2,5,2',5'-Tetrachlorobiphenyl impairs the bioenergetic functions of isolated rat liver mitochondria

The effects of 2,5,2',5'-tetrachlorobiphenyl (25TCB) on parameters related to the bioenergetic functions of isolated rat liver mitochondria were investigated. State 3 respiration was inhibited by 25TCB with both succinate and glutamate/malate as the respiratory substrates. The extent of inhibition with succinate was larger than that observed with glutamate/malate. The concentration of 25TCB required to cause 50% inhibition for succinate was 51 microM, but with glutamate/malate, only 53% inhibition was observed at 200 microM. 25TCB stimulated state 4 respiration after 1-2 min lag period; state 4 respiration in the presence of glutamate/malate was more intensely stimulated by 25TCB than in the presence of succinate. 25TCB dissipated the membrane potential across the mitochondrial membranes. Isolated rat liver mitochondria accumulate large amounts of Ca2+ at the expense of respiration-linked energy (substrate oxidation) or of that provided by the hydrolysis of ATP by the mitochondrial ATPase. The Ca2+ accumulation by mitochondria was severely depressed by 25TCB when the energy was supplied by respiration. Furthermore, the inhibition of Ca2+ accumulation by 25TCB with succinate was greater than that produced with glutamate/malate. On the other hand, with ATP as the source of energy, 25TCB inhibited Ca2+ accumulation at high concentrations. 25TCB also released Ca2+ from mitochondria that had already accumulated Ca2+, indicating that mitochondrial membrane integrity was damaged by the intercalation of 25TCB. These results show that 25TCB impairs mitochondrial energy production, and inhibits Ca2+ sequestration by mitochondria.     

Effects of bepridil on Ca2+ uptake by cardiac mitochondria

Isolated rat heart mitochondria accumulate large amounts of Ca2+ at the expense of respiration-linked energy or of that provided by the hydrolysis of ATP by the mitochondrial ATPase. At concentrations below 10 microM bepridil has no effect on the first mechanism but inhibits the second. At higher concentrations bepridil depresses both. At low concentrations bepridil decreases proton influx into mitochondria in ADP-stimulated respiration while it has no effect on proton ejection in Ca2+-stimulated respiration. A preliminary study shows that bepridil inhibits ATP hydrolysis linked to Ca2+ absorption by mitochondria. The calcium antagonists verapamil, nifedipine and diltiazem exhibit none of these effects.     

In vitro and in vivo effects of R023-6152 on heart mitochondrial calcium and energy metabolism.

Previous studies have shown that Ca2+ channel antagonists in all chemical classes can inhibit Na(+)-induced CA2+ release from mitochondria. The effects of R023-6152, a new thiazepinone Ca2+ channel antagonist, on isolated rabbit heart mitochondrial Ca2+ transport and respiratory activity were compared with those of diltiazem. Heart mitochondria were also isolated and assayed from dogs treated in vivo with either R023-6152 or diltiazem. The results indicate that R023-6152 produces half-maximal inhibition of Na(+)-induced Ca2+ release from isolated mitochondria at relatively the same concentrations (10-30 microM) as diltiazem but also produces inhibition of mitochondrial Ca2+ uptake and state 3 respiration at concentrations (25-100 microM), at which diltiazem has no effect. The greater lipophilicity of R023-6152 in gaining access to and inhibiting the phosphate transporter in the mitochondrial membrane as compared with that of diltiazem may explain these results. Heart mitochondria isolated from dogs treated with diltiazem and R023-6152 exhibited lower rates of state 3 respiration as compared with controls. We suggest that this may result from a reduction in transsarcolemmal Ca2+ flux causing a down-regulation in mitochondrial dehydrogenase activity and not from any direct intracellular effects of the two drugs.     

