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 Ca concentration may result in mitochondrial Ca (mCa) overload through the mCa uniporter, which can ultimately lead to apoptosis and graft nonfunction. We recently identified phospholipase C-dl (PLC-dl) as a novel regulator of mCa 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 microM Ca. The selective PLC inhibitor, U-73122, was added to determine the effects of PLC inhibition on mCa 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 mCa uptake (P<0.001), and increasing extramitochondrial Ca potentiated this effect. The PLC inhibitor, U-73122, decreased mCa 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 mCa uptake following CI, and that the PLC pathway may be sensitized by CI. The CI-induced increase in mitochondrial PLC-delta1 expression represents a novel mechanism whereby mCa uptake can increase independently of cytosolic conditions.

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 F(0)F(1) adenosine triphosphate (ATP) synthase reverses its activity and hydrolyzes ATP to maintain the mitochondrial transmembrane potential (mdeltapsi). 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 F(0)F(1) ATP synthase. METHODS: Rat livers were perfused with cold University of Wisconsin solution at 4 degrees C (39.2 degrees 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 microM of bongkrekic acid (BA), an ANT blocker; the nonhydrolyzable analog of ATP (adenosine 5'-beta,gamma-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 F(0)F(1) ATP synthase.

Biliary reconstruction is enhanced with a collagen-polyethylene glycol sealant.

Bile leaks occur in up to 27 per cent of liver transplant patients after biliary reconstruction. Synthetic sealants have not been investigated for these biliary procedures. We performed a randomized controlled study to evaluate a novel absorbable polyethylene glycol/collagen biopolymer sealant (CT3 Surgical Sealant) after incomplete end-to-end choledochocholedochostomy (CDCD) in pigs. Pigs (n = 18) underwent transection of the common bile duct and incomplete CDCD over a T-tube, leaving a one-sixth circumferential defect anteriorly. Animals were randomly assigned to treatment (CDCD with sealant, n = 9) or control (no sealant, n = 9). Drains were used to monitor leak volume and bilirubin (bili) concentration. Cholangiography was performed on postoperative day 3. Leaks were defined as drain bili/serum bill > 3, total drain output > 10 mL/kg, and/or extravasation on cholangiography. Animals sacrificed at 3 and 8 weeks (n = 4 and n = 5 from each group, respectively) underwent pathologic examination of the CDCD site. Statistical methods included Student's t test, chi2, linear regression, and analysis of variance procedures. The control group had a higher drain output rate over the first 4 postoperative days than the treatment group (P < 0.05, analysis of variance). Five of nine (56%) control and one of nine (11%) treatment animals had a bile leak (P < 0.05, chi2). There was no major inflammatory response to the sealant versus controls. We conclude that CT3 is effective in decreasing biliary leaks in an incomplete CDCD porcine model with no major adverse pathologic changes. This sealant should be considered for trials for biliary reconstruction in humans.

Purinergic receptor antagonism prevents cold preservation-induced cell death independent of cellular ATP levels.

BACKGROUND: Purinergic (P2Y) receptors play an important role in intracellular Ca(2+) regulation in hepatocytes. Prevention of mitochondrial Ca(2+) (mCa(2+)) overload during ischemic conditions prevents cellular cell death during the early reperfusion period. P2Y antagonists are cytoprotective in other settings. We studied the effect of P2Y receptor antagonism on mitochondrial associated cell death during the period of cold storage. METHODS: HepG2 cells were stored in UW with or without 300 muM reactive blue 2 (RB2) or 10 muM ruthenium red (RR) under either normoxic-hypothermic or hypoxic-hypothermic conditions. Cytoplasmic cytochrome c levels were studied by transfection of cytochrome c-GFP. Immunofluorescence determined the intracellular, spatio-temporal distribution of Bax, and terminal deoxynucleotidyl transferase mediated dUTP nick end labeling staining was used to evaluate cell death. Intracellular compartmental ATP levels were assayed by transfecting with luciferase vectors specific for cytoplasm (PcDNA3-luciferase-LL/V) and mitochondria (PcDNA3-COX8-luciferase). RESULTS: Bax translocation to the mitochondria occurred immediately following cold storage and was followed by cytochrome c-GFP redistribution to the cytosol during rewarming. RB2 treatment significantly attenuated Bax translocation, cytochrome c-GFP redistribution, and cell death following both storage conditions. Both RR and RB2 provided cytoprotection despite ongoing cytoplasmic ATP consumption during cold ischemia. CONCLUSION: These data indicate that the cytoprotective effects of mCa(2+) uptake inhibition and P2Y receptor antagonism are independent of cytoplasmic ATP levels during cold ischemia.

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 (mCa(2+)) overload, which triggers apoptosis; calcium also regulates mitochondrial respiration and adenosine 5'-triphosphate (ATP) production. Recently, we reported the presence of purinergic P2Y(1)- and P2Y(2)-like receptor proteins in mitochondrial membranes. Herein, we present an evaluation of the functional characteristics of these receptors. In experiments with isolated mitochondria, specific P2Y(1) and P2Y(2) 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 P2Y(1) and P2Y(2) 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 Ca(2+) uptake is regulated in part by adenine nucleotides via a P2Y-like receptor mechanism that involves mitochondrial PLC activation.

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.

Altered ATP-dependent mitochondrial Ca2+ uptake in cold ischemia is attenuated by ruthenium red.

