Biomedical subjects

Michela Capano

Publications and source records attributed to Michela Capano.

6 recordsLinked to original sources

PubMed2006

Bax translocates to mitochondria of heart cells during simulated ischaemia: involvement of AMP-activated and p38 mitogen-activated protein kinases.

The cytosolic protein Bax plays a key role in apoptosis by migrating to mitochondria and releasing proapoptotic proteins from the mitochondrial intermembrane space. The present study investigates the movement of Bax in isolated rat neonatal cardiomyocytes subjected to simulated ischaemia (minus glucose, plus cyanide), using green fluorescent protein-tagged Bax as a means of imaging Bax movements. Simulated ischaemia induced Bax translocation from the cytosol to mitochondria, commencing within 20 min of simulated ischaemia and progressing for several hours. Under the same conditions, there was an increase in the active, phosphorylated forms of p38 MAPK (mitogen-activated protein kinase) and AMPK (AMP-activated protein kinase). The AMPK activators AICAR (5-aminoimidazole-4-carboxamide ribonucleoside) and metformin also stimulated Bax translocation. Inhibition of p38 MAPK with SB203580 attenuated the phosphorylation of the downstream substrates, MAPK-activated protein kinases 2 and 3, but not that of the upstream MAPK kinase 3, nor of AMPK. Under all conditions (ischaemia, AICAR and metformin), SB203580 blocked Bax translocation completely. It is concluded that Bax translocation to mitochondria is an early step in ischaemia and that it occurs in response to activation of p38 MAPK downstream of AMPK.

AMP-Activated Protein Kinases↗

PubMed2004

Cyclophilin-D promotes the mitochondrial permeability transition but has opposite effects on apoptosis and necrosis.

Cyclophilin-D is a peptidylprolyl cis-trans isomerase of the mitochondrial matrix. It is involved in mitochondrial permeability transition, in which the adenine nucleotide translocase of the inner membrane is transformed from an antiporter to a non-selective pore. The permeability transition has been widely considered as a mechanism in both apoptosis and necrosis. The present study examines the effects of cyclophilin-D on the permeability transition and lethal cell injury, using a neuronal (B50) cell line stably overexpressing cyclophilin-D in mitochondria. Cyclophilin-D overexpression rendered isolated mitochondria far more susceptible to the permeability transition induced by Ca2+ and oxidative stress. Similarly, cyclophilin-D overexpression brought forward the onset of the permeability transition in intact cells subjected to oxidative stress. In addition, in the absence of stress, the mitochondria of cells overexpressing cyclophilin-D maintained a lower inner-membrane potential than those of normal cells. All these effects of cyclophilin-D overexpression were abolished by cyclosporin A. It is concluded that cyclophilin-D promotes the permeability transition in B50 cells. However, cyclophilin-D overexpression had opposite effects on apoptosis and necrosis; whereas NO-induced necrosis was promoted, NO- and staurosporine-induced apoptosis were inhibited. These findings indicate that the permeability transition leads to cell necrosis, but argue against its involvement in apoptosis.

Amino Acid Sequence↗

PubMed2003

Increased susceptibility of striatal mitochondria to calcium-induced permeability transition.

Mitochondria were simultaneously isolated from striatum and cortex of adult rats and compared in functional assays for their sensitivity to calcium activation of the permeability transition. Striatal mitochondria showed an increased dose-dependent sensitivity to Ca2+ compared with cortical mitochondria, as measured by mitochondrial depolarization, swelling, Ca2+ uptake, reactive oxygen species production, and respiration. Ratios of ATP to ADP were lower in striatal mitochondria exposed to calcium despite equal amounts of ADP and ATP under respiring and nonrespiring conditions. The Ca2+-induced changes were inhibited by cyclosporin A or ADP. These responses are consistent with Ca2+ activation of both low and high permeability pathways constituting the mitochondrial permeability transition. In addition to the striatal supersensitivity to induction of the permeability transition, cyclosporin A inhibition was less potent in striatal mitochondria. Immunoblots indicated that striatal mitochondria contained more cyclophilin D than cortical mitochondria. Thus striatal mitochondria may be selectively vulnerable to the permeability transition. Subsequent mitochondrial dysfunction could contribute to the initial toxicity of striatal neurons in Huntington's disease.