Effects of phenothiazine drugs on the active Ca2+ transport by sarcoplasmic reticulum

The effect of phenothiazines (trifluoperazine and chlorpromazine) on the activity of the sarcoplasmic reticulum Ca2+ pump was investigated. These drugs have a biphasic action on the ATPase activity. They inhibit the enzyme at high concentrations, but below 150 microM, they promote a significant stimulation of the ATP hydrolysis, which is accompanied by a slight increase of the Ca2+ transport. Leaking of the membrane occurs only at drug concentrations above 150 microM. The phenothiazine stimulatory effect was not found in the isolated enzyme whose activity was inhibited at all concentrations studied. These results indicate that low concentrations of phenothiazines uncouple the sarcoplasmic reticulum Ca2+ pump without disrupting the membrane and that the drug stimulatory effect on the ATPase is not due to a direct interaction with the enzyme. It is suggested that a coupling factor or a specific microenvironment surrounding the enzyme regulates the association between the Ca2+ transport and the ATP hydrolysis by sarcoplasmic reticulum of the skeletal muscle cell.     

Effects of Pb2+ added in vitro on Ca2+ movements in isolated mitochondria and slices of rat kidney cortex

We have studied the effects of Pb2+ added in vitro on the movements of Ca2+ in renal cortical mitochondria and tissue slices. The isolated mitochondria rapidly accumulated 45Ca2+ at 25 degrees by a mechanism that was dependent on respiration and inhibited 96% by ruthenium red. A concentration of 10 microM Pb2+ inhibited the Ca2+ accumulation at least as effectively as did ruthenium red. About 20% of the Ca2+ accumulation persisted at 1 degrees with a similar sensitivity to inhibitors, including 60% inhibition by Pb2+. Similar results were obtained when the accumulation of Ca2+ at 25 degrees was measured by means of a calcium-sensitive electrode, Pb2+ inhibiting by 80%. Calcium that had been accumulated by mitochondria at 25 degrees was released completely by the ionophore A23187 or by 10 microM Pb2+. The release induced by Pb2+ was greatly inhibited by ruthenium red. The Ca2+ content of tissue slices of renal cortex increased 4-fold during incubation at 1 degree while the Ca2+ content of mitochondria within the slices more than doubled, the latter being determined by isolation of mitochondria from the slices after incubation. The presence of Pb2+ (200 microM) in the incubation medium of the slices substantially reduced the entry of Ca2+ into the whole slices and into mitochondria within the slices. When the slices preincubated at 1 degree were warmed to 25 degrees in oxygenated medium, they brought about a net extrusion of Ca2+, some of which was derived from the mitochondria; Pb2+ did not alter the final level of Ca2+ then attained in the slices, but it caused a significant decrease in the quantity retained in the mitochondria. We conclude that Pb2+ both inhibits the uptake of Ca2+ by renal cortical mitochondria and displaces Ca2+ from them, these effects occurring whether the mitochondria are isolated or in situ.     

Studies on the mechanism of the cardiotonic activity of MDL 19205: effects on several biochemical systems

MDL 19205, 4-ethyl-1,3-dihydro-5-(4-pyridinyl-carbonyl)-2H-imidazol-2-one, is a new drug with cardiotonic properties. Its effects on several biochemical systems considered to be important in myocardial contraction were investigated. Cyclic nucleotide phosphodiesterases (PDEs) from dog hearts were separated into three isoenzymes, F I, F II, and F III, and effect of the drug on these enzymes was tested. MDL 19205 inhibited F III PDE specifically and produced little or no inhibition of F I and F II PDEs. The IC50 for inhibition of F III PDE was 8.6 microM when 0.5 microM cyclic AMP (cAMP) was used, whereas no more than 10% inhibition of F I and 18% of F II PDEs occurred at drug concentrations up to 200 microM when 1 microM cAMP was used. Concentrations of MDL 19205 up to 100 microM had no effect on Ca2+-adenosine triphosphatase (ATPase) or Ca2+ uptake by dog cardiac sarcoplasmic reticulum. At 100 microM, the drug produced a weak (18%) inhibition of Na+,K+-ATPase. It is suggested that inhibition of F III PDE may be the primary mechanism by which MDL 19205 produces its cardiotonic effect. Inhibition of Na+,K+-ATPase may also be involved at very high concentrations of this drug.     

