V.W.M. van Hinsbergh

Evidence for the presence of two different fibrinolytic inhibitors in human endothelial cell conditioned medium

In human umbilical artery and vein endothelial cell conditioned medium fibrinolytic inhibitors have been detected by two different techniques. A fast-acting inhibitor of tissue-type plasminogen activator (t-PA) and urokinase has been detected and quantified by its capacity to neutralize the above-mentioned plasminogen activators in a kinetic assay. By reverse fibrin autography after SDS-polyacrylamide gel electrophoresis a fibrinolytic inhibitor can be detected with a molecular mass of 52 kDa. The mutual relationship between these two inhibitors was studied. Neutralization of the fast-acting inhibitor by t-PA results in the formation of a complex with a molecular mass of 100 kDa. The t-PA added to endothelial cell conditioned medium in excess of the fast-acting inhibitor is fully stable. However, the inhibitor that is detected by SDS-polyacrylamide gel electrophoresis and reverse fibrin autography is not affected by complete neutralization of the fast-acting inhibitor, and removal of the formed complexes by immune adsorption with immobilized anti-t-PA IgG. This suggests that the inhibitor that is detected by SDS-polyacrylamide gel electrophoresis and reverse fibrin autography does not react with t-PA. Moreover, endothelial cell conditioned medium that is depleted of the fast-acting inhibitor does not show lysis resistance when directly applied to the reverse fibrin autography indicator gel (without previous electrophoresis), although the inhibitor is still present in the zymogram after SDS-polyacrylamide gel electrophoresis. This suggests that the inhibitor is induced by the SDS treatment. Heating the endothelial cell conditioned medium for 15 min at 70 degrees C fully destroys the fast-acting inhibitory activity, but leaves the inhibitor that is detected by SDS-polyacrylamide gel electrophoresis and reverse fibrin autography unaffected. Moreover, at least one additional fibrinolytic inhibitor is detected in the zymogram after SDS-polyacrylamide gel electrophoresis. We conclude that the fast-acting inhibitor is not the same as the inhibitor that is detected by SDS-polyacrylamide gel electrophoresis and reverse fibrin autography; the latter inhibitor is not operational in endothelial cell conditioned medium, but is induced by SDS-polyacrylamide gel electrophoresis.

Incomplete palmitate oxidation in cell-free systems of rat and human muscles

The palmitate oxidation capacity was determined in whole homogenates, postnuclear fractions and mitochondrial fractions of various rat and human muscles and in rat liver, kidney, brain and lung. The oxidation rate (production of 14CO2 and 14C-labeled acid-soluble intermediates) was [1-14C]palmitate greater than [U-14C]palmitate greater than [16-14C]palmitate in all cell-free systems. Oxidation rates were highest in rat heart and liver, intermediate in kidney, diaphragm and m. quadriceps, and low in brain and lung. The capacity of human heart was much lower than that of rat heart and about twice that of human skeletal muscles. Omission of L-carnitine and addition of malonyl-CoA, KCN or antimycin A decreased the oxidation rates in whole homogenates and mitochondrial fractions. Antimycin or KCN increased and malonyl-CoA decreased the ratio of the oxidation rates with [1-14C]- and [16-14C]palmitate. The carnitine concentration had no significant effect on the ratio. 14C-labeled dodecanoic and tetradecanoic acids were identified in homogenates and mitochondrial fractions of m. quadriceps and liver of rat as acid-insoluble intermediates of [16-14C]palmitate oxidation in the presence and absence of antimycin A. Their amounts recovered can account for the differences in oxidation rates found with [1-14C]- and [16-14C]palmitate. The incomplete palmitate oxidation in cell-free systems appears to be mainly caused by an inadequate mitochondrial degradation of peroxisomal oxidation products.

Palmitate oxidation by rat skeletal muscle mitochondria: Comparison of polarographic and radiochemical experiments

Abstract Oxidation of palmitate by rat skeletal muscle mitochondria was determined polarographically and radiochemically under state 3 conditions. Maximal oxidation rate is reached at 4 μ m palmitate, palmitoyl-CoA, or palmitoyl- l -carnitine. At palmitoyl-CoA concentrations higher than 30 μ m oxidation is inhibited. At limiting substrate concentrations as used in polarographic experiments palmitate is totally degraded to CO 2 . At higher concentrations the palmitate molecule is only partially degraded, due to the accumulation of intermediates. Citric acid cycle intermediates, especially 2-oxoglutarate, accumulate during oxidation of palmitate in the presence of malate. It is suggested that this accumulation is stimulated by dicarboxylate exchange. The rate of formation of 14 CO 2 and 14 C-labeled perchloric acid-soluble products is higher from [1- 14 C]palmitate than that from [ U - 14 C]palmitate. This difference, which is enhanced by higher carnitine concentrations indicates incomplete oxidation during the β-oxidation in state 3. The simultaneous determination of 14 CO 2 production and 14 C-labeled perchloric acid-soluble products appears to be a more accurate and sensitive method for measuring 14 C-fatty acid oxidation than that of 14 CO 2 production alone.

