Effects of sodium and potassium channel antagonists on the energy metabolism of rat liver during cold storage.
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Animals↗
Biomedical subjects
B J Fuller
Publications and source records attributed to B J Fuller.
The effect of cooling and hypertonic exposure on murine oocyte function, fertilization, and development.
Several individual but related steps are involved in the cryopreservation process, including the addition of cryoprotectants at various temperatures, cooling to subzero temperatures, and long-term storage. The process is completed by rewarming and removal of cryoprotectants prior to a return to physiological conditions. In this series of experiments we have attempted to distinguish the effects of some of these procedures. Control, untreated ovulated mouse oocytes showed 95% in vitro fertilization (190/200) and 92% subsequent development to hatching blastocyst (184/200). Exposure of oocytes to either isotonic or hypertonic media at 37 degrees C did not significantly change the rate of fertilization (90%, 108/120; and 89%, 154/174, respectively) or subsequent embryonic development (85%, 102/120; and 82%, 143/174, respectively). Slow cooling in isotonic medium (-3 degrees C/min) to 0 degree C had no effect on the rate of fertilization (83%, 103/124), but rapid cooling (> 1000 degrees C/min) to 0 degree C resulted in a significant reduction in fertilization rate to 75% (151/202). When oocytes suspended in a hypertonic solution were cooled using slow or rapid rates, there were marked decreases in fertilization to 26% (61/231) and 56% (156/278), respectively. Subsequent embryonic growth was reduced to 15% (34/231) after slow cooling and 26% (72/278) after rapid cooling. Exposure of oocytes to glycerol at 37 degrees C and dimethyl sulfoxide at 0 degree C reduced the fertilization rate to 57% (67/118) and 73% (103/145), respectively, with a corresponding reduction in embryonic growth to 52% (61/118) and 65% (94/145), but there were no additional effects of cooling or hypertonic exposure after addition of cryoprotectants.
Animals↗
The importance of calcium-related effects on energetics at hypothermia: effects of membrane-channel antagonists on energy metabolism of rat liver.
During normothermic metabolism, the active pumping of Ca2+ across the cell membrane, mitochondria, and specialized sequestration organelles accounts for a large proportion of total energy expenditure in the cell. This study was designed to determine the effects of Ca2+ channel antagonists (chlorpromazine, verapamil, nifedipine, prenylamine, and nisoldipine) on energy metabolism and levels of glycolytic substrate (glucose) and anaerobic endproduct (lactate) during cold ischemia in rat livers. We hypothesized that if the passive channels were blocked during cold ischemia, then the ATP requirement of active ion pumping would be reduced and ATP levels and energy charge ratios would remain higher throughout the ischemic period; thus, viability of the liver would also be increased after prolonged ischemia. The most positive effect on energy metabolism was observed in the chlorpromazine-treated livers, followed by verapamil treatment. In the chlorpromazine treatment, total adenylate (TA) contents were 0.5-1.0 mumol/g (P < 0.05) higher than the sham group for most of the 24-h time course. Energy charge (EC) ratios were 0.05-0.07 higher than the sham values up to 4-10 h ischemia. Verapamil treatment was less effective, but still exhibited positive effects on TA levels at several time points (20 min, 10 h, and 24 h) throughout the entire 24-h period. In both of these groups, TA values by 24 h ischemia were similar to levels at 10 h in the sham group (3.1 mumol/g), thus showing a considerable effect in maintaining adenylate levels. Despite similar pharmacological antagonist activities, ATP levels in the nifedipine, prenylamine, and nisoldipine treatment groups were 1.0-1.5 mumol/g (P < 0.05) less than the corresponding sham group (without Ca2+ antagonists) over the first 1 h ischemia. The decreases in high energy adenylate levels were reflected in lower EC ratios in these three groups; values were 0.06-0.17 (P < 0.05) lower than corresponding sham values. Finally, it was an unexpected finding that the sham injection (0.5 mg/kg ethanol+PEG400) resulted in the sustained elevation of ATP, total adenylates, and EC values over the first h; EC ratios remained at initial (t = 0) values (EC = 0.71 +/- 0.01) up to 1 h.
Adenosine Monophosphate↗
Hypothermic perfusion preservation of liver: the role of phosphate in stimulating ATP synthesis studied by 31P NMR.