Effect of the antianginal drug bepridil on intracellular Ca2+ release and extracellular Ca2+ influx in human neutrophils

To understand more fully the effects of bepridil, an antiarrhythmic and antianginal drug, on myocardial ischemia-reperfusion injury and systemic immune responses, its effect on intracellular Ca2+ levels ([Ca2+]i) in human neutrophils was investigated by using fura-2 as a fluorescent probe. Bepridil (10-200 microM) increased [Ca2+]i in a concentration-dependent fashion. This signal was partly inhibited by removal of extracellular Ca2+. In a Ca(2+)-free medium, pretreatment with bepridil (100 microM) abolished the Ca2+ release induced by thapsigargin (1 microM), an endoplasmic reticulum Ca2+ pump inhibitor, and by carbonylcyanide m-chlorophenylhydrazone (2 microM), a mitochondrial uncoupler. Pretreatment with carbonylcyanide m-chlorophenylhydrazone and thapsigargin, respectively, partly inhibited bepridil-induced Ca2+ release. Addition of Ca2+ (3 mM) increased [Ca2+]i after pretreatment with bepridil (100 microM) in a Ca(2+)-free medium. Bepridil (100 microM)-induced Ca2+ release was not altered when phospholipase C was inhibited by U73122 (2 microM). Both Ca2+ release and Ca2+ entry induced by bepridil (100 microM) were augmented by activating protein kinase C with phorbol 12-myristate 13-acetate (10 nM), and were suppressed by inhibiting protein kinase C with GF 109203X (2 microM). Treatment with bepridil (10-20 microM) for 30 min increased the production of reactive oxygen intermediates (ROI) by more than 50%. Collectively, it was found that bepridil increased [Ca2+]i concentration-dependently in human neutrophils by releasing Ca2+ from the endoplasmic reticulum, mitochondria and, possibly, other compartments in a phospholipase C-independent manner. Bepridil also activated Ca2+ influx. The activity of protein kinase C may regulate bepridil-induced Ca2+ release and Ca2+ entry.     

Uptake of inorganic lead in vitro by isolated mitochondria and tissue slices of rat renal cortex

Slices of rat renal cortex were shown to take up Pb2+ during incubation in vitro; Pb2+ was also shown to enter mitochondria within the slices. The uptake of Pb2+ by isolated mitochondria was inhibited by N-3, La3+ and ruthenium red. A steady state of uptake was attained within 60 sec. The concentration dependence of uptake was complex; maximum uptake was attained at 25 microM and inhibition ensued at higher concentrations. A substantial inhibitor-resistant component of Pb2+ uptake was noted, especially at medium Pb2+ concentrations greater than 25 microM, and these concentrations also inhibited respiration state 3. The effects on respiration were reduced if the mitochondria had been preincubated with ruthenium red. Slices of renal cortex incubated at 1 degree in medium with various concentrations of Pb2+ showed two fractions of uptake, one saturating at 50-100 microM external Pb2+ and the other at 150-200 microM. Subsequent incubation for 60 min at 25 degrees led to further uptake at all concentrations. Upon isolation of mitochondria from incubated slices, significant amounts of Pb2+ were detected in the mitochondria within 5 min of addition of Pb2+ (200 microM), with maximum attained at 30 min. Electron microscopy of slices showed electron-dense particles, apparently of Pb2+, in the cortical cells but the greatest concentration was deposited in the basement membranes. The results indicate the importance of the basement membrane in limiting access of Pb2+ to cortical cells, and of mitochondria in accumulating Pb2+ once it is in the cells. They also illustrate the importance of interactions between Pb2+ and Ca2+.     

