A comparative study of the effects of Pr<Superscript>3+</Superscript> and La<Superscript>3+</Superscript> ions on calcium dependent processes in frog cardiac muscle and rat heart mitochondria

The inotropic effect of Pr³⁺ and La³⁺ ions on the heart muscle of frog Rana ridibunda, as well as the influence of the ions on respiration, swelling, and the potential (ΔΨ_mito) on the inner membrane of Ca²⁺- loaded rat heart mitochondria, energized by glutamate and malate or succinate in the presence of rotenone were studied. It was found that 2 mM Pr³⁺ in Ringer’s solution reduces the force of spontaneous contractions and those induced by electrical stimulation in the heart; it had a negative chronotropic effect, decreasing the frequency of spontaneous contractions. Pr³⁺ and La³⁺ prevented a decrease in the 2,4-dinitrophenol (DNP)- uncoupled respiration of energized rat heart mitochondria, swelling of these organelles in salt media, and a reduction in ΔΨ_mito on the inner mitochondrial membrane that were induced by Ca²⁺ ions. Retardation by Pr³⁺ and La³⁺ ions of these calcium-induced effects may suggest that in the inner mitochondrial membrane these metals inhibit the opening of the mitochondrial permeability transition pore caused by Ca²⁺ overload of mitochondria. The data we obtained are important for a better understanding of the mechanisms of the damaging action of rare-earth elements on Ca²⁺-dependent processes in the vertebrate myocardium.     

Effect of oxidative processes in mitochondria on contractility of heart muscle of the frog <Emphasis Type="Italic">Rana temporaria</Emphasis>. Actions of Cd<Superscript>2+</Superscript>

The inotropic Cd²⁺ action on frog heart is studied with taking into account its toxic effects upon mitochondria. Cd²⁺ at concentrations of 1, 10, and 20 mM is established to decrease dose dependently (21.3, 50.3, and 72.0%, respectively) the muscle contraction amplitude; this is explained by its competitive action on the potential-controlled Na²⁺-channels of the L-type (Ca_v 1.2). In parallel experiments on isolated rat heart mitochondria (RHM) it was shown that Cd²⁺ at concentrations of 15 and 25 mM produces swelling of non-energized and energized mitochondria in isotonic (with KNO₂ and NH₂NO₃) and hypoosmotic (with 25 mM CH₃COOK) media. Study of oxidative processes in RHM by polarographic method has shown 20 mM Cd²⁺ to disturb activity of respiratory mitochondrial chain. The rate of endogenous respiration of isolated mitochondria in the medium with Cd²⁺ in the presence of malate and succinate was approximately 5 times lower than in control. In experimental preparations, addition into the medium of DNP—uncoupler of oxidation and phosphorylation did not cause an increase of the oxygen consumption rate. Thus, the obtained data indicate that a decrease in the cardiac muscle contractility caused by Cd²⁺ is due not only to its direct blocking action on Ca²⁺-channels, but also is mediated by toxic effect on rat heart mitochondria, which was manifested as an increase in ion permeability of the inner mitochondrial membrane (IMM), acceleration of the energy-dependent K⁺ transport into the matrix of mitochondria, and inhibition of their respiratory chain.     

