine on the regulation of Ca2+ transport by subcellular organelles from rat liver, heart, and brain were investigated using ion-sensitive minielectrodes and a 45Ca2+ tracer method. Spermine stimulated Ca2+ uptake by mitochondria but not by microsomes. In the presence of spermine, isolated mitochondria could maintain a free extramitochondrial Ca2+ concentration of 0.3-0.2 microM. Stimulation of the initial rates of Ca2+ uptake and 45Ca2+ cycling of mitochondria by spermine shows that this was accomplished through a decrease of the apparent Km for Ca2+ uptake by the Ca2+ uniporter. The half maximally effective concentration of spermine (50 microM) was in the range of physiological concentrations of this polyamine in the cell. Spermidine was five times less effective. Putrescine was ineffective. The stimulation of mitochondrial Ca2+ uptake by spermine was inhibited by Mg2+ in a concentration-dependent manner. However, the diminished contribution of the mitochondria to the regulation of the free extraorganellar Ca2+ concentration could mostly be compensated for by microsomal Ca2+ uptake. Spermine also reversed ruthenium red-induced Ca2+ efflux from mitochondria. It is concluded that spermine is an activator of the mitochondrial Ca2+ uniporter and Mg2+ an antagonist. By this mechanism, the polyamines can confer to the mitochondria an important role in the regulation of the free cytoplasmic Ca2+ concentration in the cell and of the free Ca2+ concentration in the mitochondrial matrix.     

A role of H+ flux in active Ca2+ transport into sarcoplasmic reticulum vesicles. II. H+ ejection during Ca2+ uptake

Proton efflux during Ca2+ transport into sarcoplasmic reticulum vesicles was examined. Although a rapid H+ ejection was observed during the initial phase of Ca2+ uptake and the amount of the liberated H+ was more than that due to hydrolysis of ATP, generation of a pH difference as a result of the H+ efflux could not be detected by direct pH measurement with a pH meter. Alkalinization of the inside of the vesicles during Ca2+ uptake was more precisely examined by flow dialysis assay and a significant uptake of acetate or salicylate into the vesicles was found, suggesting the generation of a small pH difference across the SR membrane. From these results, it was concluded that counter-transport of H+ was operative in Ca2+ uptake but that only a relatively small pH difference was generated as a result of the H+ efflux. The intrinsic buffering capacity of sarcoplasmic reticulum vesicles was measured and a relatively large value (130 nmol H+/pH unit/mg at pH 6.2) was obtained.     

Effects of adenosine diphosphate on Ca2+ fluxes and Ca2+ accumulation of sarcoplasmic reticulum

Ca2+ transport by sarcoplasmic reticulum vesicles was examined by incubating sarcoplasmic reticulum vesicles (0.15 mg/ml) at 37 degrees C in, either normal medium that contained 0.15 M sucrose, 0.1 M KCl, 60 microM CaCl2, 2.5 mM ATP and 30 mM Tes at pH 6.8, or a modified medium for elimination of ADP formed from ATP hydrolysis by including, in addition, 3.6 mM phosphocreatine and 33 U/ml of creatine phosphokinase. In normal medium, Ca2+ uptake of sarcoplasmic reticulum vesicles reached a plateau of about 100 nmol/mg. In modified medium, after this phase of Ca2+ uptake, a second phase of Ca2+ accumulation was initiated and reached a plateau of about 300 nmol/mg. The second phase of Ca2+ accumulation was accompanied by phosphate uptake and could be inhibited by ADP. Since, under these experimental conditions, there was no significant difference of the rates of ATP hydrolysis in normal medium and modified medium, extra Ca2+ uptake in modified medium but not in normal medium could not be explained by different phosphate accumulation in the two media. Unidirectional Ca2+ influx of sarcoplasmic reticulum near steady state of Ca2+ uptake was measured by pulse labeling with 45Ca2+. The Ca2+ efflux rate was then determined by subtracting the net uptake from the influx rate. At the first plateau of Ca2+ uptake in normal medium, Ca2+ influx was balanced by Ca2+ efflux with an exchange rate of 240 nmol/mg per min. This exchange rate was maintained relatively constant at the plateau phase. In modified medium, the Ca2+ exchange rate at the first plateau of Ca2+ uptake was about half of that in normal medium. When the second phase of Ca2+ uptake was initiated, both the influx and efflux rates started to increase and reached a similar exchange rate as observed in normal medium. Also, during the second phase of Ca2+ uptake, the difference between the influx and efflux rates continued to increase until the second plateau phase was approached. In conditions where the formation of ADP and inorganic phosphate was minimized by using a low concentration of sarcoplasmic (7.5 micrograms/ml) and/or using acetyl phosphate instead of ATP, the second phase of Ca2+ uptake was also observed. These data suggest that the Ca2+ load attained by sarcoplasmic reticulum vesicles during active transport is modulated by ADP accumulated from ATP hydrolysis. ADP probably exerts its effect by facilitating Ca2+ efflux, which subsequently stimulates Ca2+ exchange.     