Inhibition of mitochondrial Ca2+ release diminishes the effectiveness of methyl mercury to release acetylcholine from synaptosomes.

The interaction of methyl mercury (MeHg) with nerve-terminal mitochondria as a potential mechanism for its effects on the release of acetylcholine (ACh) was studied using rat brain synaptosomes. The primary goal was to assess the relative contribution of extracellular Ca2+ and Ca2+ released from nerve-terminal mitochondria to the previously described stimulatory effects of MeHg on spontaneous release of ACh. A secondary goal was to address possible mechanisms by which MeHg might interact with nerve-terminal mitochondria to elicit Ca2+ discharge and subsequent release of ACh. MeHg depressed the high-affinity uptake of [3H]choline into synaptosomes by approximately 25 and 45% when synaptosomes were incubated with [3H]choline in the presence of 10 and 100 microM MeHg, respectively. In Ca(2+)-containing solutions, 10 and 100 microM MeHg increased the release of [3H]ACh from [3H]choline-loaded synaptosomes by 10 and 30%, respectively; this effect was maximal at 10 sec. Excluding Ca2+ from the reaction medium diminished the effectiveness of both 10 and 100 microM MeHg for inducing [3H]ACh release by about 30 and 25%, respectively, from that of Ca(2+)-containing solutions; however, significant increases still occurred in nominally Ca(2+)-free solutions. Ruthenium red (RR), an inhibitor of mitochondrial Ca2+ transport, was tested for its ability to disrupt MeHg-induced release. RR alone increased [3H]ACh release by 8-10 and 10-13% at 20 and 60 microM, respectively. RR-induced release was attenuated only slightly in Ca(2+)-free solutions. Preincubation of [3H]choline-loaded synaptosomes with RR reduced the stimulatory effect of MeHg on release of [3H]ACh both in the presence and in the absence of Ca2+. The fluorescent potentiometric carbocyanine dye diS-C2(5) was used to assess the ability of RR to prevent MeHg-induced depolarization of intrasynaptosomal mitochondria. RR (20 microM) itself did not depolarize the mitochondrial membrane potential, nor did it prevent MeHg from depolarizing the mitochondria. The results indicate that extracellular Ca2+ contributes only partially to MeHg-induced spontaneous release of ACh. The results with RR suggest that MeHg interacts with mitochondria to induce release of bound intraterminal Ca2+ stores, resulting ultimately in stimulated release of ACh. The ability of RR to prevent release of mitochondrial Ca2+ and, subsequently, ACh is not due to prevention of access of MeHg to the mitochondria, nor to stabilization of the mitochondrial membrane. Finally, MeHg reduces choline uptake into nerve terminals. Thus, MeHg could interfere with cholinergic neurotransmission by affecting the regulatory step in ACh synthesis and by increasing the spontaneous release of transmitter.(ABSTRACT TRUNCATED AT 400 WORDS)     

Fenoverine inhibition of calcium channel currents in single smooth muscle cells from rat portal vein and myometrium.

1. The effects of fenoverine, an antispasmodic drug, have been studied on the Ca2+ channel currents of isolated cells from rat portal vein and pregnant myometrium by the patch-clamp technique (whole-cell configuration). 2. Fenoverine inhibited both fast and slow Ca2+ channel currents in a concentration-dependent manner. Half-inhibition of fast Ca2+ channel current (holding potential of -70 mV) and slow Ca2+ channel current (holding potential of -40 mV) in portal vein smooth muscle were obtained at concentrations of 7.5 and 1.9 microM, respectively. In myometrium, the fenoverine concentration which blocked 50% of the slow Ca2+ channel current (holding potential of -70 mV) was 2.3 microM. 3. Administration of fenoverine at rest reduced both Ca2+ channel currents. Currents activated repetitively, at a rate between 0.05 and 0.1 Hz, were inhibited equally which indicates an absence of use-dependent inhibition. 4. When cells held at depolarized membrane potentials at which fast or slow Ca2+ channel currents were strongly inactivated, the inhibitory effects of fenoverine were enhanced on both Ca2+ channel currents which indicates that the fenoverine-induced inhibition was voltage-dependent. The fenoverine concentrations which blocked the inactivated Ca2+ channels were 5-7 times lower than those which blocked the resting Ca2+ channels. 5. Our results show that fenoverine depresses inward currents through fast and slow Ca2+ channels. This effect may be explained by the preferential binding of fenoverine to resting Ca2+ channels. In addition, fenoverine has a higher affinity for inactivated Ca2+ channels than for resting channels.     