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 (Ca(2+)), which has been linked to activation of proapoptotic factors. We hypothesized that cold ischemia increases mitochondrial Ca(2+) uptake in a concentration dependent fashion and that ruthenium red (RR) will attenuate these changes by inhibiting the mitochondrial Ca(2+) uniporter. METHODS: Rat livers perfused with cold University of Wisconsin (UW) solution (4 degrees C) with or without RR (10 microM) via the portal vein (n = 3 per group) were processed immediately (no ischemia) or after 24 h cold-storage (24 h cold ischemia). Mitochondria were separated by differential centrifugation, and adenosine triphosphate (ATP)-dependent (45)Ca(2+) uptake was determined in the presence of ATP (5 mM), adenosine diphosphate (ADP), or adenosine 5'-beta,gamma-imidotriphosphate (AMP-PNP); variable concentrations of extramitochondrial (45)Ca(2+) were used. All measurements were performed in triplicate. Student's t test with P < 0.05 was taken as significant. RESULTS: Our data demonstrate the following: 1) ATP-dependent (45)Ca(2+) uptake in mitochondria separated from livers following 24 h of cold ischemia in UW alone was higher than in mitochondria isolated from non-ischemic livers; the increased uptake was dependent on the concentration of (45)Ca(2+) in the incubation buffer. 2) There was no difference in ATP-dependent (45)Ca(2+) uptake between nonischemic mitochondria and those separated from livers stored in UW-RR for 24 h. 3) (45)Ca(2+) uptake in mitochondria from livers subjected to 24 h of cold ischemia in UW-RR was significantly lower compared to those from livers stored in UW alone when (45)Ca(2+) concentrations were greater than 1 microM. CONCLUSION: 1) Cold ischemia affects mitochondrial Ca(2+) handling, especially when it is challenged by high extramitochondrial Ca(2+) concentrations. 2) The addition of RR in preservation solution attenuates the effects of cold ischemia on mitochondrial Ca(2+) handling. 3) Inhibition of mitochondrial Ca(2+) uniporter with RR protects mitochondria from Ca(2+) overload at high Ca(2+) concentrations. These findings may offer a potentially effective strategy for prevention of ischemia-reperfusion injury in liver transplantation.

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 degrees C) or hypoxic (< 0.1% O2)-hypothermic Belzer storage solution. Cells were stored for 6 h, with or without 10 microM ruthenium red (mCa + 2 uniporter inhibitor) followed by rewarming in oxygenated media at 37 degrees 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.

Novel role of phospholipase C-delta1: regulation of liver mitochondrial Ca2+ uptake.

Mitochondrial Ca2+ (mCa2+) handling is an important regulator of liver cell function that controls events ranging from cellular respiration and signal transduction to apoptosis. Cytosolic Ca2+ enters mitochondria through the ruthenium red-sensitive mCa2+ uniporter, but the mechanisms governing uniporter activity are unknown. Activation of many Ca2+ channels in the cell membrane requires PLC. This activation commonly occurs through phosphitidylinositol-4,5-biphosphate (PIP2) hydrolysis and the production of the second messengers inositol 1,4,5-trisphosphate [I(1,4,5)P3] and 1,2-diacylglycerol (DAG). PIP2 was recently identified in mitochondria. We hypothesized that PLC exists in liver mitochondria and regulates mCa2+ uptake through the uniporter. Western blot analysis with anti-PLC antibodies demonstrated the presence of PLC-delta1 in pure preparations of mitochondrial membranes isolated from rat liver. In addition, the selective PLC inhibitor U-73122 dose-dependently blocked mCa2+ uptake when whole mitochondria were incubated at 37 degrees C with 45Ca2+. Increasing extra mCa2+ concentration significantly stimulated mCa2+ uptake, and U-73122 inhibited this effect. Spermine, a uniporter agonist, significantly increased mCa2+ uptake, whereas U-73122 dose-dependently blocked this effect. The inactive analog of U-73122, U-73343, did not affect mCa2+ uptake in any experimental condition. Membrane-permeable I(1,4,5)P3 receptor antagonists 2-aminoethoxydiphenylborate and xestospongin C also inhibited mCa2+ uptake. Although extra mitochondrial I(1,4,5)P3 had no effect on mCa2+ uptake, membrane-permeable DAG analogs 1-oleoyl-2-acetyl-sn-glycerol and DAG-lactone, which inhibit PLC activity, dose-dependently inhibited mCa2+ uptake. These data indicate that PLC-delta1 exists in liver mitochondria and is involved in regulating mCa2+ uptake through the uniporter.

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 P2Y(1)-like and P2Y(2)-like receptors in hepatocyte mitochondria (mP2Y(1) and mP2Y(2)), which regulate mCa(2+) uptake though the uniporter. Mitochondrial P2Y(1) activation stimulates mCa(2+) uptake; whereas, mP2Y(2) activation inhibits mCa(2+) uptake. ATP acts preferentially on mP2Y(2) receptors, while ADP and AMP-PNP stimulate both the mP2Y(1) and mP2Y(2). PPADS inhibits ADP stimulated mP2Y(1)-mediated mCa(2+) uptake. In addition, UTP, a selective P2Y(2) agonist, strongly inhibits mCa(2+) uptake. The newly discovered presence and function of these receptors is significant because it explains increased mCa(2+) 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 mCa(2+) uptake.