Hypothermic perfusion of rat livers was investigated by 31phosphorus nuclear magnetic resonance (31P NMR) spectroscopy using a temperature-controlled module that allowed data acquisition at various time points during a 48-h period. The livers were perfused with an oxygenated lactobionate/raffinose-based solution containing adenosine and inorganic phosphate, and changes in tissue oedema were monitored by direct on-line measurements of liver weight changes. Liver tissue ATP concentrations, determined by fluorimetric assay, were low immediately after organ removal, probably reflecting metabolic stress during the removal period, and these increased slightly during the next 3 h. This was reflected by changes in the 31P NMR spectra. However, by 24 h ATP levels had increased significantly, and these were maintained for up to 48 h, suggesting a shift in the balance between energy production and consumption. When inorganic phosphate was replaced by another anion (citrate), ATP was maintained at a constant lower level during perfusion for 48 h. Tissue weight changes were similar in both groups, suggesting that volume control was not affected by the different ATP contents of the livers. By combining the temperature-controlled module with a separate perfusion circuit, NMR spectroscopy can provide a sensitive method for following energy metabolism in the same organ over long periods during hypothermic perfusion.
Adenosine Triphosphate↗
An underlying mechanism for improved liver preservation with a combined histidine-lactobionate-raffinose flush solution.
In previous experimental liver transplant studies, it was possible to extend cold ischaemic time (CIT) by using a flush/storage solution combining histidine, lactobionate and raffinose (HLR). In this study, energy metabolism, glycolytic substrate (glucose) and anaerobic end-product (lactate) were examined in rat liver over 24 h of cold storage to determine the mechanism of action of the HLR solution. In livers subjected to simple flush and storage with the HLR solution, levels of ATP and ADP were considerably higher than livers stored with modified UW throughout 24 h of storage; at 4 h of storage, ATP and ADP levels were 1.1 and 3.1 mumol/g for HLR solution versus 0.18 and 0.81 mumol/g for UW solution. Total adenylate contents (TA = ATP + ADP + AMP) also remained 1-2 mumol/g higher in HLR-treated livers than those preserved in UW; TA values ranged from 3.8 to 5.7 mumol/g. Glucose increased to 20-35 mumol/g by 10-24 h of storage (similar to the UW group). Lactate rose to almost twice that in livers stored in UW; total lactate accumulation was approximately 10.0 mumol/g. This study demonstrated that the combined HLR solution is able to prolong the maximum 'safe' CIT by increasing anaerobic metabolism and consequently preserving liver energetics. The second part of the experiment examined the effect of continuous perfusion (with/without O2) over the 1st h of cold ischaemia. Under current methods of liver flushing and excision, the 1st h of cold storage may be the critical time of metabolic 'adjustment' since most of the pH and ATP changes occur during this period.(ABSTRACT TRUNCATED AT 250 WORDS)
Adenosine↗
Resuscitation of cardiac energy metabolism in the rabbit heart by brief hypothermic reperfusion after preservation studied by 31P NMR spectroscopy.
Rabbit hearts were subjected to 24-h cold ischaemic storage (at 0 degree-2 degrees C in melting ice) after initial flushing with either St Thomas' cardioplegic solution (STS) or modified lactobionate/raffinose solution (LR), and the status of phosphorylated energy metabolites was measured by 31phosphorus nuclear magnetic resonance (P NMR) spectroscopy. In both groups signals for ATP and phosphocreatine (PCr) were still detectable by 31P NMR after 24 h, and there was significantly more ATP in the LR group (P < 0.01). The hearts were then subjected to coronary reperfusion via an aortic cannula using the same storage solution (either STS or LR) at 6 degrees-8 degrees C, which was oxygenated. In both groups PCr recovered within 30 min of cold reperfusion, and by 60 min PCr was significantly higher in the LR group (P < 0.001). Also, levels of ATP were maintained at higher values during cold reperfusion i the LR group. These studies suggest two important points: (1) the general supply of phosphorylated high-energy intermediates of hearts during cold ischaemic storage is better preserved using LR, and (2) brief cold reperfusion may be used to restore energy metabolism in hearts before re-implantation.
Adenosine Triphosphate↗
Protective properties of amino acids in liver preservation: effects of glycine and a combination of amino acids on anaerobic metabolism and energetics.