Activation of calcium uptake in rat liver mitochondria by aminoglucoside antibiotics

Ca uptake in rat liver mitochondria is accelerated by various aminoglucoside antibiotics and, to a lesser degree, by triethylenetetramine and by protamine. With 1 mM Mg2+ and at a concentration of Ca2+ = 2 microM maximal (about six-fold) activation is achieved with 20 microM neomycin; at higher concentrations of the antibiotic the velocity of Ca uptake decreases. Activation to the same degree by gentamicin, kanamycin or streptomycin requires higher concentrations of the antibiotics. The reason for the acceleration of Ca uptake by neomycin is an allosteric alteration of the kinetics corresponding to that previously observed in the presence of spermine. The conformity with effects of spermine and possible inferences of that conformity are discussed.     

Reye's syndrome: salicylates and mitochondrial functions

The effects of aspirin (acetylsalicylate, ASA) and related compounds in the presence of Ca2+ on the oxidative metabolism of isolated rat liver mitochondria were studied. Intact mitochondrial preparations preincubated with ASA + Ca2+ exhibited a transient stimulation of the state 4 respiratory rate with NAD+-linked substrates, followed by an inhibition which could not be released by the addition of ADP or uncoupler. Maximum respiratory rates were achieved by subsequent addition of NAD+ or succinate. The Ca2+-transport inhibitors ruthenium red and ethylene glycol-bis-(beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA) prevented these effects. Five brands of commercial aspirin were tested and were as effective as purified ASA. Tylenol (acetaminophen) could reproduce these effects only at much higher (greater than or equal to 10-fold) concentrations. Other salicyl derivatives showed results qualitatively similar to ASA, with potencies in the order: acid much much greater than ASA much greater than alcohol greater than or equal to catechol greater than amide, salicylate being approximately 10-fold more potent than ASA. The magnitude of the effect seen depended on the Ca2+ (endogenous + exogenous) and salicylate concentrations/mg mitochondrial protein, and on the length of the preincubation. Added inorganic phosphate was also required. That salicylate + Ca2+ induces an increase in the permeability of the mitochondrial inner membrane was demonstrated by the observation that 90% of the intramitochondrial NAD(P)+ was released into the surrounding medium upon preincubation of intact mitochondria with these agents. Salicylate + Ca2+ had virtually no effect on respiration with succinate (+ rotenone) as substrate at salicylate concentrations which markedly affected NAD+-linked substrate oxidation. The presence of rotenone in the preincubation mixture prevented the damaging effects of salicylate + Ca2+ on the mitochondrial membrane, suggesting that the redox state of intramitochondrial pyridine nucleotides can modulate these effects. The results reported here are similar to those reported previously by our laboratory for the effects of Reye's plasma and allantoin + Ca2+, and indicate that, like these agents, salicylate and salicyl compounds can potentiate the Ca2+-induced damage to the mitochondrial inner membrane and may be another factor responsible for Reye's syndrome.     

