Beata Torocsik

Anisomycin uses multiple mechanisms to stimulate mitogen-activated protein kinases and gene expression and to inhibit neuronal differentiation in PC12 phaeochromocytoma cells

Treatment of PC12 cells with nerve growth factor (NGF) stimulates extracellular signal‐regulated kinases (ERKs), as well as stress‐activated c‐Jun N‐terminal kinases (JNKs) and p38 kinase, and induces neuronal differentiation. While the pivotal role of ERKs in NGF‐induced morphological differentiation is well established, the contribution of JNK‐ and p38‐pathways is less clear. The role of the JNK‐ and p38‐pathway in PC12 cells was analysed by using anisomycin, a protein synthesis inhibitor that activates JNKs and p38. Non‐toxic concentrations of anisomycin were found to stimulate these enzyme activities as well as the expression of the early response genes c‐jun, c‐fos and zif268, and to inhibit NGF‐induced neurite formation. These effects of anisomycin appear to be mediated by the generation of reactive oxygen species (ROS), which in turn act through both TrkA/Ras‐dependent and ‐independent signalling pathways. In addition, cross‐talk between the p38‐ and ERK‐pathways appears to play a role in the action of anisomycin.

Forward operation of adenine nucleotide translocase during F0F1-ATPase reversal: critical role of matrix substrate-level phosphorylation

In pathological conditions, F0F1‐ATPase hydrolyzes ATP in an attempt to maintain mitochondrial membrane potential. Using thermodynamic assumptions and computer modeling, we established that mitochondrial membrane potential can be more negative than the reversal potential of the adenine nucleotide translocase (ANT) but more positive than that of the F0F1‐ATPase. Experiments on isolated mitochondria demonstrated that, when the electron transport chain is compromised, the F0F1‐ATPase reverses, and the membrane potential is maintained as long as matrix substrate‐level phosphorylation is functional, without a concomitant reversal of the ANT. Consistently, no cytosolic ATP consumption was observed using plasmalemmal KATP channels as cytosolic ATP biosensors in cultured neurons, in which their in situ mitochondria were compromised by respiratory chain inhibitors. This finding was further corroborated by quantitative measurements of mitochondrial membrane potential, oxygen consumption, and extracellular acidification rates, indicating nonreversal of ANT of compromised in situ neuronal and astrocytic mitochondria; and by bioluminescence ATP measurements in COS‐7 cells transfected with cytosolicor nuclear‐targeted luciferases and treated with mitochondrial respiratory chain inhibitors in the presence of glycolytic plus mitochondrial vs. only mitochondrial substrates. Our findings imply the possibility of a rescue mechanism that is protecting against cytosolic/nuclear ATP depletion under pathological conditions involving impaired respiration. This mechanism comes into play when mitochondria respire on substrates that support matrix substrate‐level phosphorylation.—Chinopoulos, C, Gerencser, A A., Mandi, M., Mathe, K., Töröcsik, B., Doczi, J., Turiak, L., Kiss, G., Konràd, C, Vajda, S., Vereczki, V., Oh, R. J., Adam‐Vizi, V. Forward operation of adenine nucleotide translocase during F0F1‐ATPase reversal: critical role of matrix substrate‐level phosphorylation. FASEB J. 24, 2405–2416 (2010). www.fasebj.org

Impaired Regulation of pH Homeostasis by Oxidative Stress in Rat Brain Capillary Endothelial Cells

