Early CD3 peripheral blood chimerism predicts the long-term engrafting unit following myeloablative double-cord blood transplantation.

After double-cord blood transplantation, long-term hematopoietic dominance of a single-cord blood donor graft is established in the majority of patients; however, the mechanism behind this observation remains largely unknown. Beginning at day 7 posttransplantation, we prospectively measured weekly lineage-specific peripheral blood donor chimerisms in patients undergoing myeloablative double-cord blood transplantation to evaluate whether the degree of early donor contribution to specific lineage(s) would predict the long-term engrafting unit. Our results demonstrate that the donor unit with higher CD3 chimerism at day 7 became the dominant engrafting unit in 26 of 31 evaluable patients (P = .0002) and in 34 of 34 evaluable patients at day 14 (P < .0001). Similarly, higher donor unit CD33 chimerism was associated with dominant engraftment in 8 of 8 (day 7) and in 31 of 32 (day 14) evaluable patients. No statistically significant correlation between the dominant unit and order of infusion, infused total nucleated cells, CD34, or CD3 cell doses, unit viability, or HLA disparity was observed. The correlation of higher early posttransplantation donor CD3 peripheral blood chimerism with the dominant unit suggests a rapid immune-mediated response as a primary mechanism of action for long-term single-donor dominance. This finding may have clinical implications for early selection of the winning unit after double-cord blood transplantation and for novel cord blood manipulation strategies.

Improved immunomagnetic enrichment of CD34(+) cells from umbilical cord blood using the CliniMACS cell separation system.

BACKGROUND AIMS: CD34(+) enrichment from cord blood units (CBU) is used increasingly in clinical applications involving ex vivo expansion. The CliniMACS instrument from Miltenyi Biotec is a current good manufacturing practice (cGMP) immunomagnetic selection system primarily designed for processing larger numbers of cells: a standard tubing set (TS) can process a maximum of 60 billion cells, while the larger capacity tubing set (LS) will handle 120 billion cells. In comparison, most CBU contain only 1-2 billion cells, raising a question regarding the optimal tubing set for CBU CD34(+) enrichment. We compared CD34(+) cell recovery and overall viability after CliniMACS processing of fresh CBU with either TS or LS. METHODS: Forty-six freshly collected CBU (≤ 36 h) were processed for CD34(+) enrichment; 22 consecutive units were selected using TS and a subsequent 24 processed with LS. Cell counts and immunophenotyping were performed pre- and post-selection to assess total nucleated cells (TNC), viability and CD34(+) cell content. RESULTS: Two-sample t-tests of mean CD34(+) recovery and viability revealed significant differences in favor of LS (CD34(+) recovery, LS = 56%, TS = 45%, P = 0.003; viability, LS = 74%, TS = 59%, P = 0.011). Stepwise linear regression, considering pre-processing unit age, viability, TNC and CD34(+) purity, demonstrated statistically significant correlations only with the tubing set used and age of unit. CONCLUSIONS: For CD34(+) enrichment from fresh CBU, LS provided higher post-selection viability and more efficient recovery. In this case, a lower maximum TNC specification of TS was not predictive of better performance. The same may hold for smaller scale enrichment of other cell types with the CliniMACS instrument.

Cryopreservation of umbilical cord blood with a novel freezing solution that mimics intracellular ionic composition.

