Reduced cytochrome C is an essential regulator of sustained insulin secretion by pancreatic islets

Influx of calcium is an essential but insufficient signal in sustained nutrient-stimulated insulin secretion, and increased metabolic rate of the beta cell is also required. The aim of the study was to test the hypothesis that the reduced state of cytochrome c is a metabolic co-factor necessary for insulin secretion, over and above its participation in the ATP-generating function of electron transport/oxidative phosphorylation. We found that nutrient stimulation of insulin secretion by isolated rat islets was strongly correlated with reduced cytochrome c, and agents that acutely and specifically reduced cytochrome c led to increased insulin secretion, even in the face of decreased oxygen consumption and calcium influx. In contrast, neither sites 1 nor 4 of the electron transport chain were both necessary and essential for the stimulation of insulin secretion to occur. Importantly, stimulation of islets with glucose, α-ketoisocaproate, or glyceraldehyde resulted in the appearance of cytochrome c in the cytosol, suggesting a pathway for the regulation of exocytotic machinery by reduction of cytochrome c. The data suggest that the metabolic factor essential for sustained calcium-stimulated insulin secretion to occur is linked to reduction and translocation of cytochrome c.     

Ferrocyanide as electron donor to cytochrome aa3. Cytochrome c requirement for oxygen uptake

1. In the absence of cytochrome c, ferrocyanide or ferrous sulphate reduces cytochrome c oxidase (EC 1.9.3.1), but no continuous oxygen uptake ensues, as it does with N,N,N′,N′-Tetramethyl-p-phenylenediamine or reduced phenazine methosulphate as reductants, unless a substoichiometric amount of cytochrome c or an excess of clupein is present. Cytochrome c cannot be replaced by porphyrin cytochrome c.2. Cytochrome c, porphyrin cytochrome c and clupein all stimulate the reduction of cytochrome aa₃ by ferrocyanide.3. A model is proposed to explain these findings in which a high-affinity site for cytochrome c on the oxidase regulates the access of hydrophilic electron donors to a low-affinity site, and reduction via the high-affinity site is required for continuous oxygen uptake.4. Furthermore, it is shown that upon reaction of oxidase with ferrocyanide, cyano-oxidase is formed.     

Reactions of mercaptans with cytochrome c oxidase and cytochrome c

1. The steady-state oxidation of ferrocytochrome c by dioxygen catalyzed by cytochrome c oxidase, is inhibited non-competitively towards cytochrome c by methanethiol, ethanethiol, 1-propanethiol and 1-butanethiol with K_i values of 4.5, 91, 200 and 330 μM, respectively.2. The inhibition constant K_i of ethanethiol is found to be constant between pH 5 and 8, which suggests that only the neutral form of the thiol inhibits the enzyme.3. The absorption spectrum of oxidized cytochrome c oxidase in the Soret region shows rapid absorbance changes upon addition of ethanethiol to the enzyme. This process is followed by a very slow reduction of the enzyme. The fast reaction, which represents a binding reaction of ethanethiol to cytochrome c oxidase, has a k₁ of 33 M^?1 · s^?1 and dissociation constant K_d of 3.9 mM.4. Ethanethiol induces fast spectral changes in the absorption spectrum of cytochrome c, which are followed by a very slow reduction of the heme. The rate constant for the fast ethanethiol reaction representing a bimolecular binding step is 50 M^?1 · s^?1 and the dissociation constant is about 2 mM. Addition of up to 25 mM ethanethiol to ferrocytochrome c does not cause spectral changes.5. EPR (electron paramagnetic resonance) spectra of cytochrome c oxidase, incubated with methanethiol or ethanethiol in the presence of cytochrome c and ascorbate, show the formation of low-spin cytochrome a₃-mercaptide compounds with g values of 2.39, 2.23, 1.93 and of 2.43, 2.24, 1.91, respectively.     

