Search PubMed⌕ Search

Biomedical subjects

W Kunz

Publications and source records attributed to W Kunz.

At least 91 records · Page 5Linked to original sources
PubMed1985

Effect of various substrates on mitochondrial and cellular energy state of intact spermatozoa.

Ejaculated bull spermatozoa separated from seminal plasma by repeated washing were used to study the effects of various substrates on respiration, mitochondrial energization, cellular ATP/ADP ratios, and motility. The respiratory rate in the presence of lactate, pyruvate, acetate or glycerol was about two to three times higher compared to cells oxidizing amino acids, fatty acids, or endogenous substrates. When well oxidizable substrates were present, the uncoupler FCCP produced a marked increase in respiration (two to three times at optimal FCCP concentrations). Mitochondrial energization was monitored in intact spermatozoa by spectrophotometry with the dye safranin. Lactate and pyruvate yielded more pronounced mitochondrial energization than did alanine or endogenous substrates. Cellular ATP/ADP ratios, with lactate, pyruvate, acetate or glycerol, were found to range from 6 to 10, whereas endogenous substrates and amino acids were able to establish ATP/ADP quotients as low as about 1 only. The capacity to oxidize various substrates was clearly reflected by the amount of motile cells. Motility in the presence of lactate, acetate or glycerol was seen in more than 60% of spermatozoa, while spermatozoa in the presence of alanine or butyrate showed a motility not above the endogenous level (approx. 30% motile cells). The experiments demonstrated that cellular performance is highly dependent on the energy state as made possible by the capacity of substrate utilization.

Acetates↗
PubMed1985

Mitochondrial respiratory function as indicator of the ischemic injury of the rat kidney.

The function of mitochondria was considered to be an indicator of ischemic injury of the rat kidney, and the respiration of isolated mitochondria was measured in different metabolic states. The respiratory control index (RCI) was chosen as a parameter of mitochondrial intactness. The results indicate that normothermic in vitro ischemia leads to a rapid decrease of RCI and an almost complete loss of respiratory control within 45 min. Hypothermia during ischemic exposure proved to be an effective protection against mitochondrial injury as indicated by a prolonged coupled respiration and an RCI of about 2, even after 24 h of ischemic kidney storage. The ischemia-induced decrease of RCI was mainly due to a progressive decrease of active-state respiration, whereas the alterations of resting-state respiration were only small. The decline of active-state respiration was paralleled by a decrease of an uncoupled respiration rate. After termination of in vivo ischemia the RCI increased very slowly. Normal RCI values were not obtained until 7 days after onset of blood reflow, which supports the assumption of long-term damage due to ischemia.

Adenosine Triphosphate↗
PubMed1984

On the role of superoxide anion radicals in skin tumour promotion.

The effect of phorbol ester tumour promoters on the release of superoxide anion radicals .O2- by human peripheral leukocytes and the role of such radicals in tumour promotion of mouse skin was studied. No significant difference was found between complete [12-O-tetradecanoylphorbol-13-acetate (TPA)] as compared with incomplete [12-O-retinoylphorbol-13-acetate (RPA), 12-O-(2Z,4E,6,8)tetradecatetraenoylphorbol-13-acetate (Ti8), mezerein] tumour promoters upon induction of .O2- when measured by the reduction of ferricytochrome c. The semisynthetic phorbol esters 12-O-ethacrynylphorbol-13-acetate (EPA) and 4-O-methyl-TPA were less active, and phorbol diacetate, phorbol and ionophore A 23187 were found to be inactive in stimulating superoxide anion radicals. TPA-induced .O2- release from leukocytes was strongly inhibited by Cu(II)-(diisopropylsalicylate)2 (CuDIPS), and, to a lesser extent, by ethacrynic acid, nordihydroguaiaretic acid and quercetin. Retinoic acid exhibited only a moderate inhibitory effect. No .O2- release was observed in epidermal cell cultures upon TPA treatment. When analysed by the alkaline elution technique, TPA-induced .O2- release from leukocytes did not lead to measurable DNA damage in co-cultivated keratinocytes even in the presence of DNA repair inhibitors. In multi-stage-tumourigenesis experiments including two-stage promotion, retinoic acid, ethacrynic acid and CuDIPS were unable to inhibit tumour promotion in mouse skin when applied in combination with TPA in first stage promotion. gamma-Irradiation at a dose level shown to cause DNA damage in vitro could not replace TPA as a stage I-promoting agent. It is concluded that superoxide anion radicals--if related to promotion at all--may play a role in stage II rather than in stage I of mouse skin tumour promotion.

