Cyclosporin A-sensitive decrease in the transmembrane potential across the inner membrane of liver mitochondria induced by low concentrations of fatty acids and Ca2+

At low Ca²⁺ concentrations the pore of the inner mitochondrial membrane can open in substates with lower permeability (Hunter, D. R., and Haworth, R. A. (1979) Arch. Biochem. Biophys., 195, 468-477). Recently, we showed that Ca²⁺ loading of mitochondria augments the cyclosporin A-dependent decrease in transmembrane potential () across the inner mitochondrial membrane caused by 10 M myristic acid but does not affect the stimulation of respiration by this fatty acid. We have proposed that in our experiments the pore opened in a substate with lower permeability rather than in the classic state (Bodrova, M. E., et al. (2000) IUBMB Life, 50, 189-194). Here we show that under conditions lowering the probability of classic pore opening in Ca²⁺-loaded mitochondria myristic acid induces the cyclosporin A-sensitive decrease and mitochondrial swelling more effectively than uncoupler SF6847 does, though their protonophoric activities are equal. In the absence of P_i and presence of succinate and rotenone (with or without glutamate) cyclosporin A either reversed or only stopped decrease induced by 5 M myristic acid and 5 M Ca²⁺. In the last case nigericin, when added after cyclosporin A, reversed the decrease, and the following addition of EGTA produced only a weak (if any) increase. In P_i-containing medium (in the presence of glutamate and malate) cyclosporin A reversed the decrease. These data show that the cyclosporin A-sensitive decrease in by low concentrations of fatty acids and Ca²⁺ cannot be explained by specific uncoupling effect of fatty acid. We propose that: 1) low concentrations of Ca²⁺ and fatty acid induce the pore opening in a substate with a selective cation permeability, and the cyclosporin A-sensitive decrease results from a conversion of to pH gradient due to the electrogenic cation transport in mitochondria; 2) the ADP/ATP-antiporter is involved in this process; 3) higher efficiency of fatty acid compared to SF6847 in the Ca²⁺-dependent pore opening seems to be due to its interaction with the nucleotide-binding site of the ADP/ATP-antiporter and higher affinity of fatty acids to cations.     

Copper sensitizes the mitochondrial permeability transition to carboxyatractyloside and oleate

Addition of 5 M copper to rat kidney mitochondria enhances the effect of carboxyatractyloside and oleate on pore opening, in a cyclosporin A-sensitive fashion. The effects of the pair copper-carboxyatractyloside were observed on matrix Ca²⁺ efflux, mitochondrial swelling and on the transmembrane electric gradient. The effect of Cu²⁺ emphasizes the importance of membrane thiol groups located, probably, in the ADP/ATP translocase (ANT), on permeability transition. It was also found that Cu²⁺ does not block the fluorescent label of ANT by eosin 5-maleimide, but abolishes the inhibition by CAT on the labeling. This suggests that the binding of Cu²⁺ to cysteine residues of ANT promotes a conformational change in the carrier, strengthening the effect of CAT and oleate on membrane leakage.     

Mitochondria in Ca2+ Signaling and Apoptosis

Cellular Ca²⁺ signals are crucial in the control of most physiological processes, cell injuryand programmed cell death; mitochondria play a pivotal role in the regulation of such cytosolicCa²⁺ ([Ca²⁺]_c) signals. Mitochondria are endowed with multiple Ca²⁺ transport mechanismsby which they take up and release Ca²⁺ across their inner membrane. These transport processesfunction to regulate local and global [Ca²⁺]_c, thereby regulating a number of Ca²⁺-sensitivecellular mechanisms. The permeability transition pore (PTP) forms the major Ca²⁺ effluxpathway from mitochondria. In addition, Ca²⁺ efflux from the mitochondrial matrix occursby the reversal of the uniporter and through the inner membrane Na⁺/Ca²⁺ exchanger. Duringcellular Ca²⁺ overload, mitochondria take up [Ca²⁺]_c, which, in turn, induces opening of PTP,disruption of mitochondrial membrane potential (_m) and cell death. In apoptosis signaling,collapse of ;_m and cytochrome c release from mitochondria occur followed by activationof caspases, DNA fragmentation, and cell death. Translocation of Bax, an apoptotic signalingprotein from the cytosol to the mitochondrial membrane, is another step during thisapoptosis-signaling pathway. The role of permeability transition in the context of cell death in relationto Bcl-2 family of proteins is discussed.     