Regulation of transmembrane ion transport by reaction products of phospholipase A2. I. Effects of lysophospholipids on mitochondrial Ca2+ transport

Lysophospholipids inhibited mitochondrial Ca2+ uptake, induced a net Ca2+ efflux, and thereby increased the extramitochondrial Ca2+ concentration. The inhibitory potency decreased in the order lysophosphatidylcholine (LPC) = lysophosphatidylglycerol (LPG) greater than lysophosphatidylinositol (LPI) greater than lysophosphatidylserine (LPS) much greater than lysophosphatidylethanolamine (LPE). This relative order is in inverse relation to the ability of the various phospholipid head-groups to build up intermolecular hydrogen bonds with neighbouring membrane lipids. This indicates that changes in Ca2+ transport induced by lysophospholipids are mediated by the interaction of the lysophospholipids with the mitochondrial membrane bilayer structure. The mitochondrial membrane potential, which is the main driving force for mitochondrial Ca2+ uptake, was affected in the same order by the various lysophospholipids. This reduction of the mitochondrial membrane potential may be the underlying cause for the inhibition of the mitochondrial Ca2+ uniport and the resulting release of Ca2+ from the mitochondria.     

Ruthenium red-insensitive Ca2+ uptake and release by mitochondria

Calcium uptake by rat liver mitochondria driven by an artificial pH gradient is ruthenium red insensitive, electrically neutral, and inhibited by the local anesthetic, nupercaine. This pH-driven Ca2+ transport is also inhibited by NH3, Pi, and acetate. Direct measurements of Pi indicate it is not translocated with Ca2+ during pH-driven Ca2+ uptake. Calcium is therefore not transported by a Ca2+-Pi symport mechanism. Ruthenium red-insensitive Ca2+ efflux is similar in its inhibition by nupercaine and its kinetics, and is also electroneutral. This suggests that the Ca2+ uptake described here occurs via reversal of the principal pathway of mitochondrial Ca2+ release. From the available data, pH-driven Ca2+ uptake (and presumably Ca2+ efflux) is hypothesized to occur by Ca2+ symport with unidentified anions. Protons may move counter to Ca2+ or reversibly dissociate from cotransported anions, which therefore couples Ca2+ transport to the pH gradient.     

Ca2+,Mg2+-ATPase of microsomal membranes from bovine aortic smooth muscle. Identification and characterization of an acid-stable phosphorylated intermediate of the Ca2+,Mg2+-ATPase