Bivalent Metal Ions Modulate Cd2+ Effects on Isolated Rat Liver Mitochondria

We have studied Cd²⁺-induced effects on mitochondrial respiration and swelling in various media as a function of the [Cd²⁺] in the presence or absence of different bivalent metal ions or ruthenium red (RR). It was confirmed by monitoring oxygen consumption by isolated rat liver mitochondria that, beginning from 5 M, Cd²⁺ decreased both ADP and uncoupler-stimulated respiration and increased their basal respiration when succinate was used as respiratory substrate. At concentrations higher than 5 M, Cd²⁺ stimulated ion permeability of the inner mitochondrial membrane, which was monitored in this study by swelling of both nonenergized mitochondria in 125 mM KNO₃ or NH₄NO₃ medium and succinate-energized mitochondria incubated in a medium containing 25 mM K-acetate and 100 mM sucrose. We have found substantial changes in the above-mentioned Cd²⁺ effects on mitochondria treated in sequence with 100 M of Ca²⁺, Sr²⁺, Mn²⁺ or Ba²⁺(Me²⁺) and 7.5 M RR, as well as the alterations in Cd²⁺ action on the uptake of ¹³⁷Cs⁺ by succinate-energized mitochondria in the presence or absence of valinomycin in acetate medium (50 mM Tris-acetate and 140 mM sucrose) with or without Ca²⁺ or RR. The evidence obtained indicate that Ca²⁺ exhibits a synergestic action on all Cd²⁺ effects examined, whereas Sr²⁺ and Mn²⁺, conversely, are antagonistic. In the presence of RR, the Cd²⁺ effects on respiration [stimulation of State 4 respiration and inhibition of 2,4-dinitrophenol (DNP)-uncoupled respiration] still exist, but are observed at concentrations of cadmium more than one order higher; the inhibition of State 3 respiration by Cd²⁺, conversely, takes place under even lower cadmium concentrations than those determined without RR in the medium. In addition, RR added simultaneously with cadmium in the incubation medium prevents any swelling in the nitrate media, but induces an increment both in Cd²⁺-stimulated swelling and ¹³⁷Cs⁺ (analog of K⁺) uptake in the acetate media. For the first time, we have shown that Cd²⁺-induced swelling in all media under study is susceptible to cyclosporin A (CSA), a high-potency inhibitor of the mitochondrial permeability transition (PT) pore. The observations are interpreted in terms of a dual effect of cadmium on respiratory chain activity and permeability transition.     

Duality of effect of La3+ on mitochondrial permeability transition pore depending on the concentration

In order to explore the role of mitochondria in proliferation promotion and/or apoptosis induction of lanthanum, the mutual influences between La³⁺ and Ca²⁺ on mitochondrial permeability transition pore (PTP) opening were investigated with isolated mitochondria from rat liver. The experimental results revealed that La³⁺ influence the state of mitochondria in a concentration-dependent biphasic manner. La³⁺ in nanomolar concentrations, acting as a Ca²⁺ analog, entered mitochondrial matrix via the RuR sensitive Ca²⁺ channel and elevated ROS level, leading to opening of PTP indicated by mitochondrial swelling, reduction of ΔΨ_m and cytochrome c release. Inhibition of PTP with 10 μM CsA attenuated the effects of La³⁺. However, micromolar concentrations La³⁺ acted mainly as a Ca²⁺ antagonist, inhibiting PTP opening induced by Ca²⁺. We postulated that this action of La³⁺ on mitochondria through interaction with Ca²⁺ might be involved in the proliferation-promoting and apoptosis induction by La³⁺.     

Effect of yttrium on calcium-dependent processes in vertebrate myocardium

Inoptopic effect of yttrium acetate (Y³⁺) on myocardium of the marsh frog Rana ridibunda and its effect on ion transport across the inner mitochondrial membrane (IMM) of rat heart was studied. Y³⁺ was found to decrease the rate of heart contractions and to stimulate ion transport in the rat heart mitochondria in media with 10 mM glutamate and 2 mM malate. Presence of Y³⁺ induced inhibition of energy-dependent Ca²⁺ transport into mitochondria, which was expressed as a marked decrease of their swelling in the media containing 125 mM NH₄NO₃ and Ca²⁺ or 25 mM potassium acetate, 100 mM sucrose and Ca²⁺. It is suggested that the Y³⁺-induced decrease in rat muscle contractions is determined not only by direct suppressing effect of Y³⁺ on potential-modulated Ca²⁺-channels of pacemaker and contractile cardiomyocytes (CM), but also by its indirect effect on Ca²⁺-carrier in IMM. The data confirming that Y³⁺ activates energy-dependent K⁺ transport catalyzed by mitochondrial uniporter and blocks Ca²⁺-channels in the mitochondrial membrane are important for more complete understanding of mechanisms of the Y³⁺ action on vertebrates and human CM.     