Amyloid beta-peptide promotes permeability transition pore in brain mitochondria

In this work the effect of the neurotoxic amino acid sequence, A_25–35, on brain mitochondrial permeability transition pore (PTP) was studied. For the purpose, the mitochondrial transmembrane potential (m), mitochondrial respiration and the calcium fluxes were examined. It was observed that A_25–35, in the presence of Ca²⁺, decreased the m, the capacity of brain mitochondria to accumulate calcium and led to a complete uncoupling of the respiration. However, the reverse sequence of the peptide A_25–35 (A_35–25) did not promote the PTP. The alterations promoted by A_35–25 and/or Ca²⁺ could be reversed when Ca²⁺ was removed by EGTA or when ADP plus oligomycin were present. The pre-treatment with CsA or ADP plus oligomycin prevented the m drop and preserved the capacity of mitochondria to accumulate Ca²⁺. These results suggest that A_25–35 can promote the PTP induced by Ca²⁺.     

Metabolic formation of dodecanedioic acid from n-dodecane by a mutant of Candida tropicalis

Summary These experiments studied the metabolic formation of a,-dodecanedioic acid using the mutant S 76 developed from the wild strain Candida tropicalis 1230 (capable of producing large amounts of a,-dodecanedioic acid).Our results show for the first time that 12-hydroxydodecanoic acid was excreted into the medium as a free acid.n-Dodecanol and n-dodecanoic acid were also detected in the n-dodecane medium. The mutant S 76 was able to produce a,-dodecanedioic acid using either n-dodecanol or dodecanoic acid as the sole carbon source. Quantitative cahnges in the concentrations of 12-hydroxy-dodecanoic acid and other intermediates were recorded during the formation of a,-dodecanedioic acid. S 76 was rapidly able to convert large amounts of 12-hydroxy-dodecanoic acid to a,-dodecanedioic acid.The formation of a,-dodecanedioic acid from n-dodecane via the sequence n-dodecanoln-dodecanoic acid 12-hydroxy-dodecanoic acid was confirmed.     

Mitochondrial function as a determinant of recovery or death in cell response to injury

Many pathological conditions can be the cause or the consequence of mitochondrial dysfunction. For instance anoxia, which is initiated by a critical reduction of oxygen availability for mitochondrial oxidations, is followed by a wide variety of mitochondrial alterations. A crucial role in the evolution of cell injury is to be attributed to the direction of operation of the F₀F₁ ATPase, which may turn mitochondria into the major consumers of cellular ATP in the futile attempt to restore the proton electrochemical gradient. On the other hand, functional mitochondria can paradoxically accelerate or exacerbate cell damage. This concept is particularly relevant for the ischemic myocardium. Indeed, inhibition of the respiratory chain or addition of uncouplers of oxidative phosphorylation can both limit the extent of enzyme release in the intact heart and prevent the onset of irreversible morphological changes in isolated myocytes. From studies on different tissues in a variety of pathological conditions a general consensus emerges on the role of intracellular Ca²⁺ overload as a pivotal link between cellular alterations and mitochondrial dysfunction. Oxidative phosphorylation is reduced by a massive mitochondrial uptake of Ca²⁺, resulting in a vicious cycle whereby the reduced ATP availability is followed by a failure of the mechanisms which extrude Ca²⁺ from the sarcoplasm. In addition, the rise in [Ca²⁺]_i could promote opening of the cyclosporin-sensitive mitochondrial permeability transition pore, leading to a sudden _m dissipation. Here, we review the changes in intracellular and intramitochondrial ionic homeostasis occurring during ischemia and reperfusion. In particular, we evaluate the potential contribution of the permeability transition pore to cellular damage and discuss the mechanisms which can determine the cellular fate from a mitochondrial point of view.     