BACKGROUND/AIMS/METHODS: In this study, we investigated the hepatoprotective effects of three storage solutions containing glycine (180 mM), glycylglycine (180 mM), and a mixture of 20 amino acids (combined concentration of 180 mM) on energy metabolism and levels of glucose and lactate (as an index of glycolytic flux) in rat livers. All effects were compared to those of livers flushed/stored with a modified University of Wisconsin solution. RESULTS: Glycine-treatment showed no improvement in liver energetics (ATP, ADP, AMP) and lactate accumulation; this solution had the lowest buffering capacity of the four tested (approximately 30% of the University of Wisconsin solution). The glycylglycine solution had the highest buffering capacity of the four solutions tested (including University of Wisconsin solution). Complete titration of the glycine-, combined amino acids-, and University of Wisconsin solutions (from 8.0 to pH = 6.0) resulted in a minor decrease in glycylglycine buffer pH; pH dropped by 0.2 pH units. In glycylglycine-treated livers, energetics showed an improvement over the first 1 h cold storage; ATP and 'energy charge' values remained high and ADP levels (and consequently total adenylate contents) were 0.7-2.4 micro mol/g greater than livers stored in University of Wisconsin solution. A 2-fold increase in lactate accumulation suggested that the improvement in liver energetics for the glycylglycine buffer was due to maintained flux through glycolysis brought about by enhanced buffering capacity. The solution containing a combination of amino acids exhibited maximum maintenance of liver energetics via increased glycolytic flux, despite its slightly inferior buffering capacity (85% of University of Wisconsin solution). ATP levels were maintained over the first 2 h storage and ADP levels (and consequently, total adenylate contents) were 1.2-2.1 micro mol/g greater than University of Wisconsin solution-treated livers during the entire 24 h storage period. Energy charge values for livers treated with the combination of amino acids were also significantly higher than with glycine-, glycylglycine- and University of Wisconsin solution-treatment; even at 24 h, energy charge was 0.36 (comparable to only 4 h storage in University of Wisconsin solution). CONCLUSIONS: Our data suggest that a combination of amino acids may be required for maximum protection of the liver, and furthermore there may be several independent mechanisms, including buffering capacity, responsible for cytoprotection of the liver during cold storage.
Adenosine↗
Restoration of hepatic metabolism by cold reperfusion.
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Adenine Nucleotides↗
Iron chelation, respiratory chain function and tubular necrosis in renal transplantation.
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Animals↗
Vitrification of human oocytes following minimal exposure to cryoprotectants; initial studies on fertilization and embryonic development.
Investigations were made into the low temperature preservation of pre-ovulatory human oocytes by vitrification using a method of brief exposure of the oocytes to the vitrification solution at room temperature. Assessments of morphological survival, fertilization and embryonic development were recorded. All those oocytes exposed to the vitrification solution alone were morphologically normal and 86% of them were fertilized after incubation with spermatozoa. All the fertilized ova (86%) underwent cell division. Following cooling to -196 degrees C, morphological survival (65%) and fertilization (45%) rates remained high. However, in all vitrified oocytes, embryonic cell division and further development were inhibited. From our study it appears that fresh human oocytes can be vitrified using only brief exposure to cryoprotective agents and survive to undergo fertilization. However, progress remains to be made in achieving further embryonic development.
Cell Survival↗
Metabolic considerations of histidine-based solutions for liver storage.
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Adenosine↗
Scanning electron microscopic changes in morphology of pulmonary endothelium in rat lung isografts following hypothermic ischaemic storage and transplantation.
Endothelial monolayer integrity is a critical factor limiting vascular permeability of solid organs in transplantation. Several in vitro, ex vivo and in vivo studies suggest that damage to endothelial cells (EC) due to hypothermia and ischaemia-reperfusion injury causes morphological and functional damage to the endothelium leading to parenchymal oedema and haemorrhage. Aiming to study morphological changes to arterial pulmonary EC subjected to transplantation procedures, random scanning electron micrographs of vascular endothelium of rat lungs were taken. Forty-eight rat lungs were hypothermically stored for 48 or 72 hours in two different preservation solutions and studied either at the end of the cold storage period, or 5 min, 24 h or 4 weeks following transplantation. After 5 minutes of revascularization, micrographs showed EC shape variations, bleb formation and cell retraction with intercellular gap formation. Twenty-four hours after transplantation loss of monolayer continuity was widely extended. Four weeks of revascularization resulted in either well preserved specimens with nearly normal endothelium, or badly preserved arteries with fibrotic degeneration of the luminal vessel wall. The morphological disruptions found in this study help to explain the alterations in permeability control and vascular dysfunction observed in lung transplantation.
Animals↗
Morphological changes in rat single lung isografts after long-term survival.
