Kunio Tagawa

Correlation between cellular ATP level and bile excretion in the rat liver

The influence of the cellular level of adenosine triphosphate (ATP) in the liver on bile excretion was studied in rats. In ischemia, the cellular ATP level decreased rapidly--and, concomitantly, bile flow stopped within 5 min. Administration of L-ethionine i.p. to rats reduced the bile flow rate with decrease in the cellular ATP level. The correlation between the bile flow rate and the cellular ATP level was confirmed in a liver perfusion system. On anoxic perfusion, the ATP level and bile flow rate changed in the same manner as in ischemia. The recovery rates of both on reoxygenation decreased with increase in the anoxic perfusion period. During perfusion under oxygenated conditions, decrease in cellular ATP to various levels by infusion of various concentrations of potassium cyanide, an inhibitor of respiration, resulted in corresponding and concomitant suppression of bile excretion. Kinetic analysis of the bile flow rate revealed a Michaelis-Menten-type curve for the cellular ATP level. The apparent Kms for ATP of bile flow rate in L-ethionine-treated rat liver and liver perfused with potassium cyanide were 1.0 and 1.6 mM, and their Vmax values were 4.1 and 2.5 microliter/min/g liver, respectively. The concentrations of main bile components, such as phospholipids, cholesterol, and taurocholate increased, but their total outputs decreased with decrease in the ATP level, and returned to the normal range with recovery of the ATP level. Thus, it was shown experimentally that the extent of hepatic injury can be assessed simply by monitoring the bile flow rate, which reflects the cellular level of ATP.

Transport of glutathione across the mitochondrial membranes.

Transport of glutathione (GSH) into mitochondria was observed when mitochondria in state 4 respiration were incubated with high concentrations of GSH. This transport was suppressed by antimycin A or dicyclohexyl-carbodiimide, or in state 3 respiration. Upon dissipation of the proton gradient by a proton ionophore, mitochondrial GSH was released into the medium. GSH moved freely across the proton-permeated mitochondrial membrane, its movement depending only on the GSH gradient across the inner membrane. These results indicate that there is a transport system for GSH in the mitochondrial membrane, and that a proton gradient is necessary to maintain GSH in the matrix, and to transport GSH into mitochondria.

ATTENUATION OF WARM ISCHEMIC INJURY OF RAT LUNG BY INFLATION WITH ROOM AIR-ASSESSMENT OF CELLULAR COMPONENTS AND THE SURFACTANT IN THE BRONCHOALVEOLAR LAVAGE FLUID IN RELATION TO CHANGES IN CELLULAR ADENOSINE TRIPHOSPHATE

Studies were made on the effects in rat lungs of aerobic and anaerobic conditions on the intracellular levels of adenosine triphosphate and its related metabolites, the releases of intracellular enzymes, and the secretion of pulmonary surfactant. After warm ischemia for 120 min, the ATP content of lungs inflated with air was significantly higher (8.0±1.2 μmol/g dry weight) than those of deflated lungs and lungs inflated with nitrogen (0.8±0.7 μmol/g dry weight and 2.0±0.7 μmol/g dry weight, respectively; P<0.001). The amounts of intracellular enzymes, such as lactate dehydrogenase, cyto-solic and mitochondrial aspartate aminotransferase, and protein in the bronchoalveolar lavage fluid (BALF) of air-inflated lungs were significantly less than those in BALFs of deflated and nitrogen-inflated lungs (P<0.001). The BALF-contents of dipalmitoyl phosphatidylcholine (DPPC), the main component of alveolar surfactant of aerobic and anaerobic ischemic lung were, however, similar. During 120-min warm ischemia after lavage, air-inflated lungs secreted significantly more DPPC into the alveolar space than nitrogen-inflated lungs did (P<0.001). We conclude that cell membranes in the lungs are damaged under anaerobic conditions, but that inflation of ischemic lungs with air is effective for protecting them from cell injury and for maintaining the intracellular level of ATP and the ability of the cells to secrete pulmonary surfactant.