Effects of local anesthetics on phospholipid topology and dopamine uptake and release in rat brain synaptosomes

Summary The effects of local anesthetics on the topology of aminophospholipids and on the release and uptake of dopamine in rat brain synaptosomes have been examined. A metabolically intact preparation of synaptosomes was prepared which maintains aminophospholipid asymmetry and the capacity for sodium-driven uptake and depolarization-dependent release of dopamine. Incubation of synaptosomes with local anesthetics at 37°C induced perturbations in the topology of aminophospholipids as determined by their reactivities to the covalent probe trinitrobenzenesulfonic acid. The reaction of trinitrobenzenesulfonate with phosphatidylethanolamine and phosphatidylserine was inhibited 10–20% by low concentrations of tetracaine (1–100 m) and enhanced by high concentrations (0.3–1.0mm). Other local anesthetics showed a similar biphasic effect with a potency order of dibucaine>tetracaine>lidocaineprocaine. K⁺-stimulated, Ca²⁺-dependent release of [³H]dopamine was inhibited significantly at low concentrations of tetracaine (1–10 m) but enhanced at higher concentrations (0.1–1.0mm). Dibucaine and procaine had a similar biphasic effect on the dopamine release. For each of the local anesthetics tested, the inhibition of the reaction of phosphatidylethanolamine and phosphatidylserine with trinitrobenzenesulfonate occurred at concentrations which were shown also to inhibit the release of [³H]dopamine. Local anesthetics were shown to inhibit uptake of [³H]dopamine with a potency order which reflects their potency in producing anesthesia. The inhibition of dopamine uptake by dibucaine, tetracaine, lidocaine, or procaine was characterized by inhibitory constants (K _I ) of 1.8±0.4 m, 27±5 m, 190 m and 0.5mm, respectively.Abbreviations TNBS 2,4,6-trinitrobenzene sulfonate - PE phosphatidylethanolamine - PS phosphatidylserine - ESR electron spin resonance - TLC thin-layer chromatography - DA dopamine     

The precursor of the F1β subunit of the ATP synthase is covalently modified upon binding to plant mitochondria

We present evidence for a unique covalent modification of a nuclear-encoded precursor protein targeted to plant mitochondria. We investigated the early events of in vitro import for the mitochondrial precursor of the ATP synthase F₁ subunit from Nicotiana plumbaginifolia (pF₁) into plant mitochondria. When pF₁ of 59 kDa was incubated with mitochondria isolated from different higher-plant species, a band of 61 kDa was generated. The 61 kDa protein was a covalently modified form of the 59 kDa pF₁. The modification was dependent on the 25 amino acid long N-terminal region of the presequence of pF₁. The modification was catalysed by an enzyme located in the outer mitochondrial membrane which was specific for higher plants and could not be washed off from the membrane by urea, KCl or EDTA. The modification was ATP- and Ca²⁺-dependent, but it was not affected by inhibitors of protein kinases. No inhibition of the modification was observed with phosphatase, methylation or acylation inhibitors. The modification occurs prior to translocation through the mitochondrial outer membrane. Inhibition of the modification process does not affect the import of the precursor protein, hence precursor modification was not a prerequisite for import. Both the modified and the unmodified pF₁ proteins were strongly associated with the mitochondrial outer membrane.     

