The effect of new proteasome inhibitors, belactosin A and C, on protein metabolism in isolated rat skeletal muscle.

The proteasome inhibitors are used as research tools to study of the ATP-dependent ubiquitin-proteasome system. Some of them are at present undergoing clinical trials to be used as therapeutic agents for cancer or inflammation. These diseases are often accompanied by muscle wasting. We herein demonstrate findings about new proteasome inhibitors, belactosin A and C, and their direct effect on protein metabolism in rat skeletal muscle. M. soleus (SOL) and m. extensor digitorum longus (EDL) were dissected from both legs of male rats (40-60 g) and incubated in a buffer containing belactosin A or C (30 microM) or no inhibitor. The release of amino acids into the medium was estimated using high performance liquid chromatography to calculate total and myofibrillar proteolysis. Chymotrypsin-like activity (CTLA) of proteasome and cathepsin B, L activity were determined by fluorometric assay. Protein synthesis and leucine oxidation were detected using specific activity of L-[1-14C] leucine added to medium. Inhibited and control muscles from the same rat were compared using paired t-test. The results indicate that after incubation with both belactosin A and C total proteolysis and CTLA of proteasome decreased while cathepsin B, L activity did not change in both SOL and EDL. Leucine oxidation was significantly enhanced in SOL, protein synthesis decreased in EDL. Myofibrillar proteolysis was reduced in both muscles in the presence of belactosin A only. In summary, belactosin A and C affected basic parameters of protein metabolism in rat skeletal muscle. The response was both muscle- and belactosin-type-dependent.

The effect of new proteasome inhibitors, belactosin A and C, on protein metabolism in isolated rat skeletal muscle

The proteasome inhibitors are used as research tools to study of the ATP-dependentubiquitin-proteasome system. Some of them are at present undergoing clinicaltrials to be used as therapeutic agents for cancer or inflammation. These diseases areoften accompanied by muscle wasting. We herein demonstrate findings about newproteasome inhibitors, belactosin A and C, and their direct effect on protein metabolismin rat skeletal muscle. M. soleus (SOL) and m. extensor digitorum longus(EDL) were dissected from both legs of male rats (40-60g) and incubated in a buffercontaining belactosin A or C (30 ìM) or no inhibitor. The release of amino acids intothe medium was estimated using high performance liquid chromatography to calculatetotal and myofibrillar proteolysis. Chymotrypsin-like activity (CTLA) of proteasomeand cathepsin B, L activity were determined by fluorometric assay. Proteinsynthesis and leucine oxidation were detected using specific activity of L-[1-14C]leucine added to medium. Inhibited and control muscles from the same rat were comparedusing paired t-test. The results indicate that after incubation with both belactosinA and C total proteolysis and CTLA of proteasome decreased while cathepsinB, L activity did not change in both SOL and EDL. Leucine oxidation was significantlyenhanced in SOL, protein synthesis decreased in EDL. Myofibrillar proteolysiswas reduced in both muscles in the presence of belactosin A only. In summary,belactosin A and C affected basic parameters of protein metabolism in rat skeletalmuscle. The response was both muscle- and belactosin-type-dependent(AU)

Direct effects of proteasome inhibitor AdaAhx3L3VS on protein and amino acid metabolism in rat skeletal muscle.

Proteasome inhibitors are novel potential drugs for therapy of many diseases, and their effects are not fully understood. We investigated direct effects of peptide vinylsulfone inhibitor AdaAhx3L3VS on protein and amino acids metabolism in rat skeletal muscle. Soleus and extensor digitorum longus muscles were incubated in a medium containing 30 micromol/l AdaAhx3L3VS or no inhibitors. Total proteolysis was determined according to the rates of tyrosine release into the medium during incubation. The rates of leucine oxidation and protein synthesis were evaluated during incubation in medium containing L-[1-14C]leucine. Amino acid concentrations in the medium were measured using HPLC. AdaAhx3L3VS decreased tyrosine release into the medium by 21 and 19 %, decreased leucine incorporation into proteins by 22 and 12 %, and increased leucine oxidation by 24 and 19 % in soleus and extensor digitorum longus muscles, respectively. The release of amino acids into the medium was reduced. We conclude that AdaAhx3L3VS significantly decreased proteolysis and protein synthesis and increased leucine oxidation.