Impaired Ca2+ Signaling in β-Cells Lacking Leptin Receptors by Cre-loxP Recombination

Obesity is a major risk factor for diabetes and is typically associated with hyperleptinemia and a state of leptin resistance. The impact of chronically elevated leptin levels on the function of insulin-secreting β-cells has not been elucidated. We previously generated mice lacking leptin signaling in β-cells by using the Cre-loxP strategy and showed that these animals develop increased body weight and adiposity, hyperinsulinemia, impaired glucose-stimulated insulin secretion and insulin resistance. Here, we performed several in vitro studies and observed that β-cells lacking leptin signaling in this model are capable of properly metabolizing glucose, but show impaired intracellular Ca²⁺ oscillations and lack of synchrony within the islets in response to glucose, display reduced response to tolbutamide and exhibit morphological abnormalities including increased autophagy. Defects in intracellular Ca²⁺ signaling were observed even in neonatal islets, ruling out the possible contribution of obesity to the β-cell irregularities observed in adults. In parallel, we also detected a disrupted intracellular Ca²⁺ pattern in response to glucose and tolbutamide in control islets from adult transgenic mice expressing Cre recombinase under the rat insulin promoter, despite these animals being glucose tolerant and secreting normal levels of insulin in response to glucose. This unexpected observation impeded us from discerning the consequences of impaired leptin signaling as opposed to long-term Cre expression in the function of insulin-secreting cells. These findings highlight the need to generate improved Cre-driver mouse models or new tools to induce Cre recombination in β-cells.     

Apoptosis of lymphoma cells is abolished due to blockade of cytochrome <Emphasis Type="Italic">c</Emphasis> release despite Nur77 mitochondrial targeting

Nur77 is reported to undergo translocation to mitochondria in response to apoptotic signaling in a variety of cancer cell lines. It was shown that on the mitochondrial membrane, Nur77 interacts with Bcl-2, leading to the conversion of this protein from a protector to a killer with subsequent release of cytochrome c to the cytosol. Here it is shown that in thymic lymphoma cells resistant to calcium-mediated apoptosis, cytochrome c release is abolished despite of Nur77 mitochondrial targeting. However, cytochrome c release and apoptosis can be restored by treatment with FK506. Hence, the molecular target regulation of the sensitivity of lymphoma cells to calcium signaling is associated with cytochrome c release and is FK506 sensitive. These results provide new insight into the role of FK506-sensitive factors as a critical link between calcium signaling and resistance of lymphoma cells to death.     

Stress-induced dissociations between intracellular calcium signaling and insulin secretion in pancreatic islets

In healthy pancreatic islets, glucose-stimulated changes in intracellular calcium ([Ca²⁺]_i) provide a reasonable reflection of the patterns and relative amounts of insulin secretion. We report that [Ca²⁺]_i in islets under stress, however, dissociates with insulin release in different ways for different stressors. Islets were exposed for 48 h to a variety of stressors: cytokines (low-grade inflammation), 28 mM glucose (28G, glucotoxicity), free fatty acids (FFAs, lipotoxicity), thapsigargin (ER stress), or rotenone (mitochondrial stress). We then measured [Ca²⁺]_i and insulin release in parallel studies. Islets exposed to all stressors except rotenone displayed significantly elevated [Ca²⁺]_i in low glucose, however, increased insulin secretion was only observed for 28G due to increased nifedipine-sensitive calcium-channel flux. Following 3–11 mM glucose stimulation, all stressors substantially reduced the peak glucose-stimulated [Ca²⁺]_i response (first phase). Thapsigargin and cytokines also substantially impacted aspects of calcium influx and ER calcium handling. Stressors did not significantly impact insulin secretion in 11 mM glucose for any stressor, although FFAs showed a borderline reduction, which contributed to a significant decrease in the stimulation index (11:3 mM glucose) observed for FFAs and also for 28G. We also clamped [Ca²⁺]_i using 30 mM KCl + 250 μM diazoxide to test the amplifying pathway. Only rotenone-treated islets showed a robust increase in 3–11 mM glucose-stimulated insulin secretion under clamped conditions, suggesting that low-level mitochondrial stress might activate the metabolic amplifying pathway. We conclude that different stressors dissociate [Ca²⁺]_i from insulin secretion differently: ER stressors (thapsigargin, cytokines) primarily affect [Ca²⁺]_i but not conventional insulin secretion and ‘metabolic’ stressors (FFAs, 28G, rotenone) impacted insulin secretion.     

