Liver mitochondrial pyrophosphate concentration is increased by Ca2+ and regulates the intramitochondrial volume and adenine nucleotide content.

1. The matrix pyrophosphate (PPi) content of isolated energized rat liver mitochondria incubated in the presence of ATP, Mg2+, Pi and respiratory substrate was about 100 pmol/mg of protein. 2. After incubation with sub-micromolar [Ca2+], this was increased by as much as 300%. There was a correlation between the effects of Ca2+ on PPi and on the increase in matrix volume reported previously [Halestrap, Quinlan, Whipps & Armston (1986) Biochem. J. 236, 779-787]. Half-maximal effects were seen at 0.3 microM-Ca2+. 3. Coincident with these effects, the total adenine nucleotide content increased in a carboxyatractyloside-sensitive manner. 4. Incubation with 0.2-0.5 mM-butyrate induced similar but smaller effects on mitochondrial swelling and matrix PPi and total adenine nucleotide content. Addition of butyrate after Ca2+, or vice versa, caused Ca2+-induced mitochondrial swelling to stop or reverse, while matrix PPi increased 30-fold. 5. Addition of atractyloside or the omission of ATP from incubations greatly enhanced swelling induced by Ca2+ without increasing matrix PPi. 6. Swelling of mitochondria incubated under de-energized conditions in iso-osmotic KSCN was progressively enhanced by the addition of increasing concentrations of PPi (1-20 mM) or valinomycin. 7. In iso-osmotic potassium pyrophosphate swelling was slow initially, but accelerated with time. This acceleration was inhibited by ADP, whereas carboxyatractyloside induced rapid swelling. Swelling in other iso-osmotic PPi salts showed that the rate of entry decreased in the order NH4+ greater than K+ greater than Na+ greater than Li+, whereas choline, tetramethylammonium and Tris did not enter. It is suggested that the adenine nucleotide translocase transports small univalent cations when PPi is bound and that PPi can also be transported when the transporter is in the conformation induced by carboxyatractyloside. 8. It is concluded that Ca2+ and butyrate cause swelling of energized mitochondria through this effect of PPi on K+ permeability of the mitochondrial inner membrane. 9. Freeze-clamped livers from rats treated with glucagon or phenylephrine show 30-50% increases in tissue PPi. It is proposed that Ca2+-mediated increases in mitochondrial PPi are responsible for the increase in matrix volume and total adenine nucleotide content observed after hormone treatment.     

Inhibition of mitochondrial-matrix inorganic pyrophosphatase by physiological [Ca2+], and its role in the hormonal regulation of mitochondrial matrix volume.

1. The pyrophosphatase activity in cytosolic and mitochondrial fractions of rat liver was 1.7 and 0.26 units/mg of protein respectively when assayed at 37 degrees C in the presence of physiological [Mg2+] (0.3 mM). 2. Approx. 80% of the mitochondrial pyrophosphatase was inaccessible to extramitochondrial PPi, of which 40% represented soluble matrix enzyme (0.38 unit/mg of matrix protein). 3. Ca2+ inhibited the soluble matrix enzyme; the effective K0.5 for inhibition increased as [Mg2+], an essential cofactor of the enzyme, increased. Measured values were 0.39, 1.15, 3.7, 8.3 and 12.5 microM at 0.04 mM-, 0.1 mM-, 0.3 mM-, 0.6 mM- and 1 mM-Mg2+ respectively. 4. The data were analysed by a kinetic model similar to that for yeast pyrophosphatase, which assumes the substrate to be MgPPi (Km 5 microM) with Mg2+ also activating at an additional site (K0.5 23 microM). Ca2+ inhibits through the formation of CaPPi, a strong competitive inhibitor (Ki 0.067 microM). 5. Heart mitochondria also contain a soluble matrix pyrophosphatase of similar activity to that of liver mitochondria and with the same sensitivity to [Ca2+]. 6. The data provide an explanation for the increase in mitochondrial PPi, mediated by Ca2+, which is responsible for the increase in matrix volume induced by gluconeogenic hormones [Davidson & Halestrap (1988) Biochem. J. 254, 379-384].     

