Mitochondrial Physiology of Cellular Redox Regulations.

Mitochondria (mt) represent the vital hub of the molecular physiology of the cell, being decision-makers in cell life/death and information signaling, including major redox regulations and redox signaling. Now we review recent advances in understanding mitochondrial redox homeostasis, including superoxide sources and H2O2 consumers, i.e., antioxidant mechanisms, as well as exemplar situations of physiological redox signaling, including the intramitochondrial one and mt-to-cytosol redox signals, which may be classified as acute and long-term signals. This review exemplifies the acute redox signals in hypoxic cell adaptation and upon insulin secretion in pancreatic beta-cells. We also show how metabolic changes under these circumstances are linked to mitochondrial cristae narrowing at higher intensity of ATP synthesis. Also, we will discuss major redox buffers, namely the peroxiredoxin system, which may also promote redox signaling. We will point out that pathological thresholds exist, specific for each cell type, above which the superoxide sources exceed regular antioxidant capacity and the concomitant harmful processes of oxidative stress subsequently initiate etiology of numerous diseases. The redox signaling may be impaired when sunk in such excessive pro-oxidative state.

Silymarin component 2,3-dehydrosilybin attenuates cardiomyocyte damage following hypoxia/reoxygenation by limiting oxidative stress.

Ischemic postconditioning and remote conditioning are potentially useful tools for protecting ischemic myocardium. This study tested the hypothesis that 2,3-dehydrosilybin (DHS), a flavonolignan component of Silybum marianum, could attenuate cardiomyocyte damage following hypoxia/reoxygenation by decreasing the generation of reactive oxygen species (ROS). After 5-6 days of cell culture in normoxic conditions the rat neonatal cardiomyocytes were divided into four groups. Control group (9 h at normoxic conditions), hypoxia/reoxygenation group (3 h at 1 % O2, 94 % N2and 5 % CO2followed by 10 min of 10 micromol·l⁻¹DHS and 6 h of reoxygenation in normoxia) and postconditioning group (3 h of hypoxia, three cycles of 5 min reoxygenation and 5 min hypoxia followed by 6 h of normoxia). Cell viability assessed by propidium iodide staining was decreased after DHS treatment consistent with increased levels of lactatedehydrogenase (LDH) after reoxygenation. LDH leakage was significantly reduced when cardiomyocytes in the H/Re group were exposed to DHS. DHS treatment reduced H2O2production and also decreased the generation of ROS in the H/Re group as evidenced by a fluorescence indicator. DHS treatment reduces reoxygenation-induced injury in cardiomyocytes by attenuation of ROS generation, H2O2and protein carbonyls levels. In addition, we found that both the postconditioning protocol and the DHS treatment are associated with restored ratio of phosphorylated/total protein kinase C epsilon, relative to the H/Re group. In conclusion, our data support the protective role of DHS in hypoxia/reperfusion injury and indicate that DHS may act as a postconditioning mimic.

Antioxidant and regulatory role of mitochondrial uncoupling protein UCP2 in pancreatic beta-cells.

Research on brown adipose tissue and its hallmark protein, mitochondrial uncoupling protein UCP1, has been conducted for half a century and has been traditionally studied in the Institute of Physiology (AS CR, Prague), likewise UCP2 residing in multiple tissues for the last two decades. Our group has significantly contributed to the elucidation of UCP uncoupling mechanism, fully dependent on free fatty acids (FFAs) within the inner mitochondrial membrane. Now we review UCP2 physiological roles emphasizing its roles in pancreatic beta-cells, such as antioxidant role, possible tuning of redox homeostasis (consequently UCP2 participation in redox regulations), and fine regulation of glucose-stimulated insulin secretion (GSIS). For example, NADPH has been firmly established as being a modulator of GSIS and since UCP2 may influence redox homeostasis, it likely affects NADPH levels. We also point out the role of phospholipase iPLA2 isoform gamma in providing FFAs for the UCP2 antioxidant function. Such initiation of mild uncoupling hypothetically precedes lipotoxicity in pancreatic beta-cells until it reaches the pathological threshold, after which the antioxidant role of UCP2 can be no more cell-protective, for example due to oxidative stress-accumulated mutations in mtDNA. These mechanisms, together with impaired autocrine insulin function belong to important causes of Type 2 diabetes etiology.

Mitochondrial phospholipase A2 activated by reactive oxygen species in heart mitochondria induces mild uncoupling.