Structural Re-arrangement and Peroxidase Activation of Cytochrome c by Anionic Analogues of Vitamin E,Tocopherol Succinate and Tocopherol Phosphate

Cytochrome c is a multifunctional hemoprotein in the mitochondrial intermembrane space whereby its participation in electron shuttling between respiratory complexes III and IV is alternative to its role in apoptosis as a peroxidase activated by interaction with cardiolipin (CL), and resulting in selective CL peroxidation. The switch from electron transfer to peroxidase function requires partial unfolding of the protein upon binding of CL, whose specific features combine negative charges of the two phosphate groups with four hydrophobic fatty acid residues. Assuming that other endogenous small molecule ligands with a hydrophobic chain and a negatively charged functionality may activate cytochrome c into a peroxidase, we investigated two hydrophobic anionic analogues of vitamin E, α-tocopherol succinate (α-TOS) and α-tocopherol phosphate (α-TOP), as potential inducers of peroxidase activity of cytochrome c. NMR studies and computational modeling indicate that they interact with cytochrome c at similar sites previously proposed for CL. Absorption spectroscopy showed that both analogues effectively disrupt the Fe-S(Met⁸⁰) bond associated with unfolding of cytochrome c. We found that α-TOS and α-TOP stimulate peroxidase activity of cytochrome c. Enhanced peroxidase activity was also observed in isolated rat liver mitochondria incubated with α-TOS and tBOOH. A mitochondria-targeted derivative of TOS, triphenylphosphonium-TOS (mito-VES), was more efficient in inducing H₂O₂-dependent apoptosis in mouse embryonic cytochrome c^+/+ cells than in cytochrome c^−/− cells. Essential for execution of the apoptotic program peroxidase activation of cytochrome c by α-TOS may contribute to its known anti-cancer pharmacological activity.     

Determination of the equilibrium constant for the binding of ferricytochrome c to phospholipid vesicles and the effect of binding on the reduction rate of cytochrome c

The rate of reduction of cytochrome c by 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropteridine was examined as a function of binding to liposomes prepared from mixed soybean phospholipids, asolectin, and from various purified phospholipids. Binding of cytochrome c to asolectin liposomes caused an increase in the rate of reduction by the pteridine derivative from 2900 to 16 000 M^?1 · s^?1 at pH 7. At low ionic strength (0.003 M) the binding stoichiometry between cytochrome c and asolectin vesicles is 15 ± 2 phosphospolipid/cytochrome c (mole ratio), determined by monitoring the change in reduction rate of cytochrome c by pteridine as cytochrome c is bound to the vesicles. A stoichiometry of 14 phospholipid/cytochrome c was obtained from gel filtration studies. Equilibrium association constants for the binding of cytochrome c to sites on the asolectin vesicles varied from 2.2 · 10⁶ to 1.8 · 10³ M^?1 between 0.02 and 0.10 M ionic strength, respectively. In general, liposomes prepared from purified phospholipids resulted in less binding of cytochrome c per mole of phospholipid and lower reduction rates than those prepared from asolectin.     