Inhibition of Ca2(+)-induced large-amplitude swelling of liver and heart mitochondria by cyclosporin is probably caused by the inhibitor binding to mitochondrial-matrix peptidyl-prolyl cis-trans isomerase and preventing it interacting with the adenine nucleotide translocase.

1. Isolated rat liver and heart mitochondria incubated in 150 mM-KSCN or sucrose medium in the presence of respiratory-chain inhibitors showed a large increase in swelling when exposed to 250 microM-Ca2+. Swelling was inhibited by bongkrekic acid and cyclosporin A in both media and by ADP in KSCN medium; the effect of ADP was reversed by carboxyatractyloside. These results demonstrate that this is a suitable technique with which to study the opening of the Ca2(+)-induced non-specific pore of the mitochondrial inner membrane and implicate the adenine nucleotide carrier in this process. 2. Titration of the rate of swelling with increasing concentrations of cyclosporin showed the number of cyclosporin-binding sites (+/- S.E.M.) in liver and heart mitochondria to be respectively 113.7 +/- 5.0 (n = 9) and 124.3 +/- 11.2 (n = 10) pmol/mg of protein, with a Ki of about 5 nM. 3. Liver and heart mitochondrial-matrix fractions were prepared free of membrane and cytosolic contamination and shown to contain cyclosporin-sensitive peptidyl-prolyl cis-trans isomerase (cyclophilin) activity. Titration of isomerase activity with cyclosporin gave values (+/- S.E.M.) of 110.6 +/- 10.1 (n = 5) and 165.4 +/- 15.0 (n = 3) pmol of enzyme/mg of liver and heart mitochondrial protein respectively, with a Ki of 2.5 nM. The similarity of these results to those from the swelling experiments suggest that the isomerase may be involved in the Ca2(+)-induced swelling. 4. The rapid light-scattering change induced in energized heart mitochondria exposed to submicromolar Ca2+ [Halestrap (1987) Biochem. J. 244, 159-164] was inhibited by ADP and bongkrekic acid, the former effect being reversed by carboxyatractyloside. These results suggest an interaction of Ca2+ with the adenine nucleotide carrier when the 'c' conformation. 5. A model is proposed in which mitochondrial peptidyl-prolyl cis-trans isomerase interacts with the adenine nucleotide carrier in the presence of Ca2+ to cause non-specific pore opening. The model also explains the involvement of the adenine nucleotide translocase in the PPi-mediated cyclosporin-insensitive increase in K+ permeability described in the preceding paper [Davidson & Halestrap (1990) Biochem. J. 268, 147-152]. 6. The physiological and pathological implications of the model are discussed in relation to reperfusion injury and cyclosporin toxicity.     

Inorganic pyrophosphate is located primarily in the mitochondria of the hepatocyte and increases in parallel with the decrease in light-scattering induced by gluconeogenic hormones, butyrate and ionophore A23187.

1. The effects of a variety of hormones on the PPi content and light-scattering of isolated rat liver cells was studied. 2. The basal PPi content was about 130 pmol/mg of cell protein, and increased after hormone addition, in parallel with a decrease in light-scattering which we have observed previously [Quinlan, Thomas, Armston & Halestrap (1983) Biochem. J. 214, 395-404]. 3. The mean increases in PPi content with the agonists shown (as pmol/mg of protein) were: 0.1 microM-glucagon, 25; 20 microM-phenylephrine, 30; 25 nM-vasopressin, 127; glucagon + phenylephrine, 115; glucagon + vasopressin, 382; 100 microM-ADP, 50; 15 microM-A23187, 72; 1 mM-butyrate, 80. 4. In the absence of extracellular Ca2+, vasopressin had little effect on either the PPi content or the light-scattering of hepatocytes. 5. The magnitude of the increase in PPi content correlated with that of the decrease in light-scattering irrespective of the stimulating agent, provided that the PPi did not exceed 300 pmol/mg of protein. Above this value little additional change in light-scattering was observed. 6. Subcellular fractionation showed that over 90% of the cellular PPi was intramitochondrial in both control and stimulated cells. 7. The data support the conclusions of previous experiments using isolated liver mitochondria [Davidson & Halestrap (1987) Biochem. J. 246, 715-723] that hormones increase the mitochondrial matrix volume through a Ca2+-induced rise in matrix [PPi]. 8. It is further proposed that this increase in mitochondrial [PPi] allows entry of ADP into the mitochondria in exchange for PPi and is therefore responsible for the increase in total mitochondrial adenine nucleotides observed after hormone treatment.     

