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W Halangk

Publications and source records attributed to W Halangk.

At least 37 records · Page 2Linked to original sources
PubMed1998

Energy metabolism in rat pancreatic acinar cells during anoxia and reoxygenation.

Mitochondrial energy metabolism was studied in isolated pancreatic acinar cells during anoxia up to 90 min and reoxygenation for 60 min. To identify critical alterations leading to the known postanoxic impairments in structure and function of acinar cells, adenine nucleotide levels and the rates of phosphorylating and non-phosphorylating respiration were determined. ATP levels and the total amount of adenine nucleotides strongly decreased as early as after 30 min of anoxia. The cells partially restored ATP and the total adenine nucleotides within 60 min of reoxygenation. Long-term anoxia caused an increase in the oligomycin-insensitive part of oxygen consumption. The respiratory capacity measured as uncoupled respiration progressively declined to 40% of controls after 90 min of anoxia. Fluorescence measurements showed that flavoproteins and mitochondrial pyridine nucleotides in reoxygenated cells after short-term anoxia were in a more reduced state than in aerobic controls, and were not fully oxidizable by uncoupling. It is concluded that long-term anoxia produces an irreversible loss of respiratory capacity leading to a limited ATP production. This functional impairment and the progressive damage to acinar cells may be relevant for pancreatic injuries such as acute pancreatitis or post-transplantation pancreatitis.

Adenine Nucleotides↗
PubMed1998

Occurrence of oxidatively modified proteins: an early event in experimental acute pancreatitis.

Free radical-mediated injury is believed to play a key role in the pathogenesis of acute pancreatitis (AP). Therefore, oxidative damage of proteins may be an important event in the development of AP. The present study was performed to investigate oxidative protein modification, quantified as 2,4-dinitrophenylhydrazine-reactive protein-carbonyls, during the time course of taurocholate-induced pancreatitis of the rat and to analyze oxidatively modified proteins by Western blotting. Protein modification in pancreatic homogenates was found as early as 30 min after induction of severe AP with 3% taurocholate preceding the elevation of serum amylase activity and the increase of malondialdehyde in the tissue. A correlation of protein-carbonyl contents to a score of pancreatic macroscopic alterations (r = .69) and to the wet weight/dry weight ratio (r = .65) was found. Infusion of 5% taurocholate resulted in fulminant AP with high lethality during the 24 h of the experiment. However, rats surviving showed significantly lower level of protein-carbonyls than animals that died between 20-24 h after AP induction. The quantitative data were confirmed by the intensity of immunostained protein-carbonyls. The present data show a rather uniform increase in the staining pattern not revealing single, selectively damaged proteins. The aldehydic product of lipid peroxidation 4-hydroxynonenal (HNE) is known for its reactivity towards proteins. Interestingly, an antibody raised against protein-bound HNE did not indicate an increased protein modification by this aldehyde. In conclusion, experimental AP is characterized by an early oxidative protein modification, possibly contributing to functional impairment of the pancreas. This protein alteration may not be mediated by HNE.

Acute Disease↗
PubMed1998

Effect of supramaximal cerulein stimulation on mitochondrial energy metabolism in rat pancreas.

Hyperstimulation with the cholecystokinin analogue cerulein induces mild edematous pancreatitis in rats. It is believed that an impaired energy metabolism diminishes the cellular defense capacity in the inflamed pancreatic tissue and, therefore, contributes to the injuries in acinar cells. In the present study, changes in the capacity of oxidative phosphorylation were quantified within the first 24 h after subcutaneous cerulein injections. Serum amylase level and pancreatic water content were maximally elevated 5 h after the first injection. The capacity of mitochondrial respiration was reduced in isolated acinar cells to 69 and 44% at 5 and 24 h, respectively, compared to that in saline controls. Simultaneously, glutamate dehydrogenase (GLDH) activity dropped to 70 and 46%. The respiration rates of acinar cells and of isolated mitochondria related to GLDH activities were not different from controls. This suggests that the major portion of the mitochondrial population within the acinar cell is inactivated in the course of cerulein treatment. After 24 h, the reduced population of functionally intact mitochondria restricted the rate of phosphorylating respiration in acinar cells (52%), which resulted in a diminution of cellular ATP to 57%. It is concluded that cerulein hyperstimulation induces a drastic and long-lasting reduction of the capacity for mitochondrial ATP production which may adversely affect energy-requiring reactions of the gland during regeneration.

