The effect of Ca2+ and acyl coenzyme A:lysophospholipid acyltransferase inhibitors on permeability properties of the liver mitochondrial inner membrane

We have reported previously that a number of metabolites and toxins which cause Ca2+ release from mitochondria do so by increasing the permeability of the inner membrane. The metabolic basis of this permeability change is proposed to be perturbation of a phospholipid deacylation-reacylation cycle which results in an accumulation of free fatty acids and lysophospholipids (see Broekemeier, K. M., Schmid, P. C., Schmid, H. H. O., and Pfeiffer, D. R. (1985) J. Biol. Chem. 260, 105-113 and references therein). This hypothesis predicts that inhibitors of acyl-CoA:lysophospholipid acyltransferase would be among those agents which increase membrane permeability and that their effects on permeability could occur in the absence of pyridine nucleotide oxidation or of an accumulation of glutathione disulfide. The hypolipidemic drugs WY-14643 and clofibric acid inhibit the mitochondrial acyl-CoA:lysophospholipid acyltransferase and have the predicted effects on mitochondrial permeability properties. The development of increased permeability due to WY-14643 and clofibric acid requires accumulated Ca2+ specifically, is sensitive to inhibitors of phospholipase A2, and results in a pattern of solute release and swelling which is typical of other Ca2+-releasing agents. Neither agent promotes pyridine nucleotide nor sulfhydryl glutathione oxidation in the absence of Ca2+. In addition, the swelling response to hypolipidemic drugs is not significantly inhibited by dithiothreitol. In the presence of Ca2+, both agents promote an accumulation of free fatty acids. The composition of these lipid degradation products suggests that mitochondria treated with hypolipidemic drugs retain an active lysophospholipase whereas this enzyme is inactivated by Ca2+-releasing agents which alter mitochondrial sulfhydryl groups.     

Formation of monohydroxy derivatives of arachidonic acid, linoleic acid, and oleic acid during oxidation of low density lipoprotein by copper ions and endothelial cells.

An important event in the formation of atherosclerotic lesions is the uptake of modified low density lipoprotein (LDL) by macrophages via scavenger receptors. Modification of LDL, which results in its recognition by these receptors, can be initiated by peroxidation of LDL lipids. The first step in this process is the formation of monohydroperoxy derivatives of fatty acids, which are subsequently degraded to the corresponding monohydroxy compounds, or to a variety of secondary oxidation products. In order to understand this process more completely, we have developed a mass spectrometric procedure to measure the amounts of specific hydroperoxy/hydroxy fatty acids formed by oxidation of the major unsaturated fatty acids in human LDL, oleic acid, linoleic acid, and arachidonic acid. Oxidation of human LDL in the presence of a relatively strong stimulus (20 microM CuSO4) resulted in very large increases in the amounts of the major monohydroxy derivatives of linoleic acid (9- and 13-hydroxy derivatives) and arachidonic acid (5-, 8-, 9-, 11-, 12-, and 15-hydroxy derivatives) in LDL lipids in the early stages of the reaction. After 20 h, the amounts of these products declined due to substrate depletion, but large amounts of monohydroxy derivatives of oleic acid (8-, 10-, and 11-hydroxy derivatives) were detected. Although thiobarbituric acid-reactive substances clearly increased under these conditions, the changes were not nearly so dramatic as those observed for monohydroxy fatty acids. Oxidation of LDL in the presence of endothelial cells, a much milder stimulus, resulted in 2.5- to 3-fold increases in the amounts of monohydroxy derivatives of linoleic and arachidonic acids, as well as thiobarbituric acid-reactive substances, with more modest increases in the amounts of hydroxylated derivatives of oleic acid. There was little positional specificity in the oxidation of the above fatty acids in the presence of either stimulus, suggesting that the formation of these products proceeds primarily by lipid peroxidation, rather than by catalysis by lipoxygenases. However, an important role for lipoxygenases in the initiation of these reactions cannot be excluded. In conclusion, oxidation of LDL in the presence of copper ions or endothelial cells results in the formation of a large number of monohydroxy derivatives of oleic, linoleic, and arachidonic acids. The relative amounts of products formed from each of these fatty acids depends on the strength of the stimulus as well as the incubation time.     

Incorporation of lipoxygenase products into cholesteryl esters by acyl-CoA:cholesterol acyltransferase in cholesterol-rich macrophages.