Animals↗
PubMed1984

Promoting effect of 4-dimethylaminoazobenzene on enzyme altered foci induced in rat liver by N-nitrosodiethanolamine.

Female Wistar rats were treated sequentially with 4-dimethylaminoazobenzene (4-DAB) and N-nitrosodiethanolamine ( NDEOL ) for periods of 6 weeks. One group received first 4-DAB (0.06% in the diet) and NDEOL (2000 p.p.m. in the drinking water) thereafter, while the second group was treated in the reversed sequence; control groups received the single agent alone. The extent of foci negative for adenosine-triphosphatase (ATPase) or positive for gamma-glutamyl-transpeptidase (gamma-GT)-activity was quantitated in liver as a means to assess carcinogenic efficacy. A very low response was obtained in rats treated first with 4-DAB and then with NDEOL whereas a strong increase in number and especially in size of foci was observed when 4-DAB was given after NDEOL . The response in this latter group was clearly over-additive. Treatment of rats with either carcinogen alone resulted in similar pattern of increases in the volumetric fraction of liver occupied by ATPase-deficient foci. A differential behaviour, however, was observed with respect to islet size. NDEOL produced large numbers of small foci whereas with 4-DAB only few foci were obtained which grew rapidly in the presence of the carcinogen. These findings are consistent with the hypothesis that 4-DAB, besides acting as an initiator, has very strong promoting activity as was to be expected from the characteristic relationship between carcinogen dose and time of liver tumour induction.

Adenosine Triphosphatases↗
PubMed1984

Influence of octanoate on the rate of oxidative phosphorylation and the associated extramitochondrial ATP/ADP ratios studied with isolated rat liver mitochondria oxidizing pyruvate.

The regulation of oxidative phosphorylation by the extramitochondrial ATP/ADP ratio was investigated with pyruvate (malate) or pyruvate (malate) plus octanoate as substrates in experiments with isolated rat liver mitochondria. Steady states in the supply of non-saturating substrate concentrations and the activity of oxidative phosphorylation were adjusted by means of a perifusion technique which is based on immobilisation of mitochondria on glass filters. Michaelis-Menten parameters of the pyruvate oxidation in active state respiration were determined both in the absence and presence of octanoate. Octanoate yielded only slight Km value increase, whereas the maximal rate of pyruvate oxidation was diminished to one half. Therefore, it is concluded that the inhibitory action of octanoate on pyruvate oxidation comes close to a non-competitive mechanism. The inhibitory effect of octanoate is completely reversible. When a change in substrate supply was accomplished by substituting pyruvate (malate) plus octanoate for pyruvate (malate) under conditions of a simulated extramitochondrial energy demand, respiration was stimulated and ATP/ADP ratios generated outside mitochondria were shifted to a higher value. Two different responses of the mitochondrial respiration to the extramitochondrial ATP/ADP ratios were found as a function of the presence or absence of octanoate.

Adenosine Diphosphate↗
PubMed1984

The mechanism of cocarcinogenic action of ethanol in rat liver.

The effects of chronic alcohol consumption on nitrosamine metabolism in vivo, DNA synthesis and repair, and carcinogen-induced preneoplasia were studied in rat liver. Following a single injection of different doses of 14C-N-nitrosodimethylamine, there was no significant difference between controls and ethanol-pretreated rats in the alkylation pattern of cellular protein nor in the levels of the alkylation products 7-methylguanine and O6-methylguanine isolated from liver DNA. O6-Methylguanine-specific DNA repair was also unchanged. An increase in the number and size of foci staining negative for adenosine triphosphatase and/or positive for gamma-glutamyltranspeptidase was observed in rats treated intermittently with ethanol and N-nitrosomorpholine. The numbers of clear-cell and mixed-cell foci were also increased. An ethanol-mediated enhancement of DNA synthesis, which was ascertained by different methods, may be related to this cocarcinogenic action of the alcohol. Ethanol, however, failed to demonstrate promoting activity. Long-term treatment of carcinogen pretreated rats with ethanol, according to the classical initiation-promotion protocol, had no effect on the incidence of preneoplastic foci in liver.