Effects of Zn2+ and Cu2+ on growth,lignin degradation and ligninolytic enzymes in Phanerochaete chrysosporium

The influence of Zn²⁺ (6.0 × 10^–3 –18.0 × 10^–3 M) and Cu²⁺ (4 × 10^–4 –1.2 × 10^–4 M) in the basal medium on mycelial growth (dry weight), activities of lignin peroxidase (Lip), manganese peroxidase (Mnp), solubilization, and mineralization (¹⁴CO₂ evolution) of lignin during a period of 3 weeks was studied in Phanerochaete chrysosporium strain MTCC-787. Highest mycelial growth was obtained at 0.6 M Zn²⁺ and 0.4 M Cu²⁺ levels. Enzyme activities were found to increase up to the highest levels of both the trace elements. However, Zn²⁺ had a relatively more stimulatory effect on Lip production and the reverse was true in case of Cu²⁺. [¹⁴C]Lignin solubilization was also promoted by higher levels of both trace elements. Mineralization of [¹⁴C]lignin was optimal at 6.0 M Zn²⁺ and 1.2 M Cu²⁺. The stimulatory effect of Zn²⁺ on Lip production was correlated with higher rates of [¹⁴C]lignin mineralization.     

Mitochondrial Dysfunction in the Pathogenesis of Necrotic and Apoptotic Cell Death

Mitochondria are frequently the target of injury after stresses leading to necrotic and apoptoticcell death. Inhibition of oxidative phosphorylation progresses to uncoupling when opening ofa high conductance permeability transition (PT) pore in the mitochondrial inner membraneabruptly increases the permeability of the mitochondrial inner membrane to solutes of molecularmass up to 1500 Da. Cyclosporin A (CsA) blocks this mitochondrial permeability transition(MPT) and prevents necrotic cell death from oxidative stress, Ca²⁺ ionophore toxicity,Reye-related drug toxicity, pH-dependent ischemia/reperfusion injury, and other models of cell injury.Confocal fluorescence microscopy directly visualizes onset of the MPT from the movementof green-fluorescing calcein into mitochondria and the simultaneous release from mitochondriaof red-fluorescing tetramethylrhodamine methylester, a membrane potential-indicatingfluorophore. In oxidative stress to hepatocytes induced by tert-butylhydroperoxide, NAD(P)Hoxidation, increased mitochondrial Ca²⁺, and mitochondrial generation of reactive oxygen speciesprecede and contribute to onset of the MPT. Confocal microscopy also shows directly thatthe MPT is a critical event in apoptosis of hepatocytes induced by tumor necrosis factor-.Progression to necrotic and apoptotic cell killing depends, at least in part, on the effect theMPT has on cellular ATP levels. If ATP levels fall profoundly, necrotic killing ensues. If ATPlevels are at least partially maintained, apoptosis follows the MPT. Cellular features of bothapoptosis and necrosis frequently occur together after death signals and toxic stresses. A newterm, necrapoptosis, describes such death processes that begin with a common stress or deathsignal, progress by shared pathways, but culminate in either cell lysis (necrosis) or programmedcellular resorption (apoptosis) depending on modifying factors such as ATP.     

Minireview: Characterization of the Yeast Mitochondria Unselective Channel: A Counterpart to the Mammalian Permeability Transition Pore?

Large and unselective permeabilities through the inner membrane of yeast mitochondria have been observed for more than 20 years, but the characterization of these permeabilities, leading to hypothesize the existence of a large-conductance unselective channel in yeast inner mitochondrial membrane, was done only recently by several groups. This channel has been tentatively identified as a yeast counterpart to the mammalian permeability transition pore, the crucial role of which is now well-documented in physiopathological phenomena, such as Ca²⁺ homeostasis, ischemic damages, or programmed cell death. The aim of this review is to make a point on the known characteristics of this yeast mitochondrial unselective channel (YMUC) and to analyze whether or not it can be considered as a yeast permeability transition pore.     

Recent progress on regulation of the mitochondrial permeability transition pore; a cyclosporin-sensitive pore in the inner mitochondrial membrane