Differentiation of calcium antagonists with respect to their effects in normal and skinned taenia caeci preparations

In taenia preparations, depolarized by a K+-rich medium, Ca2+ caused contraction and cinnarizine (0.4-100 microM), trifluoperazine (2-100 microM) and verapamil (0.02-10 microM) caused concentration-dependent antagonism of Ca2+, displacing the Ca2+ log concentration-effect curve to the right and depressing the maximal response. Equieffective (IC75) antispasmogenic concentrations were selected. The antispasmogenic effects of verapamil were readily offset by removing the drug from the bathing fluid but those of the other drugs were not. The calcium antagonists (antispasmogenic IC75) were then tested for spasmolytic activity in tissues generating tension in response to the EC80 of Ca2+. Verapamil was more effective in producing spasmolysis than cinnarazine or trifluoperazine. In skinned taenia preparations, verapamil (100 microM) did not inhibit Ca2+-induced contractions. High concentrations of cinnarizine (100 microM) and trifluoperazine (100 microM) inhibited Ca2+-induced activation of the contractile proteins. However, antispasmogenic IC75s from intact taenia were not able to produce this effect on skinned preparations. It is concluded that there are differences between calcium antagonists. The action of verapamil on intact taenia is mainly exerted on the plasma membrane. Cinnarizine and trifluoperazine act both on the plasma membrane and upon the intracellular contractile machinery.     

Antidiabetic sulphonylureas activate mitochondrial permeability transition in rat skeletal muscle

Antidiabetic sulphonylureas can bind to various intracellular organelles including mitochondria. The aim of this study was to monitor the influence of antidiabetic sulphonylureas on membrane permeability in mitochondria isolated from rat skeletal muscle. The effects of glibenclamide (and other sulphonylurea derivatives) on mitochondrial function were studied by measuring mitochondrial swelling, mitochondrial membrane potential, respiration rate and Ca2+ transport into mitochondria. We observed that glibenclamide induced mitochondrial swelling (EC50 = 8.2 +/- 2.5 microM), decreased the mitochondrial membrane potential and evoked Ca2+ efflux from the mitochondrial matrix. These effects were blocked by 2 microM cyclosporin A, an inhibitor of the mitochondrial permeability transition. Moreover, 30 microM glibenclamide accelerated the respiratory rate in the presence of glutamate/malate, substrates of complex I of the mitochondrial respiratory chain. In conclusion, we postulate that the antidiabetic sulphonylureas activate the mitochondrial permeability transition in skeletal muscle by increasing its sensitivity to Ca2+.     

Changing surface charge with salicylate differentiates between subgroups of calcium-antagonists.

Sodium salicylate (5-10 mM) has been used to distinguish the effects of the three calcium-antagonist subgroups which had been previously differentiated in functional studies. Sodium salicylate (10 mM) reduced the antagonistic effects of verapamil and diltiazem on Ca2+-induced contractions of K+ (40 mM)-depolarized taenia preparations from the guinea-pig caecum. In contrast, salicylate had no effect on the potency of nifedipine and increased the inhibitory effects of cinnarizine and flunarizine. Sodium salicylate (10 mM) had little effect on Ca2+-induced contractions per se. In preparations pretreated with calcium-antagonists and recontracted with high concentrations of Ca2+, salicylate (5 mM) caused an additional contraction when the preparations had been pretreated with verapamil or diltiazem but had no effect in control or nifedipine-treated preparations. In contrast, salicylate relaxed Ca2+-induced contractions in tissues which had been pretreated with cinnarizine, flunarizine, pimozide, bepridil, fendiline, perhexiline and with the calmodulin antagonist W-7. The mechanism of action of salicylate was investigated. Inhibition of prostaglandin biosynthesis or of oxidative phosphorylation by salicylate was not responsible for these effects because indomethacin (28 microM) and 2,4-dinitrophenol (20 microM) did not differentiate between calcium antagonists. The effects of salicylate are ascribed to an increase in negative surface charge on the membrane because other agents changing surface charge (3,5-dichlorosalicylate, 0.3 mM; benzoate, 20 mM) have similar effects and their potency is dependent on their affinity for lipid membranes. Furthermore, salicylate increased the effectiveness of the cationic local anaesthetic, (+)-propranolol (100 microM), but did not change the effects of the neutral local anesthetic, benzocaine (1 mM). It is argued that salicylate increases the effectiveness of cinnarizine by increasing accumulation of this drug in the cell membrane or at intracellular sites whereas the reduced effectiveness of verapamil and diltiazem is secondary to a change in the state of the Ca2+ channel.     