Animals↗
PubMed1983

Control of mitochondrial respiration. The contribution of the adenine nucleotide translocator depends on the ATP- and ADP-consuming enzymes.

The consequence of the complexity of the metabolic network on the amount of control strength of adenine nucleotide translocator was investigated with isolated rat liver mitochondria. Two experimental systems were compared: (i) mitochondria in the presence of yeast hexokinase (hexokinase system) and (ii) the same system plus additional pyruvate kinase (pyruvate kinase system). In both systems the control strength was analysed for the adenine nucleotide translocator by inhibitor titration studies with carboxyatractyloside and for the hexokinase or pyruvate kinase by changing their relative activities. Experimental results were compared with computer simulation of these systems and that of a third one, where the extramitochondrial ATP/ADP ratio was held constant by perifusion (perifusion system). The results demonstrate quite different flux-dependent control strength of the translocator in the three systems. In the hexokinase system the control strength of the translocator on mitochondrial respiration was zero up to respiration rates of about 60 nmol O2/mg protein per min. For higher rates, the control strength increased until the maximum value (0.45) was reached in the fully active state. Here, the same value was also found in the pyruvate kinase system. In all other states of respiration the translocator exerts a higher control strength in the pyruvate kinase system than in the hexokinase system. This different behaviour was attributed to the various changes in the adenine nucleotide pattern caused by partial inhibition of the translocator in the hexokinase and pyruvate kinase system. The data clearly show that the sharing of control strength depends not only on the respiration rate but also on the complexity of the metabolic system.

Adenosine Diphosphate↗
PubMed1983

Control of mitochondrial respiration.

The control theory of Kacser and Burns [in: Rate Control of Biological Processes (Davies, D.D. ed) pp. 65-104, Cambridge University Press, London, 1973] and Heinrich and Rapoport [Eur. J. Biochem. (1974) 42, 97-105] has been used to quantify the amount of control exerted by different steps on mitochondrial oxidative phosphorylation in rat-liver mitochondria. Inhibitors were used to manipulate the amount of active enzyme. The control strength of the adenine nucleotide translocator was measured by carrying out titrations with carboxyatractyloside. In state 4, the control strength of the translocator was found to be zero. As the rate of respiration was increased by adding hexokinase, the control strength of the translocator increased to a maximum value of approximately 30% at approximately 80% of state 3 respiration. In state 3, control of respiration is distributed between a number of steps, including the adenine nucleotide translocator, the dicarboxylate carrier and cytochrome c oxidase. The measured values for the distribution of control agree very well with those calculated with the aid of a model for mitochondrial oxidative phosphorylation developed by Bohnensack et al. [Biochim. Biophys. Acta (1982) 680, 271-280].

Adenosine Diphosphate↗
PubMed1983

Determination of the region of rDNA involved in polytenization in salivary glands of Drosophila hydei.

During the formation of polytene chromosomes in salivary glands of Drosophila hydei, the genes for ribosomal RNA (rDNA) are underreplicated relative to the rest of the genome. We have measured the number of rRNA genes with and without intervening sequences (ivs+ and ivs- genes) in polytene chromosomes of different genotypes. In the group of genotypes having a large number of ivs- rRNA genes polytenization only occurs within the cluster of ivs- genes. In each of these genotypes rDNA polytenization reaches a constant level of 150 ivs- genes per two chromatid sets (2C); X/X constitutions having two nucleolus organizers (NOs) in the diploid set polytenize the same amount of rDNA as X/O constitutions. In the group of genotypes with small ivs- gene numbers, the rDNA region involved in polytenization is longer and has an average length of 1,700 kb per NO, which is constant in these genotypes. Polytenization of rDNA is extended into the cluster of ivs+ genes, in spite of the fact that these genes appear to be nonfunctional. The smaller the number of ivs- genes, the greater the number of ivs+ genes that are polytenized in the NO. In these genotypes, X/X females replicate twice as much rDNA as X/O males, suggesting that both NOs of the diploid set are polytenized. A comparison of the pattern of spacer length heterogeneity in hybrids between different stocks also demonstrates that both NOs are replicated during polytenization.