An accurate and sensitive assay of long-chain fatty acid oxidation in human skeletal muscle.

Palmitate oxidation rates by mitochondria and homogenates of human skeletal muscle and heart were determined and expressed in relation to protein content and the activities of cytochrome c oxidase and monoamine oxidase. 14CO2 production from [14C]palmitate was very variable, depending on palmitate concentration, label position and the palmitate/albumin ratio in the system. Only 2 to 20% of the palmitate oxidation rate, calculated from the rate of oxygen consumption, was reflected in the rate of 14CO2 production. Estimation of the total of 14CO2 and 14C-labeled perchloric acid-soluble products from [U-14C]palmitate appeared to be a more accurate assay of palmitate oxidation rate, since this value closely agreed with the rate obtained by polarographic estimation. Values obtained by the latter assay expressed per unit of cytochrome c oxidase activity appeared to be less variable than those from 14CO2 production. Because of the reduced variability and greater sensitivity the method may be more favorable for screening the long-chain fatty acid oxidation capacity of muscle of patients with neuromuscular disease.

Role of carnitine in leucine oxidation by mitochondria of rat muscle

Branched-chain amino acids are preferentially oxidized in muscle, in contrast to other amino acids, which are mainly oxidized in liver [ 1 ] . In muscular tissue carnitine concentration is considerably higher than in other tissues, except for kidney [2]. It may therefore be physiologically important that carnitine stimulates the oxidation of branched-chain amino acids and their 0x0 acids in mitochondria and homogenates of rat skeletal muscle [3] and in mitochondria of rat heart [4]. Since the products of branchedchain 0x0 acid decarboxylation are branched-chain acyl-CoA esters, carnitine might be involved in the transport of their acyl-residues across the mitochondrial membrane. On the other hand, carnitine might stimulate branched-chain amino acid oxidation indirectly by removal of long-chain acyl-CoA esters, which are known to be potent inhibitors of various enzymes [5]. In this study we excluded this latter possibility. It is also shown that carnitine stimulates 2-oxoisocaproate oxidation in intact mitochondria of skeletal muscle and heart, but not in those of liver, kidney cortex and brain, nor in broken mitochondria of skeletal muscle. The stimulation is accompanied by an accumulation of isovaleryl-carnitine. It is proposed that carnitine plays a role in leucine oxidation in muscle by removal of isovaleryl-residues out of the mitochondrion, and that these isovaleryl-residues have to be oxidized in other tissues.

Effect of L-carnitine on the oxidation of leucine and valine by rat skeletal muscle.

Abstract An accurate and sensitive method was developed for measuring the oxidation rates of leucine, valine, and α-ketoisocaproate in skeletal muscle homogenates and mitochondria. 14CO2 production from 1-14C-labeled substrates was markedly enhanced by coenzyme A, α-ketoglutarate, and particularly l -carnitine. Thiamine pyrophosphate did not affect oxidation, whereas pyruvate was inhibitory. Carnitine may stimulate oxidation by transporting branched-chain acyl groups out of the mitochondria, by which coenzyme A remains available for oxidative decarboxylation.

Cytochrome c oxidase activity and fatty acid oxidation in various types of human muscle

Cytochrome c oxidase activity, carnitine concentration and oxidation rates of pyruvate and palmitate were determined in homogenates of various types of human skeletal muscle. Cytochrome c oxidase activity appeared to be closely related to the pyruvate oxidation rate, but its correlation with palmitate oxidation was less distinct. Trunk muscles oxidize less palmitate and have a lower cytochrome c oxidase activity per mg homogenate protein than leg muscles; soleus muscle biopsies showed higher activities than those of other leg muscles. Based on cytochrome c oxidase activity no large differences are found in palmitate oxidation rate between various types of human muscle. Cytochrome c oxidase activity and palmitate oxidation rate of muscles do not show an age dependency. The carnitine concentration is similar in all kinds of human skeletal muscle.

Effect of carnitine and branched-chain acylcarnitines on the 2-oxo acid dehydrogenase activity in intact mitochondria of rat muscle.