Summary1. Endothelial cells are permanently challenged by altering pH in the blood, and oxidative damage could also influence the intracellular pH (pHi) of the endothelium. Cerebral microvascular endothelial cells form the blood–brain barrier (BBB) and pHi regulation of brain capillary endothelial cells is important for the maintenance of BBB integrity. The aim of this study was to address the pH regulatory mechanisms and the effect of an acute exposure to hydrogen peroxide (H2O2) on the pH regulation in primary rat brain capillary endothelial (RBCE) cells. The RBCE monolayers were loaded with the fluorescent pH indicator BCECF and pHi was monitored by detecting the fluorescent changes.2. The steady-state pHi of RBCE cells in HEPES-buffer (6.83 ± 0.1) did not differ significantly from that found in bicarbonate-buffered medium (6.90 ± 0.08). Cells were exposed to NH4Cl to induce intracellular acidification and then the recovery to resting pH was studied. Half-recovery time after NH4Cl prepulse-induced acid load was significantly less in the bicarbonate-buffered medium than in the HEPES-medium, suggesting that in addition to the Na+/H+ exchanger, HCO3−/Cl− exchange mechanism is also involved in the restoration of pHi after an intracellular acid load in primary RBCE cells. We used RT-PCR-reactions to detect the isoforms of Na+/H+ exchanger gene family (NHE). NHE-1 -2, -3 and -4 were equally present, and there was no significant difference in the relative abundance of the four transcripts in these cells.3. No pHi recovery was detected when the washout after an intracellular acid load occurred in nominally Na+-free HEPES-buffered medium or in the presence of 10 μM 5-(N-ethyl-N-isopropyl)amiloride (EIPA), a specific inhibitor of Na+/H+ exchanger. The new steady-state pHi were 6.37 ± 0.02 and 6.60 ± 0.02, respectively.4. No detectable change was observed in the steady-state pHi in the presence of 100 μM H2O2; however, recovery from NH4Cl prepulse-induced intracellular acid load was inhibited when H2O2 was present in 50 or 100 μM concentration in the HEPES-buffered medium during NH4Cl washout. These data suggest that H2O2 is without effect on the activity of Na+/H+ exchanger at rest, but could inhibit the function of the exchanger after an intracellular acid load.

Modulation of F0F1-ATP synthase activity by cyclophilin D regulates matrix adenine nucleotide levels

Cyclophilin D was recently shown to bind to and decrease the activity of F0F1‐ATP synthase in submitochondrial particles and permeabilized mitochondria [Giorgio V et al. (2009) J Biol Chem, 284, 33982–33988]. Cyclophilin D binding decreased both ATP synthesis and hydrolysis rates. In the present study, we reaffirm these findings by demonstrating that, in intact mouse liver mitochondria energized by ATP, the absence of cyclophilin D or the presence of cyclosporin A led to a decrease in the extent of uncoupler‐induced depolarization. Accordingly, in substrate‐energized mitochondria, an increase in F0F1‐ATP synthase activity mediated by a relief of inhibition by cyclophilin D was evident in the form of slightly increased respiration rates during arsenolysis. However, the modulation of F0F1‐ATP synthase by cyclophilin D did not increase the adenine nucleotide translocase (ANT)‐mediated ATP efflux rate in energized mitochondria or the ATP influx rate in de‐energized mitochondria. The lack of an effect of cyclophilin D on the ANT‐mediated adenine nucleotide exchange rate was attributed to the ∼ 2.2‐fold lower flux control coefficient of the F0F1‐ATP synthase than that of ANT, as deduced from measurements of adenine nucleotide flux rates in intact mitochondria. These findings were further supported by a recent kinetic model of the mitochondrial phosphorylation system, suggesting that an ∼ 30% change in F0F1‐ATP synthase activity in fully energized or fully de‐energized mitochondria affects the ADP–ATP exchange rate mediated by the ANT in the range 1.38–1.7%. We conclude that, in mitochondria exhibiting intact inner membranes, the absence of cyclophilin D or the inhibition of its binding to F0F1‐ATP synthase by cyclosporin A will affect only matrix adenine nucleotides levels.

Stimulation of reactive oxygen species generation by disease-causing mutations of lipoamide dehydrogenase

We investigated pathogenic mutations relevant in dihydrolipoamide dehydrogenase (LADH; gene: Dld) deficiency, a severe human disease, to elucidate how they alter reactive oxygen species (ROS) generation and associated biophysical characteristics of LADH. Twelve known disease-causing mutants of human LADH have been expressed and purified to homogeneity from E. coli. Detailed biophysical and biochemical characterization of the mutants has been performed applying circular dichroism (CD) spectroscopy, nano-spray mass spectrometry (MS), calibrated gel filtration and flavin adenine dinucleotide-content analysis. Functional analyses revealed that four of the pathogenic mutations significantly stimulated the ROS-generating activity of LADH and also increased its sensitivity to an acidic shift in pH. LADH activity was reduced by variable extents in the mutants exhibiting excessive ROS generation. It is remarkable that in the P453L mutant, enzyme activity was nearly completely lost with a ROS-forming activity becoming dominant, whereas the G194C mutation, common among Ashkenazi Jews, resulted in no alteration in LADH activity but a gain in the ROS-generating activity. There have been neither major conformational alterations nor monomerization of the functional homodimer of LADH associated with the higher ROS-generating capacity as measured by CD spectroscopy and size-exclusion chromatography combined with nano-spray MS, respectively. The excessive ROS generation of selected LADH mutants could be an important factor in the pathology and clinical presentation of human LADH deficiency and raises the possibility of an antioxidant therapy in the treatment of this condition.