Homeostasis of reactive oxygen species (ROS) in cardiomyocytes is critical for elucidation of normal heart physiology and pathology. Mitochondrial phospholipases A2 (mt-PLA2) have been previously suggested to be activated by ROS. Therefore, we have attempted to elucidate physiological role of such activation. We have found that function of a specific i-isoform of mitochondrial phospholipase A2 (mt-iPLA2) is activated by tert-butylhydroperoxide in isolated rat heart mitochondria. Isoform specificity was judged from the inhibition by bromoenol lactone (BEL), a specific iPLA2 inhibitor. Concomitant uncoupling has been caused by free fatty acids, since it was inhibited by bovine serum albumin. The uncoupling was manifested as a respiration burst accompanied by a slight decrease in mitochondrial inner membrane potential. Since this uncoupling was sensitive to carboxyatractyloside and purine nucleotide di- and tri-phosphates, we conclude that it originated from the onset of fatty acid cycling mediated by the adenine nucleotide translocase (major contribution) and mitochondrial uncoupling protein(s) (minor contribution), respectively. Such a mild uncoupling may provide a feedback downregulation of oxidative stress, since it can further attenuate mitochondrial production of ROS. In conclusion, ROS-induced function of cardiac mt-iPLA2 may stand on a pro-survival side of ischemia-reperfusion injury.

Mitochondrial uncoupling proteins--facts and fantasies.

Instead of a comprehensive review, we describe the basic undisputed facts and a modest contribution of our group to the fascinating area of the research on mitochondrial uncoupling proteins. After defining the terms uncoupling, leak, protein-mediated uncoupling, we discuss the assumption that due to their low abundance the novel mitochondrial uncoupling proteins (UCP2 to UCP5) can provide only a mild uncoupling, i.e. can decrease the proton motive force by several mV only. Contrary to this, the highly thermogenic role of UCP1 in brown adipose tissue is not given only by its high content (approximately 5 % of mitochondrial proteins) but also by the low ATP synthase content and high capacity respiratory chain. Fatty acid cycling mechanism as a plausible explanation for the protonophoretic function of all UCPs and some other mitochondrial carriers is described together with the experiments supporting it. The phylogenesis of all UCPs, estimated UCP2 content in several tissues, and details of UCP2 activation are described on the basis of our experiments. Functional activation of UCP2 is proposed to decrease reactive oxygen species (ROS) production. Moreover, reaction products of lipoperoxidation such as cleaved hydroperoxy-fatty acids and hydroxy-fatty acid can activate UCP2 and promote feedback down-regulation of mitochondrial ROS production.

Mechanism of uncoupling protein action.

Two competing models of uncoupling protein (UCP) transport mechanism agree that fatty acids (FAs) are obligatory for uncoupling, but they disagree about which ion is transported. In Klingenberg's model, UCPs conduct protons. In Garlid's model, UCPs conduct anions, like all members of this gene family. In the latter model, UCP transports the anionic FA head group from one side of the membrane to the other, and the cycle is completed by rapid flip-flop of protonated FAs across the bilayer. The head groups of the FA analogues, long-chain alkylsulphonates, are translocated by UCP, but they cannot induce uncoupling, because these strong acids cannot be protonated for the flip-flop part of the cycle. We have overcome this limitation by ion-pair transport of undecanesulphonate with propranolol, which causes the sulphonate to deliver protons across the membrane as if it were an FA. Full GDP-sensitive uncoupling is seen in the presence of propranolol and undecanesulphonate. This result confirms that the mechanism of UCP uncoupling requires transport of the anionic FA head group by UCP and that the proton transport occurs via the bilayer and not via UCP.

Alkylsulfonates as probes of uncoupling protein transport mechanism. Ion pair transport demonstrates that direct H(+) translocation by UCP1 is not necessary for uncoupling.

The mechanism of fatty acid-dependent uncoupling by mitochondrial uncoupling proteins (UCP) is still in debate. We have hypothesized that the anionic fatty acid head group is translocated by UCP, and the proton is transported electroneutrally in the bilayer by flip-flop of the protonated fatty acid. Alkylsulfonates are useful as probes of the UCP transport mechanism. They are analogues of fatty acids, and they are transported by UCP1, UCP2, and UCP3. We show that undecanesulfonate and laurate are mutually competitive inhibitors, supporting the hypothesis that fatty acid anion is transported by UCP1. Alkylsulfonates cannot be protonated because of their low pK(a), consequently, they cannot catalyze electroneutral proton transport in the bilayer and cannot support uncoupling by UCP. We report for the first time that propranolol forms permeant ion pairs with the alkylsulfonates, thereby removing this restriction. Because a proton is transported with the neutral ion pair, the sulfonate is able to deliver protons across the bilayer, behaving as if it were a fatty acid. When ion pair transport is combined with UCP1, we now observe electrophoretic proton transport and uncoupling of brown adipose tissue mitochondria. These experiments confirm that the proton transport of UCP-mediated uncoupling takes place in the lipid bilayer and not via UCP itself. Thus, UCP1, like other members of its gene family, translocates anions and does not translocate protons.

How do uncoupling proteins uncouple?