Macrophages which were incubated with acetylated low-density lipoproteins, resulting in cholesteryl ester accumulation, incorporated the monohydroxyeicosatetraenoic acids (5-, 15-, and 12-HETEs) into cholesteryl esters. The esterification of these hydroxy fatty acids to cholesterol by total membrane preparations of cholesterol-rich macrophages was dependent on the synthesis of the fatty acyl-CoA derivative, and was catalysed by acyl-CoA:cholesterol acyltransferase (ACAT). Stimulation of membrane ACAT activity by 25-hydroxycholesterol increased the synthesis of cholesteryl 12-HETE by 40%. In contrast, inhibiting ACAT activity by progesterone and compound 58-035 decreased cholesteryl 12-HETE production by 60% and 90% respectively. Although 5-, 15- and 12-HETE were esterified to cholesterol by ACAT, these monohydroxy fatty acids were less optimal as substrates compared with oleic acid or arachidonic acid. The hydrolysis and release of 12-HETE and the other monohydroxyeicosatetraenoic acids from intracellular cholesteryl esters and phospholipids occurred at a faster rate than for the more conventional fatty acids, oleate and arachidonate. Cholesteryl esters which contain hydroxy fatty acids therefore provide only a transient storage for lipoxygenase products, as these fatty acids are released into the medium as readily as hydroxy fatty acids found in phospholipids and triacylglycerols. The data provide evidence, for the first time, of an ACAT-dependent esterification of the lipoxygenase products 5-, 15- and 12-HETEs to cholesterol in the macrophage-derived foam cell. The channelling of these monohydroxy fatty acids to cholesteryl esters provides a mechanism which can alter the amount of lipoxygenase products incorporated into cellular phospholipids, thus averting deleterious changes to cell membranes. ACAT, by catalysing the esterification of monohydroxyeicosatetraenoic acids to cholesterol, could play a key role in regulating the amount of lipoxygenase products in the pericellular space of the cholesterol-enriched macrophage.     

Interaction between free fatty acids and Ca2+-dependent ATPase from sarcoplasmic reticulum membranes

The interaction between free fatty acids and Ca2+-dependent ATPase, an intrinsic protein of sarcoplasmic reticulum membranes, was studied with relevance to the changes in membrane permeability induced by free fatty acids. It was found that only unsaturated fatty acids increase the permeability of reticulum membranes for Ca2+, this effect being completely reversible. The increase in the membrane permeability by fatty acids is coupled to a generation of a channel for Ca2+ efflux under effect of Ca2+-dependent ATPase. The interaction between fatty acids and Ca2+-dependent ATPase was demonstrated by the protein fluorescence and electron paramagnetic resonance methods, using spin-labelled fatty acid derivatives. A model demonstrating the increase of sarcoplasmic reticulum membrane permeability for Ca2+ in the presence of the fatty acid-Ca2+-dependent ATPase complex is proposed.     

Cholesteryl ester acyl oxidation and remodeling in murine macrophages: formation of oxidized phosphatidylcholine

Cholesterol is an essential component of eukaryotic cell membranes, regulating fluidity and permeability of the bilayer. Outside the membrane, cholesterol is esterified to fatty acids forming cholesterol esters (CEs). Metabolism of CEs is characterized by recurrent hydrolysis and esterification as part of the CE cycle; however, since recombinant 15-lipoxygenase (15-LO) was shown to oxidize cholesteryl linoleate of LDL, there has been interest in CE oxidation, particularly in the context atherogenesis. Studies of oxidized CE (oxCE) metabolism have focused on hydrolysis and subsequent reverse cholesterol transport with little emphasis on the fate the newly released oxidized fatty acyl component. Here, using mass spectrometry to analyze lipid oxidation products, CE metabolism in murine peritoneal macrophages was investigated. Ex vivo macrophage incubations revealed that cellular 15-LO directly oxidized multiple CE substrates from intracellular stores and from extracellular sources. Freshly harvested murine macrophages also contained 15-LO-specific oxCEs, suggesting the enzyme may act as a CE-oxidase in vivo. The metabolic fate of oxCEs, particularly the hydrolysis and remodeling of oxidized fatty acyl chains, was also examined in the macrophage. Metabolism of deuterated CE resulted in the genesis of deuterated, oxidized phosphatidylcholine (oxPC). Further experiments revealed these oxPC species were formed chiefly from the hydrolysis of oxidized CE and subsequent reacylation of the oxidized acyl components into PC.     