Abstract 1. 1. Studies were made of the effect of carnitine and branched-chain acylcarnitines on branched-chain 2-oxo acid oxidation by intact mitochondria of rat skeletal muscle. 2. 2. l -carnitine increased the oxidation of the branched-chain 2-oxo acids, derived from leucine and valine, but its effect was markedly dependent on its concentration. Apparent concentrations of l -carnitine which led to half-maximal activation were 186 and 50 μM for oxidation of 4-methyl-2-oxopentanoate and 3-methyl-2-oxobutanoate, respectively. 3. 3. Maximal oxidation rates decreased in the order 3-methyl-2-oxobutanoate > 3-methyl-2-oxopentanoate 4-methyl-2-oxopentanoate in the presence of 2 mM l -carnitine. Half-maximal rates of α-decarboxylation were observed at 0.18mM 3-methyl-2-oxobutanoate and 0.23 mM 3-methyl-2-oxopentanoate. 4. 4. Considerable amounts of branched-chain acylcarnitines accumulated in the incubation system during oxidation of the 2-oxo acids. 5. 5. 3-Methyl-butyrylcarnitine inhibited the mitochondrial decarboxylation of all three branched-chain 2-oxo acids, but had no effect on pyruvate oxidation. 2-Methyl-propionylcarnitine and 2-methyl-butyrylcarnitine had less inhibitory effect. The inhibition by 3-methyl-butyrylcarnitine is competitively counteracted by carnitine. 6. 6. Hexanoyl-, octanoyl- and decanoylcarnitine caused also inhibition of 3-methyl-2-oxobutanoate oxidation in contrast to acetyl- and butyrylcarnitine. 7. 7. Carnitine and 3-methyl-butyrylcarnitine did not or only slightly influence branehed-chain 2-oxo acid oxidation by mitochondria of liver, brain and kidney cortex, but had a marked effect on this process in heart mitochondria. 8. 8. It is suggested that the intramitochondrial ratio of acylcarnitine and acyl-CoA ester affects the activity of the branched-chain 2-oxo acid dehydrogenase for all three branched-chain 2-oxo acids. The release of branched-chain residues from skeletal muscle to be used in liver is discussed.

The active and the inactive plasminogen activator inhibitor from human endothelial cell conditioned medium are immunologically and functionally related to each other

In human endothelial cell conditioned medium a fast-acting inhibitor of tissue-type plasminogen activator and urokinase has been detected. Moreover, an inactive inhibitor of these plasminogen activators is present, that can be activated by denaturing agents such as sodium dodecyl sulphate (SDS). The mutual relationship between these inhibitors was studied. The fast-acting plasminogen activator inhibitor from human endothelial cell conditioned medium was purified in a complex with tissue-type plasminogen activator by immune adsorption, using an immobilized anti-tissue-type plasminogen activator antibody. With the complex as an antigen, specific antibodies were raised against this inhibitor in rabbits. The antiserum immunoreacted with both the inactive and the fast-acting plasminogen activator inhibitor. Endothelial cell conditioned medium (containing the inactive plasminogen activator inhibitor) was treated with SDS and the inhibitory activity that emerged was purified. The SDS-generated product formed complexes with tissue-type plasminogen activator with the same molecular mass as those formed with the fast-acting inhibitor. Moreover, the inhibitory activity generated by SDS treatment showed the same kinetic behaviour with tissue-type plasminogen activator as did the fast-acting inhibitor. These data show that the fast-acting and the inactive plasminogen activator inhibitor are immunologically and functionally related to each other, and probably represent different molecular forms of the same protein.

Degradation of branched-chain amino acids and 2-oxo acids in human and rat muscle

Abstract Studies were made at physiological substrate concentrations on the transamination and oxidative decarboxylation of branched-chain amino acids and on the oxidation of branched-chain 2-oxo acids in homogenates, 600 g supernatants, and mitochondria of various types of human skeletal muscle and of rat m. quadriceps and liver. l -Carnitine increased the oxidation of 4-methyl-2-oxopentanoate and 3-methyl-2-oxobutanoate in mitochondria of human muscle, but to a lesser degree than it did in mitochondria of rat muscle. It had no effect on the oxidation of 3-methyl-2-oxopentanoate. Half-maximal rates of α-decarboxylation of the 2-oxo acids were observed at similar concentrations in muscle mitochondria of man and rat, but the maximal rates were much higher in rat muscle mitochondria. Rates of transamination of valine and leucine were about similar in four types of human muscle, but lower than those in rat m. quadriceps. The oxidation rate of the branched-chain 2-oxo acids appears to vary for the different types of human muscle, but is markedly lower than that in rat muscle. In homogenates of human muscle CO 2 was only produced from 4-methyl-2-oxopentanoate by oxidative decarboxylation and the oxidation appears to proceed no further.