Delineation of glutamate pathways and secretory responses in pancreatic islets with β-cell–specific abrogation of the glutamate dehydrogenase

In pancreatic β-cells, glutamate dehydrogenase (GDH) modulates insulin secretion, although its function regarding specific secretagogues is unclear. This study investigated the role of GDH using a β-cell–specific GDH knockout mouse model, called βGlud1^−/−. The absence of GDH in islets isolated from βGlud1^–/– mice resulted in abrogation of insulin release evoked by glutamine combined with 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid or l-leucine. Reintroduction of GDH in βGlud1^–/– islets fully restored the secretory response. Regarding glucose stimulation, insulin secretion in islets isolated from βGlud1^–/– mice exhibited half of the response measured in control islets. The amplifying pathway, tested at stimulatory glucose concentrations in the presence of KCl and diazoxide, was markedly inhibited in βGlud1^–/– islets. On glucose stimulation, net synthesis of glutamate from α-ketoglutarate was impaired in GDH-deficient islets. Accordingly, glucose-induced elevation of glutamate levels observed in control islets was absent in βGlud1^–/– islets. Parallel biochemical pathways, namely alanine and aspartate aminotransferases, could not compensate for the lack of GDH. However, the secretory response to glucose was fully restored by the provision of cellular glutamate when βGlud1^–/– islets were exposed to dimethyl glutamate. This shows that permissive levels of glutamate are required for the full development of glucose-stimulated insulin secretion and that GDH plays an indispensable role in this process.     

Low-pH-induced apoptosis: role of endoplasmic reticulum stress-induced calcium permeability and mitochondria-dependent signaling

The acidic microenvironment around tumor cells is a major determinant in cancer growth, metabolism, and metastasis. However, its role in cancer physiology is still not clearly understood. In the present investigation, an attempt has been made to explore the effect of acidic environment on physiology of cancer cells. Exposure of Raji cells to extracellular acidic environment was associated with enhanced cytosolic calcium level and endoplasmic reticulum stress response. X-box binding protein 1 (XBP1) splicing, CCAAT/enhancer-binding protein homologous protein (CHOP), and glucose-regulated protein 78 kDa (GRP78) upregulation suggested endoplasmic reticulum stress generation. On the other hand, real-time-based upregulation of Bax gene expression and flow cytometric analysis of cytochrome c release as well as enhanced active caspase-3 further confirmed mitochondrion-mediated events leading to induction of apoptosis. The expression of TP53 and p21 was upregulated. These observations collectively strongly suggest that both endoplasmic reticulum stress-mediated calcium release and Bax targeting might be altering mitochondrion membrane potential which in turn could induce secondary apoptotic signals; subsequently, endoplasmic reticulum stress can also lead to nuclear localization of Nuclear factor-κB (NF-κB) which in turn favors p53 mediated apoptotic signals.

Electronic supplementary material

The online version of this article (doi:10.1007/s12192-014-0568-6) contains supplementary material, which is available to authorized users.     

The reaction of cytochrome aa3 with (porphyrin) cytochrome c as studied by pulse radiolysis

(1) Using the pulse-radiolysis and stopped-flow techniques, the reactions of iron-free (porphyrin) cytochrome c and native cytochrome c with cytochrome aa₃ were investigated. The porphyrin cytochrome c anion radical (generated by reduction of porphyrin cytochrome c by the hydrated electron) can transfer its electron to cytochrome aa₃. The bimolecular rate constant for this reaction is 2·10⁷ M^?1·s^?1 (5 mM potassium phosphate, 0.5% Tween 20, pH 7.0, 20°C). (2) The ionic strength dependence of the cytochrome c-cytochromeaa₃ interaction was measured in the ionic strength range between 40 and 120 mM. At ionic strengths below 30 mM, a cytochrome c-cytochrome aa₃ complex is formed in which cytochrome c is no longer reducible by the hydrated electron. A method is described by which the contributions of electrostatic forces to the reaction rate can be determined. (3) Using the stopped-flow technique, the effect of the dielectric constant (?) of the reaction medium on the reaction of cytochrome c with cytochrome aa₃ was investigated. With increasing ? the second-order rate constant decreased.     