[Reperfusion injury in the isolated rat liver after hypothermic preservation]. / Reperfuzní poskození izolovaných jater potkanu po hypotermním skladování.

Histological changes which appear as a result of reperfusion injury of cold-preserved rat liver were studied at intervals of 0 hr, 3 hr, 24 hr and 48 hr of cold storage. The isolated livers were stored in a UW solution (University of Wisconsin), which is used in human liver transplantations. Computer image analysis of light microscopic sections (methyl green-pyronin stained) was used for the study and quantification of injured cells. The method of TUNEL was performed to prove possible apoptosis of sinusoidal endothelial cells and heptocytes. Bile production during reperfusion and ALT, AST, LDH and ACP were measured in the reperfusion medium at the end of the 90 min reperfusion. It has been confirmed that prolongation of the cold storage of liver results in extensive changes in the liver structure and increased injury of liver cells. Sinusoidal endothelial cells were damaged more and earlier than hepatocytes. It has been shown that methyl green-pyronin stained sections are advantageous for the study of these morphological changes, allowing the strongest view of these changes. The appearance of TUNEL positive cells and an increase in the levels of biochemical parameters, e.g. AST or ALT, indicate earlier cell injury. The methodology described in this article can be used for the study of reperfusion injury of the liver and for the study of this phenomenon in other experiments.

Leucine and protein metabolism in rats with chronic renal insufficiency.

The aim of this study was to evaluate the effect of chronic uremia induced by 5/6 nephrectomy (5/6NX) on changes in protein and branched-chain amino acid (BCAA; valine, leucine and isoleucine) metabolism. The control group consisted of sham operated rats. Twenty eight weeks after surgery the parameters of protein and amino acid metabolism were evaluated using a primed constant intravenous infusion of L-[1-(14)C]leucine. A drop in BCAA levels and a significant increase in urea, creatinine and cholesterol were observed in plasma of all 5/6NX rats. However, severe uremia with acidosis developed only in one third of rats with 5/6NX. In 5/6NX rats with acidosis significant increases in proteolysis, leucine oxidation, leucine oxidized fraction, and leucine clearance were observed in comparison with the control group and rats with 5/6NX without acidosis. In addition, in 5/6NX rats with acidosis a significant decrease in valine concentration in gastrocnemius muscle was found. We conclude that marked activation of proteolysis occurs in severe chronic renal failure and is probably caused by metabolic changes related to acidosis development.

Metabolism of branched-chain amino acids in starved rats: the role of hepatic tissue.

Parameters of branched-chain amino acids (BCAA; leucine, isoleucine and valine) and protein metabolism were evaluated using L-[1-(14)C]leucine and alpha-keto[1-(14)C]isocaproate (KIC) in the whole body and in isolated perfused liver (IPL) of rats fed ad libitum or starved for 3 days. Starvation caused a significant increase in plasma BCAA levels and a decrease in leucine appearance from proteolysis, leucine incorporation into body proteins, leucine oxidation, leucine-oxidized fraction, and leucine clearance. Protein synthesis decreased significantly in skeletal muscle and the liver. There were no significant differences in leucine and KIC oxidation by IPL. In starved animals, a significant increase in net release of BCAA and tyrosine by IPL was observed, while the effect on other amino acids was non-significant. We conclude that the protein-sparing phase of uncomplicated starvation is associated with decreased whole-body proteolysis, protein synthesis, branched-chain amino acid (BCAA) oxidation, and BCAA clearance. The increase in plasma BCAA levels in starved animals results in part from decreased BCAA catabolism, particularly in heart and skeletal muscles, and from a net release of BCAA by the hepatic tissue.

Leucine and protein metabolism after bilateral nephrectomy in rats: the role of hepatic tissue.