Comparative study of Y<Superscript>3+</Superscript> effect on calcium-dependent processes in frog cardiac muscle and mitochondria of rat cardiomyocytes

Inotropic effects of yttrium acetate (Y³⁺) on contractions of myocardium preparations of the frog Rana ridibunda, as well as on respiration and the inner membrane potential (ΔΨ_mito) of isolated rat heart mitochondria were studied. 2 mM yttrium in Ringer solution was found to significantly reduce the amplitude of myocardium contractions, evoked by electric stimulation, and increase the half-relaxation time (n = 5). In experiments with Ca²⁺, Y³⁺ decreased the Ca²⁺-dependent basal respiration rate in rat heart mitochondria, energized by glutamate and malate, impeded the reduction in respiration of these mitochondria operating in state 3 after Chance or uncoupled by 2,4-dinitrophenol, and inhibited a Ca²⁺-induced reduction in their inner membrane potential. The data obtained are important for better understanding the mechanism underlying Y³⁺ effects on the myocardial Ca²⁺-dependent processes. Possible mechanisms of the negative inotropic effect of Y³⁺ on myocardium and its influence on the Ca²⁺-dependent processes in rat mitochondria are discussed.     

Ursodeoxycholic acid,in contrast to other bile acids,induces Ca<Superscript>2+</Superscript>-dependent cyclosporin A-insensitive permeability transition in liver mitochondria in the presence of potassium chloride

The effects of hydrophobic and hydrophilic bile acids as inducers of Ca²⁺-dependent permeability of the inner membrane were studied on isolated liver mitochondria. It is shown that in the absence of the inorganic phosphate (P_i)–a modulator of the mitochondrial pore–hydrophobic bile acids (lithocholic, deoxycholic, chenodeoxycholic) at concentrations of 20–50 μM, as well as a hydrophilic cholic acid at a concentration of 800 μM, induce swelling of liver mitochondria loaded with Ca²⁺. This effect is completely eliminated by a specific inhibitor of mitochondrial pore cyclosporin A (CsA). The effect of the bile acids as inducers of Ca²⁺-dependent CsA-sensitive mitochondrial pore is not associated with the modulation of the P_i effects. In contrast to other tested bile acids, a hydrophilic ursodeoxycholic acid (UDCA) at a concentration of 400 μM is able to induce Ca²⁺-dependent CsA-sensitive pore opening in liver mitochondria only in the presence of P_i or in the absence of potassium chloride in the incubation medium. In the presence of potassium chloride but in the absence of P_i, UDCA effects associated with the induction of the inner membrane permeability (swelling of mitochondria, drop in Δψ, and Ca²⁺ release from the matrix) are also observed in the presence of CsA. This Ca²⁺-dependent permeability of the inner membrane, in contrast to the “classical” CsA-sensitive pore, is characterized by a lower intensity of the mitochondrial swelling, a total drop in Δψ, and Ca²⁺ release from the matrix and is blocked by P_i. We suggest that the induction of the CsA-insensitive permeability in the inner mitochondrial membrane by UDCA is associated with activation of electrophoretic influx of K⁺ into the matrix and Ca²⁺ release from the matrix in exchange to H+. The effect of P_i as a blocker of such permeability is discussed.     

The mechanism of calcium transport in mitochondria isolated from the marine mussel, Mytilus edulis (L.)

Isolated mussel mitochondria produced a less pronounced transient stimulation of respiration upon the addition of Ca²⁺ in a reaction medium containing Pi and a slower rate of Ca²⁺ transport compared to rat liver mitochondria. The initial rates of Ca²⁺ transport in the absence of Pi were more similar and both types of mitochondria possessed a sigmoidal relationship between the initial rate of Ca²⁺ transport and the free Ca²⁺ concentration $\text{(‘Km’ ? 5μM)}$. Ruthenium red produced an equal maximal inhibition of the initial rate of Ca²⁺ transport in both types of mitochondria but mussel mitochondria were rather more resistant to the inhibitor. The major difference found was that approximately 15 nmoles La³⁺ mg protein^?1 was required to produce maximal inhibition of the initial rate of Ca²⁺ transport in mussel mitochondria compared to approximately 1.0 nmole La³⁺ mg protein^?1 in rat liver mitochondria. It is concluded that mussel mitochondria possess a comparable Ca²⁺ transporter to vertebrate mitochondria and possible reasons for resistance to La³⁺ are discussed.     