Studies of Ca2+ -dependent smooth muscle mitochondria swelling using flow cytometry and spermine effects on this process

In the experiments, which were carried out using flow cytometry on isolated uterus mitochondria of nonpregnant rats, conditions for studying Ca2+-induced increase in nonspecific permeability of mitochondial membrane were tested. Fluorescent probe nonyl acridin orange was used to determine the purity of isolated mitochondria. Mitochondrial swelling induced by addition of Ca2+ or alamethicin was detected as a decrease in light side scattering (SS). It was shown that mitochondrial swelling in the presence of 100 microM Ca plus 1 microM A23187 was 86 +/- 4% compared with maximal response after alamethicin treatment. The calcium-induced swelling of mitochondria was inhibited by the addition of 5 microM cyclosporin plus 40 microM ADP. Mitochondrial swelling was inhibited by spermine at a dose of 0.1 micromol/mg or induced at a dose of 10 micromol/mg. It was supposed that the experimental approach proposed in this paper can be useful for mitochondrial pore modulating effectors screening.     

Regulation of the mitochondrial matrix volume in vivo and in vitro. The role of calcium.

The ability of alpha-adrenergic agonists and vasopressin to increase the mitochondrial volume in hepatocytes is dependent on the presence of extracellular Ca2+. Addition of Ca2+ to hormone-treated cells incubated in the absence of Ca2+ initiates mitochondrial swelling. In the presence of extracellular Ca2+, A23187 (7.5 microM) induces mitochondrial swelling and stimulates gluconeogenesis from L-lactate. Isolated liver mitochondria incubated in KCl medium in the presence of 2.5 mM-phosphate undergo energy-dependent swelling, which is associated with electrogenic K+ uptake and reaches an equilibrium when the volume has increased to about 1.3-1.5 microliter/mg of protein. This K+-dependent swelling is stimulated by the presence of 0.3-1.0 microM-Ca2+, leading to an increase in matrix volume at equilibrium that is dependent on [Ca2+]. Ca2+-activated K+-dependent swelling requires phosphate and shows a strong preference for K+ over Na+, Li+ or choline. It is not associated with either uncoupling of mitochondria or any non-specific permeability changes and cannot be produced by Ba2+, Mn2+ or Sr2+. Ca2+-activated K+-dependent swelling is not prevented by any known inhibitors of plasma-membrane ion-transport systems, nor by inhibitors of mitochondrial phospholipase A2. Swelling is inhibited by 65% and 35% by 1 mM-ATP and 100 microM-quinine respectively. The effect of Ca2+ is blocked by Ruthenium Red (5 micrograms/ml) at low [Ca2+]. Spermine (0.25 mM) enhanced the swelling seen on addition of Ca2+, correlating with its ability to increase Ca2+ uptake into the mitochondria as measured by using Arsenazo-III. Mitochondria derived from rats treated with glucagon showed less swelling than did control mitochondria. In the presence of Ruthenium Red and higher [Ca2+], the mitochondria from hormone-treated animals showed greater swelling than did control mitochondria. These data imply that an increase in intramitochondrial [Ca2+] can increase the electrogenic flux of K+ into mitochondria by an unknown mechanism and thereby cause swelling. It is proposed that this is the mechanism by which alpha-agonists and vasopressin cause an increase in mitochondrial volume in situ.     

A possible role for calmodulin in Ca2+-induced swelling of mitochondria

Ca2+-induced mitochondrial swelling was inhibited by a low concentration of calmodulin antagonists. Two affinities of Ca2+ to mitochondrial swelling were observed: high (2-5 microM) and low (more than 100 microM) systems. The high-affinity change was inhibited by micromolar level of trifluoperazine and W-7, but not by W-5. The possible mechanism of this inhibition and the role of CaM in mitochondria are discussed.     