An acid-stable phosphoprotein was formed in a microsomal membrane fraction isolated from bovine aortic smooth muscle in the presence of Mg2+ + ATP and Ca2+. The microsomes also showed Ca2+ uptake activity. The Ca2+ dependence of phosphoprotein formation and of Ca2+ uptake occurred over the same range of Ca2+ concentration (1-10 microM), and resembled similar findings from rabbit skeletal microsomes. The molecular weight of the phosphorylated protein, estimated by SDS-gel electrophoresis, was approximately 105,000. The phosphoprotein was labile at alkaline pH, and its decomposition was accelerated by hydroxylamine. Half-maximum incorporation of 32P in the presence of 10 microM Ca2+ occurred at 60 nM ATP. The calcium-dependent phosphoprotein formation was not affected by 5 mM NaN3, but was inhibited in a dose-dependent fashion by ADP with a 50% inhibition occurring at 180 microM. Fifty mM MgCl2 was required for the maximal phosphorylation. The rate of phosphoprotein decomposition after adding 2 mM EGTA was accelerated by varying the Mg2+ concentration from 10 microM to 3 mM. Alkaline pH (9.0) slowed the rate of phosphoprotein decay. Optimal Ca2+-dependent phosphoprotein occurred at 15 degrees C over a broad pH range (6.4 to 9.0). The activation energy of EGTA-induced phosphoprotein decomposition was 25.6 kcal/mol between 0 and 16 degrees C and 14.6 kcal/mol between 16 and 30 degrees C. The phosphoprotein formed by aortic microsomes was thus quite similar to the acid-stable phosphorylated intermediate of the Ca2+-transport ATPase of sarcoplasmic reticulum from skeletal and cardiac muscle. These data suggest that the Ca2+-dependent phosphoprotein is a reaction intermediate of the Ca2+,Mg2+-ATPase of the aortic microsomes.     

Effects of spermine on mitochondrial Ca2+ transport and the ranges of extramitochondrial Ca2+ to which the matrix Ca2+-sensitive dehydrogenases respond.

1. Spermine has previously been reported to be an activator of mitochondrial Ca2+ uptake [Nicchitta & Williamson (1984) J. Biol. Chem. 259, 12978-12983]. This is confirmed in the present studies on rat heart, liver and kidney mitochondria by using the activities of the Ca2+-sensitive intramitochondrial dehydrogenases (pyruvate, NAD+-isocitrate and 2-oxoglutarate dehydrogenases) as probes for matrix Ca2+, and also, for the heart mitochondria, by using entrapped fura-2. 2. As also found previously [Damuni, Humphreys & Reed (1984) Biochem. Biophys. Res. Commun. 124, 95-99], spermine activated extracted pyruvate dehydrogenase phosphate phosphatase. However, it was found to have no effects at all on the extracted NAD+-isocitrate or 2-oxoglutarate dehydrogenases. It also had no effects on activities of the enzymes in mitochondria incubated in the absence of Ca2+, or on the Ca2+-sensitivity of the enzymes in uncoupled mitochondria. 3. Spermine clearly activated 45Ca uptake by coupled mitochondria, but had no effect on Ca2+ egress from mitochondria previously loaded with 45Ca. 4. Spermine (with effective Km values of around 0.2-0.4 mM) caused an approx. 2-3-fold decrease in the effective ranges of extramitochondrial Ca2+ in the activation of the Ca2+-sensitive matrix enzymes in coupled mitochondria from all of the tissues. The effects of spermine appeared to be largely independent of the other effectors of mitochondrial Ca2+ transport, such as Mg2+ (inhibitor of uptake) and Na+ (promoter of egrees). 5. In the most physiological circumstance, coupled mitochondria incubated with Na+ and Mg2+, the presence of saturating spermine (2 mM) resulted in an effective extramitochondrial Ca2+ range for matrix enzyme activation of from about 30-50 nM up to about 800-1200 nM, with half-maximal effects around 250-400 nM-Ca2+. The implications of these findings for the regulation of matrix and extramitochondrial Ca2+ are discussed.     

Temperature dependence of Ca2+-activated K+ currents in the membrane of human erythrocytes

The currents through single Ca2+-activated K+ channels were studied in excised inside-out membrane patches of human erythrocytes. The effects of temperature on single-channel conductance, on channel gating and on activation by Ca2+ were investigated in the temperature range from 0 up to 47 degrees C. The single-channel conductance shows a continuous increase with increasing temperature; an Arrhenius plot of the conductance gives the activation energy of 29.6 +/- 0.4 kJ/mol. Reducing the temperature alters channel-gating kinetics which results in a significant increase of the probability of the channel being open (Po). The calcium dependence of Po is affected by temperature in different ways; the threshold concentration for activation by Ca2+ is not changed, the Ca2+ concentration of half-maximal channel activation is reduced from 2.1 mumol/l at 20 degrees C to 0.3 mumol/l at 0 degrees C, the saturation level of the dependence is reduced for temperatures higher then about 30 degrees C. The relevance of the obtained data for the interpretation of the results known from flux experiments on cells in suspensions is discussed.     