According to the proton buffering model, introduced by Klingenberg, UCP1 conducts protons through a hydrophilic pathway lined with fatty acid head groups that buffer the protons as they move across the membrane. According to the fatty acid protonophore model, introduced by Garlid, UCPs do not conduct protons at all. Rather, like all members of this gene family, they are anion carriers. A variety of anions are transported, but the physiological substrates are fatty acid (FA) anions. Because the carboxylate head group is translocated by UCP, and because the protonated FA rapidly diffuses across the membrane, this mechanism permits FA to behave as regulated cycling protonophores. Favoring the latter mechanism is the fact that the head group of long-chain alkylsulfonates, strong acid analogues of FA, is also translocated by UCP.

Transport function and regulation of mitochondrial uncoupling proteins 2 and 3.

Uncoupling protein 1 (UCP1) dissipates energy and generates heat by catalyzing back-flux of protons into the mitochondrial matrix, probably by a fatty acid cycling mechanism. If the newly discovered UCP2 and UCP3 function similarly, they will enhance peripheral energy expenditure and are potential molecular targets for the treatment of obesity. We expressed UCP2 and UCP3 in Escherichia coli and reconstituted the detergent-extracted proteins into liposomes. Ion flux studies show that purified UCP2 and UCP3 behave identically to UCP1. They catalyze electrophoretic flux of protons and alkylsulfonates, and proton flux exhibits an obligatory requirement for fatty acids. Proton flux is inhibited by purine nucleotides but with much lower affinity than observed with UCP1. These findings are consistent with the hypothesis that UCP2 and UCP3 behave as uncoupling proteins in the cell.

The mechanism of proton transport mediated by mitochondrial uncoupling proteins.

The effort to understand the mechanism of uncoupling by UCP has devolved into two models - the fatty acid protonophore model and the proton buffering model. Evidence for each hypothesis is summarized and evaluated. We also evaluate the obligatory requirement for fatty acids in UCP1-mediated uncoupling and the question of fatty acid affinity for UCP1. The structural bases of UCP transport function and nucleotide inhibition are discussed in light of recent mutagenesis studies and in relationship to the sequences of newly discovered UCPs.

State-dependent inhibition of the mitochondrial KATP channel by glyburide and 5-hydroxydecanoate.

The mitochondrial KATP channel (mitoKATP) is hypothesized to be the receptor for the cardioprotective effects of K+ channel openers (KCO) and for the blocking of cardioprotection by glyburide and 5-hydroxydecanoate (5-HD). Studies on glyburide have indicated that this drug is inactive in isolated mitochondria. No studies of the effects of 5-HD on isolated mitochondria have been reported. This paper examines the effects of glyburide and 5-HD on K+ flux in isolated, respiring mitochondria. We show that mitoKATP is completely insensitive to glyburide and 5-HD under the experimental conditions in which the open state of the channel is induced by the absence of ATP and Mg2+. On the other hand, mitoKATP became highly sensitive to glyburide and 5-HD when the open state was induced by Mg2+, ATP, and a physiological opener, such as GTP, or a pharmacological opener, such as diazoxide. In these open states, glyburide (K1/2 values 1-6 microM) and 5-HD (K1/2 values 45-75 microM) inhibited specific, mitoKATP-mediated K+ flux in both heart and liver mitochondria from rat. These results are consistent with a role for mitoKATP in cardioprotection and show that different open states of mitoKATP, although catalyzing identical K+ fluxes, exhibit very different susceptibilities to channel inhibitors.

The nucleotide regulatory sites on the mitochondrial KATP channel face the cytosol.

The mitochondrial KATP channel (mitoKATP) is richly endowed with regulatory sites for metabolites and drugs, but the topological location of these sites is unknown. Thus, it is not known whether ATP, GTP and acyl CoA esters regulate mitoKATP from the matrix or cytosolic side of the inner membrane, nor whether they all act from the same side. The experiments reported in this paper provide an unambiguous answer to these questions. Electrophysiological experiments in bilayer membranes containing purified mitoKATP showed that current is blocked asymmetrically by ATP. K+ flux experiments using proteoliposomes containing purified mitoKATP showed that mitoKATP is unipolar with respect to regulation by Mg2+, ATP, GTP, and palmitoyl CoA and that all of these ligands react on the same pole of the protein. This demonstration was made possible by the new finding that mitoKATP is 85-90% oriented inward or outward in liposomes, depending on the presence or absence of Mg2+ in the reconstitution buffer. K+ flux experiments in respiring rat liver mitochondria showed that mitoKATP was inhibited by palmitoyl CoA and activated by GTP when these agents were added to the external medium. Given that the inner membrane is impermeant to these ligands and that mitoKATP is unipolar with respect to nucleotide regulation, it follows that the regulatory sites on mitoKATP face the cytosol.