Aya Wakata

Mitochondrial Calcium Uptake Regulates Cold Preservation‐Induced Bax Translocation and Early Reperfusion Apoptosis

Mitochondrial calcium (mCa + 2) overload occurs during cold preservation and is an integral part of mitochondrial‐dependent apoptotic pathways. We investigated the role of mCa + 2 overload in cell death following hypothermic storage using HepG2 cells stored in normoxic‐hypothermic (4 °C) or hypoxic (< 0.1% O2)‐hypothermic Belzer storage solution. Cells were stored for 6 h, with or without 10 μM ruthenium red (mCa + 2 uniporter inhibitor) followed by rewarming in oxygenated media at 37 °C. Cytoplasmic cytochrome c levels were studied by Western analysis and by fluorescent microscopy after transfection of cytochrome c‐GFP expression plasmid. Immunofluorescence determined the intracellular, spatio‐temporal distribution of Bax, and TUNEL staining was used to evaluate cell death after 180 min of rewarming. Caspase activation was evaluated using Western analysis and a caspase 3 activity assay. Bax translocation, cytochrome c release, and early rewarming cell death occurred following hypothermic storage and were exacerbated by hypoxia. Caspase 3 activation did not occur following hypothermic storage. Blockade of mCa + 2 uptake prevented Bax translocation, cytochrome c release, and early rewarming cell death. These studies demonstrate that mCa + 2 uptake during hypothermic storage, both hypoxic and normoxic, contributes to early rewarming apoptosis by triggering Bax translocation to mitochondria and cytochrome c release.

Mitochondrial P2Y-Like receptors link cytosolic adenosine nucleotides to mitochondrial calcium uptake.

ATP is a known extracellular ligand for cell membrane purinergic receptors. Intracellular ATP can work also as a regulatory ligand via binding sites on functional proteins. We report herein the existence of P2Y1‐like and P2Y2‐like receptors in hepatocyte mitochondria (mP2Y1 and mP2Y2), which regulate mCa2+ uptake though the uniporter. Mitochondrial P2Y1 activation stimulates mCa2+ uptake; whereas, mP2Y2 activation inhibits mCa2+ uptake. ATP acts preferentially on mP2Y2 receptors, while ADP and AMP‐PNP stimulate both the mP2Y1 and mP2Y2. PPADS inhibits ADP stimulated mP2Y1‐mediated mCa2+ uptake. In addition, UTP, a selective P2Y2 agonist, strongly inhibits mCa2+ uptake. The newly discovered presence and function of these receptors is significant because it explains increased mCa2+ uptake in the setting of low cytosolic [ATP] and, therefore, establishes a mechanism for direct feedback in which cytosolic [ATP] governs mitochondrial ATP production through regulation of mCa2+ uptake.

Mitochondrial calcium transport is regulated by P2Y1‐ and P2Y2‐like mitochondrial receptors

Ischemia‐reperfusion injury remains a major clinical problem in liver transplantation. One contributing factor is mitochondrial calcium (mCa2+) overload, which triggers apoptosis; calcium also regulates mitochondrial respiration and adenosine 5′‐triphosphate (ATP) production. Recently, we reported the presence of purinergic P2Y1‐ and P2Y2‐like receptor proteins in mitochondrial membranes. Herein, we present an evaluation of the functional characteristics of these receptors. In experiments with isolated mitochondria, specific P2Y1 and P2Y2 receptors ligands: 2‐methylthio‐adenosine 5′‐diphosphate (2meSADP) and uridine 5′‐triphosphate (UTP), respectively, were used, and mitochondrial calcium uptake was measured. 2meSADP and UTP had a maximum effect at concentrations in the range of the known P2Y1 and P2Y2 receptors. The P2Y inhibitor phosphate‐6‐azophenyl‐2′,4′‐disulfonate (PPADS) blocked the effects of both ligands. The phospholipase C (PLC) antagonist U73122 inhibited the effect of both ligands while its inactive analog U73343 had no effect. These data strongly support the hypothesis that mitochondrial Ca2+ uptake is regulated in part by adenine nucleotides via a P2Y‐like receptor mechanism that involves mitochondrial PLC activation. J. Cell. Biochem. 99: 1165–1174, 2006.