Effects of lysophospholipids on Ca2+ transport in rat liver mitochondria incubated at physiological Ca2+ concentrations in the presence of Mg2+, phosphate and ATP at 37 degrees C.

Lysophospholipids caused the release of 45Ca2+ from isolated rat liver mitochondria incubated at 37 degrees C in the presence of low concentrations of free Ca2+, ATP, Mg2+, and phosphate ions. The concentrations of lysophosphatidylethanolamine, lysophosphatidylcholine, lysophosphatidic acid and lysophosphatidylinositol which gave half-maximal effects were 5, 26, 40 and 56 microM, respectively. The effects of lysophosphatidylethanolamine were not associated with a significant impairment of the integrity of the mitochondria as monitored by measurement of membrane potential and the rate of respiration. Lysophosphatidylethanolamine did not induce the release of Ca2+ from a microsomal fraction, or enhance Ca2+ inflow across the plasma membrane of intact cells, but did release Ca2+ from an homogenate prepared from isolated hepatocytes and incubated under the same conditions as isolated mitochondria. The proportion of mitochondrial 45Ca2+ released by lysophosphatidylethanolamine was not markedly affected by altering the total amount of Ca2+ in the mitochondria, the concentration of extramitochondrial Mg2+, by the addition of Ruthenium Red, or when oleoyl lysophosphatidylethanolamine was employed instead of the palmitoyl derivative. The effects of 5 microM-lysophosphatidylethanolamine were reversed by washing the mitochondria. The possibility that lysophosphatidylethanolamine acts to release Ca2+ from mitochondria in intact hepatocytes following the binding of Ca2+-dependent hormones to the plasma membrane is briefly discussed.     

The entrapment of the Ca2+ indicator arsenazo III in the matrix space of rat liver mitochondria by permeabilization and resealing. Na+-dependent and -independent effluxes of Ca2+ in arsenazo III-loaded mitochondria.

The permeabilization-resealing technique [Al-Nasser & Crompton, Biochem. J. (1986) 239, 19-29] has been applied to the entrapment of arsenazo III in the matrix compartment of rat liver mitochondria. The addition of 10 mM-arsenazo III to mitochondria permeabilized with Ca2+ partially restores the inner-membrane potential (delta psi) and leads to the recovery of 3.9 nmol of arsenazo III/mg of protein in the matrix when the mitochondria are washed three times. The recovery of entrapped arsenazo III is increased 2-fold by 4 mM-Mg2+, which also promotes repolarization. ATP with or without Mg2+ decreased arsenazo III recovery. Under all conditions, less arsenazo III than [14C]sucrose is entrapped, in particular in the presence of ATP. The amount of arsenazo III entrapped is proportional to the concentration of arsenazo III used as resealant, and is equally distributed between heavy and light mitochondria. Arsenazo III-loaded permeabilized and resealed (PR) mitochondria develop delta psi values of 141 +/- 3 mV. PR mitochondria retain arsenazo III and [14C]sucrose for more than 2 h at 0 degrees C. At 25 degrees C, and in the presence of Ruthenium Red, PR mitochondria lose arsenazo III and [14C]sucrose at equal rates, but Ca2+ efflux is more rapid; this indicates that Ca2+ is released by an Na+-independent carrier in addition to permeabilization. The Na+/Ca2+ carrier of PR mitochondria is partially (60%) inhibited by extramitochondrial free Ca2+ stabilized with Ca2+ buffers; maximal inhibition is attained with 2 microM free Ca2+. A similar inhibition occurs in normal mitochondria with 3.5 nmol of matrix Ca2+/mg of protein, but the inhibition is decreased by increased matrix Ca2+. The data suggest the presence of Ca2+ regulatory sites on the Na+/Ca2+ carrier that change the affinity for matrix free Ca2+.     