Animals↗
PubMed1983

Effect of ethanol on early stages in nitrosamine carcinogenesis in rat liver.

The effect of chronic alcohol consumption on the extent of adenosine triphosphatase(ATPase)-deficient preneoplastic lesions in rat liver induced by either diethylnitrosamine (DEN) (3 mg/kg, p.o.) or N-nitrosomorpholine (NNM) (40 ppm in the drinking water) was studied. Carcinogens were administered on 4 days in every week for 11 (DEN) and 15 (NNM) weeks, respectively. Ethanol was given at a concentration of 10% (w/v) in the drinking water either during carcinogen treatment or after withdrawal of carcinogen. An increase in both number and size of ATPase-deficient foci in liver was observed when the alcohol was given during the period of carcinogen administration. This increase may be associated with the known toxic action of ethanol which leads to single cell necrosis and liver regeneration. In contrast, when ethanol (10% in the drinking water for 16 weeks) was given after cessation of carcinogen treatment following a tumor-promotion feeding protocol, no such enhancement in preneoplastic response was obtained. Ethanol alone was ineffective in inducing ATPase-deficient foci. In liver, ethanol thus appears to possess, under certain conditions, co-carcinogenic but not tumor-promoting capacity.

Adenosine Triphosphatases↗
PubMed1983

The effect of pre- and post-treatment with phenobarbital on the extent of gamma-glutamyl transpeptidase positive foci induced in rat liver by N-nitrosomorpholine.

Male Sprague-Dawley rats were treated with a combination of N-nitrosomorpholine (NNM) and phenobarbital and the extent of foci positive for gamma-glutamyl transpeptidase (gamma-GT), as demonstrated histochemically in liver, was quantitated. Phenobarbital (0.05% in the diet) was given either alone, before, or after a 7-week period of treatment with NNM (80 ppm in the drinking water). When given after the carcinogen treatment, phenobarbital enhanced the formation of gamma-GT-positive foci in liver compared with rats treated with NNM alone. No such increase, however, was observed when phenobarbital was administered before treatment with NNM. Phenobarbital alone did not produce any apparent amount of foci in liver. These data suggest that the enhancement of carcinogenic response mediated by phenobarbital as observed under the present experimental conditions is only operative in the presence of pre-existing initiated hepatocytes.

Animals↗
PubMed1983

Involvement of intramitochondrial adenine nucleotides and inorganic phosphate in oxidative phosphorylation of extramitochondrially added adenosine-5'-diphosphate.

Possible direct, product/substrate mediated interactions between the phosphate carrier and/or the adenine nucleotide translocator and the mitochondrial H+-ATPase were investigated by tracer flux experiments. For this purpose the specific radioactivity of ATP synthesized was measured and compared with those of intra- and extramitochondrial precursors. Two experimental strategies were applied: 1) preloading of mitochondria with [32P]Pi before starting oxidative phosphorylation of external ADP; 2) simultaneous addition of tracer amounts of [3H]ADP and [32P]Pi to phosphorylating mitochondria under steady-state conditions. The observed participation of the intramitochondrial Pi pool in oxidative phosphorylation excludes a directly functional interaction between Pi carrier and H+-ATPase. At 5 degrees C an at least partial compartmentation of intramitochondrial ADP during oxidative phosphorylation was found, whereas at 25 degrees C a complete equilibrium of intra- and extramitochondrial [32P]Pi and [3H]ADP could be observed, excluding a functionally important compartmentation of these species in the intramitochondrial space under physiological conditions.