Salicylate-induced kidney mitochondrial permeability transition is prevented by cyclosporin A

The effect of salicylate, the active metabolite of aspirin (acetyl salicylic acid) in the presence of Ca2+ and phosphate on mitochondrial permeability transition (MPT) was studied. MPT is often associated with opening of a Ca2+ -induced pore. The opening of this pore leads to swelling, loss of mitochondrial membrane potential and release of accumulated Ca2+. In freshly isolated rat kidney mitochondria, salicylate (400 microM) in the presence of 20 nmol Ca2+/mg protein and 0.1 mM phosphate induced swelling, loss of mitochondrial membrane potential and release of accumulated Ca2+. All these changes were eliminated when cyclosporin A (1 microM), (a pore inhibitory agent) was included in the incubation medium. Unlike salicylate, unhydrolyzed aspirin (400 microM) induced these changes slightly. We concluded that salicylate acts as an activator of Ca2+ and phosphate in promoting the opening of kidney inner mitochondrial membrane pore. As a result a great consideration should be given to its toxicological effect.     

Mechanisms underlying the activation of large conductance Ca2+-activated K+ channels by nordihydroguaiaretic acid

The mechanisms underlying the activation of large conductance Ca2+-activated K+ (BK) channel by nordihydroguaiaretic acid (NDGA) were examined in human embryonic kidney (HEK293) cells, where BK channel alpha (BKalpha) or a plus beta1 subunit (BKalphabeta1) was heterologously expressed, and also in freshly isolated porcine coronary arterial smooth muscle cells (PCASMCs). The activity of both BKalpha and BKalphabeta1 channels was increased by 10 microM NDGA in similar manners, indicating the selective action on the a subunit to increase Ca2+ sensitivity. The application of NDGA to PCASMCs induced outward current and hyperpolarization under voltage and current clamp, respectively, in a concentration-dependent manner (> or = 3 microM). These effects were blocked by 100 nM iberiotoxin. Electrical events induced by NDGA (> or = 10 microM) were, unexpectedly, associated with the increase in [Ca2+]i. After the treatment with caffeine and ryanodine, the [Ca2+]i increase by NDGA was markedly reduced and the hyperpolarization by NDGA was attenuated. The Ca2+ release by 10 microM NDGA was preceded by membrane depolarization of mitochondria. These results indicate that BK channel opening by NDGA in PCASMCs is due to the direct action on a subunit and also to Ca2+ release from sarcoplasmic reticulum, presumably via, at least in part, the inhibition of mitochondria respiration.     

Cyclosporin A protects hepatocytes against prooxidant-induced cell killing. A study on the role of mitochondrial Ca2+ cycling in cytotoxicity.

Cyclosporin A (CsA) is a potent inhibitor of the prooxidant-induced release of Ca2+ from isolated mitochondria. In this investigation, pretreatment of hepatocytes with CsA before exposure to the prooxidants tert-butyl hydroperoxide (tBH), cumene hydroperoxide or 3,5-dimethyl-N-acetyl-p-benzoquinone imine (3,5-Me2-NAPQI) prevented the loss of cell viability. HPLC analysis of adenine and pyridine nucleotide concentrations in hepatocytes treated with 3,5-Me2-NAPQI showed a rapid depletion of ATP prior to the loss of cell viability versus the maintenance of near control levels of ATP in hepatocytes treated with CsA before 3,5-Me2-NAPQI. In 3,5-Me2-NAPQI-exposed hepatocytes there was also a rapid loss of cellular NAD+ which could be accounted for initially by a transient increase in NADP+. Measurement of the intracellular Ca2+ pools showed an early depletion of the mitochondrial Ca2+ pool in hepatocytes exposed to 3,5-Me2-NAPQI, tBH or cumene hydroperoxide; this loss was prevented by CsA. In conclusion, these results show that CsA protected hepatocytes from prooxidant injury by preventing mitochondrial Ca2+ cycling and subsequent mitochondrial dysfunction. This suggests that in prooxidant injury, excessive Ca2+ cycling is an early and important event leading to mitochondrial damage and subsequently to cell death.     