Regulatory proteins of F1F0-ATPase: Role of ATPase inhibitor

An intrinsic ATPase inhibitor inhibits the ATP-hydrolyzing activity of mitochondrial F1F0-ATPase and is released from its binding site on the enzyme upon energization of mitochondrial membranes to allow phosphorylation of ADP. The mitochondrial activity to synthesize ATP is not influenced by the absence of the inhibitor protein. The enzyme activity to hydrolyze ATP is induced by dissipation of the membrane potential in the absence of the inhibitor. Thus, the inhibitor is not responsible for oxidative phosphorylation, but acts only to inhibit ATP hydrolysis by F1F0-ATPase upon deenergization of mitochondrial membranes. The inhibitor protein forms a regulatory complex with two stabilizing factors, 9K and 15K proteins, which facilitate the binding of the inhibitor to F1F0-ATPase and stabilize the resultant inactivated enzyme. The 9K protein, having a sequence very similar to the inhibitor, binds directly to F1 in a manner similar to the inhibitor. The 15K protein binds to the F0 part and holds the inhibitor and the 9K protein on F1F0-ATPase even when one of them is detached from the F1 part.

Levels of purine compounds in a perfusate as a biochemical marker of ischemic injury of cold-preserved liver.

Biochemical markers of ischemic injury of rat liver were studied in an extracorporeal perfusion system. During anoxic perfusion, purine compounds appeared in the perfusate as soon as they were formed in the liver and their recovery in the perfusate balanced the loss of adenine nucleotides from the liver. In contrast, cytosolic aspartate aminotransferase did not appear in the perfusate at slow rates of liver perfusion or during hypothermic perfusion. The production of purine compounds was further investigated in hypothermically preserved liver in connection with the restoration of some metabolic functions of liver. The amount of purine compounds released into the perfusate was found to be closely related to the degrees of damage of the hepatic functions of gluconeogenesis, ureogenesis, and mitochondrial respiration on reperfusion. These results indicate that release of purine compounds into the perfusate is a good marker of ischemic damage.

Peroxidative injury of the mitochondrial respiratory chain during reperfusion of hypothermic rat liver.

Mitochondrial dysfunction in ischemic liver has been demonstrated to be due to decrease in the intramitochondrial level of ATP and the subsequent disruption of the proton barrier of the inner membrane (Watanabe, F., Hashimoto, T. and Tagawa, K. (1985) J. Biochem. 97, 1229-1234). In this study, another injury process, impairment of the electron-transfer system, which occurred during reoxygenation of ischemic liver, was studied during reperfusion of cold preserved liver and during cold incubation of isolated rat-liver mitochondria. The sites of the respiratory chain that were sensitive to peroxidative damage were ubiquinone-cytochrome c oxidoreductase and NADH-ubiquinone oxidoreductase. These enzymic activities decreased with increase in lipid peroxidation. Incubation of submitochondrial particles with t-butyl hydroperoxide or with an NADPH-dependent peroxidation system decreased the enzymic activities of the electron-transport system. These data strongly suggested that lipid peroxidation during reoxygenation of ischemic liver impaired the electron-transfer system. Thus, mitochondria of ischemic liver suffer from two different types of injury: increase in proton permeability during anoxia, and decrease in enzymic activities of the electron-transport system during reoxygenation.

Ca2+-induced, phospholipase-independent injury during reoxygenation of anoxic mitochondria