The interaction of La 3+ with mitochondria in relation to respiration-coupled Ca 2+ transport

La³⁺ inhibits the respiration-dependent accumulation of Ca²⁺ by rat liver mitochondria when added in very small amounts (0.1–l.0 nmole per mg protein). However, La³⁺ itself does not activate respiration. With the use of ¹⁴⁰La³⁺ it was found that La³⁺ is very rapidly bound to rat liver mitochondria in a respiration-independent process accompanied by loss of H⁺ to the medium. When both La³⁺ and Ca²⁺ are added to mitochondria simultaneously, most of the La³⁺ but little Ca²⁺ are bound. La³⁺ added to mitochondria previously loaded with Ca²⁺ is tightly bound without discharge of Ca²⁺. Conversely, when Ca²⁺ is added to La³⁺-loaded mitochondria it is not bound nor is the La³⁺ discharged. La³⁺ inhibits both high-affinity and low-affinity respiration-independent Ca²⁺ binding. Isotopic experiments showed that La³⁺ is, in fact, bound to the same high-affinity sites as Ca²⁺, in both intact mitochondria and in mitochondrial extracts. It is concluded (1) that La³⁺ binds to and inhibits the Ca²⁺ carrier; (2) that La³⁺ is not transported by the Ca²⁺ carrier; and (3) that La³⁺ is, in addition, bound to a large number of external sites on mitochondria for which Ca²⁺ is not a strong competitor.     

Physiological modulators of cyclosporin A-insensitive nonspecific permeability of the inner membrane of liver mitochondria induced by calcium ions and α,ω-hexadecanedioic acid

Long-chain saturated α,ω-dioic acids can induce nonspecific permeability of the inner membrane (pore opening) of liver mitochondria loaded with Ca²⁺ or Sr²⁺ by the mechanism insensitive to cyclosporin A (CsA). In this work we found that 200 μM Ca²⁺ and 20 μM α,ω-hexadecanedioic acid (HDA) in the presence of 1 μM CsA induced high-amplitude swelling of liver mitochondria (pore opening) only in the presence of succinate as oxidation substrate. Under these conditions protonophore uncoupler of oxidative phosphorylation 2,4-dinitrophenol at the concentration of 75 μM, which is optimal for its uncoupling activity, inhibited mitochondrial swelling induced by Ca²⁺ and HDA, despite the presence of succinate in the incubation medium. Natural uncouplers of oxidative phosphorylation, oleic and linoleic acids, produced a similar effect. These data suggest that energization of organelles, which promotes Ca²⁺ transport into the matrix, is one of the basic requirements of pore opening in liver mitochondria induced by Ca²⁺ and HDA. It is shown that ATP at the physiological concentration of 2 mM inhibits HDA-induced high-amplitude swelling of mitochondria by reducing free Ca²⁺ concentration in the medium. ADP at the same concentration had a similar effect. This modulating effect of nucleotides apparently is attributable to their ability to chelate calcium ions. Polycation spermine, which is known as an inhibitor of the classical CsA-sensitive pore, at the physiological concentration of 1 mM inhibited CsA-insensitive swelling of liver mitochondria induced by sequential addition of Ca²⁺ and HDA. It is assumed that such action of spermine is due to its ability to shield the negative surface charges on the inner membrane of mitochondria. Bovine serum albumin (BSA), which is able to bind free fatty acids and thus prevent the induction of Ca²⁺-dependent pore, inhibited HDA-induced swelling of mitochondria. However, at the same BSA/fatty acid molar ratio inhibitory effect of BSA was much less pronounced if HDA was used as the pore inducer instead of palmitic acid. Apparently, this can be accounted by the fact that BSA binds α,ω-dioic acids weaker than their monocarboxylic analogues.     