Metabolism of linoleic and arachidonic acids in VX2 carcinoma tissue: Identification of monohydroxy octadecadienoic acids and monohydroxy eicosatetraenoic acids

The metabolism of arachidonic and linoleic acids by VX₂ carcinoma tissue was determined. Prostaglandin E₂ was the major metabolic product of arachidonic acid in the neoplastic tissue. Minor products accounting for 3– 8% of arachidonic acid metabolism were 11-hydroxy-5, 8, 12, 14-eicosatetraenoic acid (11-HETE) and 15-hydroxy-5, 8, 11, 13-eicosatetraenoic acid (15-HETE). Linoleic acid was converted to a mixture of 9-hydroxy-10, 12-octadecadienoic acid (9-HODD) and 13-hydroxy-9, 11-octadecadienoic acid (13-HODD). The conversion of linoleic acid to monohydroxy C-18 fatty acids varied from 40–80% 9-HODD and 20–60% 13-HODD in tumor tissue harvested from different animals. The quantity of monohydroxy C-18 fatty acids biosynthesized by VX₂ carcinoma tissue from endogenous linoleic acid equals or exceeds that of prostaglandin E₂ biosynthesis from endogenous arachidonic acid. The presence of a hydroxyl group adjacent to a conjugated diene suggest that the monohydroxy C-18 and monohydroxy C-20 fatty acids were formed via the action of lipoxygenase-like enzymes. These lipoxygenase-like reactions are inhibited by indomethacin in a concentration-dependent fashion similar to the inhibition of prostaglandin E₂ biosynthesis. The enzymes catalyzing the lipoxygenase-like reactions of linoleic and arachidonic acids are localized in the microsomal fraction of VX₂ carcinoma tissue. These data suggest that the lipoxygenase-like reactions are catalyzed by fatty acid cyclooxygenase and that there are two major pathways of fatty acid cyclooxygenase metabolism of polyenoic fatty acids in the neoplastic tissue. One pathway involves the formation of prostaglandin E₂ via cyclic endoperoxy intermediates. The second pathway involves the formation of monohydroxy C-18 fatty acids from linoleic acid via lipoxygenase-like reactions.     

Continuous fluorometric assay of Ca2+ transport by liposomes with Quin 2 entrapped: effect of phospholipase A2 and unsaturated long-chain fatty acids

The amount of free calcium in the cytoplasm is important in stimulation coupled with a number of cellular functions. The putative ionophoretic action of membrane lipid metabolites on Ca2+ offers convenient explanation of the stimulation-coupled mobilization of cytoplasmic Ca2+. To analyze the ionophoretic action of the lipid metabolites, we devised a sensitive method to study Ca2+ transport that uses liposome-entrapped Quin 2. A calcium ionophore, A23187, increased the fluorescence intensity of the Ca2+-Quin 2 complex as a function of Ca2+ transport into liposomes. A similar Ca2+ flux into the liposomes was induced by phospholipase A2 (PLA2) and by various long-chain fatty acids in liposomes that consist of phospholipids containing unsaturated fatty acids. The potencies of the fatty acids for Ca2+ transport is inversely correlated with their melting points. The oxidized products of the unsaturated fatty acids increased the Ca2+ and nonspecific permeability of the biological membranes. These results suggest that stimulation-coupled PLA2 activation might mediates the mobilization of cytoplasmic Ca2+.     

Bacteriorhodopsin: photochemistry and energy conversion

This study examined effects of fatty acids on the metabolism of 1,3 diphenylisobenzofuran (DPBF) and benzo(α) pyrene (BP) by rat or human colonic mucosal microsomes. Arachidonate, linoleate (25 μM) or their hydroperoxides increased oxidation of DPBF or BP 4 to 5-fold, whereas saturated fatty acids and NADPH had no effect. Studies of the influence of O₂ exclusion and indomethacin on DPBF and BP oxidation were consistent with the existence of both cyclooxygenase dependent and independent pathways for fatty acid stimulation of colonic microsomal drug oxidation. These results may have a bearing on the increased prevalence of colon cancer in populations with high fat intakes.     

Both membrane stretch and fatty acids directly activate large conductance Ca(2+)-activated K+ channels in vascular smooth muscle cells.

