Influence of cholesterol on the formation of palmitate/Ca<Superscript>2+</Superscript>-activated pores in mitochondria and liposomes

The influence of cholesterol on the formation of a mitochondrial cyclosporin A-insensitive palmitate/Ca²⁺-activated pore has been studied. Loading of mitochondrial membranes with cholesterol increases the rate of mitochondrial swelling induced by palmitic acid (≥20 μM) and Ca²⁺ (30 μM). This effect is not related to changes in the functional activity of organelles, since cholesterol does not influence the mitochondrial respiration in different metabolic states. At the same time, palmitate/Ca²⁺-induced permeabilization of azolectin/cholesterol liposomes is more pronounced than that of azolectin liposomes. In the liposomal membrane, Ca²⁺ induces phase separation of palmitic acid into distinct membrane domains; the presence of cholesterol in membranes enhances this effect.     

Physiological aspects of the mitochondrial cyclosporin A-insensitive palmitate/Ca<Superscript>2+</Superscript>-induced pore: tissue specificity,age profile and dependence on the animal’s adaptation to hypoxia

Earlier we found that being added to rat liver mitochondria, palmitic acid (Pal) plus Ca²⁺ opened a cyclosporin A-insensitive pore, which remained open for a short time. Apparently, this pore is involved in the Pal-induced apoptosis and may also take part in the mitochondrial Ca²⁺ recycling as a Ca²⁺ efflux system (Belosludtsev et al. J Bioenerg Biomembr 38:113–120, 2006; Mironova et al. J. Bioenerg. Biomembr. 39:167–174, 2007). In this paper, we continue studying physiological and regulatory aspects of the pore. The following observations have been made. (1) Cardiolipin has been found to facilitate the Ca²⁺-induced formation of pores in the Pal-containing liposomal membranes. (2) The opening of Pal/Ca²⁺-induced pore is accompanied by the release of apoptosis-induced factor (AIF) from mitochondria. (3) The rate of Pal/Ca²⁺-induced swelling of rat liver mitochondria increases substantially with the age of animals. (4) Although the Pal/Ca²⁺-induced pore opens both in the liver and heart mitochondria, the latter require higher Pal concentrations for the pore to open. (5) The pore opening depends on the resistance of animals to hypoxia: in the highly resistant to hypoxia rats, the mitochondrial Pal/Ca²⁺-induced pore opens easier than in the low resistant animals, this being opposite for the classical, cyclosporin A-sensitive MPT pore. The adaptation of the low resistant rats to oxygen deficiency increases the sensitivity of their mitochondria to PalCaP inductors. The paper also discusses a possible role of the mitochondrial Pal/Ca²⁺-induced pore in the protection of tissues against hypoxia.     

Peptide Pheromone Plantaricin A Produced by Lactobacillus plantarum Permeabilizes Liver and Kidney Cells

Certain antimicrobial peptides from multicellular animals kill a variety of tumor cells at concentrations not affecting normal eukaryotic cells. Recently, it was reported that also plantaricin A (PlnA), which is a peptide pheromone with strain-specific antibacterial activity produced by Lactobacillus plantarum, permeabilizes cancerous rat pituitary cells (GH₄ cells), whereas normal rat anterior pituitary cells are resistant to the peptide. To examine whether the preferential permeabilization of cancerous cells is a general feature of PlnA, we studied its effect on primary cultures of cells from rat liver (hepatocytes, endothelial, and Kupffer cells) and rat kidney cortex, as well as two epithelial cell lines of primate kidney origin (Vero cells from green monkey and human Caki-2 cells). The Vero cell line is derived from normal cells, whereas the Caki-2 cell line is derived from a cancerous tumor. The membrane effects were studied by patch clamp recordings and microfluorometric (fura-2) monitoring of the cytosolic concentrations of Ca²⁺ ([Ca²⁺]_i) and fluorophore. In all the tested cell types except Kupffer cells, exposure to 10–100 μM PlnA induced a nearly instant permeabilization of the membrane, indicated by the following criteria: increased membrane conductance, membrane depolarization, increased [Ca²⁺]_i, and diffusional loss of fluorophore from the cytosol. At a concentration of 5 μM, PlnA had no effect on any of the cell types. The Kupffer cells were permeabilized by 500 μM PlnA. We conclude that the permeabilizing effect of PlnA is not restricted to cancerous cells.     