The mitochondrial permeability transition pore allows solutes with a m.w. 1500 to equilibrate across the inner membrane. A closed pore is favored by cyclosporin A acting at a high-affinity site, which may be the matrix space cylophilin isozyme. Early results obtained with cyclosporin A analogs and metabolites support this hypothesis. Inhibition by cyclosporin does not appear to require inhibition of calcineurin activity; however, it may relate to inhibition of cyclophilin peptide bond isomerase activity. The permeability transition pore is strongly regulated by both the membrane potential () and pH components of the mitochondrial protonmotive force. A voltage sensor which is influenced by the disulfide/sulhydryl state of vicinal sulfhydryls is proposed to render pore opening sensitive to . Early results indicate that this sensor is also responsive to membrane surface potential and/or to surface potential gradients. Histidine residues located on the matrix side of the inner membrane render the pore responsive to pH. The pore is also regulated by several ions and metabolites which act at sites that are interactive. There are many analogies between the systems which regulate the permeability transition pore and the NMDA receptor channel. These suggest structural similarities and that the permeability transition pore belongs to the family of ligand gated ion channels.     

Use of mitochondrial inhibitors to differentiate kinetic properties of the ATP-dependent Ca2+ uptake system in synaptic membranes

Synaptosomal membranes accumulate 3–6 times more Ca²⁺ in the presence of ATP (50–1000 M) than basal Ca²⁺ accumulation (-ATP). The location of this Ca²⁺ accumulation appears to reside on the cytosolic face of the synaptosome since lysed synaptosomes accumulate 4-times more Ca²⁺ than intact synaptosomes. The inclusion of mitochondrial inhibitors, oligomycin (0.7 g/ml), sodium azide (100 M) and dinitrophenol (100 M) differentiate mitochondrial from nonmitochondrial Ca²⁺ accumulation under conditions that are [Ca²⁺]- and ATP-dependent. In the presence of low concentrations of ATP (<150 M) and Ca _free ²⁺ (2.5 or 6.8 M), Ca²⁺ accumulation occurs as one process in both lysed synaptosomal membranes and purified synaptic plasma membranes in the presence and/or absence of MI. When ATP levels are increased (>200 M), the Ca²⁺ accumulation process remains independent of the presence of mitochondrial inhibitors when Ca _free ²⁺ =2.5 M. When Ca _free ²⁺ is increased to 6.8 M, mitochondrial inhibitors differentiate mitochondrial from nonmitochondrial accumulation. These studies suggest that optimal conditions for the measurement of Ca²⁺ accumulating mechanisms in synaptosomal membranes depend on both [Ca²⁺] and ATP. Use of these assay conditions provide evidence that ATP-dependent Ca²⁺ uptake may be a viable mechanism for the regulation of synaptosomal Ca²⁺ levels.     

Inactivation of Mitochondrial Permeability Transition Pore by Octylguanidine and Octylamine

Mitochondrial permeability transition occurs through a Ca²⁺-dependent opening of atransmembrane pore, whose identity has been attributed to that of the adenine nucleotide translocase(ANT). In this work, we induced permeability transition by adding 0.5 M carboxyatractyloside.The process was evaluated analyzing Ca²⁺ efflux, a drop in transmembrane electric gradient,and swelling. We found that the amphiphyllic cations octylguanidine and octylamine, at theconcentration of 100 M, inhibited, almost completely, nonspecific membrane permeability.Hexylguanidine, hexylamine, as well as guanidine chloride and hydroxylamine failed to doso. The inhibition was reversed after the addition of 40 mM Li⁺, Na⁺ K⁺,Rb⁺, or Cs⁺; K⁺ wasthe most effective. We propose that the positive charge of the amines interact with negativecharges of membrane proteins, more likely the ADP/ATP carrier, while the alkyl chain penetratesinto the hydrophobic milieu of the inner membrane, fixing the reagent.     