Induction of Ca2+-dependent cyclosporin a-insensitive nonspecific permeability of the inner membrane of liver mitochondria and cytochrome c release by α,ω-hexadecanedioic acid in media of varying ionic strength

In liver mitochondria loaded with Ca²⁺ or Sr²⁺, α,ω-hexadecanedioic acid (HDA) can induce nonspecific permeability of the inner membrane (mitochondrial pore) by the mechanism insensitive to cyclosporin A (CsA). In this work we studied the effect of ionic strength of the incubation medium on the kinetics of the processes that accompany Ca²⁺-dependent induction of the mitochondrial pore by fatty acid: organelle swelling, Ca²⁺ release from the matrix, changes in transmembrane potential (Δψ) and rate of oxygen consumption, and the release of cytochrome c from the intermembrane space. Two basic incubation media were used: sucrose medium and isotonic ionic medium containing KCl without sucrose. We found that 200 μM Ca²⁺ and 20 μM HDA in the presence of CsA effectively induce high-amplitude swelling of mitochondria both in the case of sucrose and in the ionic incubation medium. In the presence of CsA, mitochondria can rapidly absorb Ca²⁺ and retain it in the matrix for a while without reducing Δψ. Upon incubation in the ionic medium, mitochondria retain most of the added Ca²⁺ in the matrix for a short time without reducing the Δψ. In both cases the addition of HDA to the mitochondria 2 min after the introduction of Ca²⁺ leads to the rapid release of these ions from the matrix and total drop in Δψ. The mitochondrial swelling induced by Ca²⁺ and HDA in non-ionic medium is accompanied by almost maximal stimulation of respiration. Under the same conditions, but during incubation of mitochondria in the ionic medium, it is necessary to add cytochrome c for significant stimulation of respiration. The mitochondrial swelling induced by Ca²⁺ and HDA leads to the release of cytochrome c in a larger amount in the case of ionic medium than for the sucrose medium. We conclude that high ionic strength of the incubation medium determines the massive release of cytochrome c from mitochondria and liberates it from the respiratory chain, which leads to blockade of electron transport along the respiratory chain and consequently to disruption of the energy functions of the organelles.     

Induction of calcium-dependent nonspecific permeability of the inner membrane in liver mitochondria of mammals and birds: A comparative study

The kinetics of the processes accompanying the induction of Ca²⁺-dependent permeability (pore opening) of the inner membrane—swelling of organelles and Ca²⁺ release from the matrix—was studied in isolated liver mitochondria of mammals (mice, rats, and rabbits) and birds (pigeons and guinea fowls). It was found that the mitochondria of rats, pigeons, and guinea fowls of the gray-speckled population (GSP) are similar in terms of respiration and oxidative ATP synthesis, whereas mitochondria of rabbits exhibit a greater degree of coupling of respiration and ATP synthesis, and mitochondria of mice and Zagorskaya White breed (ZWB) guinea fowls, a lower degree of coupling. It was established that mammalian mitochondria energized by succinate oxidation and incubated with 1 mM of inorganic phosphate are able to swell upon the addition of 125 nmol of CaCl₂ per 1 mg protein. Under these conditions, mitochondria of GSP and ZWB guinea fowls and pigeons are capable of swelling upon addition of at least 875, 875 and 1000 nmol of CaCl₂ per 1 mg protein, respectively. Cyclosporin A (CsA, 1 μM) inhibits mitochondrial swelling. It was shown that mitochondria of mammalians and guinea fowls but not of pigeons are able to effectively absorb and retain Ca²⁺ in the matrix. Calcium retention capacity of mitochondria from rats, mice, rabbits, GSP, and ZWB guinea fowls were, respectively, 70, 57, 38, 844 and 793 nmol of CaCl₂ per 1 mg of protein. In the presence of an oxidizing agent tert-butylhydroperoxide (TBH), the induction of the Ca²⁺-dependent pore in the mitochondria was observed upon addition of CaCl₂ in substantially smaller quantities. TBH was most effective in the case of rabbit mitochondria and had the lowest efficiency in the case of guinea fowl and pigeon mitochondria.     