Mitochondrial lipid pore in the mechanism of glutamate-induced calcium deregulation of brain neurons

The work examines the mechanism of central nerve cell death upon stimulation of brain NMDA receptors with the stimulatory mediator glutamate. A prolonged stimulation of neurons with glutamate is known to result in the disorder of Ca²⁺ homeostasis and severe mitochondrial depolarization followed by cell death. It has been shown that the overload of mitochondria with Sr²⁺ leads to the release of the cation, medium alkalization, decrease of membrane potential and mitochondrial swelling, indicating a nonspecific permeabilization of the mitochondrial membrane. The permeabilization, in our opinion, is caused by the activation of Ca²⁺/Sr²⁺-dependent phospholipase A₂ (PLA₂), resulting in the formation of free palmitic and stearic acids in the mitochondrial membrane. These fatty acids bind Ca²⁺ with high affinity and the process of binding is accompanied by the formation of a transient lipid pore—a phenomenon demonstrated earlier on both artificial and mitochondrial membranes. The inhibitors of PLA₂ have been shown to suppress permeabilization of mitochondrial membranes. In the culture of granular cerebellum neurons, the PLA₂ inhibitors prolonged the lag of the delayed Sr²⁺ deregulation and membrane depolarization. On the basis of data obtained on isolated mitochondria and neurons we suppose that the initial stages of glutamate-induced Ca²⁺ deregulation of neurons are underlain by the opening of lipid pores in brain mitochondria.     

Characteristics of non-specific permeability and H+-ATPase inhibition induced in the plasma membrane of Nitella flexilis by excessive Cu2+

Effects of Cu²⁺ on a non-specific conductance and H⁺-ATPase activity in the plasma membrane of the freshwater alga Nitella flexilis L. Agardh was studied using a conventional microelectrode voltage-clamp technique. We show that a Cu²⁺-induced increase in the non-specific conductance is related to the formation of pores in the plasma membrane. Pore formation is the result of unidentified chemical reactions, since the Q₁₀ for the rate of increase of conductance over time was about 3. Various oxidants and antioxidants (10 mmol/l H₂O₂, 10 mmol/l ascorbate, 100 μg/ml superoxide dismutase, and 100 μg/ml catalase) did not alter Cu²⁺-induced changes in the plasma membrane conductance, suggesting that the effect of Cu²⁺ was unrelated to peroxidation of plasma-membrane lipids. In contrast, organic and inorganic Ca²⁺-channel antagonists (nifedipine, Zn²⁺, Cd²⁺, Fe²⁺, Ni²⁺) inhibited the Cu²⁺-induced non-specific conductance increase. This suggests that changes in Ca²⁺ influx underlie this effect of Cu²⁺. Decreasing the pH or the ionic strength of external solutions also inhibited the Cu²⁺-induced plasma-membrane conductance increase. Copper was also found to inhibit plasma-membrane H⁺-ATPase activity with half-maximal inhibition occurring at about 5–20 μmol/l and full inhibition at about 100–300 μmol/l. The Hill coefficient of Cu²⁺ inhibition of the H⁺-ATPase was close to two. Received: 8 December 1999 / Accepted: 16 August 2000     

Involvement of palmitate/Ca2 +(Sr2 +)-induced pore in the cycling of ions across the mitochondrial membrane