Adenosine Triphosphate↗
PubMed1997

Trypsinogen activation in rat pancreatic acinar cells hyperstimulated by caerulein.

Inappropriate trypsinogen activation is discussed as an early intracellular event in the secretagogue-induced model of acute pancreatitis. However, the mechanisms by which trypsinogen is activated are not well characterized. In the present work, trypsinogen activation was studied in intact acinar cells using bis-(CBZ-arginyl)-Rhodamine 110 [(CBZ-Arg)2-Rho 110] as a cell-permeant substrate for trypsin and also independently via the formation of trypsinogen activation peptide (TAP). Preincubation with 10 nM caerulein increased the Rho 110-substrate cleavage more than threefold. This proteolytic activity was fully sensitive to a benzamidine (BA)-type serine protease inhibitor. The appearance of enzymatic activity was paralleled by the formation of TAP. The lack of effect of the high-molecular soybean trypsin inhibitor indicates an intracellular substrate cleavage. The cathepsin B inhibitor CA-074 prevented neither the caerulein-induced formation of TAP nor the (CBZ-Arg)2-Rho 110-cleaving activity. BA inhibited the Rho 110-substrate cleavage and significantly reduced the TAP formation. These results show that trypsinogen activation in caerulein-hyperstimulated acinar cells may occur independently of the activity of cathepsin B. On the contrary, the effect of BA suggests the role of a serine protease in trypsinogen activation.

Animals↗
PubMed1997

[Modification of energy supply by pancreatic mitochondria in acute experimental pancreatitis].

A disturbed energy metabolism in pancreatic acinar cells is discussed as factor contributing to the development of acute pancreatitis (AP). In this study, we investigated to what extent the mitochondrial ATP producing capacity is impaired in the pancreatic tissue of rats with experimental AP. For preparation of mitochondria from rat pancreas, routine isolation procedures (tissue homogenization and differential centrifugation) were applied. Mitochondria were isolated from rats with edematous pancreatitis produced by hyperstimulation with caerulein, and from rats with mild necrotizing acute pancreatitis. The latter form of AP was induced by a temporary occlusion of the biliary pancreatic duct accompanied by a simultaneous intravenous injection of caerulein plus secretin and an intraabdominal administration of ethanol. As functional parameters of oxidative phosphorylation, the respiration rate, the mitochondrial membrane potential, and the activity of the complex I of the respiratory chain were determined. Mitochondria from rats with caerulein AP showed an enhanced respiration (61% vs. saline control) and a diminished membrane potential (-17 mV) if respiring with succinate in the non-phosphorylating state. This indicates an increased proton leak across the mitochondrial inner membrane. In the mild necrotizing AP, mitochondria were characterized by a decreased respiration with NAD(+)-linked substrates (-33% vs. sham-operated animals). This inhibition of respiration was confirmed by the reduced activity measured for the NADH-cytochrome c reductase (-32%). In both models of experimental AP the potency of mitochondria to produce ATP was significantly diminished. The stronger impairment of mitochondrial functions were found in the necrotizing form of AP. Reactive oxygen species may lead to the observed alterations--to the enhanced permeability of the mitochondrial inner membrane as well as to the inhibition of the complex I of the respiratory chain.

Acute Disease↗
PubMed1996

Interrelation between mitochondrial respiration, substrate supply and redox ratio in perifused permeabilized rat hepatocytes.