Reye's syndrome: mitochondrial swelling and Ca2+ release induced by Reye's plasma, allantoin, and salicylate

The effects of Reye's plasma, allantoin, and salicylates on mitochondrial structure and Ca2+ transport have been investigated. Measurements of Ca2+ transport showed that when 20-30 microM Ca2+ was added to isolated rat liver mitochondria preincubated with one of these agents, Ca2+ uptake was followed by its spontaneous release into the medium. This was accompanied by large-amplitude swelling; the onset preceded the Ca2+ release. No further Ca2+ release was induced by uncoupler or the Ca2+ ionophore, A23187. The mitochondria continued to swell even after all of the Ca2+ had been released. The time between the addition of Ca2+ and the onset of swelling (or Ca2+ release) depended on the concentration of the agent added and the preincubation time; the extent of swelling did not. These effects were prevented, but not reversed, by ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid, ruthenium red, rotenone, or adenine nucleotides. The massive swelling and membrane disruption were confirmed by electron microscopy of the treated vs untreated mitochondria. Similar results concerning swelling and Ca2+ release were also seen with Ca2+ alone, but the time scale was much longer (i.e., greater than 3-4 min), indicating that these agents act by potentiating Ca2+-induced alterations in mitochondrial structure, as suggested by our earlier work (T.Y. Segalman and C.P. Lee (1982) Arch. Biochem. Biophys. 214, 522-530; M.E. Martens and C.P. Lee (1984) Biochem. Pharmacol. 33, 2869-2876). Our data show, therefore, that allantoin, salicylates, and the "toxic" agent in Reye's plasma severely limit the ability of isolated rat liver mitochondria to maintain their structural integrity under conditions of limited Ca2+ loading.     

Possible Mechanism for Formation and Regulation of the Palmitate-Induced Cyclosporin A-Insensitive Mitochondrial Pore

The mechanism of the palmitate-induced opening of the mitochondrial Ca2+-dependent cyclosporin A (CsA)-insensitive pore was studied, as well as the influence on this process of well-known modulators of the CsA-sensitive Ca2+-dependent pore. Palmitic acid, which can bind Ca2+ with high affinity, induced the cyclosporin A-insensitive swelling of mitochondria, whereas palmitoleic and 2-bromopalmitic acids, which have no such affinity for Ca2+, failed to induce the pore opening. The palmitate-induced Ca2+-dependent swelling of mitochondria was not affected by a well-known inhibitor of the CsA-sensitive pore (ADP) and an activator of this pore (inorganic phosphate, P(i)). However, this swelling was inhibited by physiological concentrations of ATP ([I]50 = 1.3 mM), but 100 microM ATP increased by 30% the rate of mitochondria swelling if Ca2+ had been added earlier. The effects of ATP (inhibition and activation) manifested themselves from different sides of the inner mitochondrial membrane. Mg2+ inhibited the palmitate-induced Ca2+-dependent swelling of mitochondria with [I]50 = 0.8 mM. It is concluded that palmitic acid induces the opening of the CsA-insensitive pore due to its ability for complexing with Ca2+. A possible mechanism of the pore formation and the influence of some modulators on this process are discussed.     

Zinc as an inducer of the membrane permeability transition in rat liver mitochondria

It is shown that 2-10 microM Zn2+ induces swelling of rat liver mitochondria incubated in a buffered sucrose medium either with valinomycin or with FCCP, Ca2+, ionophore A23187, oligomycin, and nigericin. This swelling was associated with the release of GSH from mitochondria. Both processes were sensitive to known inhibitors of the mitochondrial permeability transition (MPT), cyclosporin A, and Mg2+. Mitochondrial swelling induced by Zn2+ was also inhibited by rotenone, antymycin A, N-ethylmaleimide, butylhydroxytoluene, and spermine, whereas it was stimulated by tert-butyl hydroperoxide, diamide, and monobromobimane. It did not require the addition of phosphate. The same sensitivity to pH of the mitochondrial swelling induced by Zn2+ and by phenylarsine oxide suggests the same site of the interaction, namely, thiol groups. The ability of Zn2+ to induce mitochondrial swelling gradually decreased along with its increasing concentration above 10 microM. It is concluded that micromolar Zn2+ induces the MPT presumably by the interaction with cysteinyl residues. This process is independent of the mitochondrial membrane potential.     