The palmitate/Ca^2 +-induced (Pal/Ca^2 +) pore, which is formed due to the unique feature of long-chain saturated fatty acids to bind Ca^2 + with high affinity, has been shown to play an important role in the physiology of mitochondria. The present study demonstrates that the efflux of Ca^2 + from rat liver mitochondria induced by ruthenium red, an inhibitor of the energy-dependent Ca^2 + influx, seems to be partly due to the opening of Pal/Ca^2 + pores. Exogenous Pal stimulates the efflux. Measurements of pH showed that the Ca^2 +-induced alkalization of the mitochondrial matrix increased in the presence of Pal. The influx of Ca^2 + (Sr^2 +) also induced an outflow of K⁺ followed by the reuptake of the ion by mitochondria. The outflow was not affected by a K⁺/H⁺ exchange blocker, and the reuptake was prevented by an ATP-dependent K⁺ channel inhibitor. It was also shown that the addition of Sr^2 + to mitochondria under hypotonic conditions was accompanied by reversible cyclic changes in the membrane potential, the concentrations of Sr^2 + and K⁺ and the respiratory rate. The cyclic changes were effectively suppressed by the inhibitors of Ca^2 +-dependent phospholipase A₂, and a new Sr^2 + cycle could only be initiated after the previous cycle was finished, indicating a refractory period in the mitochondrial sensitivity to Sr^2 +. All of the Ca^2 +- and Sr^2 +-induced effects were observed in the presence of cyclosporin A. This paper discusses a possible role of Pal/Ca^2 + pores in the maintenance of cell ion homeostasis.     

Ca2 +-dependent permeabilization of mitochondria and liposomes by palmitic and oleic acids: A comparative study

In the present work, we examine and compare the effects of saturated (palmitic) and unsaturated (oleic) fatty acids in relation to their ability to cause the Ca^2 +-dependent membrane permeabilization. The results obtained can be summarized as follows. (1) Oleic acid (OA) permeabilizes liposomal membranes at much higher concentrations of Ca^2 + than palmitic acid (PA): 1 mM versus 100 μM respectively. (2) The OA/Ca^2 +-induced permeabilization of liposomes is not accompanied by changes in the phase state of lipid bilayer, in contrast to what is observed with PA and Ca^2 +. (3) The addition of Ca^2 + to the PA-containing vesicles does not change their size; in the case of OA, it leads to the appearance of larger and smaller vesicles, with larger vesicles dominating. This can be interpreted as a result of fusion and fission of liposomes. (4) Like PA, OA is able to induce a Ca^2 +-dependent high-amplitude swelling of mitochondria, yet it requires higher concentrations of Ca^2 + (30 and 100 μM for PA and OA respectively). (5) In contrast to PA, OA is unable to cause the Ca^2 +-dependent high-amplitude swelling of mitoplasts, suggesting that the cause of OA/Ca^2 +-induced permeability transition in mitochondria may be the fusion of the inner and outer mitochondrial membranes. (6) The presence of OA enhances PA/Ca^2 +-induced permeabilization of liposomes and mitochondria. The paper discusses possible mechanisms of PA/Ca^2 +- and OA/Ca^2 +-induced membrane permeabilization, the probability of these mechanisms to be realized in the cell, and their possible physiological role.     

The SR Ca2+ ATPase of the Antarctic scallop Adamussium colbecki: cold adaptation and heavy metal effects