BACKGROUND: Cryopreservation protocols have remained relatively unchanged since the first umbilical cord blood banking program was established. This study evaluated the preservation efficacy of a novel intracellular-like cryopreservation solution (CryoStor, BioLife Solutions, Inc.), the rate of addition of two cryopreservation solutions to cord blood units (CBUs), and reduced final dimethyl sulfoxide (DMSO) concentration of 5%. STUDY DESIGN AND METHODS: Split-sample CBUs were cryopreserved with either an in-house 20% DMSO-based cryopreservation solution or CryoStor CS10 at a rate of 1 mL/min (n = 10; i.e., slow addition) or as a bolus injection (n = 6; i.e., fast addition). Infrared images of exothermic effects of the cryopreservation solutions were monitored relative to the rate of addition. Prefreeze and postthaw colony-forming unit assays, total nucleated cells, and CD34+ cell counts were compared. RESULTS: Maximum temperature excursions observed were less than 6°C, regardless of the rate of solution addition. Fast addition resulted in peak excursions approximately twice that of slow addition but the magnitude and duration were minimal and transient. Slow addition of CryoStor CS10 (i.e., final concentration ≤ 5% DMSO) resulted in significantly better postthaw CD34+ cell recoveries; no other metrics were significantly different. Fast addition of CryoStor resulted in similar postthaw metrics compared to slow addition of the in-house solution. CONCLUSION: Slow and fast addition of cryopreservation solutions result in mean temperature changes of approximately 3.3 to 4.45°C. Postthaw recoveries with CryoStor were equivalent to or slightly better than with the in-house cryopreservation solution. CryoStor also provides several advantages including reduced processing time, formulation consistency, and reduced DMSO in the frozen product (≤ 5%).

Improved post-thaw recovery of peripheral blood stem/progenitor cells using a novel intracellular-like cryopreservation solution.

BACKGROUND AIMS: Peripheral blood stem cells (PBSC) have become the preferred stem cell source for autologous hematopoietic transplantation. A critical aspect of this treatment modality is cryopreservation of the stem cell products, which permits temporal separation of the PBSC mobilization/collection phase from the subsequent high-dose therapy. While controlled rate-freezing and liquid nitrogen storage have become 'routine' practice in many cell-processing facilities, there is clearly room for improvement as current cryopreservation media formulations still result in significant loss and damage to the stem/progenitor cell populations essential for engraftment, and can also expose the patients to relatively undefined serum components and larger volumes of dimethylsulfoxide (DMSO) that can contribute to the morbidity and mortality of the transplant therapy. METHODS: This study compared cryopreservation of PBSC in a novel intracellular-like, fully defined, serum- and protein-free preservation solution, CryoStor (BioLife Solutions Inc.), with a standard formulation used by the Fred Hutchinson Cancer Research Center (FHCRC). Briefly, human PBSC apheresis specimens were collected and 5 x 10(7) cells/1 mL sample vial were prepared for cryopreservation in the following solutions: (a) FHCRC standard, Normosol-R, 5% human serum albumin (HAS) and 10% DMSO; and (b) CryoStor CS10 (final diluted concentration of 5% DMSO). A standard controlled-rate freezing program was employed, and frozen vials were stored in the vapor phase of a liquid nitrogen freezer for a minimum of 1 week. Vials were then thawed and evaluated for total nucleated cell count (TNC), viability, CD34 and granulocytes by flow cytometry, along with colony-forming activity in methylcellulose. RESULTS: The PBSC samples frozen in CryoStor CS10 yielded significantly improved post-thaw recoveries for total viable CD34(+), colony-forming units (CFU) and granulocytes. Specifically, relative to the FHCRC standard formulation, cryopreservation with CS10 resulted in an average 1.8-fold increased recovery of viable CD34(+) cells (P=0.005), a 1.5-fold increase in CFU-granulocyte-macrophage (GM) numbers (P=0.030) and a 2.3-fold increase in granulocyte recovery (P=0.045). CONCLUSIONS: This study indicates that use of CryoStor for cryopreservation can yield significantly improved recovery and in vitro functionality of stem/progenitor cells in PBSC products. In addition, it is important to note that these improved recoveries were obtained while not introducing any extra serum or serum-derived proteins, and reducing the final concentration/volume of DMSO by half. Further in vitro and in vivo studies are clearly necessary; however, these findings imply use of CryoStor for cryopreservation could result in improved engraftment for those patients with a lower content of CD34(+) cells in their PBSC collections, along with reducing the requirement for additional apheresis collections and decreasing the risk of adverse infusion reactions associated with higher exposure to DMSO.