Reoxygenation of rat-liver mitochondria after anoxic incubation induced release of matrix proteins. As assessed by release of a matrix enzyme, it was proportional to the rate of H2O2 production. The release was not observed with low concentrations of extramitochondrial free Ca2+, indicating a Ca(2+)-dependent pathway. Phospholipase A2 was not involved in the reoxygenation injury, because non-esterified fatty acids did not increase on reoxygenation even when re-acylation was inhibited and because inhibitors of phospholipase A2 had little effect on enzyme release. Cyclosporin A, ATP, ADP and inhibitors of pyridine nucleotide oxidation had a protective effect, strongly suggesting involvement of so-called Ca(2+)-dependent permeability transition. Ca2+ was also released from reoxygenated mitochondria and inhibition of reuptake of released Ca2+ attenuated the enzyme release. Similar releases of aspartate aminotransferase and Ca2+ were observed with mitochondria in an oxygen radical-generating system, hypoxanthine and xanthine oxidase. In this system, lecithin-cardiolipin liposomes also released entrapped Ca2+ without disruption of the membrane. From these results, we conclude that during reoxygenation, Ca2+ release and subsequent reuptake induced permeability transition of mitochondria, resulting in reoxygenation injury.

Enzyme release from mitochondria during reoxygenation of rat liver

Reoxygenation-induced release of mitochondrial aspartate aminotransferase (mAST) into the cytosol was studied using perfused rat liver. As the absolute activity of mAST in the perfusate did not indicate the degree of mitochondrial enzyme release, the following 3 methods were applied: measurement of the mAST to total AST ratio in the efferent perfusate, the digitonin infusion method, and measurement of mAST activity in the cytosolic compartment isolated from perfused livers. The results by all 3 methods were consistent and showed that mitochondrial injury occurs on reoxygenation. The mitochondrial Ca2+ content was proportional to the extent of mAST release during reoxygenation, indicating involvement of Ca2+ in the enzyme release. CsA, a potent inhibitor of Ca(2+)-induced increase in permeability of the mitochondrial membrane, completely prevented mAST release on reoxygenation. We conclude that during reoxygenation of hypoxic liver, mAST leaks into the cytosol in a Ca(2+)-dependent, CsA-sensitive manner.

Protection of cellular and mitochondrial functions against anoxic damage by fructose in perfused liver

In anoxic perfused liver, conversion of fructose to lactate was greatly increased to about 3 mumol/min per g liver. This increase in lactate implied that the same amount of ATP was also produced. The rate of metabolism of glucose was less than 10% of that of fructose, as judged by rate of production of lactate. In anoxic liver perfused with fructose, the ATP levels of both the tissue and mitochondria remained high, despite lack of oxygen, thus preventing enzyme leakage and preserving processes requiring ATP, such as bile excretion and urea formation. The mitochondrial oxidative phosphorylation capacity of anoxic liver perfused with fructose was also unimpaired. Spectral analysis of light transmitted through the liver revealed that the mitochondrial electron transfer system was in the completely reduced state during anoxia, indicating that the mitochondria were incapable of synthesizing ATP. These results suggest that fructose metabolism during anoxia resulted in sufficient production of ATP for maintaining the physiological functions of the cells and the oxidative phosphorylation capacity of their mitochondria.

Different patterns of leakage of cytosolic and mitochondrial enzymes

The mechanisms of leakage of intracellular enzymes, and especially the cytosolic and mitochondrial isozymes of aspartate aminotransferase (AST), in ischemic rat liver were studied. On recirculation of ischemic liver, cytosolic AST (cAST) promptly appeared in the blood. Release of cytosolic enzymes, including cAST and lactic dehydrogenase, resulted from disruption of blebs that protruded from parenchymal cells into the sinusoidal space. When these blebs were formed in ischemic liver, mitochondria still remained in core regions of the injured cells and were not found in the blebs. Consistent with this fact, mitochondrial AST (mAST) did not leak into the circulation from ischemic liver until most of the cAST had leaked out. This delayed leakage of mitochondrial enzymes was also consistent with the fact that the mitochondrial membranes maintained a diffusion barrier against matrix enzymes even after anoxia for 2 h, when their oxidative phosphorylation capacity had been lost. These results indicate that mitochondrial enzymes are liberated into the blood only after appreciable disintegration of the cells, probably necrosis, and that the cumulative activity of mAST in the blood should reflect the extent of necrosis in ischemic organs better than that of cAST.