Large conductance Ca(2+)-activated K+ channels in rabbit pulmonary artery smooth muscle cells are activated by membrane stretch and by arachidonic acid and other fatty acids. Activation by stretch appears to occur by a direct effect of stretch on the channel itself or a closely associated component. In excised inside-out patches stretch activation was seen under conditions which precluded possible mechanisms involving cytosolic factors, release of Ca2+ from intracellular stores, or stretch induced transmembrane flux of Ca2+ or other ions potentially capable of activating the channel. Fatty acids also directly activate this channel. Like stretch activation, fatty acid activation occurs in excised inside-out patches in the absence of cytosolic constituents. Moreover, the channel is activated by fatty acids which, unlike arachidonic acid, are not substrates for the cyclo-oxygenase or lypoxygenase pathways, indicating that oxygenated metabolites do not mediate the response. Thus, four distinct types of stimuli (cytosolic Ca2+, membrane potential, membrane stretch, and fatty acids) can directly affect the activity of this channel.     

Proinflammatory properties of unsaturated fatty acids and their monohydroxy metabolites

The proinflammatory effects of unsaturated fatty acids and, where appropriate, their monohydroxy derivatives, have been investigated both by application to human skin and with respect to human polymorphonuclear leukocyte (PMN) migration. Of the fatty acids applied to the skin only eicosapentaenoic and arachidonic acids (EPA; AA) produced consistent, measurable erythema. The monohydroxy derivatives of the two fatty acids also caused erythema, the 12-hydroxy isomers being the most potent. Chemokinetic activity towards PMNs was observed in the presence of AA, EPA and alpha-linolenic acid using an agarose microdroplet chemokinesis assay. In contrast to their in vivo properties, the 5-hydroxy isomers of AA and EPA were the most potent, being approximately 10 times more chemokinetically active than the other isomers. Quantification of the hydroxyeicosatetraenoic and hydroxyeicosapentaenoic acids (HETEs; HEPEs) in the lesional skin of psoriatic patients demonstrated that, of the metabolites measured, 12-HETE was present in the greatest amounts. Twenty five times more 12-HETE than 12- or 15-HEPE was detected, these being the most abundant of the HEPEs formed. The monohydroxy derivatives of AA and EPA may contribute to the inflammatory changes observed in psoriasis. The HETEs appear to be of greater importance than the HEPEs in view of the relative amounts present.     

Differences in partitioning of meal fatty acids into blood lipid fractions: a comparison of linoleate, oleate, and palmitate

There has been much interest in the health effects of dietary fat, but few studies have comprehensively compared the acute metabolic fate of specific fatty acids in vivo. We hypothesized that different classes of fatty acids would be variably partitioned in metabolic pathways and that this would become evident over 24 h. We traced the fate of fatty acids using equal amounts of [U-(13)C]linoleate, [U-(13)C]oleate, and [U-(13)C]palmitate given in a test breakfast meal in 12 healthy subjects. There was a tendency for differences in the concentrations of the tracers in plasma chylomicron-triacylglycerol (TG) (oleate > palmitate > linoleate). This pattern remained in plasma nonesterified fatty acid (NEFA) and very low-density lipoprotein (VLDL)-TG (P 相似文献   

Thermodynamic parameters of free oxidation pathways in liver mitochondria

Two pathways of free oxidation in liver mitochondria were examined. One of these pathways is determined by the protonophoric action of free fatty acids, and the other pathway, by passive proton leakage in the absence of fatty acids. According to the model of the proton futile cycle of mitochondria, the protonophoric activity of fatty acids was defined as a quotient of the division of the acceleration of respiration by fatty acid by the coefficient of respiration control for the proton leakage. The temperature dependence of the palmitate protonophoric activity on the Arrhenius plot has a break at 22 degrees C and is characterized by the transition of activation energy from 120 to 60 kJ/mol. The dependence of the respiration rate in state 4 on the Arrhenius plot is linear and, the activation energy is 17 kJ/mol. It was concluded that the first pathway of free oxidation is determined by the cyclic transport of fatty acids with the participation of metabolic carriers, and this process depends on the membrane fluidity; the second pathway is determined by passive leakage of protons through membrane channels, without fatty acids and this process is independent on membrane fluidity.     