Cell calcium is accumulated in intracellular stores by sarco-endoplasmic reticulum Ca²⁺ ATPases functionally interacting with the membrane lipid environment. Cold adaptations of membrane lipids in Antarctic Sea organisms suggest possible adaptive effects also on sarco-endoplasmic reticulum Ca²⁺ ATPases. We investigated the SR Ca²⁺ ATPase of an Antarctic scallop, Adamussium colbecki, by characterising the enzyme activity and studying temperature effects. Ca²⁺ ATPase, assayed by following ATP hydrolysis, was thapsigargin- and vanadate-sensitive, showed maximum activity under 2 μM Ca²⁺, 200 mM KCl and pH 7.2, and had a K _M for ATP of 22 ± 7 μM. Temperature effects showed an Arrhenius inversion between −1.8 and 0°C, indicating cold adaptation, an Arrhenius break at 10°C, and a collapse above 20°C. A. colbecki accumulates high amounts of cadmium in the digestive gland; heavy metal effects on sarco-endoplasmic reticulum Ca²⁺ ATPases were therefore tested, finding an IC₅₀ = 0.9 μM for Hg²⁺ and 3 μM for Cd²⁺. Finally, SDS-PAGE analysis showed a main band at about 100 kDa, which was identified as sarco-endoplasmic reticulum Ca²⁺ ATPase after trypsin digestion, and accounted for 60% total protein. Accepted: 10 December 1998     

Modulation of the Permeability Transition Pore by Inhibition of the Mitochondrial KATP Channel in Liver vs. Brain Mitochondria

Inhibition of the mitochondrial K_ATP (mitoK_ATP) channel abrogates the beneficial effects of preconditioning induced by a brief episode of sublethal ischemia. We studied the effect of 5-hydroxydecanoate, a well-known inhibitor of the mitoK_ATP channel, on swelling of isolated liver and brain mitochondria. Volume changes were determined by measurement of light absorbance at 540 nm. Mitochondrial swelling induced by adding Ca²⁺ ions correlated with opening of the permeability transition pore as shown by modulation by 1 μM cyclosporin A. In brain mitochondria, 5-hydroxydecanoate did not significantly affect Ca²⁺-induced swelling. In contrast, 50 or 500 μM 5-hydroxydecanoate increased swelling of liver mitochondria by 9.7 ± 5.1% (n = 6, P = 0.057) and 29.4 ± 1.4% (n = 5, P < 0.0001), respectively. The effect of 5-hydroxydecanoate was blocked by cyclosporin A and was dependent on the presence of potassium in the medium. In medium containing 200 μM ATP to inhibit the mitoK_ATP channel, 5–hydroxydecanoate did not further increase Ca²⁺-induced swelling. We conclude that inhibition of the mitoK_ATP channel exerts its detrimental effect by facilitation of permeability transition pore opening.     

Mitochondrial Ca<Superscript>2+</Superscript> cycle mediated by the palmitate-activated cyclosporin a-insensitive pore

Earlier we found that in isolated rat liver mitochondria the reversible opening of the mitochondrial cyclosporin A-insensitive pore induced by low concentrations of palmitic acid (Pal) plus Ca²⁺ results in the brief loss of Δψ [Mironova et al., J Bioenerg Biomembr (2004), 36:171–178]. Now we report that Pal and Ca²⁺, increased to 30 and 70 nmol/mg protein respectively, induce a stable and prolonged (10 min) partial depolarization of the mitochondrial membrane, the release of Ca²⁺ and the swelling of mitochondria. Inhibitors of the Ca²⁺ uniporter, ruthenium red and La³⁺, as well as EGTA added in 10 min after the Pal/Ca²⁺-activated pore opening, prevent the release of Ca²⁺ and repolarize the membrane to initial level. Similar effects can be observed in the absence of exogeneous Pal, upon mitochondria accumulating high [Sr²⁺], which leads to the activation of phospholipase A₂ and appearance of endogenous fatty acids. The paper proposes a new model of the mitochondrial Ca²⁺ cycle, in which Ca²⁺ uptake is mediated by the Ca²⁺ uniporter and Ca²⁺ efflux occurs via a short-living Pal/Ca²⁺-activated pore.     