A one-step perifusion technique is described for studying the regulation of energy metabolism in intact hepatocytes and in mitochondria of the same cells after their permeabilization by digitonin. Cell count and activities of glutamate dehydrogenase, the latter being used as an indicator of mitochondrial integrity, were found to be nearly unchanged after permeabilization and perifusion for at least 40 min at 37 degrees C. The residual activity of lactate dehydrogenase after permeabilized indicated that permeabilized cells were almost depleted of soluble cytosolic components. The composition of the perifusion medium was chosen so that various metabolic states could be adjusted of both intact and permeabilized hepatocytes without the need to change the perfusion medium. Oxidative phosphorylation of mitochondria within permeabilized hepatocytes remained intact throughout the perifusion as indicated by the response of respiration to the addition of ADP, carboxyatractyloside and uncoupler. The application of the perifusion technique allows us to sample indicator metabolites in the effluent medium like acetoacetate (AcAc) and 3-hydroxybutyrate (HB) for calculating the mitochondrial redox ratios and rates of ketogenesis. In the presence of octanoate and ADP, an improvement of substrate supply by glutamate and malate led to increases in the intramitochondrial HB/AcAc ratio and the respiration rate. Glutamate/malate concentrations of 1 mM resulted in maximal respiration rates, whereas concentrations of 5 mM further enhanced the HB/AcAc ratio. Mitochondria responded to increasing ATP/ADP ratios in the perifusion medium by decreased respiration rates at higher HB/AcAc ratios. By comparing respiration rates and redox ratios of mitochondria in permeabilized cells with those before permeabilization (gluconeogenic conditions of hepatocytes), it is concluded that in the intact cell oxidative phosphorylation is limited with respect to substrate supply as well as by the ATP demand.

3-Hydroxybutyric Acid↗
PubMed1995

Cytochrome-c oxidase in developing rat heart. Enzymic properties and amino-terminal sequences suggest identity of the fetal heart and the adult liver isoform.

Perinatal development of cytochrome-c oxidase (complex IV) and ubiquinol-cytochrome-c reductase (complex III) was investigated in rat heart and liver by analysing catalytic properties, protein amounts, and subunit isoforms during the transition from the fetal to the adult state. The total amounts of complexes from milligram quantities of tissue, and the portions of isoforms of complex IV, were quantified densitometrically after isolation of the native complexes by blue native polyacrylamide gel electrophoresis and separation of the protein subunits by Tricine/SDS/PAGE [Schägger, H. & von Jagow, G. (1991) Anal. Biochem. 199, 223-231]. A parallel increase of protein amounts and catalytic activities during perinatal development was observed in heart and liver for complex III, but only in liver for complex IV. In heart, both a doubling of the turnover number of complex IV and a lowered Km for cytochrome c were observed. The altered enzymic properties correlated with the increase of heart type subunits VIa and VIII. The fetal enzymes from heart and liver seem to be identical to the adult liver isoform, as deduced from their enzymic properties and identical aminoterminal sequences of subunits VIa and VIII.

Amino Acid Sequence↗
PubMed1995

Acinar cell respiration in experimental acute pancreatitis.

The early pathogenetic steps that finally lead to acinar cell necrosis in acute pancreatitis have been characterized only scarcely as yet. Among a lot of hypotheses, one concept favors disturbances of cellular energy metabolism as a major factor that contributes to preterm cell decline. To investigate, whether an experimental acute pancreatitis alters cell respiration, the respiratory capacities of acinar cells isolated from rats with acute pancreatitis were measured. Acute pancreatitis was induced using Popper's model, i.e., a combination of duct obstruction, secretory stimulation, and mesenteric short-term ischemia with subsequent reperfusion. Acinar cells were isolated using a collagenase digestion technique. The respiratory rates of the isolated cells in suspension were measured at 37 degrees C in 100% oxygen-saturated N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid-buffered Eagle's-minimal essential medium. Resting respiration of the acinar cells uniformly amounted to about 60 pmol of O2/s x 10(6) cells in both the control and the pancreatitis group. Cellular respiration could significantly be stimulated by stepwise uncoupling of oxidative phosphorylation by means of 2,4-dinitrophenol in all cell suspensions investigated. The maximum rate of stimulated respiration was diminished in the cells isolated from rats with acute pancreatitis as compared with the controls (79.3 +/- 5.0 vs. 160.2 +/- 15.5 pmol of O2/s x 10(6) cells, p < .05), however. This reduced respiratory load capacity of the acinar cells in acute pancreatitis reflects the restricted ability of the cells to increase respiration on enhanced cellular demand. Since mitochondrial respiration is coupled to oxidative phosphorylation, an altered energy-transforming potential of the acinar cells in acute pancreatitis becomes evident.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute Disease↗
PubMed1993