The Ca2+-induced membrane transition in mitochondria. II. Nature of the Ca2+ trigger site.

The permeability of isolated mitochondria which have undergone the Ca²⁺-induced transition can be modulated over a wide range simply by adjusting the concentration of free Ca²⁺ in the medium. The effect varies sigmoidally with respect to Ca²⁺ concentration, with an apparent K_m of 16 μm at pH 7.0. It is concluded that the trigger site (by “trigger site” we mean the site of binding of Ca²⁺ which, when Ca²⁺ is bound, will allow the transition in permeability to occur) is possibly also the site for high-affinity Ca²⁺ uptake. Added ADP, NADH and Mg²⁺ inhibit the Ca²⁺-induced permeability of mitochondria which have undergone the Ca²⁺-induced transition. Mg²⁺ and other ions, including H⁺, act like competitive inhibitors of the Ca²⁺ effect. In the presence of Ca²⁺, both neutral and charged molecules of molecular weight <1000 pass readily through the membrane. This response to Ca²⁺ is interpreted as a gating effect at the internal end of hydrophilic channels which span the inner membrane.     

Brain mitochondria from rats treated with sulforaphane are resistant to redox-regulated permeability transition

Oxidative stress promotes Ca²⁺-dependent opening of the mitochondrial inner membrane permeability transition pore (PTP), causing bioenergetic failure and subsequent cell death in many paradigms, including those related to acute brain injury. One approach to pre-conditioning against oxidative stress is pharmacologic activation of the Nrf2/ARE pathway of antioxidant gene expression by agents such as sulforaphane (SFP). This study tested the hypothesis that administration of SFP to normal rats increases resistance of isolated brain mitochondria to redox-sensitive PTP opening. SFP or DMSO vehicle was administered intraperitoneally to adult male rats at 10 mg/kg 40 h prior to isolation of non-synaptic brain mitochondria. Mitochondria were suspended in medium containing a respiratory substrate and were exposed to an addition of Ca²⁺ below the threshold for PTP opening. Subsequent addition of tert-butyl hydroperoxide (tBOOH) resulted in a cyclosporin A-inhibitable release of accumulated Ca²⁺ into the medium, as monitored by an increase in fluorescence of Calcium Green 5N within the medium, and was preceded by a decrease in the autofluorescence of mitochondrial NAD(P)H. SFP treatment significantly reduced the rate of tBOOH-induced Ca²⁺ release but did not affect NAD(P)H oxidation or inhibit PTP opening induced by the addition of phenylarsine oxide, a direct sulfhydryl oxidizing agent. SFP treatment had no effect on respiration by brain mitochondria and had no effect on PTP opening or respiration when added directly to isolated mitochondria. We conclude that SFP confers resistance of brain mitochondria to redox-regulated PTP opening, which could contribute to neuroprotection observed with SFP.     

Diacylglycerols Activate Mitochondrial Cationic Channel(s) and Release Sequestered Ca<Superscript>2+</Superscript>

Mitochondria contribute to cytosolic Ca²⁺ homeostasis through several uptake and release pathways. Here we report that 1,2-sn-diacylglycerols (DAGs) induce Ca²⁺ release from Ca²⁺-loaded mammalian mitochondria. Release is not mediated by the uniporter or the Na⁺/Ca²⁺ exchanger, nor is it attributed to putative catabolites. DAGs-induced Ca²⁺ efflux is biphasic. Initial release is rapid and transient, insensitive to permeability transition inhibitors, and not accompanied by mitochondrial swelling. Following initial rapid release of Ca²⁺ and relatively slow reuptake, a secondary progressive release of Ca²⁺ occurs, associated with swelling, and mitigated by permeability transition inhibitors. The initial peak of DAGs-induced Ca²⁺ efflux is abolished by La³⁺ (1 mM) and potentiated by protein kinase C inhibitors. Phorbol esters, 1,3-diacylglycerols and 1-monoacylglycerols do not induce mitochondrial Ca²⁺ efflux. Ca²⁺-loaded mitoplasts devoid of outer mitochondrial membrane also exhibit DAGs-induced Ca²⁺ release, indicating that this mechanism resides at the inner mitochondrial membrane. Patch clamping brain mitoplasts reveal DAGs-induced slightly cation-selective channel activity that is insensitive to bongkrekic acid and abolished by La³⁺. The presence of a second messenger-sensitive Ca²⁺ release mechanism in mitochondria could have an important impact on intracellular Ca²⁺ homeostasis.     