A permeability transition in liposomes induced by the formation of Ca/palmitic acid complexes

Formation of palmitic acid/Ca²⁺ (PA/Ca²⁺) complexes was suggested to play a key role in the non-classical permeability transition in mitochondria (NCPT), which seems to be involved in the PA-induced apoptosis of cardiomyocytes. Our previous studies of complexation of free fatty acids (FFA) with Ca²⁺ showed that long-chain (C:16-C:22) saturated FFA had an affinity to Ca²⁺, which was much higher than that of other FFA and lipids. The formation of FFA/Ca²⁺ complexes in the black-lipid membrane (BLM) was demonstrated to induce a nonspecific ion permeability of the membrane. In the present work, we have found that binding of Ca²⁺ to PA incorporated into the membrane of sulforhodamine B (SRB)-loaded liposomes results in an instant release of a part of SRB, with the quantity of SRB released depending on the concentration of PA and Ca²⁺. The pH-optimum of this phenomenon, similar to that of PA/Ca²⁺ complexation, is in the alkaline range. The same picture of SRB release has been revealed for stearic, but not for linoleic acid. Along with Ca²⁺, some other bivalent cations (Ba²⁺, Sr²⁺, Mn²⁺, Ni²⁺, Co²⁺) also induce SRB release upon binding to PA-containing liposomes, while Mg²⁺ turns out to be relatively ineffective. As revealed by fluorescence correlation spectroscopy, the apparent size of liposomes does not alter after the addition of PA, Ca²⁺ or their combination. So it has been supposed that the cause of SRB release from liposomes is the formation of lipid pores. The effect of FFA/Ca²⁺-induced permeabilization of liposomal membranes has several analogies with NCPT, suggesting that both these phenomena are of similar nature.     

Kinetic Analysis of the Calcium- and Cadmium-Induced Development of Nonspecific Permeability of the Mitochondrial Inner Membrane

We studied the Са²⁺- and Cd²⁺-induced development of the nonspecific permeability of the mitochondrial inner membrane in preparations obtained from rat liver tissue, which is accompanied by swelling of these organelles and intensification of light dispersion of their suspension. Addition of 5 to 100 μM Са²⁺ or 1 to 50 μM Сd²⁺ to the medium caused swelling of the mitochondria. With increase in concentrations of Са²⁺ and Cd²⁺, the latency of the effect decreased, and the rate of swelling of these organelles increased. Upon isolated action of Са²⁺, the intensity of the process (amplitude of changes) did not depend significantly on the concentration of the above ions, while upon isolated action of Cd²⁺, it was the maximum at the concentration of 1 mM and noticeably decreased with increase in the concentration. The dependence of the rate of Са²⁺- and Cd²⁺-induced swelling of the mitochondria on the concentration of these ions was described by power and sigmoid functions, respectively. The calculated maximum rate and the constant of 50% saturation of these processes were equal to 0.609 and 1.084 extinction units/min⋅mg protein and 19.85 and 7.28 μM for Са²⁺- and Cd²⁺-induced swelling of the mitochondria, respectively. Cyclosporine A (10 μM) suppressed completely the Са²⁺-induced swelling of the mitochondria and decreased only partly the Cd²⁺-induced swelling. Dithiothreitol (1 mM) inhibited completely the latter effect but did not influence significantly the Са²⁺-stimulated process. Therefore, the distinctions between the kinetics of Са²⁺- and Cd²⁺-induced swelling of the mitochondria, as well as the different sensitivity of these processes to cyclosporine A and dithiothreitol, prove that the mechanisms underlying interactions between the cations of the above metals and the inner mitochondrial membrane in the course of the development of nonspecific permeability of these organelles are dissimilar. *Deceased     

Boric acid inhibits stored Ca<Superscript>2+</Superscript> release in DU-145 prostate cancer cells