A 1H-NMR longitudinal relaxation study of the interaction between cytochrome c and cytochrome c oxidase

The interaction between the oxidized forms of cytochrome c and cytochrome c oxidase (EC 1.9.3.1) has been investigated by ¹H-NMR longitudinal relaxation measurements. It is found that relaxation of methyl groups on the heme ring of cytochrome c markedly deviates from a simple exponential behavior in the presence of small amounts of cytochrome oxidase. A comparison with the relaxation behavior of cytochrome c modified by 4-carboxy-3,5-dinitrophenyl at Lys-13 shows that the oxidase induces a conformation in native cytochrome c that is closely related to that of the derivative. It is suggested that this change in conformation consists of a rupture of the salt bridge between Lys-13 and Glu-90 and a concomitant perturbation of the methionine ligand.     

Characterisation of ionisations that influence the redox potential of mitochondrial cytochrome c and photosynthetic bacterial cytochromes c2

Several cytochromes c₂ from the Rhodospirillaceae show a pH dependence of redox potential in the physiological pH range which can be described by equations involving an ionisation in the oxidised form (pK_o) and one in the reduced form (pK_r). These cytochromes fall into one of two groups according to the degree of separation of pK_o and pK_r. In group A, represented here by the Rhodomicrobium vannielii cytochrome c₂, the separation is approx. one pH unit and the ionisation is that of a haem propionic acid. Members of this group are unique among both cytochromes c₂ and mitochondrial cytochromes c in lacking the conserved residue Arg-38. We propose that the role of Arg-38 is to lower the pK of the nearby propionic acid, so that it lies out of the physiological pH range. Substitution of this residue by an uncharged amino acid leads to a raised pK for the propionic acid. In group B, represented here by Rhodopseudomonas viridis cytochrome c₂, the separation between pK_o and pK_r is approx. 0.4 pH unit and the ionisable group is a histidine at position 39. This was established by NMR spectroscopy and confirmed by chemical modification. Only a few other members of the cytochrome c₂/mitochondrial cytochrome c family have a histidine at this position and of these, both Crithidia cytochrome c-557 and yeast cytochrome c were found to have a pH-dependent redox potential similar to that of Rps. viridis cytochrome c₂. Using Coulomb's law, it was found that the energy required to separate pK_o and pK_r could be accounted for by simple electrostatic interactions between the haem iron and the ionisable group.     

Inhibition of Pancreatic β-Cell Ca2+/Calmodulin-dependent Protein Kinase II Reduces Glucose-stimulated Calcium Influx and Insulin Secretion,Impairing Glucose Tolerance

Glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells is caused by Ca²⁺ entry via voltage-dependent Ca²⁺ channels. CaMKII is a key mediator and feedback regulator of Ca²⁺ signaling in many tissues, but its role in β-cells is poorly understood, especially in vivo. Here, we report that mice with conditional inhibition of CaMKII in β-cells show significantly impaired glucose tolerance due to decreased GSIS. Moreover, β-cell CaMKII inhibition dramatically exacerbates glucose intolerance following exposure to a high fat diet. The impairment of islet GSIS by β-cell CaMKII inhibition is not accompanied by changes in either glucose metabolism or the activities of K_ATP and voltage-gated potassium channels. However, glucose-stimulated Ca²⁺ entry via voltage-dependent Ca²⁺ channels is reduced in islet β-cells with CaMKII inhibition, as well as in primary wild-type β-cells treated with a peptide inhibitor of CaMKII. The levels of basal β-cell cytoplasmic Ca²⁺ and of endoplasmic reticulum Ca²⁺ stores are also decreased by CaMKII inhibition. In addition, CaMKII inhibition suppresses glucose-stimulated action potential firing frequency. These results reveal that CaMKII is a Ca²⁺ sensor with a key role as a feed-forward stimulator of β-cell Ca²⁺ signals that enhance GSIS under physiological and pathological conditions.     