Mersalyl prevents the Tl+-induced permeability transition pore opening in the inner membrane of Ca2+-loaded rat liver mitochondria

It was earlier shown that the calcium load of rat liver mitochondria in medium containing TlNO₃ and KNO₃ resulted in the Tl⁺-induced mitochondrial permeability transition pore (MPTP) opening in the inner membrane. This opening was accompanied by an increase in swelling and membrane potential dissipation and a decrease in state 3, state 4, and 2,4-dinitrophenol-uncoupled respiration. This respiratory decrease was markedly leveled by mersalyl (MSL), the phosphate symporter (PiC) inhibitor which poorly stimulated the calcium-induced swelling, but further increased the potential dissipation. All of these effects of Ca²⁺ and MSL were visibly reduced in the presence of the MPTP inhibitors (ADP, N-ethylmaleimide, and cyclosporine A). High MSL concentrations attenuated the ability of ADP to inhibit the MPTP. Our data suggest that the PiC can participate in the Tl⁺-induced MPTP opening in the inner membrane of Ca²⁺-loaded rat liver mitochondria.     

Metformin promotes isolated rat liver mitochondria impairment

Metformin, a drug widely used in the treatment of type 2 diabetes, has recently received attention due to the new and contrasting findings regarding its effects on mitochondrial function. In the present study, we evaluated the effect of metformin in isolated rat liver mitochondria status. We observed that metformin concentrations ≥8 mM induce an impairment of the respiratory chain characterized by a decrease in RCR and state 3 respiration. However, only metformin concentrations ≥10 mM affect the oxidative phosphorylation system by decreasing the mitochondrial transmembrane potential and increasing the repolarization lag phase. Moreover, our results show that metformin does not prevent H₂O₂ production, neither protects against lipid peroxidation induced by the pro-oxidant pair ADP/Fe²⁺. In addition, we observed that metformin exacerbates Ca²⁺-induced permeability transition pore opening by decreasing the capacity of mitochondria to accumulate Ca²⁺ and increasing the oxidation of thiol groups. Taken together, our results show that metformin can promote liver mitochondria injury predisposing to cell death. Cristina Carvalho and Sónia Correia contributed equally to this work.     

The differentiation inducer,dimethyl sulfoxide,transiently increases the intracellular calcium ion concentration in various cell types

Dimethyl sulfoxide (DMSO) initiates a coordinated differentiation program in various cell types but the mechanism(s) by which DMSO does this is not understood. In this study, the effect of DMSO on intracellular calcium ion concentration ([Ca²⁺]_i) was determined in primary cultures of chicken ovarian granulosa cells from the two largest preovulatory follicles of laying hens, and in three cell lines: undifferentiated P19 embryonal carcinoma cells, 3T3-L1 fibroblasts, and Friend murine erythroleukemia (MEL) cells. [Ca²⁺]_i was measured in cells loaded with the Ca²⁺ -specific fluoroprobe Fura-2. There was an immediate (i.e., within 5 sec), transient, two to sixfold increase in [Ca²⁺]_i after exposing all cell types to 1% DMSO. DMSO was effective between 0.2 and 1%. The prompt DMSO-induced [Ca²⁺]_i spike in all of the cell types was not prevented by incubating the cells in Ca²⁺ -free medium containing 2 mM EGTA or by pretreating them with the Ca²⁺-channel blockers methoxyverapamil (D600; 100 μM), nifedipine (20 μM), or cobalt (5 mM). However, when granulosa cells, 3T3-L1 cells, or MEL cells were pretreated with lanthanum (La³⁺; 1 mM), which blocks both Ca²⁺ channels and membrane Ca²⁺ pumps, there was a sustained increase in [Ca²⁺]_i in response to 1% DMSO. By contrast, pretreating P19 cells with La³⁺ (1 mM) did not prolong the DMSO-triggered [Ca²⁺]_i transient. In all cases, the DMSO-induced [Ca²⁺]_i surge was unaffected by pretreating the cells with the inhibitors of inositol phospholipid hydrolysis, neomycin (1.5 mM) or U-73, 122 (2.5 μM). These results suggest that DMSO almost instantaneously triggers the release of Ca²⁺ from intracellular stores through a common mechanism in cells in primary cultures and in cells of a variety of established lines, but, this release is not mediated through phosphoinositide breakdown. This large, DMSO-induced Ca²⁺ spike may play a role in the induction of cell differentiation by DMSO. © 1993 Wiley-Liss, Inc.     