Boron (B) is a developmental and reproductive toxin. It is also essential for some organisms. Plants use uptake and efflux transport proteins to maintain homeostasis, and in humans, boron has been reported to reduce prostate cancer. Ca²⁺ signaling is one of the primary mechanisms used by cells to respond to their environment. In this paper, we report that boric acid (BA) inhibits NAD⁺ and NADP⁺ as well as mechanically induced release of stored Ca²⁺ in growing DU-145 prostate cancer cells. Cell proliferation was inhibited by 30% at 100μM, 60% at 250μM, and 97% at 1,000μM BA. NAD⁺-induced Ca²⁺ transients were partly inhibited at 250μM BA and completely at 1,000μM BA, whereas both NADP⁺ and mechanically induced transients were inhibited by 1,000μM BA. Expression of CD38 protein increased in proportion to BA exposure (0–1,000μM). In vitro mass spectrometry analysis showed that BA formed adducts with the CD38 products and Ca²⁺ channel agonists cyclic adenosine diphosphate ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP). Vesicles positive for the Ca²⁺ fluorophore fluo-3 acetoxymethyl ester accumulated in cells exposed to 250 and 1,000μM BA. The BA analog, methylboronic acid (MBA; 250 and 1,000μM), did not inhibit cell proliferation or NAD⁺, NADP⁺, or mechanically stimulated Ca²⁺ store release. Nor did MBA increase CD38 expression or cause the formation of intracellular vesicles. Thus, mammalian cells can distinguish between BA and its synthetic analog MBA and exhibit graded concentration-dependent responses. Based on these observations, we hypothesize that toxicity of BA stems from the ability of high concentrations to impair Ca²⁺ signaling.     

Involvement of P2X receptors in the NAD+-induced rise in [Ca2+]i in human monocytes

In the present study, we show that the extracellular addition of nicotinamide adenine dinucleotide (NAD⁺) induces a transient rise in [Ca²⁺]_i in human monocytes caused by an influx of extracellular calcium. The NAD⁺-induced Ca²⁺ response was prevented by adenosine triphosphate (ATP), suggesting the involvement of ATP receptors. Of the two subtypes of ATP receptors (P2X and P2Y), the P2X receptors were considered the most likely candidates. By the use of subtype preferential agonists and antagonists, we identified P2X₁, P2X₄, and P2X₇ receptors being engaged in the NAD⁺-induced rise in [Ca²⁺]_i. Among the P2X receptor subtypes, the P2X₇ receptor is unique in facilitating the induction of nonselective pores that allow entry of ethidium upon stimulation with ATP. In monocytes, opening of P2X₇ receptor-dependent pores strongly depends on specific ionic conditions. Measuring pore formation in response to NAD⁺, we found that NAD⁺ unlike ATP lacks the ability to induce this pore-forming response. Whereas as little as 100 μM ATP was sufficient to activate the nonselective pore, NAD⁺ at concentrations up to 2 mM had no effect. Taken together, these data indicate that despite similarities in the action of extracellular NAD⁺ and ATP there are nucleotide-specific variations. So far, common and distinct features of the two nucleotides are only beginning to be understood.     

Inhibition of ATP-Induced Ca<Superscript>2+</Superscript> Influx by Corticosterone in Dorsal Root Ganglion Neurons

In addition to the classic genomic effects, it is well known that glucocorticoids also have rapid, nongenomic effects on neurons. In the present study, the effect of corticosterone (CORT) on ATP-induced Ca²⁺ mobilization in cultured dorsal root ganglion (DRG) neurons were detected with confocal laser scanning microscopy using fluo-4/AM as a calcium fluorescent indicator that could monitor real-time alterations of intracellular calcium concentration ([Ca²⁺]i). ATP, an algesic agent, caused [Ca²⁺]i increase in DRG neurons by activation of P2X receptor. Pretreatment with CORT (1 nM–1 μM for 5 min) inhibited ATP-induced [Ca²⁺]i increase in DRG neurons. The rapid inhibition of ATP-induced Ca²⁺ response by CORT was concentration-dependent, reversible and could be blocked by glucocorticoid receptor antagonist RU38486 (10 μM). Furthermore, the inhibitory effect of CORT was abolished by protein kinase A inhibitor H89 (10 μM), but was not influenced by protein kinase C inhibitor Chelerythrine chloride (10 μM). On the other hand, membrane-impermeable bovine serum albumin-conjugated corticosterone had no effect on ATP-induced [Ca²⁺]i transients. These observations suggest that a nongenomic pathways may be involved in the effect of CORT on ATP-induced [Ca²⁺]i transients in cultured DRG neurons.     