On the relation between rapid light-induced Ca2+ release and proton uptake in rod outer segment disk membranes

In this paper we review our experiments on the light-induced Ca2+ release and proton uptake at the rod outer segment (ROS) disk membrane using flash-spectrophotometry and the indicating dyes arsenazo III and bromcresol purple. We used three different ROS preparations in order to locate the intracellular site of Ca2+ release. The ionophore A23187 was required to communicate the Ca2+ release to the indicator located in the external medium in both ROS with an intact and with a leaky plasma membrane. A23187 was also required to observe the Ca2+ released in the interior of vesicles prepared by sonication of ROS. From this we conclude that the site of Ca2+ release is located at the luminal side of the disk membrane, whereas this Ca2+ was not transported across the disk membrane under our experimental conditions and on the time scale of our experiments (20 s). Light-induced Ca2+ release was inhibited by electrolysis in the suspension medium provided that the electrolytes gained access to the compartment where Ca2+ was released. The effectivity to inhibit Ca2+ release markedly increased from monovalent to divalent to trivalent cations. The results strongly suggest that electrolytes (cations) act by screening the electrostatic potential at the disk membrane surface due to the presence of a net fixed negative surface charge. The surface potential controls the free Ca2+ concentration at the membrane surface and, therefore, controls the amount of Ca2+ bound to the disk membrane. The kinetics of light-induced Ca2+ release and proton uptake showed a similar dependence on the structural status of the ROS. In sonicated ROS almost linear Arrhenius plots were observed for metarhodopsin II formation, Ca2+ release and proton uptake (energy of activation 150 kJ/mol). In intact ROS both Ca2+ release and proton uptake showed a nonlinear Arrhenius plot with rate constants up to 30-fold slower than metarhodopsin II formation. At temperatures above 10 degrees C a process other than metarhodopsin II formation rate limited both ligh-induced proton uptake and Ca2+ release (energy of activation 42 kJ/mol). A model is discussed in which metarhodopsin II formation triggers the uptake of proton(s) into the disk membrane lowering the surface potential. A reduction potential of the surface in turn decreases the free Ca2+ concentration at the surface thereby causing the release of part of the bound Ca2+.     

Structural requirements of lysophospholipid-regulated mitochondrial Ca2+ transport

Analogues of lysophosphatidylcholine, including PAF (platelet-activating-factor) and HePC (an experimental anticancer drug), were studied for their influence on mitochondrial Ca2+ transport and membrane potential. Lysophospholipids released Ca2+ from mitochondria and reduced the maximal Ca2+ uptake. The structure-activity relations indicate that deprotonated head groups like phosphocholines yield active compounds while partially protonated head groups like phosphoethanolamines are essentially inactive. Structural requirements for the apolar part of the molecules were acyl or alkyl chain lengths of less than 18 carbon atoms at the C1-position of the glycerol backbone and residues of small size and/or low polarity at the C2-position. Choline lysophospholipids, but not ethanolamine lysophospholipids, may therefore induce mitochondrial Ca2+ efflux and become mediators of ischaemic tissue damage where dysregulated phospholipase A2 activity and an impairment of mitochondrial function are supposed to play a crucial role.     

Temperature dependence of the atractyloside-induced mitochondrial Ca2+ release

1. Mitochondrial Ca2+, accumulated by succinate oxidation was released by addition of 50 microM atractyloside. Beside this Ca2+ efflux, a large oxidation of pyridine nucleotides and sustained membrane depolarization occurs. An absolute requirement for acetate to support Ca2+ release is demonstrated. 2. Membrane de-energization, NAD(P)H oxidation, and Ca2+ efflux as induced by atractyloside were temperature-dependent, since it occurs when mitochondria are incubated at 22 degrees C and was abolished at 4 degrees C. 3. Taking into account this latter, the effects of atractyloside on mitochondrial Ca2+ release appears not to be a simple result of the binding of the inhibitor to adenine nucleotide translocase. 4. It is proposed that the mechanism involved in atractyloside-driven membrane permeability to Ca2+ must be related with the transference of the conformational change of the carrier, to another membrane structure responsible for the maintenance permeability to ions.