Proinflammatory properties of unsaturated fatty acids and their monohydroxy metabolites

The proinflammatory effects of unsaturated fatty acids and, where appropriate, their monohydroxy derivatives, have been investigated both by application to human skin and with respect to human polymorphonuclear leukocyte (PMN) migration. Of the fatty acids applied to the skin only eicosapentaenoic and arachidonic acids (EPA;AA) produced consistent, measurable erythema. The monohydroxy derivatives of the two fatty acids also caused erythema. the 12-hydroxy isomers being the most potent. Chemokinetic activity towards PMNs was observed in the presence of AA, EPA and α-linolenic acid using an agarose microdroplet chemokinesis assay. In contrast to their properties, the 5-hydroxy isomers of AA and EPA were the most potent, being approximately 10 times more chemokinetically active than the other isomers. Quantification of the hydroxyeicosatetraenoic and hydroxyeicosapentaenoic acids (HETEs;HEPEs) in the lesional skin of psoriatic patients demonstrated that, of the metabolites measured, 12-HETE was present in the greatest amounts. Twenty five times more 12-HETE than 12- or 15-HEPE was detected, these being the most abundant of teh HEPEs formed. The monohydroxy derivatives of AA and EPA may contribute to the inflammatory changes observed in psoriasis. The HETEs appear to be of greater importance than the HEPEs in view of the relative amounts present.     

Plant peroxisomal solute transporter proteins

Plant peroxisomes are unique subcellular organelles which play an indispensable role in several key metabolic pathways, including fatty acid b-oxidation,photorespiration, and degradation of reactive oxygen species. The compartmentalization of metabolic pathways into peroxisomes is a strategy for organizing the metabolic network and improving pathway efficiency. An important prerequisite, however, is the exchange of metabolites between peroxisomes and other cell compartments. Since the first studies in the 1970s scientists contributed to understanding how solutes enter or leave this organelle.This review gives an overview about our current knowledge of the solute permeability of peroxisomal membranes described in plants, yeast, mammals and other eukaryotes. In general, peroxisomes contain in their bilayer membrane specific transporters for hydrophobic fatty acids(ABC transporter) and large cofactor molecules(carrier for ATP, NAD and CoA). Smaller solutes with molecular masses below 300–400 Da, like the organic acids malate, oxaloacetate, and 2-oxoglutarate, are shuttled via non-selective channels across the peroxisomal membrane.In comparison to yeast, human, mammals and other eukaryotes, the function of these known peroxisomal transporters and channels in plants are discussed in this review.     

Ca2+ translocation across liposomal membranes enhanced by unsaturated long-chain fatty acids

The putative ionophoretic action of phosphatidic acid or arachidonic acid metabolites for Ca2+ has offered an attractive explanation for stimulation-coupled mobilization of cytoplasmic Ca2+. We have examined the effects of Ca2+ ionophore and long-chain unsaturated fatty acids on the translocation of Ca2+ across the liposomal membrane by using Quin II-entrapped liposomes, a sensitive assay system for ionophoresis of Ca2+. A23187 increased Quin II fluorescence intensity corresponding to the translocation of Ca2+ into liposomes. Similar translocation was observed with unsaturated long-chain fatty acids but not with saturated fatty acids. Thus, when phospholipases of cell membrane are activated by certain stimuli, unsaturated long-chain fatty acids are liberated and might mediate the mobilization of cytoplasmic Ca2+.     

Degradation of trilinolein by laccase enzymes

Laccase enzymes were investigated for their potential to catalyze the oxidation of trilinolein and methyl linoleate. This study demonstrates that laccase enzymes can oxidize unsaturated fatty acid esters and their associated lipids. The reaction products resulting from laccase-catalyzed reactions with trilinolein were analyzed using combined reversed-phase high-performance liquid chromatography and mass spectrometry via an atmospheric pressure chemical ionization source. The dominant oxidation products detected were monohydroperoxides, bishydroperoxides, and epoxides. This paper presents the first detailed investigation into the interaction between laccase enzymes and lipids containing unsaturated fatty acids.     

Bacillus megaterium CYP102A1 oxidation of acyl homoserine lactones and acyl homoserines