Testing Pancreatic Islet Function at the Single Cell Level by Calcium Influx with Associated Marker Expression

Studying the response of islet cells to glucose stimulation is important for understanding cell function in healthy and disease states. Most functional assays are performed on whole islets or cell populations, resulting in averaged observations and loss of information at the single cell level. We demonstrate methods to examine calcium fluxing in individual cells of intact islets in response to multiple glucose challenges. Wild-type mouse islets predominantly contained cells that responded to three (out of three) sequential high glucose challenges, whereas cells of diabetic islets (db/db or NOD) responded less frequently or not at all. Imaged islets were also immunostained for endocrine markers to associate the calcium flux profile of individual cells with gene expression. Wild-type mouse islet cells that robustly fluxed calcium expressed β cell markers (INS/NKX6.1), whereas islet cells that inversely fluxed at low glucose expressed α cell markers (GCG). Diabetic mouse islets showed a higher proportion of dysfunctional β cells that responded poorly to glucose challenges. Most of the failed calcium influx responses in β cells were observed in the second and third high glucose challenges, emphasizing the importance of multiple sequential glucose challenges for assessing the full function of islet cells. Human islet cells were also assessed and showed functional α and β cells. This approach to analyze islet responses to multiple glucose challenges in correlation with gene expression assays expands the understanding of β cell function and the diseased state.     

Calcium and pancreatic β-cell function: 4. Evidence that glucose and phosphate stimulate calcium-45 incorporation into different intracellular pools

β-Cell-rich pancreatic islets were microdissected from ob/ob-mice and used for studies of ⁴⁵Ca uptake and washout. Irrespective of whether the experiments were performed at 21 or 37°C both glucose and phosphate stimulated the net uptake of lanthanum-nondisplaceable ⁴⁵Ca. The stimulatory effect of phosphate was additive to that produced by glucose. ⁴⁵Ca incorporated in response to phosphate differed from that taken up in the presence of 20 mM glucose in being easily washed out although it was not affected by the glucose concentration of the washing medium. The efflux of ⁴⁵Ca was reduced after introducing phosphate into a medium used to perifuse islets which had accumulated ⁴⁵Ca in response to 20 mM glucose. This suggests that the outward calcium transport can be influenced also by intracellular trapping of the cation. The glucose-stimulated insulin release was inhibited by phosphate; an effect reversed by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. It is concluded that a common effect of glucose and phosphate is to trap calcium in the pancreatic β-cells but that there are fundamental differences between their effects on intracellular distribution of calcium and on insulin release.     

Electron transfer between hydrogenases and mono- and multiheme cytochromes in Desulfovibrio ssp

 A comparative study of electron transfer between the 16 heme high molecular mass cytochrome (Hmc) from Desulfovibrio vulgaris Hildenborough and the [Fe] and [NiFe] hydrogenases from the same organism was carried out, both in the presence and in the absence of catalytic amounts of cytochrome c ₃. For comparison, this study was repeated with the [NiFe] hydrogenase from D. gigas. Hmc is very slowly reduced by the [Fe] hydrogenase, but faster by either of the two [NiFe] hydrogenases. In the presence of cytochrome c ₃, in equimolar amounts to the hydrogenases, the rates of electron transfer are significantly increased and are similar for the three hydrogenases. The results obtained indicate that the reduction of Hmc by the [Fe] or [NiFe] hydrogenases is most likely mediated by cytochrome c ₃. A similar study with D. vulgaris Hildenborough cytochrome c ₅₅₃ shows that, in contrast, this cytochrome is reduced faster by the [Fe] hydrogenase than by the [NiFe] hydrogenases. However, although catalytic amounts of cytochrome c ₃ have no effect in the reduction by the [Fe] hydrogenase, it significantly increases the rate of reduction by the [NiFe] hydrogenases. Received: 14 April 1998 / Accepted: 25 June 1998     