Flufenamic acid as an inducer of mitochondrial permeability transition

To assess the mechanism by which mitochondrial permeability transition (MPT) is induced by the nonpolar carboxylic acids, we investigated the effects of flufenamic acid (3-trifluoromethyl diphenylamine-2-carboxylic acid, FA) on mitochondrial respiration, electrical transmembrane potential difference (), osmotic swelling, Ca²⁺ efflux, NAD(P)H oxidation and reactive oxygen species (ROS) generation. Succinate-energized isolated rat liver mitochondria incubated in the absence or presence of 10 M Ca²⁺, 5 M ruthenium red (RR) or 1 M cyclosporin A (CsA) were used. The dose response-curves for both respiration release and dissipation were nearly linear, presenting an IC₅₀ of approximately 10 M and reaching saturation within 25-50 M, indicating that FA causes mitochondrial uncoupling by a protonophoric mechanism. Within this same concentration range FA showed the ability to induce MPT in energized mitochondria incubated with 10 M Ca²⁺, followed by dissipation and Ca²⁺ efflux, and even in deenergized mitochondria incubated with 0.5 mM Ca²⁺. ADP, Mg²⁺, trifluoperazine (TFP) and N-ethylmaleimide (NEM) reduced the extent of FA-promoted swelling in energized mitochondria by approximately one half, whereas dithiothreitol (DTT) slightly enhanced it. NAD(P)H oxidation and ROS generation (H₂O₂ production) by mitochondria were markedly stimulated by FA; these responses were partly prevented by CsA, suggesting that they may be implicated as both a cause and effect of FA-induced MPT. FA incubated with mitochondria under swelling assay conditions caused a decrease of approximately 40% in the content of protein thiol groups reacting with 5,5-dithiobis(2-nitrobenzoic acid) (DTNB). The present results are consistent with a ROS-intermediated sensitization of MPT by a direct or indirect FA interaction with inner mitochondrial membrane at a site which is in equilibrium with the NAD(P)H pool, namely thiol groups of integral membrane proteins.     

Effects of atrial natriuretic factor,nitroprusside and acetylcholine on cGMP immunostaining in the isolated perfused rat kidney

Summary In the present study the localization of the cGMP production in response to the vasodilators acetylcholine (ACh) and sodium nitroprusside (SNP) and to atrial natriuretic factor (ANF) was studied in the isolated perfused rat kidney using cGMP immunocytochemistry. After ACh (0.3 M) infusion increased cGMP immunoreactivity was found in kidney interlobar and segmental arteries and in glomeruli. SNP (1 M) and ANF (0.01 M) elevated cGMP staining in the same elements of the kidney as ACh. In the glomeruli ACh and SNP stimulated cGMP production in mesangial cells whereas ANF stimulated cGMP production in epithelial cells (podocytes). However, SNP at higher doses (10 M) stimulated cGMP production not only in glomeruli, but also in interstitial cells throughout the cortex. In addition SNP and ANF increased cGMP production in the medulla.     

Sensitivity of Ca2+ Transport of Mitochondria to Reactive Oxygen Species

The relationship between Ca²⁺ transport and energy transduction of myocardial mitochondria in the presence of reactive oxygen species was investigated. Following treatment with oxygen free radicals [superoxide(O ₂ ) or hydroxyl radical ()OH], lipid free radicals in myocardial mitochondrial membrane could be detected by using the method of EPR spin trap. Simultaneously there were obvious alterations in the free Ca²⁺ ([Ca²⁺]_m) in the mitochondrial matrix; the physical state of membrane lipid; the efficiency of oxidative phosphorylation (ADP/O); the value of the respiratory control ratio (RCR); and the membrane potential of the inner membrane of myocardial mitochondria. If the concentrations of reactive oxygen species were reduced by about 30%, the alterations in the physical state of the membrane lipid and energy transduction of myocardial mitochondria were not observed, but the changes in Ca²⁺ homeostasis remained. We conclude that Ca²⁺ transport by myocardial mitochondria is more sensitive to agents such as (O ₂ ) or OH, etc. than are oxidation phosphorylation and the respiratory chain.     