Inhibition by [corrected] ursolic acid of [corrected] calcium-induced mitochondrial permeability transition and release of two proapoptotic proteins

The possible inhibition by [corrected] ursolic acid (UA) of [corrected] mitochondrial permeability transition (MPT) in mouse liver was investigated to identify the mechanisms underlying the hepatoprotective effect of UA. The effect of UA on liver MPT induced by Ca2+ was assessed by measuring changes in mitochondrial volume, mitochondrial membrane potential (MMP), release of matrix Ca2+, and transfer of cytochrome c (Cyt c) and apoptosis-inducing factor (AIF) from the intermembrane space to the cytoplasm. The results showed that obvious mitochondrial swelling, loss of MMP, and release of matrix Ca2+ occurred after the addition of 50 microM Ca2+. However, preincubation with 20, 50 or 100 microg ml(-1) UA significantly blocked the above changes. Addition of 100 microg ml(-1) UA inhibited on mitochondrial swelling by 73.2% after 5 min, while the MMP dissipating and Ca2+ releasing were, respectively, suppressed by 59.3% and 54.1% after 3 min. In addition, Western blot analysis showed Cyt c and AIF transferred from mitochondrial pellet to the supernatant after the addition of 50 microM Ca2+, but the process was significantly inhibited by various concentrations of UA. The results suggest that the mechanisms underlying the hepatoprotection of UA may be related to its direct inhibitory action on MPT.     

Inhibition by cyclosporin A of a Ca2+-dependent pore in heart mitochondria activated by inorganic phosphate and oxidative stress.

The capacity of cyclosporin A to inhibit opening of a Ca2+-dependent pore in the inner membrane of heart mitochondria was investigated. Whereas in the presence of 25 nmol of Ca2+/mg of mitochondrial protein and 5 mM-Pi mitochondria were unable to maintain accumulated Ca2+, inner-membrane potential and sucrose impermeability, all three parameters were preserved when cyclosporin was included. Pore opening was assayed directly by [14C]sucrose entry and entrapment in the matrix space. [14C]Sucrose entry induced by both Ca2+ plus Pi and Ca2+ plus t-butyl hydroperoxide was almost completely inhibited by 60 pmol of cyclosporin/mg of mitochondrial protein. It is concluded that cyclosporin A is a potent inhibitor of the pore.     

The permeability transition in heart mitochondria is regulated synergistically by ADP and cyclosporin A.

Heart mitochondria respiring in a sucrose medium containing P(i) show a permeability transition when challenged with Ca2+ and an oxidant such as cumene hydroperoxide. The transition results from the opening of a Ca(2+)-dependent pore and is evidenced by loss of membrane potential (delta psi) and osmotic swelling due to uptake of sucrose and other solutes. In the absence of oxidant, high concentrations of Ca2+ (100-150 microM) are necessary to induce loss of delta psi and initiate swelling. Cyclosporin A delays the loss of delta psi but enhances swelling under these conditions, apparently by promoting better retention of accumulated Ca2+. Cyclosporin A and ADP together restore delta psi in respiring mitochondria that have undergone the permeability transition at levels that are not effective when either is added alone. When the state of the Ca(2+)-dependent pore is assessed using passive osmotic contraction in response to polyethylene glycol (Haworth, R. A., and Hunter, D. R. (1979) Arch. Biochem. Biophys. 195, 460-467), cyclosporin A is found to be a partial inhibitor of solute flow through the open pore. Cyclosporin A decreases the Vmax of passive contraction and increases the Km for Ca2+ without affecting the Hill slope. ADP in the presence of carboxyatractyloside closes the pore almost completely even in the presence of 40 microM Ca2+. ADP shows mixed type inhibition of the Ca(2+)-dependent pore, and cyclosporin A increases the affinity of the pore for ADP. It is concluded that cyclosporin A and ADP act synergistically to close the Ca(2+)-dependent pore of the mitochondrion and that the pore is probably not formed directly from the adenine nucleotide transporter.     