The aim of this study was to evaluate the effect of acute uremia on changes in leucine and protein metabolism in the whole body and in hepatic tissue. Acute renal insufficiency was induced by bilateral nephrectomy (BNX). Twenty-four hours later, parameters of protein and amino acid metabolism were evaluated in the whole body using primed constant intravenous infusion of L-[1-14C]leucine, and in isolated perfused liver (IPL) using cx-keto[1-14C]isocaproate. The control group consisted of sham-operated rats. BNX induced a marked decrease in proteolysis, protein synthesis, leucine oxidized fraction and leucine clearance. The decrease in protein synthesis was higher than in proteolysis. A significant drop in protein synthesis was observed in muscle, gut, heart and spleen. The study with IPL in BNX animals showed decreased oxidation of ketoisocaproic acid and higher concentrations of the branched-chain amino acids (BCAA) leucine, isoleucine and valine in perfusion solution. We conclude that the cause of rapid depletion of body proteins after BNX is a greater decrease in protein synthesis than in proteolysis associated with an increase in leucine oxidized fraction. The data obtained in the IPL model indicate that BNX causes metabolic changes that enable resynthesis of BCAA from corresponding branched-chain keto acids in liver.

Leucine metabolism in partially hepatectomized rats.

BACKGROUND/AIMS: We hypothesized that the decrease in plasma branched-chain amino acids (i.e. valine, leucine and isoleucine) and the increase in the oxidized leucine fraction demonstrated in cirrhotic rats in our previous study were caused by the reduced liver cell mass. In the present study we have evaluated the influence of the loss of a substantial amount of the hepatic tissue on changes in leucine metabolism. METHODS: A two-thirds partial hepatectomy (PH) was performed in male Wistar rats, weighing 210-250 g. Sham-operated rats served as controls. Whole-body leucine kinetics and ketoisocaproate oxidation rates in the isolated perfused liver were investigated using continuous infusion of [1-14C]leucine and alpha-keto[1-14C]ketoisocaproate at 0 h, 24 h and 72 h after surgery. All groups were compared by analysis of variance, and differences were considered significant at the p < 0.05 level. RESULTS: A significant decrease in the sum of branched-chain amino acids in blood plasma was observed at 24 h after PH. The decrease in whole-body leucine utilization in protein synthesis observed at 24 h after PH was associated with a decrease in protein synthesis in the gastrocnemius muscle, in the small intestine and in the liver remnant (although protein synthesis per mg of liver protein was higher than in sham-treated animals). In contrast, the rate of whole-body leucine oxidation increased immediately after PH (PH: 4.5 +/- 0.7 vs. sham: 2.4 +/- 0.4; mumol .100 g b.w.-1.h-1). As a result of the opposite changes in protein synthesis and leucine oxidation, marked increases in oxidized leucine fraction were observed immediately (14.6 +/- 1.5%) and 24 h (15.1 +/- 1.6%) after PH in comparison to the sham-treated rats (7.1 +/- 0.8%). In isolated perfused livers of PH rats, an increase in ketoisocaproate oxidation per liver weight unit was observed at 24 h and 72 h in comparison to the sham group. The loss of liver capacity for ketoisocaproate oxidation was restored at 72 h after PH, although the liver weight did not reach the preoperative value. CONCLUSIONS: We conclude that the loss of hepatic tissue results in an increase in leucine oxidized fraction that is caused by both a decrease in protein synthesis and an increase in leucine oxidation. Both the liver remnant and the extrahepatic tissues are involved in this response.

Effect of supplementation of University of Wisconsin solution with glycoproteins from psychrophilic strains of yeast on hypothermic liver storage of rats.

Extracellular yeast glycoproteins (YG) produced by Rhodosporidium toruloides have been shown to increase the survival rate of different yeast species after storage in liquid nitrogen. The purpose of this study was to investigate the effect of YG on cold-stored rat livers. Water-soluble YG produced either by Phaffia rhodozyma (G3) or by Leucosporidium antarcticum (G4) were added to a modified University of Wisconsin solution (mUW) and used for cold storage (1 degree C) of isolated livers. The functional status of each liver was then assessed under conditions of 90-min normothermic reperfusion. The 46-h cold storage in mUW without G3 and G4 resulted in serious preservation-reperfusion injury of the liver. The addition of G3 to mUW for 46-h preservation of the liver resulted in significantly higher bile flow (4.32 +/- 0.35 vs 2.35 +/- 0.49 microliters/min/10 g at 75-90 min), higher portal blood flow (10.99 +/- 0.2 vs 4.78 +/- 1.07 ml/min/g at 90 min), lower liver weight after reperfusion (102.4 +/- 1.5 vs 116.7 +/- 6.6% of weight before preservation), and lower total tissue water after reperfusion (2.49 +/- 0.05 vs 2.92 +/- 0.13 g water/g dry weight). However, the activity of ALT, AST, and LDH in perfusate was not changed. The beneficial effect of G4 was less pronounced. The 24-h storage in mUW resulted in a significant increase of AST and LDH activity in perfusate; the addition of G3 to mUW for 24-h preservation did not affect these parameters. In conclusion, the addition of 0.05% G3 or G4 to mUW was only partially beneficial in improving rat liver preservation.