A high priority in organ transplantation research is to increase the number of hours that an organ can be successfully preserved. Transplant programmes rely on hypothermia and flush solutions to maintain organ viability during the storage period. We studied long-term morphology in lungs stored for 24 or 48 hours using two modified versions of University of Wisconsin solution, one mimicking the extracellular medium and the other the intracellular medium. Four weeks after transplantation, X-ray and angiograms were used to assess the proportion of ventilating tissue, and light and electron microscopy to analyse morphology. Pulmonary tissue presented near-normal histological appearance in well preserved areas while fibrosis and chronic inflammation were found in scarring processes. Electron microscopy studies revealed some damage-related changes in tissue which appeared histologically normal. Four weeks after transplantation, quality and quantity of recovery were uniform for both solutions tested after 24 hours of storage. However, without reaching significance, after 48 hours the quantity of successfully preserved pulmonary tissue was greater in the group stored in the intracellular solution.
Animals↗
Effects of hypothermic storage on the vascular endothelium: a scanning electron microscope study of morphological change in human vein.
OBJECTIVE: The aims of this study were: i) to identify morphological changes occurring in the endothelium of human umbilical veins subjected to the typical storage procedures used in transplantation and ii) to determine the relative efficacy of preservation solutions containing intra and extracellular levels of sodium and potassium. EXPERIMENTAL DESIGN: Prospective. PROCEDURE: Scanning electron micrographs were taken pre and post cold hypoxic storage of human umbilical veins for 3 or 16 hours. RESULTS: Cold preservation resulted in severe cell detachment with subsequent loss of monolayer continuity and exposure of thrombogenic basal membrane components (highly significant after only 3 hours of cold storage, Kruskal-Wallis, p < 0.01). The morphological alterations culminated in EC with spherical shapes. Cytoplasmic membranes presented an increased number of microvilli and intercellular processes, followed by microvillous swelling and surface blebbing as damage increased. Bleb detachment was seen in severely damaged specimens. However, morphological preservation was not significantly affected by the duration of hypoxia or the ionic balance of the solution tested. CONCLUSIONS: The present results demonstrate that even short periods (3 hours) of cold storage without revascularization cause significant morphological damage to the endothelium. The ionic composition of the preservation solution did not significantly affect the process. The morphological changes seen in this study could explain storage-related problems such as loss of normal vascular permeability and increased thrombogenicity, problems often associated with transplantation procedures.
Cryopreservation↗
Inhibition of leukotriene B4 synthesis does not prevent development of acute renal failure following storage and transplantation.
Compound BW B70C, a selective 5-lipoxygenase inhibitor was tested for its ability to reduce inflammatory damage in an in vivo rabbit model of renal storage and transplantation. Kidneys were stored at 0-2 degrees C for 48 hr prior to autografting. In controls, renal vein LTB4 levels rose significantly after 30 min reperfusion but fell after 2 hr to baseline. TxB2 levels remained at baseline for the 6 hr measured. 6-k-PGF1 alpha levels rose significantly after 1 hr of reperfusion and remained elevated thereafter. Histology after 6 hr reperfusion showed moderate-to-severe cortical edema and mild congestion. Infused colloidal carbon was retained in the perivascular area in a narrow band at the corticomedullary junction, indicating a zone of vascular permeability. At 3 days after transplant, kidneys exhibited widespread tubular necrosis and calcification but little inflammation. Serum creatinine and urea peaked between days 3 and 5. 3/6 rabbits showed no symptoms of renal failure after 3 weeks. Pretreatment with BW B70C prevented the increase in LTB4 but had little effect on TxB2 and 6-k-PGF1 alpha levels. Histology showed no amelioration of cortical edema at 6 hr and congestion and hemorrhage were exacerbated. BW B70C had no effect on either colloidal carbon retention or distribution but did significantly reduce tubular necrosis and calcification at day 3. There was very little inflammatory infiltrate. BW B70C treatment did not improve the long-term viability of transplanted kidneys: 2/6 rabbits showed no symptoms of renal failure after 3 weeks. These data indicate that inhibition of LTB4 synthesis by BW B70C does not prevent the development of acute renal failure following 48 hr hypothermic storage and transplantation.
Acute Kidney Injury↗
Evaluation of cold reperfusion as an indicator of viability in stored organs: a 31P NMR study in rat liver.