Phosphate and calcium uptake by mitochondria and by perfused rat liver induced by the synergistic action of glucagon and vasopressin.

Co-administration of glucagon and vasopressin to rat liver perfused with buffer containing 1.3 mM-Ca2+ induces a 4-fold increase in Pi in the subsequently isolated mitochondria (from approx. 9 to approx. 40 nmol/mg of mitochondrial protein). This increase is not attributable to PPi hydrolysis, and is not observed if the perfusate Ca2+ is lowered from 1.3 mM to 50 microM. The increase in mitochondrial Pi closely parallels that of mitochondrial Ca2+; when the increase in Pi and Ca2+ accumulation is maximal, the molar ratio is close to that in Ca3(PO4)2. Measurement of changes in the perfusate Pi revealed that, whereas administration of glucagon or vasopressin alone brought about a rapid decline in perfusate Pi, the largest decrease (reflecting net retention of Pi by the liver) was observed when the hormone was co-administered in the presence of 1.3 mM-Ca2+. The synergistic action of glucagon plus vasopressin was nullified by lowering the perfusate Ca2+ to 50 microM. The data provide evidence that, whereas glucagon may be able to alter Pi fluxes directly in intact liver, any alterations induced by vasopressin are indirect and result only from its action of mobilizing Ca2+.     

Palmitic acid opens a novel cyclosporin A-insensitive pore in the inner mitochondrial membrane

An assortment of agents can induce mitochondria to undergo a permeability transition, which results in the inner mitochondrial membrane becoming nonselectively permeable to small (<1500 Da) solutes. This mitochondrial permeability transition (MPT) is characterized by a strict dependence on matrix Ca2+ and sensitivity to cyclosporin A (CsA). However, it is becoming increasingly clear that other experimental conditions can elicit increases in mitochondrial permeability that are distinct from this classic MPT. For example, butylated hydroxytoluene (BHT; Sokolove, P. M., and Haley, L. M. (1996) J. Bioenerg. Biomembr. 28, 199-206) and signal peptides (Sokolove, P. M., and Kinnally, K. W. (1996) Arch. Biochem. Biophys. 336, 69-76) promote increases in mitochondrial permeability that are CsA-insensitive. It has been suggested (Gudz, T., Eriksson, O., Kushnareva, Y., Saris, N.-E., and Novgorodov, S. A. (1997) Arch. Biochem. Biophys. 342, 143-156) that BHT might be opening a CsA-insensitive pore by increasing phospholipase A2 activity and thereby producing an accumulation of free fatty acids and lysophospholipids. We have therefore examined the effect of the saturated free fatty acid, palmitic acid (PA), on the permeability of isolated rat liver mitochondria. The following results were obtained: (1) In the absence of additional triggers, PA (20-60 microM) induced concentration-dependent, CsA-insensitive mitochondrial swelling. (2) Swelling required mitochondrial energization. (3) PA-induced swelling was fast and occurred without a lag. (4) Both Ca2+ and Sr2+ supported PA-induced swelling; the site of cation action was the matrix. (5) EGTA and BSA were potent inhibitors of PA-induced swelling. (6) PA opened a pore rather than disrupting mitochondrial membrane structure. (7) The pore opened by PA closed spontaneously. These results suggest that palmitic acid promotes a nonclassic permeability increase that is clearly distinguishable from the occurrence of the MPT.     