Leucine metabolism in rats with cirrhosis.

BACKGROUND/AIMS: This study aimed to investigate the pathogenesis of reduced plasma levels of branched-chain amino acids leucine, isoleucine and valine in cirrhosis. METHODS: Cirrhosis was induced by intragastric administration of 36 doses of carbon tetrachloride in olive oil over a period of 12 weeks. Rats treated with oil alone served as controls. The rates of leucine turnover, clearance, oxidation and incorporation into proteins were evaluated using [1-14C]leucine, [4,5-3H]leucine and alpha-keto[1-14C]isocaproate 3 days after the last intragastric treatment in vivo and in the isolated perfused liver. RESULTS: In animals with cirrhosis we observed a profound fall in plasma branched-chain amino acid levels and significant decreases in leucine turnover, oxidation and incorporation into tissue proteins. A more pronounced fall in leucine incorporation in proteins resulted in a significant increase in the oxidized leucine fraction in rats with cirrhosis as compared to controls. Leucine clearance was higher in the cirrhosis group. Concomitant to the fall of whole body leucine turnover, decreases of leucine incorporation into protein and of ketoisocaproic acid decarboxylation were observed in the isolated perfused liver of rats with cirrhosis. However, leucine oxidation was increased compared with control rats. CONCLUSIONS: Our results indicate that the predominant mechanism of the decrease in plasma leucine levels in rats with cirrhosis is an increase in the oxidized leucine fraction associated with a decrease in leucine turnover. An increase in leucine oxidation in the cirrhotic liver is one of the mechanisms involved.

Plasma amino acids in four models of experimental liver injury in rats.

We studied the plasma amino acid profiles in four models of hepatic injury in rats. In partially hepatectomized rats (65% of liver was removed) we observed significant increase of aromatic amino acids (AAA; i.e. tyrosine and phenylalanine), taurine, aspartate, threonine, serine, asparagine, methionine, ornithine and histidine. Branched-chain amino acids (BCAA; i.e. valine, leucine and isoleucine) concentrations were unchanged. In ischemic and carbon tetrachloride acute liver damage we observed extreme elevation of most of amino acids (BCAA included) and very low concentration of arginine. In carbon tetrachloride induced liver cirrhosis we observed increased levels of AAA, aspartate, asparagine, methionine, ornithine and histidine and decrease of BCAA, threonine and cystine. BCAA/AAA ratio decreased significantly in partially hepatectomized and cirrhotic rats and was unchanged in ischemic and acute carbon tetrachloride liver damage. We conclude that a high increase of most of amino acids is characteristic of fulminant hepatic necrosis; decreased BCAA/AAA ratio is characteristic of liver cirrhosis; and decrease of BCAA/AAA ratio may not be used as an indicator of the severity of hepatic parenchymal damage.

The effect of pretreatment with prostaglandin E2 (PGE2), verapamil, nicergoline and bromocriptine on ischemia-reperfusion injury of rat liver in vivo.

Ergot alkaloids possess some properties potentially beneficial in ischemia of organs. Therefore the effect of pretreatment by nicergoline and bromocriptine was established in ischemia-reperfusion injury of rat liver. PGE2 and verapamil were used as comparative agents. Hepatic ischemia (60 min) of anesthetized rats was induced by clamping of vessels supplying the median and left lateral lobe. Tested drugs were given i.v. 2 or 5 min prior to inducing ischemia. ALT and AST activities in serum two hours after the end of ischemia were used as markers of hepatocellular injury. Only PGE2 (0.1 mg.kg-1) pretreatment minimized the postischemic rise of both ALT and AST activities. Pretreatment with various doses of nicergoline (1 or 4 mg.kg-1), bromocriptine (1 or 4 mg.kg-1) and verapamil (0.9 or 4.5 mg.kg-1) did not influence significantly serum transaminases activities after ischemia. Bromocriptine (4 mg.kg-1) given together with PGE2 did not improve a protective effect against ischemia achieved by the administration of PGE2 (0.1 microgram.kg-1).