Quorum sensing, the ability of bacteria to sense their own population density through the synthesis and detection of small molecule signals, has received a great deal of attention in recent years. Acyl homoserine lactones (AHLs) are a major class of quorum sensing signaling molecules. In nature, some bacteria that do not synthesize AHLs themselves have developed the ability to degrade these compounds by cleaving the amide bond or the lactone ring. By inactivating this signal used by competing bacteria, the degrading microbe is believed to gain a competitive advantage. In this work we report that CYP102A1, a widely studied cytochrome P450 from Bacillus megaterium, is capable of very efficient oxidation of AHLs and their lactonolysis products acyl homoserines. The previously known substrates for this enzyme, fatty acids, can also be formed in nature by hydrolysis of the amide of AHLs, so CYP102A1 is capable of inactivating the active parent compound and the products of both known pathways for AHL inactivation observed in nature. AHL oxidation primarily takes place at the omega-1, omega-2, and omega-3 carbons of the acyl chain, similar to this enzyme's well-known activity on fatty acids. Acyl homoserines and their lactones are better substrates for CYP102A1 than fatty acids. Bioassay of the quorum sensing activity of oxidation products reveals that the subterminally hydroxylated AHLs exhibit quorum sensing activity, but are 18-fold less active than the parent compound. In vivo, B. megaterium inactivates AHLs by a CYP102A1 dependent mechanism that must involve additional components that further sequester or metabolize the products, eliminating their quorum sensing activity. Cytochrome P450 oxidation of AHLs represents an important new mechanism of quorum quenching.     

Studies on the role of vitamin E in the oxidation of blood components by fatty hydroperoxides.

Studies are reported on the oxidation of vitamin E and changes in lipid and fatty acid composition of rat blood components incubated in vitro with hydroperoxides prepared from autoxidized methyl linoleate. Red blood cells, plasma, serum, and hemoglobin free stroma were incubated at 37 °C with suspensions of linoleate hydroperoxide in Tris buffer at pH 7.4. The RBC were destroyed and substances with excitation-fluorescent properties were produced. Phosphatidylethanolamine, vitamin E and unsaturated fatty acids were oxidized in the reaction. Among the reaction products were substances that gave a positive thiobarbituric acid value, tocoquinone, and an unidentified substance isolated in the nonsaponifiable fraction of the lipid extract of the hemolyzed red cells. The reaction of linoleate hydroperoxide with stroma was similar to that with red blood cells and the same products were observed. In contrast there was little reaction of linoleate hydroperoxide with vitamin E or lipids of the serum or plasma in the absence of red blood cells. The destruction of the red blood cells appeared to be closely related to the oxidation of vitamin E indicating that the strong antioxygenic action of vitamin E in vivo was due to its particular form or structural orientation in the red cell membrane.     

Activation of rat brain protein kinase C by lipid oxidation products

The unsaturated fatty acid components of membrane lipids are susceptible to oxidation in vitro and in vivo. The initial oxidation products are hydroperoxy fatty acids that are converted spontaneously or enzymatically to a variety of products. Hydroperoxy derivatives of oleic, linoleic, or arachidonic acids stimulate the activity of protein kinase C (PKC) purified from rat brain. The hydroperoxy acids satisfy the requirement of PKC for phospholipid (e.g., phosphatidylserine). Activation is observed in the presence or absence of 1 mM Ca2+. Reduction of the hydroperoxides to alcohols or dehydration of the hydroperoxides to ketones increases the Ka for activation three- to fourfold but does not significantly reduce the maximal extent of PKC activation. The Ka's for activation by hydroperoxy acids are approximately half the values exhibited by the unoxidized fatty acids. Since oxidation of unsaturated fatty acids to hydroperoxides is the first event in lipid peroxidation, activation of PKC by hydroperoxy fatty acids may be an early cellular response to oxidative stress.     

Regulation of Ca2+ efflux from kidney and liver mitochondria by unsaturated fatty acids and Na+ ions.

The effects of fatty acids and monovalent cations on the Ca2+ efflux from isolated liver and kidney mitochondria were investigated by means of electrode techniques. It was shown that unsaturated fatty acids and saturated fatty acids of medium chain length (C12 and C14) induced a Ca2+ efflux from mitochondria which was not inhibited by ruthenium red, but was specifically inhibited by Na+ and Li+. The Ca2+-releasing activity of unsaturated fatty acids did not correlate with their uncoupling activity. In kidney mitochondria a spontaneous, temperature-dependent Ca2+ efflux was observed which was inhibited either by albumin or by Na+. It is suggested that the net Ca2+ accumulation by mitochondria depends on the operation of independent pump and leak pathways. The pump is driven by the membrane potential and can be inhibited by ruthenium red, the leak depends on the presence of unsaturated fatty acids and is inhibited by Na+ and Li+. It is suggested that the unsaturated fatty acids produced by mitochondrial phospholipase A2 can be essential in the regulation of the Ca2+ retention in and the Ca2+ release from the mitochondria.