Newcastle disease virus-induced apoptosis in PC12 pheochromocytoma cells.

The avian paramyxovirus Newcastle disease virus (NDV) causes severe infections in birds. It is essentially nonpathogenic in rodents and human beings but was found to have an oncolytic potential against certain types of human malignancies. An attenuated NDV vaccine (designated MTH-68/H) was found to cause regression of various human tumors, but the mechanism of its oncolytic action and its selectivity toward malignant cells remain poorly understood. NDV was reported to cause apoptotic death in several avian cultured cell types. Programmed cell death may thus be the basis for the oncolytic effect of NDV vaccines. To test this possibility, we chose the PC12 rat pheochromocytoma cell line, a widely used model system for apoptosis. The MTH-68/H vaccine was found to cause apoptotic death of PC12 cells in a dose-dependent manner. A brief exposure of cells to the virus was found to trigger the apoptotic response. Cell death induced by the vaccine was not accompanied by significant alterations in the major mitogen-activated protein kinase pathways of these cells. Apoptotic DNA fragmentation was not affected by stimulating growth factor pathways or signaling mechanisms mediated by protein kinase C or the second messenger, calcium. In contrast, stimulation of protein kinase A by cyclic adenosine monophosphate analogs gave partial protection against the virus. PC12 cells thus provide a useful model system to study the effects of NDV on cell survival at the molecular level.

Complex contribution of cyclophilin D to Ca2+-induced permeability transition in brain mitochondria, with relation to the bioenergetic state

Cyclophilin d (cypD)-deficient mice exhibit resistance to focal cerebral ischemia and to necrotic but not apoptotic stimuli. To address this disparity, we investigated isolated brain and in situ neuronal and astrocytic mitochondria from cypD-deficient and wild-type mice. Isolated mitochondria were challenged by high Ca2+, and the effects of substrates and respiratory chain inhibitors were evaluated on permeability transition pore opening by light scatter. In situ neuronal and astrocytic mitochondria were visualized by mito-DsRed2 targeting and challenged by calcimycin, and the effects of glucose, NaCN, and an uncoupler were evaluated by measuring mitochondrial volume. In isolated mitochondria, Ca2+ caused a large cypD-dependent change in light scatter in the absence of substrates that was insensitive to Ruthenium red or Ru360. Uniporter inhibitors only partially affected the entry of free Ca2+ in the matrix. Inhibition of complex III/IV negated the effect of substrates, but inhibition of complex I was protective. Mitochondria within neurons and astrocytes exhibited cypD-independent swelling that was dramatically hastened when NaCN and 2-deoxyglucose were present in a glucose-free medium during calcimycin treatment. In the presence of an uncoupler, cypD-deficient astrocytic mitochondria performed better than wild-type mitochondria, whereas the opposite was observed in neurons. Neuronal mitochondria were examined further during glutamate-induced delayed Ca2+ deregulation. CypD-knock-out mitochondria exhibited an absence or a delay in the onset of mitochondrial swelling after glutamate application. Apparently, some conditions involving deenergization render cypD an important modulator of PTP in the brain. These findings could explain why absence of cypD protects against necrotic (deenergized mitochondria), but not apoptotic (energized mitochondria) stimuli.

