Andrey E. Belous

Modulation of mitochondrial calcium management attenuates hepatic warm ischemia-reperfusion injury.

Hepatic warm ischemia and reperfusion (IR) injury occurs in many clinical situations and has an important link to subsequent hepatic failure. The pathogenesis of this injury involves numerous pathways, including mitochondrial‐associated apoptosis. We studied the effect of mitochondrial calcium uptake inhibition on hepatic IR injury using the specific mitochondrial calcium uptake inhibitor, ruthenium red (RR). Rats were subjected to 1 hour of 70% warm hepatic ischemia following RR pretreatment or vehicle injection. Sham‐operated animals served as controls. Analysis was performed at 15 minutes, 1 hour, 3 hours, or 6 hours after reperfusion. Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) concentrations were determined. Terminal deoxynucleotidyl transferase‐mediated deoxyuridine triphosphate nick‐end labeling (TUNEL) staining was performed to assess apoptosis, and hepatocellular necrosis was semiquantitated from hematoxylin and eosin–stained tissue sections. RR pretreatment significantly decreased both AST and ALT serum levels after 6 hours of reperfusion (AST: 1,556 ± 181 U/L vs. 597 ± 121 U/L, P = 0.005; ALT: 1,118 ± 187 U/L vs. 294 ± 39 U/L, P = 0.005). Apoptosis was observed within 15 minutes of reperfusion in vehicle‐pretreated animals and peaked after 3 hours of reperfusion (98 ± 21 cells/high‐power field [hpf]). Apoptosis was inhibited at all time points by RR pretreatment. Histologic evidence of necrosis was not observed prior to 3 hours of reperfusion (23% ± 4%), and maximal necrosis was observed after 6 hours of reperfusion (26% ± 1% percent area). RR pretreatment significantly decreased the necrotic percent area at both the 3‐hour and the 6‐hour time points (4.2% ± 2%; 3.7% ± 1%, respectively). Hepatic IR injury resulted in both apoptotic and necrotic cell death, which were attenuated by RR pretreatment. In conclusion, these observations implicate mitochondrial calcium uptake in the pathogenesis of hepatic IR injury. (Liver Transpl 2005;11:663–668.)

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.

Reversed activity of mitochondrial adenine nucleotide translocator in ischemia-reperfusion

Background. Graft dysfunction as a result of preservation injury remains a major clinical problem in liver transplantation. This is related in part to accumulation of mitochondrial calcium. In an attempt to sustain cell and mitochondrial integrity during ischemia, intramitochondrial F0F1 adenosine triphosphate (ATP) synthase reverses its activity and hydrolyzes ATP to maintain the mitochondrial transmembrane potential (m&Dgr;&psgr;). It is not known how cytoplasmic ATP becomes available for hydrolysis by this enzyme. The authors hypothesized that mitochondrial adenine nucleotide translocator (ANT) reverses its activity during ischemia, making cytoplasmic ATP available for hydrolysis by F0F1 ATP synthase. Methods. Rat livers were perfused with cold University of Wisconsin solution at 4°C (39.2°F)through the portal vein and processed immediately or after 24 hr of cold storage. Mitochondria were separated by differential centrifugation. ATP-dependent mitochondrial calcium-45 (45Ca)2+ uptake was determined after incubation with ATP (5 mM) or adenosine diphosphate (ADP) (5 mM) with or without 15 &mgr;M of bongkrekic acid (BA), an ANT blocker; the nonhydrolyzable analog of ATP (adenosine 5′-&bgr;,&ggr;-imidotriphosphate [AMP-PNP]) served as the negative control. All measurements were performed in triplicate. Student t test, P <0.05 was taken as significant. Results. Inhibition of ANT by BA prevents mitochondrial Ca2+ accumulation in the presence of ATP and high 45Ca2+ concentrations, and increased extramitochondrial 45Ca2+ stimulated mitochondrial 45Ca2+ uptake in the presence of ATP but not ADP, AMP-PNP, or BA. Conclusions. These data demonstrate that ANT plays an important role in mitochondrial Ca2+ uptake under ischemic conditions by reversing its activity and allowing transport of extramitochondrial ATP into the matrix for hydrolysis by reversed F0F1 ATP synthase.

Inhibition of phospholipase C attenuates liver mitochondrial calcium overload following cold ischemia.

Background. Graft failure due to cold ischemia (CI) injury remains a significant problem during liver transplantation. During CI, the consumption of ATP and the increase in cellular Ca2+ concentration may result in mitochondrial Ca2+ (mCa2+) overload through the mCa2+ uniporter, which can ultimately lead to apoptosis and graft nonfunction. We recently identified phospholipase C-dl (PLC-dl) as a novel regulator of mCa2+ uptake in the liver, and now extend those studies to examine the role of mitochondrial PLC in liver CI injury. Methods. Rat livers were perfused with University of Wisconsin (UW) solution. Half was homogenized immediately; the other half was cold-stored for 24 hr in UW. Mitochondria were extracted by differential centrifugation and incubated in buffer containing ATP and 0.1 or 0.2 &mgr;M 45Ca2+. The selective PLC inhibitor, U-73122, was added to determine the effects of PLC inhibition on mCa2+ uptake following CI. Western blots and densitometry quantified mitochondrial PLC expression. Mito Tracker Red fluorescence microscopy was used to verify mitochondrial transmembrane potential. Results. Twenty-four hour CI caused a significant increase in mCa2+ uptake (P<0.001), and increasing extramitochondrial Ca2+ potentiated this effect. The PLC inhibitor, U-73122, decreased mCa2+ uptake in nonischemic mitochondria (P<0.001), and had a greater effect on CI mitochondria (P<0.001). Mitochondrial PLC-dl expression increased 175±75% following CI (P<0.05). Conclusions. These data demonstrate that PLC-dl is essential for mCa2+ uptake following CI, and that the PLC pathway may be sensitized by CI. The CI-induced increase in mitochondrial PLC-&dgr;1 expression represents a novel mechanism whereby mCa2+ uptake can increase independently of cytosolic conditions.