On the mechanism of palmitic acid-induced apoptosis: the role of a pore induced by palmitic acid and Ca2+ in mitochondria

Palmitic acid (Pal) is known to promote apoptosis (Sparagna G et al (2000) Am J Physiol Heart Circ Physiol 279: H2124–H2132) and its amount in blood and mitochondria increases under some pathological conditions. Yet, the mechanism of the proapoptotic action of Pal has not been elucidated. We present evidence for the involvement of the mitochondrial cyclosporin A-insensitive pore induced by Pal/Ca²⁺ complexes in the apoptotic process. Opening of this pore led to a fall of the mitochondrial membrane potential and the release of the proapoptotic signal cytochrome c. The addition of cytochrome c prevented these effects and recovered membrane potential, which is in contrast to the cyclosporin A-sensitive mitochondrial permeability transition pore. Oleic and linoleic acids prevented the Pal/Ca²⁺-induced pore opening in the intact mitochondria, this directly and significantly correlating with the effect of these fatty acids on Pal-induced apoptosis in cells (Hardy S et al (2003) J Biol Chem 278: 31861–31870). The specific probe for cardiolipin, 10-N-nonyl acridine orange, inhibited formation of this pore.     

Potentiation of Regulatory Volume Decrease by a P2-Like Receptor and Arachidonic Acid in American Alligator Erythrocytes

This study examined the role of a P2 receptor and arachidonic acid (AA) in regulatory volume decrease (RVD) by American alligator red blood cells (RBCs). Osmotic fragility was determined optically, mean cell volume was measured by electronic sizing, and changes in intracellular Ca²⁺ concentration were visualized using fluorescence microscopy. Gadolinium (50 μM), hexokinase (2.5 U/ml), and suramin (100 μM) increased osmotic fragility, blocked volume recovery after hypotonic shock, and prevented a rise in intracellular Ca²⁺ that normally occurs during cell swelling. The P2X antagonists PPADS (50 μM) and TNP-ATP (10 μM) also increased fragility and inhibited volume recovery. In contrast, ATPγS (10 μM), α,β-methylene-ATP (50 μM) and Bz-ATP (50 μM) had the opposite effect, whereas 2-methylthio-ATP (50 μM) and UTP (10 μM) had no effect. In addition, the phospholipase A₂ (PLA₂) inhibitors ONO-RS-082 (10 μM), chlorpromazine (10 μM), and isotetrandrine (10 μM) increased osmotic fragility and blocked volume recovery, whereas AA (10 μM) and its nonhydrolyzable analog eicosatetraynoic acid (ETYA, 10 μM) had the reverse effect. Further, AA (10 μM), but not ATPγS (10 μM), prevented the inhibitory effect of a low Ca²⁺-EGTA Ringer on RVD, whereas both AA (10 μM) and ATPγS (10 μM) caused cell shrinkage under isosmotic conditions. In conclusion, our results are consistent with the presence of a P2-like receptor whose activation stimulated RVD. In addition, AA also was important for volume recovery.     