Increase in the adenine nucleotide translocase protein contributes to the perinatal maturation of respiration in rat liver mitochondria.

An assay based on the high-affinity binding of tritium-labelled atractyloside to the adenine nucleotide translocase (ANT) was developed for estimation of its content in samples of mitochondria, cells and tissue homogenate. The assay was used to study the developmental change of the ANT protein concentration in perinatal rat liver. Within the last 3 days before birth the content of the ANT protein per mg tissue protein increased from 29 to 45% of the maximum value found 2 days after birth. A similar developmental change of the ANT protein was found in isolated mitochondria, demonstrating that the perinatal increase in the ANT protein content was due mainly to a mitochondrial differentiation process and not the result of an increase in the number of mitochondria per cell. A close proportionality between the ANT protein and the ADP-stimulated respiration of liver homogenate was found in the perinatal period from 3 days before to 2 days after birth. This finding suggests that the developmental change in the ANT protein content plays an important role in the onset of oxidative phosphorylation after birth.

Animals↗
PubMed1993

[Morphologic characteristics and ATPase activity in permeabilized epididymal bull sperm].

The treatment of bull spermatozoa from epididymal cauda with 5 micrograms digitonin per microliters cells led to the destruction of the plasma membrane which was shown by electron microscopy. Structural changes of mitochondria and of the axoneme were not found. The functional intactness was maintained. The plasma membranes of hypotonically treated spermatozoa were permeable for succinate and the mitochondria showed a diminished functional intactness. For the total ATPase activity of digitonin-treated cells, mainly representing the dynein ATPase, a maximal activity of 20.3 nmol ATP x min-1 x microliters cells-1 (mean +/- S.D.; n = 8) and an apparent half saturation constant of 0.29 mM ATP were determined. The portions of Na(+)-K(+)-ATPase and mitochondrial ATPase in the total ATP-splitting activity were found to be less than 15%. In comparison to the capacity of mitochondrial ATP production, the maximal activity of ATP consuming reactions amounted to about 70%. These results support findings that the ATP turnover of spermatozoa is more limited by the ATP consumption than by the ATP production.

Adenosine Triphosphatases↗
PubMed1991

Use of NAD(P)H and flavoprotein fluorescence signals to characterize the redox state of pyridine nucleotides in epididymal bull spermatozoa.

The redox behaviour of the NAD(P) system and flavoproteins was registered by simultaneous fluorescence measurements in epididymal bull spermatozoa. The flavoprotein fluorescence signal can nearly exclusively be attributed to an NAD-linked enzyme, alpha-lipoamide dehydrogenase (Em7.4 = -286 mV). A comparison of intact with digitonin-permeabilized spermatozoa revealed that about 50% of the total NAD(P)H fluorescence signal was of mitochondrial origin. Under equilibrium conditions, the midpoint potentials of the NAD(P)H fluorescence signal of both compartments were almost identical (-300 mV). When lactate was present as substrate, 1 mM caffeine increased respiration oxidizing the NAD(P)H system in both mitochondria and cytosol. This indicates a close relationship of the two NAD pools in spermatozoa.

Adenine Nucleotides↗
PubMed1990

Quantification of aerobic energy turnover in epididymal bull spermatozoa.