NFAT4 deficiency results in incomplete liver regeneration following partial hepatectomy.

BACKGROUND: Liver regeneration following partial hepatectomy requires the orchestration of highly regulated molecular pathways; a change in the abundance or activity of a specific gene product has the potential to adversely affect this process. The nuclear factor of activated T-cells (NFAT) transcription factors represent a family of gene transcription signaling intermediates that translate receptor-dependent signaling events into specific transcriptional responses using the Ras/Raf pathway. MATERIALS AND METHODS: Eight-week old NFAT4 knockout (KO) mice and their wild type counterparts (Balb-c) underwent two-thirds partial hepatectomy. The animals were sacrificed and their livers were harvested at specific time points during regeneration. Recovery of liver mass was measured for each time point. PCR analysis was used to analyze expression levels of the immediate early genes c-fos, c-jun and c-myc as well as downstream effectors of NFAT4 including FGF-18 and BMP-4. RESULTS: Hepatocyte proliferation and thus liver regeneration following hepatectomy was suppressed in NFAT4 knockout (KO) mice. Statistical significance was reached at 1 h, 7 d, and 10 d (P < 0.05) with a 22% median reduction in regeneration of liver mass in the NFAT4 KO mice by 10 d, at which time liver regeneration should be complete in mice. The immediate early gene c-fos was elevated in NFAT4 KO mice during early regeneration with a median value at 1 h and 1 d of 1.60E-08 and 1.09E-08 versus 6.10E-09 and 1.55E-09 in the Balb-c mice. C-jun, in contrast, was elevated during late regeneration in the NFAT4 KO mice (3.40E-09 and 5.67E-09 at 7 and 10 d, respectively) in comparison with the Balb-c mice (7.76E-10 and 1.24E-09, respectively.). NFAT2 was also up-regulated in the NFAT4 KO mice; however, no changes were detected in its downstream effectors, CCR1 and CCL3. CONCLUSIONS: We demonstrated that NFAT4 deficiency impairs hepatic regeneration in a murine model proving that NFAT4 plays an important yet unclear role in liver regeneration; its absence may be compensated by c-fos, c-jun, and NFAT2 expression changes.

Warm hepatic ischemia-reperfusion promotes growth of colorectal carcinoma micrometastases in mouse liver via matrix metalloproteinase-9 induction.

Surgical resection remains the best treatment for colorectal metastases isolated to the liver; however, 5-year survival rates following liver resection are only 40% to 50%, with liver recurrence being a significant reason for treatment failure. The ischemia-reperfusion (I/R) injury incurred during liver surgery can lead to cellular dysfunction and elevations in proinflammatory cytokines and matrix metalloproteinases (MMP). In rodents, I/R injury to the liver has been shown to accelerate the outgrowth of implanted tumors. The mechanism for increased tumor growth in the setting of liver I/R injury is unknown. To investigate the effect of I/R on tumor growth, an experimental model was used whereby small hepatic metastases form after 28 days. Mice subjected to 30 min of 70% liver ischemia at the time of tumor inoculation had significantly larger tumor number and volume, and had elevated MMP9 serum and liver tissue MMP9 as evidenced by zymography and quantitative real-time PCR. Mice treated with doxycycline, a broad-spectrum MMP inhibitor, had reduced MMP9 levels and significantly smaller tumor number and volume in the liver. MMP9-null mice were used to determine if the effects of doxycycline were due to the absence of stromal-derived MMP9. The MMP9-null mice, with or without doxycycline treatment, had reduced tumor number and volume that was equivalent to wild-type mice treated with doxycycline. These findings indicate that hepatic I/R-induced elevations in MMP9 contribute to the growth of metastatic colorectal carcinoma in the liver and that postresection MMP9 inhibition may be clinically beneficial in preventing recurrence following hepatic surgery.

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.