The role of ion-lipid interactions and lipid packing in transient defects caused by phenolic compounds

The transient disruption of membranes for the passive permeation of ions or small molecules is a complex process relevant to understanding physiological processes and biotechnology applications. Phenolic compounds are widely studied for their antioxidant and antimicrobial properties, and some of these activities are based on the interactions of the phenolic compound with membranes. Ions are ubiquitous in cells and are known to alter the structure of phospholipid bilayers. Yet, ion-lipid interactions are usually ignored when studying the membrane-altering properties of phenolic compounds. This study aims to assess the role of Ca²⁺ ions on the membrane-disrupting activity of two phenolic acids and to highlight the role of local changes in lipid packing in forming transient defects or pores. Results from tethered bilayer lipid membrane electrical impedance spectroscopy experiments showed that Ca²⁺ significantly reduces membrane disruption by caffeic acid methyl ester and caffeic acid. As phenolic acids are known metal chelators, we used UV-vis and fluorescence spectroscopy to exclude the possibility that Ca²⁺ interferes with membrane disruption by binding to the phenolic compound and subsequently preventing membrane binding. Molecular dynamics simulations showed that Ca²⁺ but not caffeic acid methyl ester or caffeic acid increases lipid packing in POPC bilayers. The combined data confirm that Ca²⁺ reduces the membrane-disrupting activity of the phenolic compounds, and that Ca²⁺-induced changes to lipid packing govern this effect. We discuss our data in the context of ion-induced pores and transient defects and how lipid packing affects membrane disruption by small molecules.     

Inhibitory Effects of Adenine Nucleotides on Brain Mitochondrial Permeability Transition

The adenine nucleotides ADP and ATP are probably the most important endogenous inhibitors of the mitochondrial permeability transition (MPT). We studied the inhibitory effects of adenine nucleotides on brain MPT by measuring mitochondrial swelling and Ca²⁺ and cytochrome c release. We observed that in the presence of either ADP or ATP, at 250 μM, brain mitochondria accumulated more than 1 μmol Ca²⁺ × mg protein⁻¹. ADP or ATP also prevented Ca²⁺-induced mitochondrial swelling and cytochrome c release. Interestingly, ATP lost most of its inhibitory effects on MPT when the experiments were carried out in the presence of ATP-regenerating systems. These results indicate that MPT inhibition observed in the presence of added ATP could be mainly due to hydrolysis of ATP to ADP. From mitochondrial swelling measurements, half-maximal inhibitory values (K _i) of 4.5 and 98 μM were obtained for ADP and ATP, respectively. In addition, a delayed mitochondrial swelling sensitive to higher ADP concentrations was observed. Mitochondrial anoxia/reoxygenation did not interfere with the inhibitory effect of ADP on Ca²⁺-induced MPT, but oxidative phosphorylation markedly decreased this effect. We conclude that ADP is a potent inhibitor of brain MPT whereas ATP is a weaker inhibitor of this phenomenon. Our results suggest that ADP can have an important protective role against MPT-mediated tissue damage under conditions of brain ischemia and hypoglycemia.     

Enhanced Glutathione Efflux from Astrocytes in Culture by Low Extracellular Ca<Superscript>2+</Superscript> and Curcumin

Efflux of glutathione (GSH) from astrocytes has been suggested as a key factor for neuroprotection by astrocytes. Here we evaluated if the Nrf2 activator curcumin affects basal and stimulated (Ca²⁺ omission) GSH efflux from cultures of astroglial cells. Stimulated efflux of GSH was observed at medium concentration of 0, 0.1 mM Ca²⁺, but not at 0.2 or 0.3 mM Ca²⁺. Astroglia treated with 30 μM curcumin increased the cellular content of GSH in parallel with elevated basal and stimulated efflux. Conversely treatment with buthionine sulfoximine lowered efflux of GSH. The efflux stimulated by Ca²⁺- omission was not affected by the P2X7-receptor antagonist Blue Brilliant G (100 nM) or the pannexin mimetic/blocking peptide ¹⁰Panx1 but inhibited by the gap junction blocker carbenoxolone (100 μM) and a hemichannel blocker Gap26 (300 μM). RNAi directed against Nrf2 partly inhibited the effect of curcumin. The results show that elevated cellular GSH by curcumin treatment enhance efflux from astroglial cells, a process which appear to be a prerequisite for astroglial mediated neuroprotection.