Influence of temperature on the quality of hypothermic preservation of isolated rat livers.

The function normothermally perfused isolated liver will be determined in our future experiments for the assessment of the quality of previous hypothermic preservation performed by means of various preservation solutions. As the opinions on the optimal storage temperature of organs differ, the aim of the present work was to find a proper storage temperature which would be suitable for our experiments. A comparison of liver preservation using simplified UW solution (UWs) at various temperatures was performed. During the isolation the liver was flushed by 50 ml of UWs through the portal vein and stored for 24 h either at 0 degree or 10 degrees C. The isolated liver was then perfused in a recirculation manner at 37 degrees C. The livers that were stored at 0 degree C showed only mild injury resulting in a slower bile flow rate in comparison with the control group without preservation; the results were only marginally significantly different. The livers that were stored at 10 degrees C showed more pronounced injury (higher portal resistance, higher ALT and AST activity in perfusate, higher water content in livers and slower bile flow) in comparison with other groups. Storage temperature of 0 degree C seems to be more convenient for our future experiments.

The effect of metipranolol and inosine on total hepatic ischemia of rats in vivo.

A protective effect of metipranolol on renal ischemia has recently been demonstrated in our laboratory. The aim of present work was to investigate the effect of this drug on a model of total hepatic ischemia. Inosine was chosen as a comparative agent. Metipranolol (1 mg.kg-1) or inosine (160 mg.kg-1) were given i.v. to rats 15 min prior to inducing of 30-min lasting hepatic ischemia. The animals were followed up for 90 min after the end of ischemia. Pretreatment with inosine almost removed the harmful effect of ischemia on bile flow. Pretreatment with metipranolol slightly minimized the post-ischemic bile flow fall, this effect having been statistically significant only at 30. min of postischemic period. Neither inosine, nor metipranolol administration influenced significantly the ALT or AST plasma activity 90 min after release of hepatic vessels occlusion.

A beneficial effect of metipranolol pretreatment on rat kidney ischemia in vivo.

D,L-propranolol protects kidney against warm ischemia. On the contrary beta-blockers without membrane stabilizing activity (timolol, L-propranolol) do not possess this protective ability. The aim of the present work was to test the potential anti-ischemic properties of metipranolol, another beta-blocker with membrane stabilizing component. Metipranolol (1 mg/kg) was given 30 min prior to inducing the ischemia of left rat kidney followed with a contralateral nephrectomy. This pretreatment lowered the creatinemia as measured 24 hr after 60 min ischemia and improved the survival of rats after 90 min renal ischemia. The results are compatible with the concept that the membrane stabilizing activity may play a crucial role in the protective action of beta-blockers.

[Kinetics of gentamycin in premature infants with a low birth weight during the first four days of postnatal life]. / Kinetika gentamycinu u nedonosených detí s nízkou porodní hmotností v prubehu prvních ctyr dnu postnatálního zivota.

The authors investigated in a group of 19 premature neonates with a low birthweight (0.65-2.1 kg) during the first four days of postnatal life the pharmacokinetics of gentamycin after indicated administration of 2 mg/kg of the antibiotic by the i.v. route by a 30-minute infusion in 18-hour intervals. Serum concentrations of gentamycin were assessed by immunoassay 0.5 hours before administration and then 0.5, 5.5, 11.5 and 17.5 hours after the 5th infusion, i.e. in a steady state. The maximum serum concentrations detected 0.5 hours after the completed infusion exceeded 10 mg/l in 21% of the neonates, while the minimal concentrations of the antibiotic before the next administration were above 2 mg/l in 42% of the infants. The calculation of pharmacokinetic parameters according to the one-compartment model revealed considerable interindividual differences of all values. The authors consider particularly important the low clearance value of the antibiotic (30.24 +/- 14.55 ml/kg.hour-1) which may lead to cumulation of gentamycin and its toxic action. Gentamycin administration to premature neonates should be associated with monitoring of serum concentrations of the drug which would make individual adjustment of the dosage possible.

Disappearance of radioactivity from perfusate of isolated rat liver after administration of different doses of tritiated DH-ergotoxine.