Conformational changes in the catalytically inactive nucleotide-binding site of CFTR

A central step in the gating of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is the association of its two cytosolic nucleotide-binding domains (NBDs) into a head-to-tail dimer, with two nucleotides bound at the interface. Channel opening and closing, respectively, are coupled to formation and disruption of this tight NBD dimer. CFTR is an asymmetric adenosine triphosphate (ATP)-binding cassette protein in which the two interfacial-binding sites (composite sites 1 and 2) are functionally different. During gating, the canonical, catalytically active nucleotide-binding site (site 2) cycles between dimerized prehydrolytic (state O1), dimerized post-hydrolytic (state O2), and dissociated (state C) forms in a preferential C→O1→O2→C sequence. In contrast, the catalytically inactive nucleotide-binding site (site 1) is believed to remain associated, ATP-bound, for several gating cycles. Here, we have examined the possibility of conformational changes in site 1 during gating, by studying gating effects of perturbations in site 1. Previous work showed that channel closure is slowed, both under hydrolytic and nonhydrolytic conditions, by occupancy of site 1 by N6-(2-phenylethyl)-ATP (P-ATP) as well as by the site-1 mutation H1348A (NBD2 signature sequence). Here, we found that P-ATP prolongs wild-type (WT) CFTR burst durations by selectively slowing (>2×) transition O1→O2 and decreases the nonhydrolytic closing rate (transition O1→C) of CFTR mutants K1250A (∼4×) and E1371S (∼3×). Mutation H1348A also slowed (∼3×) the O1→O2 transition in the WT background and decreased the nonhydrolytic closing rate of both K1250A (∼3×) and E1371S (∼3×) background mutants. Neither P-ATP nor the H1348A mutation affected the 1:1 stoichiometry between ATP occlusion and channel burst events characteristic to WT CFTR gating in ATP. The marked effect that different structural perturbations at site 1 have on both steps O1→C and O1→O2 suggests that the overall conformational changes that CFTR undergoes upon opening and coincident with hydrolysis at the active site 2 include significant structural rearrangement at site 1.

Catalyst-like modulation of transition states for CFTR channel opening and closing: New stimulation strategy exploits nonequilibrium gating

Cystic fibrosis transmembrane conductance regulator (CFTR) is the chloride ion channel mutated in cystic fibrosis (CF) patients. It is an ATP-binding cassette protein, and its resulting cyclic nonequilibrium gating mechanism sets it apart from most other ion channels. The most common CF mutation (ΔF508) impairs folding of CFTR but also channel gating, reducing open probability (Po). This gating defect must be addressed to effectively treat CF. Combining single-channel and macroscopic current measurements in inside-out patches, we show here that the two effects of 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB) on CFTR, pore block and gating stimulation, are independent, suggesting action at distinct sites. Furthermore, detailed kinetic analysis revealed that NPPB potently increases Po, also of ΔF508 CFTR, by affecting the stability of gating transition states. This finding is unexpected, because for most ion channels, which gate at equilibrium, altering transition-state stabilities has no effect on Po; rather, agonists usually stimulate by stabilizing open states. Our results highlight how for CFTR, because of its unique cyclic mechanism, gating transition states determine Po and offer strategic targets for potentiator compounds to achieve maximal efficacy.

Alterations in voltage-sensing of the mitochondrial permeability transition pore in ANT1-deficient cells

The probability of mitochondrial permeability transition (mPT) pore opening is inversely related to the magnitude of the proton electrochemical gradient. The module conferring sensitivity of the pore to this gradient has not been identified. We investigated mPT’s voltage-sensing properties elicited by calcimycin or H2O2 in human fibroblasts exhibiting partial or complete lack of ANT1 and in C2C12 myotubes with knocked-down ANT1 expression. mPT onset was assessed by measuring in situ mitochondrial volume using the ‘thinness ratio’ and the ‘cobalt-calcein’ technique. De-energization hastened calcimycin-induced swelling in control and partially-expressing ANT1 fibroblasts, but not in cells lacking ANT1, despite greater losses of mitochondrial membrane potential. Matrix Ca2+ levels measured by X-rhod-1 or mitochondrially-targeted ratiometric biosensor 4mtD3cpv, or ADP-ATP exchange rates did not differ among cell types. ANT1-null fibroblasts were also resistant to H2O2-induced mitochondrial swelling. Permeabilized C2C12 myotubes with knocked-down ANT1 exhibited higher calcium uptake capacity and voltage-thresholds of mPT opening inferred from cytochrome c release, but intact cells showed no differences in calcimycin-induced onset of mPT, irrespective of energization and ANT1 expression, albeit the number of cells undergoing mPT increased less significantly upon chemically-induced hypoxia than control cells. We conclude that ANT1 confers sensitivity of the pore to the electrochemical gradient.