Fluorescence spectroscopy accurately detects irreversible cell damage during hepatic radiofrequency ablation.

BACKGROUND: A current limitation of hepatic radiofrequency ablation (RFA) is an inability to detect ablation margins in real time. Thermal injury from RFA alters the biochemical properties governing tissue fluorescence. We hypothesized that the changes in hepatic fluorescence measured during hepatic RFA could be used to detect irreversible hepatocyte damage accurately and to determine ablation margins in real time. METHODS: RFA was performed on healthy pig livers and monitored in vivo simultaneously for fluorescence and temperature by a fiberoptic micro-interrogation probe connected to a spectroscopy system. Ablations were stopped based on previously established real-time fluorescence spectral data, not based on temperature or time. To determine where in the ablated tissue cell death occurred, biopsies for transmission electron microscopy were taken from 4 areas of 3 specimens: (1) nonablated liver, (2) hemorrhagic zone/normal liver interface, (3) hemorrhagic zone/coagulated zone interface, and (4) coagulated zone. In vitro fluorescence emission intensity was determined at each biopsy site. RESULTS: Peak hepatic fluorescence intensity occurred at 470 nm and decreased as RFA progressed. Transmission electron microscopy evidence of irreversible hepatocyte damage occurred at the interface of the coagulation zone and the hemorrhagic zone and correlated with a 87.5% +/- 9% decrease in fluorescence emission intensity. Tissue fluorescent changes from thermal injury were unaffected by tissue cooling. CONCLUSION: Fluorescence spectroscopy accurately detected hepatocellular thermal injury from RFA in real time and can detect irreversible cell damage during tissue thermal therapy.

Real-time spectroscopic assessment of thermal damage: implications for radiofrequency ablation.

Radiofrequency ablation (RFA) is an evolving technology used to treat unresectable liver tumors. Currently, there is no accurate method to determine RFA margins in real-time during the procedure. We hypothesized that a fiber-optic based spectroscopic monitoring system could detect thermal damage from RFA in real-time. Fluorescence (F) and diffuse reflectance (Rd) spectra were continuously acquired from within the expected ablation zone during canine hepatic RFA using a fiber-optic microinterrogation probe (MIP). The F and Rd spectral feedback were continuously monitored and ablations were stopped based on changes in spectra alone. After each ablation, the MIP tract was marked with India ink and the ablation zone was excised. The relationship of the MIP to the zone of ablation was examined grossly and microscopically. F and Rd spectral changes occurred in three characteristic phases as the ablation zone progresses past the MIP. Phase 1 indicates minimal deviation from normal lives. Phase 2 occurs as the MIP lies within the hemorrhagic zone of the ablated tissue. Phase 3 correlates with complete tissue coagulation. The absolute magnitude of spectral change correlates with the gross and histologic degree of thermal damage. Optical spectroscopy is a technology that allows real-time detection of thermal tissue damage. In this study, both F and Rd spectroscopy accurately defined the advancing hemorrhagic edge of the zone of ablation and the central coagulation zone. These results suggest that F and Rd spectroscopy can be used to create a real-time feedback system to accurately define RFA margins.

The use of genomics and proteomics for the recognition of transplantation rejection of solid organs.

Solid organ transplantation has saved many lives since its first success in 1954. Prior to that landmark day, the greatest obstacle to transplantation success was the recipients rejection of the transplanted organ. Although much has been learned about the immune response to transplant, organ rejection remains a prevalent clinical problem. Recent advances in the fields of genomics and proteomics have opened the door to patented new technologies for detecting rejection episodes in transplanted patients, and are even beginning to prospectively diagnose the risk of rejection based on donor and recipient biomarkers. This report briefly discusses transplant rejection, with highlights of published manuscripts that incorporate current assays utilizing genomic and or proteomic methods to detect rejection, reviews patents that focus on detection or therapy of transplant rejection, and concludes with a prospective discussion of future developments in the field of transplant rejection.

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.

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.

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 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.

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.