In ergot alkaloids a disproportion between the size of the peroral dose and the achieved area under the curve concentrations was described. This process can be explained by nonlinearity in the absorption, distribution or elimination of alkaloids. The aim of the present paper is to find whether elimination of tritiated DH-ergotoxine (3HDHE) in the liver is a linear, dose-independent process. Therefore on the model of the isolated rat liver disappearance of radioactivity in perfusate after the administration of two doses of 3HDHE, viz. 60 ng g-1 of the liver and 3030 ng g-1 of the liver, was investigated. The disappearance curves of radioactivity expressed as the percentage of the administered dose did not significantly differ between both groups. No significant changes between the groups were found either in the size of pharmacokinetic parameters, or in the portion of the administered radioactivity excreted in bile. Therefore the present authors think that disappearance of radioactivity in perfusate of the isolated liver after administration of 3HDHE is a linear process following first-order kinetics.

A study of the inhibition of adrenaline-induced vasoconstriction in the isolated perfused liver of rabbit.

We have studied the action of a series of vasoactive and antispasmodic agents on the intrahepatic vasoconstriction induced by adrenaline in the isolated perfused liver of rabbits. The arterial and portal venous resistance, oxygen consumption, liver weight and bile flow were investigated. The drugs used were as follows: nonspecific alpha-adrenergic antagonists (DH-ergocristine, dibenamine, phenoxybenzamine), vasodilators with a direct miscellaneous action (theophylline, papaverine, dipyridamole, glucagon, Aiu-cor by Instituto Gentilli, Italy [inosine, ATP, IPI, UTP]) and antispasmodics (piperylone, tropenziline, noraminophenazone). Adrenaline increased arterial and portal venous resistance followed by a diminution of oxygen consumption, liver weight and bile flow. alpha-Adrenergic antagonists inhibited the effects of adrenaline on portal venous resistance and oxygen consumption and especially the effects on hepatic arterial resistance. The most potent agent was phenoxybenzamine. In contrast to alpha-adrenoceptor blockade, the effects of other vasoactive agents were without a sustained influence on hepatic arterial resistance (excepting those of glucagon and dipyridamole). Some of them were effective as antagonists on responses in the portal venous bed (papaverine, Aiu-cor). Moreover, there were drugs exerting an enhancement of the vasoconstrictor responses of hepatic artery to low concentrations of adrenaline with no effect on the portal venous bed (piperylone, tropenziline). Theophylline and noraminophenazone exerted no effect either on the arterial or portal venous bed. No vasodilator agent antagonized the changes of the bile flow after adrenaline administration.

Effect of palmitoyl-DL-carnitine on the 3H-dihydroergotoxine intestinal absorption in rat.

Using the methods of rat intestine perfusion in situ and kinetics examination in vivo, absorption of 3H-dihydroergotoxine (3HDHE) from the gastrointestinal tract into systemic blood was investigated. The aim of the study was to increase absorption of the ergot alkaloid with palmitoyl-DL-carnitine (5 mg/rat). Low absorption of 3HDHE was demonstrated in contrast to fast and almost complete absorption of the model drug theophylline (5 mg kg-1). In the experiment this was evidenced by in situ continuous measurement of the administered activity (125 micrograms kg-1) into a reservoir. At the end of the experiment (120 min) the plasma activity reached 0.034 +/- 0.014%, retention in the intestine achieved more than 60%, uptake in the brain 0.044 +/- 0.015%, cumulative excretion in bile 1.50 +/- 0.31% of administered activity. Palmitoyl-DL-carnitine did not influenced the percentage of activity in the plasma and did not affect bile excretion, retention in the intestine and uptake in the brain. In the vivo experiment oral administration of 3HDHE into the stomach (222 micrograms kg-1) increased activity in plasma (0.069 +/- 0.020% within 24 h), cumulative excretion in urine was 6.1 +/- 3.6%, retention of the drug in the stomach and intestine 46 +/- 12%, activity of the brain 0.11 +/- 0.02%, in the kidney 0.39 +/- 0.17%, and in the liver 0.80 +/- 0.30%. After palmitoyl-DL-carnitine administration the activity of plasma reached 0.084 +/- 0.022% (NS), retention in gastrointestinal tract 39 +/- 9% (NS), activity in the liver 0.71 +/- 0.17% (NS), and activity in the kidney 0.42 +/- 0.13% (NS).(ABSTRACT TRUNCATED AT 250 WORDS)