Calcium-induced cytochrome c release from CNS mitochondria is associated with the permeability transition and rupture of the outer membrane

The mechanisms of Ca2+-induced release of Cytochrome c (Cyt c) from rat brain mitochondria were examined quantitatively using a capture ELISA. In 75 or 125 mm KCl-based media 1.4 micromol Ca2+/mg protein caused depolarization and mitochondrial swelling. However, this resulted in partial Cyt c release only in 75 mm KCl. The release was inhibited by Ru360, an inhibitor of the Ca2+ uniporter, and by cyclosporin A plus ADP, a combination of mitochondrial permeability transition inhibitors. Transmission electron microscopy (TEM) revealed that Ca2+-induced swelling caused rupture of the outer membrane only in 75 mm KCl. Koenig's polyanion, an inhibitor of mitochondrial porin (VDAC), enhanced swelling and amplified Cyt c release. Dextran T70 that is known to enhance mitochondrial contact site formation did not prevent Cyt c release. Exposure of cultured cortical neurons to 500 microM glutamate for 5 min caused Cyt c release into the cytosol 30 min after glutamate removal. MK-801 or CsA inhibited this release. Thus, the release of Cyt c from CNS mitochondria induced by Ca2+ in vitro as well as in situ involved the mPT and appeared to require the rupture of the outer membrane.     

Mechanisms of nordihydroguaiaretic acid-induced [Ca2+]i increases in MDCK cells

The effect of nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor, on Ca2+ signaling in Madin Darby canine kidney (MDCK) cells has been investigated. NDGA (10-100 microM) increased [Ca2+]i concentration-dependently. The [Ca2+]i increase comprised an initial slow rise and a plateau over a time period of 5 min. Ca2+ removal partly inhibited the Ca2+ signals induced by 25-100 microM NDGA and abolished that induced by 10 microM NDGA. In Ca(2+)-free medium, pretreatment with 0.1 mM NDGA for 12 min abolished the [Ca2+]i increase induced by the mitochondrial uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP; 2 microM) and the endoplasmic reticulum (ER) Ca2+ pump inhibitor thapsigargin (1 microM). However, 0.1 mM NDGA still increased [Ca2+]i after Ca2+ stores had been depleted by pretreating with 2 microM CCCP, 1 microM thapsigargin and 0.1 mM cyclopiazonic acid. NDGA (50 microM) activated Mn2+ quench of fura-2 fluorescence at 360 nm excitation wavelength, which was almost abolished by 50 microM La3+. This implies NDGA induced Ca2+ influx mainly via a La(3+)-sensitive pathway. Consistently, 50 microM La3+ pretreatment inhibited 0.1 mM NDGA-induced [Ca2+]i increase. Adding 3 mM Ca2+ increased [Ca2+]i in cells pretreated with 0.1 mM NDGA in Ca(2+)-free medium, suggesting NDGA activated capacitative Ca2+ entry. Pretreatment with 0.1 mM NDGA for 200 s prior to Ca2+ did not alter 1 microM thapsigargin-induced capacitative Ca2+ entry. Pretreatment with 40 microM aristolochic acid to inhibit phospholipase A2 reduced 0.1 mM NDGA-induced Ca2+ release by 65%, but inhibiting phospholipase C with 2 microM U73122 had little effect. This suggests NDGA-induced Ca2+ release was independent of inositol 1,4,5-trisphosphate (IP3), but was modulated by phospholipase A2.     

Mitochondrial injury by disulfiram: two different mechanisms of the mitochondrial permeability transition.

Disulfiram (Ds), a clinically employed alcohol deterrent of the thiuram disulfide (TD) class of compounds, is known to cause hepatitis and neuropathies. Although this drug has been shown to inhibit different thiol-containing enzymes, the actual mechanism of Ds toxicity is not clear. We have previously demonstrated that Ds impairs the permeability of inner mitochondrial membrane (IMM) [Arch. Biochem. Biophys. 356 (1998) 46]. In this report, the effect of Ds and its structural analogue thiram (Th) on mitochondrial functions was studied in detail. We found that mitochondria metabolize TDs in a NAD(P)H- and GSH-dependent manner. At the concentration above characteristic threshold, TDs induced irreversible oxidation of NAD(P)H and glutathione (GSH) pools, collapse of transmembrane potential, and inhibition of oxidative phosphorylation. The presence of Ca(2+) and exhaustion of mitochondrial glutathione (GSH+GSSG) decreased the threshold concentration of TDs. Swelling of the mitochondria and leakage of non-transported fluorescent dye BCECF from the matrix indicated that TDs induced the mitochondrial permeability transition (MPT). Mitochondrial permeabilization by TDs involves two, apparently distinct mechanisms. In the presence of Ca(2+), TDs produced cylosporin A-sensitive swelling of mitochondria, which was inhibited by ADP and accelerated by carboxyatractyloside (CATR) and phosphate. In contrast, the swelling produced by TDs in the absence of Ca(2+) was not sensitive to cyclosporin A (CsA), ADP and CATR but was inhibited by phosphate. Titration with N-ethylmaleimide revealed that these two mechanisms involve different SH-groups and probably different transport proteins on the IMM. Our findings indicate that at pharmacologically relevant concentrations TDs may cause an irreversible mitochondrial injury as a result of induction of the MPT.     