Electrostatic surface properties of plasmalemma vesicles from oat and wheat roots. Ion binding and screening investigated by 9-aminoacridine fluorescence

Right-side-out and sealed plasmalemma vesicles were isolated from roots of spring wheat (Triticum aestivum L. cv. Drabant) and oat (Avena sativa L. cv. Brighton) by two-phase partition in a medium containing sucrose (0.25 mol l^-1). Oat root plasmalemma vesicles were discovered to contain a strongly fluorescent compound with an emission maximum at 418 nm. The surface potential of the membranes was monitored by 9-aminoacridine fluorescence and the effect of protein concentration, mannitol versus sucrose, absence of osmoticum, concentrations of salt, and titrations with chelators investigated. It is concluded that i) protein concentrations of less than 50 g ml^-1 for oat and 100 g ml^-1 for wheat plasmalemma vesicles should be used to avoid serious problems with non-linearity of response of 9-aminoacridine fluorescence, ii) mannitol can be used instead of sucrose as the osmoticum, iii) the vesicles were ruptured in the absence of osmoticum allowing us to monitor both sides of the membranes, iv) plasmalemma vesicles from oat roots are more negative than vesicles from wheat roots, and v) oat and wheat root plasmalemma vesicles are isolated with about the same amounts of bound Ca²⁺ and Mg²⁺. These bound divalent cations may not, however, reflect the in-vivo conditions since the tissues were homogenised in the presence of ethylenediaminetetraacetic acid.Abbreviations EDTA ethylenediaminetetraacetic acid - EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - c1/2 value concentration at which half of the maximum effect is observed - Mops 3-(N-morpholino)propanesulfonic acid     

Modulation of brain mitochondrial membrane permeability and synaptosomal Ca2+ transport by dopamine oxidation

Effects of dopamine on the membrane permeability transition, thioredoxin reductase activity, production of free radicals and oxidation of sulfhydryl groups in brain mitochondria and the Ca²⁺ uptake by Na⁺-Ca²⁺ exchange and sulfhydryl oxidation in brain synaptosomes were examined. The brain mitochondrial swelling and the fall of transmembrane potential were altered by pretreatment of dopamine in a dose dependent manner. Depressive effect of dopamine on mitochondrial swelling was reversed by 10 g/ml catalase, and 10 mM DMSO. The activities of thioredoxin reductase in intact or disrupted mitochondria were decreased by dopamine (1-100 M), 25 M Zn²⁺ and 50 M Mn²⁺. Dopamine-inhibited enzyme activity was reversed by 10 g/ml SOD and 10 g/ml catalase. Pretreatment of dopamine decreased Ca²⁺ transport in synaptosomes, which was restored by 10 g/ml SOD and 10 mM DMSO. Dopamine (1-100 M) in the medium containing mitochondria produced superoxide anion and hydrogen peroxide, while its effect on nitrite production was very weak. The oxidation of sulfhydryl groups in mitochondria and synaptosomes were enhanced by dopamine with increasing incubation times. Results suggest that dopamine could modulate membrane permeability in mitochondria and calcium transport at nerve terminals, which may be ascribed to the action of free radicals and the loss of reduced sulfhydryl groups.     

Inhibitory ca2+-control of movement of beads coated withphysarum myosin along actin-cables inChara internodal cells

Summary The actin-activated ATPase activityPhysarum myosin was shown to be inhibited of M levels of Ca²⁺. To determine if Ca²⁺ regulates ATP-dependent movement ofPhysarum myosin on actin, latex beads coated withPhysarum myosin were introduced intoChara cells by intracellular perfusion. In perfusion solution containing EGTA, the beads moved along the parallel arrays ofChara actin filaments at a rate of 1.0–1.8 m/sec; however, in perfusion solution containing Ca²⁺, the rate reduced to 0.0–0.7 m/sec. The movement of beads coated with scallop myosin, whose actin-activated ATPase activity is activated by Ca²⁺, was observed only in the perfusion solution containing Ca²⁺, indicating that myosin is responsible for the inhibitory effect of Ca²⁺ onPhysarum myosin movement. The involvement of this myosin-linked regulation in the inhibitory effect of Ca²⁺ on the cytoplasmic streaming observed inChara internodal cell andPhysarum plasmodium was discussed.Abbreviations ATP adenosine 5-triphosphate - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - EGTA ethyleneglycolbis(-aminoethylether) N,N,N,N-tetraacetic acid - PIPES piperazine-N,N-bis(2-ethanesulfonic acid)