Rat livers were studied during hypothermic resuscitation perfusion using 31P nuclear magnetic resonance (NMR) spectroscopy as a viability index. Livers were stored for 48 h after being flushed either with a synthetic solution containing plasma-equivalent concentrations of cations, plus citrate and including gelatin polypeptides as colloid (GC solution), or with a modified lactobionate/raffinose [University of Wisconsin (UWC)] solution. After 48 h in either solution, all NMR-detectable ATP plus ADP had disappeared, inorganic phosphate had increased markedly and pH in the livers had become acidotic. During cold reperfusion, ATP was resynthesized, inorganic phosphate declined, and pH returned toward normal values. ATP recovery and decrease in tissue inorganic phosphate were significantly greater (P < 0.02 and P < 0.005, respectively) after 1 h cold reperfusion with the modified UW compared with reperfusion with the GC. 31P NMR spectroscopy was able to detect differences in the metabolic responses of livers stored in different solutions, and coupled with cold reperfusion may be a useful indicator of viability.
Adenosine↗
Glycolysis and energy metabolism in rat liver during warm and cold ischemia: evidence of an activation of the regulatory enzyme phosphofructokinase.
The current study was undertaken so that the effects of both ischemia and ischemia + hypothermia could be examined in mammalian liver. Particular reference was made to the function of glycolysis, which is the only mechanism for energy production under these conditions. The response of adenylate pools reflected the energy imbalance created during warm ischemia within minutes of organ isolation. ATP levels and energy charge values for control (freshly isolated) livers were 1.20 +/- 0.07 and 0.49 +/- 0.02 mumol/g. Within 5 min of warm ischemia, ATP levels had dropped well below control values and by 30 min warm ischemia, ATP, AMP, and E.C. values were 0.21, 2.01, and 0.17 mumol/g, respectively. Cold ischemic livers (flushed with Marshall's citrate solution and stored on ice) exhibited similar, but more protracted, patterns of adenylate depletion (ATP and ADP) and accumulation (AMP). In both warm and cold ischemic livers, levels of fructose-6-phosphate (F6P) and fructose-1,6-bisphosphate (F1,6P2) indicated a marked activation of glycolysis at the phosphofructokinase (PFK) locus after a certain time of ischemia. Although the activations occurred at different times (30 min and 10 h for warm and cold ischemic livers, respectively), the patterns of change in levels of glycolytic metabolites associated with the PFK-catalyzed reaction were similar; levels of F6P dropped and F1,6P2 increased. Changes in metabolite levels (phosphoenol pyruvate and pyruvate) associated with another key suspect regulatory enzyme, pyruvate kinase, indicated no role in regulatory control of glycolysis during warm or cold ischemia. The activation of PFK at 30 min and 10 h of warm and cold ischemia, respectively, may reflect the accumulating effects of loss of intracellular homeostasis, which leads to impending irreversible damage.
Adenine Nucleotides↗
Liver metabolism in cold hypoxia: a comparison of energy metabolism and glycolysis in cold-sensitive and cold-resistant mammals.
The effects of cold hypoxia were examined during a time-course at 2 degrees C on levels of glycolytic metabolites: glycogen, glucose, glucose-1-phosphate, glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-bisphosphate, phosphoenolpyruvate, pyruvate, lactate and energetics (ATP, ADP, AMP) of livers from rats and columbian ground squirrels. Responses of adenylate pools reflected the energy imbalance created during cold hypoxia in both rat and ground squirrel liver within minutes of organ isolation. In rat, ATP levels and energy charge values for freshly isolated livers were 2.54 mumol.g-1 and 0.70, respectively. Within 5 min of cold hypoxia, ATP levels had dropped well below control values and by 8 h storage, ATP, AMP, and energy charge values were 0.21 mumol.g-1, 2.01 mumol.g-1, and 0.17, respectively. In columbian ground squirrels the patterns of rapid ATP depletion and AMP accumulation were similar to those found in rat. In rat liver, enzymatic regulatory control of glycolysis appeared to be extremely sensitive to the decline in cellular energy levels. After 8 h cold hypoxia levels of fructose-6-phosphate decreased and fructose-1,6-bisphosphate increased, thus reflecting an activation of glycolysis at the regulatory step catalysed by phosphofructokinase fructose-1,6-bisphosphatase. Despite an initial increase in flux through glycolysis over the first 2 min (lactate levels increased 3.7 mumol.g-1), further flux through the pathway was not permitted even though glycolysis was activated at the phosphofructokinase/fructose-1,6-bisphosphatase locus at 8 h, since supplies of phosphorylated substrate glucose-1-phosphate or glucose-6-phosphate remained low throughout the duration of the 24-h period.(ABSTRACT TRUNCATED AT 250 WORDS)
Acclimatization↗