Tributyltin causes cytochrome C release from isolated mitochondria by two discrete mechanisms

Using isolated liver mitochondria we show that low concentrations of TBT (0.5 microM) cause the release of mitochondrial cytochrome c, in the presence of Ca(2+). This is reflected in a rapid loss of membrane potential (DeltaPsi(m)), and a large-amplitude swelling characteristic of mitochondrial permeability transition (MPT). Despite this, the inclusion of cyclosporin A could not prevent the release of cytochrome c. Further, in the absence of Ca(2+), low concentrations of TBT (0.5 microM) resulted in a slow sub-maximal shift of DeltaPsi(m), not characteristic of MPT, which was still paralleled by a release of cytochrome c. Further experiments showed that the loss of DeltaPsi(m) in the absence of Ca(2+) was due to a combination of inhibition of respiration and a direct uncoupling effect on the respiratory chain. Under these conditions, rapid swelling of mitochondria could be demonstrated, due to chloride exchange over the inner mitochondrial membrane. Taken together these data suggest that TBT can induce the release of cytochrome c in intact cells by at least two mechanisms. The first and critical mechanism is initiated immediately the mitochondria sense the presence of TBT and involves a slow loss of DeltaPsi(m) and induction of swelling, which allows release of cytochrome c in a relatively non-specific manner and independently from a rise in [Ca(2+)](i). The second mechanism involves the induction of formal MPT as intracellular [Ca(2+)](i) increases. These data help to explain previous observations in intact lymphocytes demonstrating TBT-induced release of mitochondrial cytochrome c in the absence of a rise in [Ca(2+)](i) (Stridh, H., Gigliotti, D., Orrenius, S., and Cotgreave, I. A. (1999) Biochem. Biophys. Res. Commun. 266, 460-465).     

Phosphorylation-dependent and -independent pathways of platelet aggregation.

We have used the non-specific inhibitor of protein kinases, staurosporine, to investigate the role of protein phosphorylation during aggregation, the mobilization of intracellular Ca2+ (Ca2+)i and intracellular pH (pHi) in thrombin-stimulated platelets. The concentration of staurosporine chosen for these studies, 1 microM, was previously reported to inhibit protein phosphorylation completely but to have no effect on the activation of phospholipase C in thrombin-stimulated human platelets [Watson, McNally, Shipman & Godfrey (1988) Biochem. J. 249, 345-350]. Aggregation induced by phorbol dibutyrate is slow (several minutes) and is inhibited completely by staurosporine. In contrast, aggregation induced by thrombin, platelet-activating factor or ionophore A23187 is rapid (occurs within 60 s), and is slowed, but not inhibited, in the presence of staurosporine. On the other hand, staurosporine causes a small potentiation of the peak [Ca2+]i signal induced by thrombin and a marked increase in the half-life of decay of this signal, but has no effect on pHi. Under conditions designed to prevent an increase in [Ca2+]i (presence of Ni2+ to prevent Ca2+ entry, and depletion of the intracellular Ca2+ stores), aggregation induced by thrombin resembles that by phorbol dibutyrate and is now inhibited completely by staurosporine. Taken together, these results provide evidence for two signalling pathways for aggregation, a relatively rapid phosphorylation-independent route mediated by Ca2+ and a slower, phosphorylation-dependent, pathway mediated by protein kinase C. Since staurosporine slows aggregation induced by thrombin, it appears that under normal conditions these pathways interact synergistically.