Concentration dependent mitochondrial effect of amiodarone

Although, the antiarrhythmic effect of amiodarone is well characterized, its effect on post-ischemic heart and cardiomyocytes, as well as the mechanism of its toxicity on extracardiac tissues is still poorly understood. In this study, we analyzed energy metabolism in situ during ischemia-reperfusion in Langendorff-perfused heart model by measuring the high-energy phosphate metabolites using 31P NMR spectroscopy. The toxicity of amiodarone on cardiomyocytes and cell lines of extracardiac origin, as well as direct effect of the drug on mitochondrial functions in isolated mitochondria was also analyzed. Amiodarone, when was present at low concentrations and predominantly in membrane bound form, protected heart and mitochondrial energy metabolism from ischemia-reperfusion-induced damages in Langendorff-perfused heart model. Toxicity of the drug was significantly higher on hepatocytes and pancreatic cells than on cardiomyocytes. In isolated mitochondria, amiodarone did not induce reactive oxygen species formation, while it affected mitochondrial permeability transition in a concentration dependent way. Up to the concentration of 10 microM, the drug considerably inhibited Ca(2+)-induced permeability transition, while at higher concentrations it induced a cyclosporin A independent permeability transition of its own. At concentrations where it inhibited the Ca(2+)-induced permeability transition (IC(50)=3.9+/-0.8 microM), it did not affect, between 6 and 30 microM it uncoupled, while, at higher concentrations it inhibited the respiratory chain. Thus, the concentration dependent nature of amiodarone's effect on permeability transition together with the different sensitivities of the tissues toward amiodarone can be involved in the beneficial cardiac and the simultaneous toxic extracardiac effects of the drug.     

Adriamycin-induced lipid peroxidation in mitochondria and microsomes

The effect of the anti-neoplastic agent adriamycin on the peroxidation of lipids from rat liver and heart mitochondria and rat liver microsomes was investigated. The extent of total lipid peroxidation was determined by assaying for malondialdehyde (MDA), while the degradation of unsaturated fatty acids was monitored using gas chromatography. For liver mitochondria and microsomes, the formation of MDA was dependent on the concentrations of adriamycin, Fe3+, and protein, as well as time. In the presence of 50 microM adriamycin and saturating amounts of NADH, 1.5 +/- 0.2 nmol MDA/mg protein/60 min was produced with liver mitochondria. Upon addition of 25 microM Fe3+, the amount of MDA generated was increased to 6.5 +/- 0.1 nmol/mg protein/60 min. Liver microsomes produced amounts which were approximately 2-fold higher under all conditions. No MDA formation could be detected in rat heart mitochondria. The addition of 50 microM chlorpromazine completely inhibited peroxidation, whereas 0.5 to 1.0 mM p-bromophenacyl bromide blocked MDA formation by 50%. Analysis of fatty acids by gas chromatography showed that there was about a 50% decrease in arachidonic and docosahexaenoic acids in liver mitochondria and microsomes, but no change in the fatty acid content of heart mitochondria when incubated with both 50 microM adriamycin and 25 microM Fe3+ for 1 hr. These results suggest that (1) therapeutic concentrations of adriamycin enhance the peroxidation of lipids in liver mitochondria and microsomes through an enzymatic mechanism, especially in the presence of Fe3+; and (2) toxicity of this drug may be related to the degradation of membrane lipids.