Turnover rates of oxidative energy metabolism were measured as oxygen consumption in untreated and caffeine-stimulated epididymal bull spermatozoa respiring with lactate. Incubation of spermatozoa with 1 mM caffeine led to an increase in respiration of approx. 60%. The rate of uncoupled respiration and the vanadate-insensitive part of oxygen consumption were not affected by caffeine. The small effect of ouabain on respiration (-10%) indicated a minor contribution of Na+/K+-ATPase to the ATP consumption. The major part of ATP turnover was caused by motility shown by the strong linear correlation between respiration and motility in untreated and caffeine-treated spermatozoa. In comparison with ejaculated spermatozoa investigated in a previous study, epididymal cells exhibited the same rates of uncoupled and ouabain-sensitive respiration. The efficiency of transforming mitochondrially-produced ATP into cell motion was the same in epididymal and ejaculated spermatozoa. The ATP-producing capacity of sperm mitochondria was utilized in untreated epididymal, in caffeine-stimulated epididymal and in ejaculated spermatozoa, by 20-25%, 40-45% and 45-50%, respectively. The results showed that the capacity of mitochondrial. ATP formation remains unchanged after ejaculation and is utilized to a higher extent by stimulated motility. Treatment with caffeine affected epididymal spermatozoa in a similar manner.

Aerobiosis↗
PubMed1990

[Respiration and motility of epididymal sperm from slaughtered bulls].

The simple method of retrograde flushing of spermatozoa from the epididymal cauda of slaughter bulls yielded an average of 2 x 10(9) spermatozoa from one cauda. The plasma membrane was intact in something between 80 and 85 percent of all epididymal spermatozoa. Respiratory rates and motility were clearly below values typical of ejaculated spermatozoa, though sizeable differences were found to exist for both parameters between individual preparations. The endogenous substrates available proved to be sufficient for such low metabolic activity, whereas higher energy turnover required the presence of exogenous substrates, such as lactate, pyruvate or fructose. Respiratory capacity, determined by uncoupling of oxidative phosphorylation in the presence of lactate, was comparable to that of ejaculated bull spermatozoa. The above findings are likely to suggest that the lower respiratory rates of epididymal spermatozoa were attributable to the fact that their consumption for motility of adenosine triphosphoric acid (ATP) was lower than that of ejaculated spermatozoa.

Abattoirs↗
PubMed1989

Characterization of mitochondrial functions in digitonin-permeabilized rat liver cells.

Mitochondrial functions were investigated in permeabilized rat liver cells. For permeabilization isolated hepatocytes were treated with digitonin using a perifusion technique. After permeabilization the cell count was almost unchanged, and the mitochondrial marker enzyme, glutamate dehydrogenase, was decreased to as little as 83%. The mitochondria in permeabilized cells were functionally intact, a finding evident from a marked stimulation of respiration by ADP, inhibition by carboxyatractyloside, and stimulation by uncoupler. The ADP-stimulated and uncoupled respiration rates with succinate as substrate were comparable to those reported for isolated mitochondria, whereas the rates with NAD(+)-dependent substrates were somewhat higher. The ratios between ADP-stimulated and carboxyatractyloside-inhibited respiration rates were in the range noted for isolated mitochondria with identical substrates. Almost unchanged ADP-stimulated and carboxyatractyloside-inhibited respiration rates were found for at least 180 min after digitonin treatment. The preparation exhibited only a low extramitochondrial ATPase activity which was partially inhibited by vanadate.

Adenosine Diphosphate↗
PubMed1987

Regulation of oxidative phosphorylation in mitochondria of epididymal bull spermatozoa.