A Syntenic Cross Species Aneuploidy Genetic Screen Links RCAN1 Expression to β-Cell Mitochondrial Dysfunction in Type 2 Diabetes

Type 2 diabetes (T2D) is a complex metabolic disease associated with obesity, insulin resistance and hypoinsulinemia due to pancreatic β-cell dysfunction. Reduced mitochondrial function is thought to be central to β-cell dysfunction. Mitochondrial dysfunction and reduced insulin secretion are also observed in β-cells of humans with the most common human genetic disorder, Down syndrome (DS, Trisomy 21). To identify regions of chromosome 21 that may be associated with perturbed glucose homeostasis we profiled the glycaemic status of different DS mouse models. The Ts65Dn and Dp16 DS mouse lines were hyperglycemic, while Tc1 and Ts1Rhr mice were not, providing us with a region of chromosome 21 containing genes that cause hyperglycemia. We then examined whether any of these genes were upregulated in a set of ~5,000 gene expression changes we had identified in a large gene expression analysis of human T2D β-cells. This approach produced a single gene, RCAN1, as a candidate gene linking hyperglycemia and functional changes in T2D β-cells. Further investigations demonstrated that RCAN1 methylation is reduced in human T2D islets at multiple sites, correlating with increased expression. RCAN1 protein expression was also increased in db/db mouse islets and in human and mouse islets exposed to high glucose. Mice overexpressing RCAN1 had reduced in vivo glucose-stimulated insulin secretion and their β-cells displayed mitochondrial dysfunction including hyperpolarised membrane potential, reduced oxidative phosphorylation and low ATP production. This lack of β-cell ATP had functional consequences by negatively affecting both glucose-stimulated membrane depolarisation and ATP-dependent insulin granule exocytosis. Thus, from amongst the myriad of gene expression changes occurring in T2D β-cells where we had little knowledge of which changes cause β-cell dysfunction, we applied a trisomy 21 screening approach which linked RCAN1 to β-cell mitochondrial dysfunction in T2D.     

Activation of protein kinase C delta following cerebral ischemia leads to release of cytochrome C from the mitochondria via bad pathway

Background

The release of cytochrome c from the mitochondria following cerebral ischemia is a key event leading to cell death. The goal of the present study was to determine the mechanisms involved in post-ischemic activation of protein kinase c delta (δPKC) that lead to cytochrome c release.

Methods/Findings

We used a rat model of cardiac arrest as an in vivo model, and an in vitro analog, oxygen glucose deprivation (OGD) in rat hippocampal synaptosomes. Cardiac arrest triggered translocation of δPKC to the mitochondrial fraction at 1 h reperfusion. In synaptosomes, the peptide inhibitor of δPKC blocked OGD-induced translocation to the mitochondria. We tested two potential pathways by which δPKC activation could lead to cytochrome c release: phosphorylation of phospholipid scramblase-3 (PLSCR3) and/or protein phosphatase 2A (PP2A). Cardiac arrest increased levels of phosphorlyated PLSCR3; however, inhibition of δPKC translocation failed to affect the OGD-induced increase in PLSCR3 in synaptosomal mitochondria suggesting the post-ischemic phosphorylation of PLSCR3 is not mediated by δPKC. Inhibition of either δPKC or PP2A decreased cytochrome c release from synaptosomal mitochondria. Cardiac arrest results in the dephosphorylation of Bad and Bax, both downstream targets of PP2A promoting apoptosis. Inhibition of δPKC or PP2A prevented OGD-induced Bad, but not Bax, dephosphorylation. To complement these studies, we used proteomics to identify novel mitochondrial substrates of δPKC.

Conclusions

We conclude that δPKC initiates cytochrome c release via phosphorylation of PP2A and subsequent dephosphorylation of Bad and identified δPKC, PP2A and additional mitochondrial proteins as potential therapeutic targets for ischemic neuroprotection.