Effect of Ca2+ on programmed death of guard and epidermal cells of pea leaves

The effect of Ca²⁺ on programmed death of guard cells (GC) and epidermal cells (EC) determined from destruction of the cell nucleus was investigated in epidermis of pea leaves. Ca²⁺ at concentrations of 1–100 μM increased and at a concentration of 1 mM prevented the CN—induced destruction of the nucleus in GC, disrupting the permeability barrier of GC plasma membrane for propidium iodide (PI). Ca²⁺ at concentrations of 0.1–1 mM enhanced drastically the number of EC nuclei stained by PI in epidermis treated with chitosan, an inducer of programmed cell death. The internucleosomal DNA fragmentation caused by CN^? was suppressed by 2 mM Ca²⁺ on 6 h incubation, but fragmentation was stimulated on more prolonged treatment (16 h). Presumably, the disruption of the permeability barrier of plasma membrane for PI is not a sign of necrosis in plant cells. Quinacrine and diphenylene iodonium at 50 μM concentration prevented GC death induced by CN^? or CN^? + 0.1 mM Ca²⁺ but had no influence on respiration and photosynthetic O₂ evolution in pea leaf slices. The generation of reactive oxygen species determined from 2′,7′-dichlorofluorescein fluorescence was promoted by Ca²⁺ in epidermal peels from pea leaves.     

Sildenafil citrate concentrations not affecting oxidative phosphorylation depress H2O2 generation by rat heart mitochondria

Sildenafil citrate (Viagra) is a potent and specific inhibitor of cyclic guanosine monophosphate (cGMP)-specific phosphodiesterase type 5 (PDE5), which exhibits cardioprotective action against ischemia/reperfusion injury in intact and isolated heart. The mechanism of its cardioprotective action is not completely understood, but some results suggested that sildenafil exerts cardioprotection through the opening of mitochondrial ATP-sensitive K⁺ channels (mitoK_ATP). However, the impact of sildenafil citrate per se on isolated heart mitochondrial function is unknown. The goal of this study was to investigate the influence of the compound on mitochondrial function (bioenergetics, Ca²⁺-induced mitochondrial permeability transition, and hydrogen peroxide (H₂O₂) generation) in an attempt to correlate its known actions with effects on heart mitochondria. It was observed that sildenafil citrate concentrations of up to 50 μM did not significantly affect glutamate/malate-supported respiration in states 2, 3, 4, oligomycin-inhibited state 3, and uncoupled respiration. The respiratory control ratio (RCR), the ADP to oxygen ratio (ADP/O), the transmembrane potential (ΔΨ), the phosphorylation rate, and the membrane permeability to H⁺, K⁺ and Ca²⁺ were not affected either. However, sildenafil citrate decreased H₂O₂ generation by mitochondria respiring glutamate/malate, and also decreased the formation of superoxide radical (O₂^•−) generated in a hypoxantine/xantine oxidase system. It was concluded that sildenafil citrate concentrations of up to 50 μM do not affect either rat heart mitochondrial bioenergetics or Ca²⁺-induced mitochondrial permeability transition, but it depresses H₂O₂ generation by acting as a superoxide dismutase (SOD)-mimetic. By preventing reactive oxygen species (ROS) generation, sildenafil citrate may preserve heart mitochondrial function.