The regulation of oxidative phosphorylation was studied with digitonin-treated epididymal bull spermatozoa in which mitochondria are directly accessible to low molecular compounds in the extracellular medium. Due to the high extramitochondrial ATPase activity in this cell preparation, it was possible to stimulate respiration to a small extent only by added hexokinase in the presence of glucose and adenine nucleotides. Added pyruvate kinase plus phosphoenol pyruvate, however, strongly suppressed the respiration. Under these conditions, the respiration was found to depend on the extramitochondrial [ATP]/[ADP] ratio in the range of 1-100. The contribution of the adenine nucleotide translocator to this dependence was determined by titration with the irreversible inhibitor carboxyatractyloside in the presence of ADP. Using lactate plus malate as substrate, the active state respiration was controlled to about 30% by the translocator, whereas 12 and 4% were determined in the presence of L-glycerol-3-phosphate and malate alone, respectively. In order to compare the results with those for intact cells, the adenine nucleotide patterns were determined in intact and digitonin-treated spermatozoa under conditions of controlled respiration in the presence of vanadate and carboxyatractyloside, respectively. About 21% of total cellular adenine nucleotides were found in digitonin-treated cells representing the mitochondrial compartment. While allowing for the intramitochondrial amount of adenine nucleotides, the cytosolic [ATP]/[ADP] ratio was estimated to be 6-times higher than the mitochondrial ratio in intact cells. It is concluded from the data presented that the principal mechanism by which oxidative phosphorylation in sperm mitochondria is regulated via the extramitochondrial [ATP]/[ADP] ratio is the same as that demonstrated for other isolated mitochondria.

Adenine Nucleotides↗
PubMed1986

Control of respiration and of motility in ejaculated bull spermatozoa.

The relations between motility and respiration were studied in ejaculated bull spermatozoa respiring with lactate. Motility was quantitatively evaluated by a turbidimetric procedure as percentage of cells moving per minute from the bottom of the cuvette into the light path. For selective inhibition of ATP-consuming reactions including motility or of mitochondrial respiration, vanadate or cyanide, respectively, were used. Both inhibitors were found to produce proportional changes in motility and respiration. The simultaneous changes in motility and respiration were linked to shifts in the cellular ATP/ADP ratio. Partial uncoupling of respiration in vanadate-inhibited cells gave similar relations between respiration and ATP/ADP ratios as stepwise inhibition of ATP-utilizing reactions by vanadate. Presuming saturation kinetics with respect to the ATP/ADP ratio, half maximum constants of 1.7 and 4.7 for the ATP/ADP ratio and maximum values of about 130% and 300% (in comparison to untreated cells) were estimated for motility and respiration, respectively. Respiration showed a much steeper dependence on the ATP/ADP ratio than motility resulting in an apparent cooperativity coefficient of 2.9. From these dependences on the ATP/ADP ratio, the shares in the control of ATP turnover in untreated cells were estimated. At sufficient supply with substrate, more than 80% of control were excreted by motility and other ATP-utilizing reactions, the rest by mitochondrial ATP production, i.e., the reactions of oxidative phosphorylation.

Adenosine Diphosphate↗
PubMed1986

Quantification of sperm motility by a turbidimetric assay. Correlation to cellular respiration.

The turbidimetric method according to SOKOLOSKI et al. (Fertil. Steril. 28, 1337-1341, 1977) was used for quantitative measurement of the motility of washed bovine spermatozoa. The method is based on the registration of the increase in absorbance (A538) which is produced by spermatozoa entering the light path after run from the bottom of a photometer cuvette. This increase in absorbance is dependent on the amount of motile cells and their average velocity. The increase in absorbance related to the total cell concentration was found to be a highly reproducible quantity (motility index M). Inhibitors of dynein ATPase (vanadate) and of the mitochondrial ATP-production (KCN, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), oligomycin) produced a diminution of the motility index M in a concentration-dependent manner. Under conditions of oxidative energy metabolism (lactate as substrate) a linear relation between respiration and M existed. Such a relationship was found under treatment of spermatozoa with KCN or vanadate as well as for untreated sperm suspensions. The linear correlation to the respiratory rate justifies to use the proposed motility index as a relative measure of the average motility of a sperm sample.

Adenosine Triphosphatases↗