Adenosine Diphosphate↗

PubMed2002

Biphasic translocation of Bax to mitochondria.

Using green fluorescent protein-tagged Bax, we demonstrate that Bax is sequestered from the cytosol of cardiomyocytes in two distinct phases following the induction of apoptosis with staurosporine. In the first phase, lasting several hours, Bax removal from the cytosol was relatively small. In the second phase, Bax was very largely removed from the cytosol and sequestered into large aggregates associated with the mitochondria. To test which of the phases involved cytochrome c release, cells were transfected with a red fluorescent protein-cytochrome c fusion. The cytochrome c fusion protein was accumulated by mitochondria of healthy cells and was released by staurosporine in phase 1. When green fluorescent protein-Bax was immunoprecipitated from extracts of cells in phase 1 and phase 2, the voltage-dependent anion channel (mitochondrial outer membrane) and the adenine nucleotide translocase (mitochondrial inner membrane) were also precipitated. These data support a two-phase model of Bax translocation in which Bax targets the mitochondrial intermembrane contact sites and releases cytochrome c in the first phase, and is then packaged into large aggregates on mitochondria in the second.

Animals↗

PubMed2002

Cyclophilin-A is involved in excitotoxin-induced caspase activation in rat neuronal B50 cells.

Glutamate and the NO donor, nitroprusside, synergistically induced the death of B50 cells from a rat CNS-derived neuroblastoma cell line. With low [nitroprusside] (10 microM) both nitroprusside and glutamate were required. Under these conditions, nuclei became pyknotic and caspases were activated. The activities of caspase-3 and caspase-6 (effector caspases) were higher than those of caspase-8 and caspase-9 (initiator caspases). The activation of all four caspases was inhibited by cyclosporin A, with the order of susceptibility caspase-8=caspase-9=caspase-6>caspase-3. To identify the possible locus of cyclosporin A action, we used an antisense oligodeoxynucleotide to suppress the level of cyclophilin-A to<5% of its control value. Cyclophilin-A suppression largely reproduced the inhibitory effects of cyclosporin A. These results provide the first indication that cyclophilin-A participates in the activation of the caspase cascade in neuronal cells, in particular in the form of cascade elicited by excitotoxic stimuli. It is concluded that neuroprotection by cyclosporin A against excitotoxin-induced apoptosis is, at least partly, due to inhibition of cyclophilin-A.

Animals↗

PubMedDate not supplied

Mitochondrial intermembrane junctional complexes and their involvement in cell death.

Mitochondria establish contact sites between the inner and outer membranes. The contact sites are held together by junctional complexes of the adenine nucleotide translocase (ANT; inner membrane) and the voltage-dependent anion channel (VDAC; outer membrane). The junctional complexes act as multifunctional recruitment centres, binding a range of proteins according to the function to be executed. Some of these, involving kinases and enzymes of lipid transfer, are readily understood as ongoing functions in energy and lipid metabolism. But the roles of other proteins recruited to the junctional complexes are less well defined. Here, we focus on the complexes formed with Bax and with cyclophilin-D, and their possible roles in apoptotic and necrotic cell death. We have isolated both types of complexes using glutathione-S-transferase fusion proteins of Bax and of cyclophilin-D. The VDAC/ANT/cyclophilin-D complex reconstitutes Ca(2+)- and cyclosporin A-sensitive permeability transition pore activity when incorporated into proteoliposomes. The complex forms readily in the absence of factors required for pore opening in isolated mitochondria, suggesting that these factors act on the preexisting complex, rather than drive its assembly, and that the complex is a physiological entity in healthy cells.

Adenosine Triphosphate↗