Adenine Nucleotides↗
PubMed1983

Influence of the beta-hydroxybutyrate/acetoacetate ratio on the redox states of mitochondrial NAD(P) and cytochrome c systems, extramitochondrial ATP/ADP ratio and the respiration of isolated liver mitochondria in the resting state.

The effect of changes in hydrogen supply on the rate of energy transformation (measured as respiration rate), on the redox state of the NAD(P) and cytochrome c couples and on the extramitochondrial ATP/ADP was investigated with isolated rat liver mitochondria. Hydrogen supply was changed by variation of the beta-hydroxybutyrate/acetoacetate ratio in the incubation medium. In the active state respiration rate was strongly diminished by lowering the beta-hydroxybutyrate/acetoacetate ratio, whereas in the resting state a minor effect was observed. This resulted in a decrease in the respiratory control index (RCI). Even under a metabolic situation of strongly diminished hydrogen supply corresponding to a beta-hydroxybutyrate/acetoacetate ratio equal to 0.5 added ADP was phosphorylated. However, the ATP/ADP ratio generated extramitochondrially amounted to half that obtained at a beta-hydroxybutyrate/acetoacetate ratio of more than 100. Nevertheless at a beta-hydroxybutyrate/acetoacetate ratio of 0.5 the generated extramitochondrial ATP/ADP ratio is near to 100. A comparison of the changes in the available free redox energy with those in the extramitochondrial phosphorylation potential points to a disequilibrium between them in the resting state, too. The experimental results are discussed in the light of the metabolic conditions in vivo.

3-Hydroxybutyric Acid↗
PubMed1982

Effect of ethanol on dimethylnitrosamine activation and DNA synthesis in rat liver.

Male Wistar rats were treated for 2 weeks with 20% ethanol in the drinking water. Twenty four hours after the termination of treatment animals were injected with different doses of [14C]dimethylnitrosamine and killed 4 h thereafter. The amounts of 7-methylguanine and O6-methylguanine present in liver DNA were determined. There was no significant difference in the levels of either DNA alkylation product between untreated controls and animals pre-treated with ethanol. The O6-methylguanine/7-methylguanine ratio was also unchanged. Specific radioactivity levels in the cellular protein of [14C]dimethylnitrosamine injected animals were slightly lowered after ethanol pre-treatment. In contrast, incorporation of labelled guanine into liver DNA was greatly enhanced at all DMN doses in ethanol pre-treated animals indicating an increase in DNA synthesis. This enhancement of DNA synthesis was confirmed at different ethanol doses, given either in the drinking water or by stomach tube, by the increase in specific radioactivity of liver DNA and the increase in the number of labelled nuclei following [3H]thymidine pulse-labelling.

Animals↗
PubMed1982

Interrelationships between hydrogen-supplying reactions, respiration rate and extramitochondrial adenine nucleotide pattern.

1. The influence of a diminished hydrogen supply on the regulation of oxidative phosphorylation of isolated rat liver mitochondria in dependence on the extramitochondrial (ATP)/(ADP) ratio was investigated. 2. The hydrogen supply was diminished by using various (beta-hydroxybutyrate)/(acetoacetate) ratios as a redox buffer and the results were compared with those of experiments using perifusion of immobilized mitochondria with non-saturating substrate concentrations. 3. In both experimental approaches the influence of a diminished hydrogen pressure on the maximum (ATP)/(ADP) ratio at minimum flux was low. An extreme decrease in the (beta-hydroxybutyrate)/(acetoacetate) ratio by more than two orders of magnetitude causes the (APT)/(ADP) ratio to decrease by about 50%. 4. The load capacity of oxidative phosphorylation (maximum flux) is considerably decreased by diminished hydrogen pressure. 5. The borderline cases of purely kinetic and thermodynamic limitations of hydrogen supply were calculated by computer simulation with respect to the regulating behaviour of oxidative phosphorylation and changes in the control strength of adenine nucleotide translocator and hydrogen supply in the overall reaction. 6. A prevalent thermodynamic influence of hydrogen supply on oxidative energy transformation in the cell is discussed in the light of experimental data.

3-Hydroxybutyric Acid↗
PubMed1982

Interrelationship between oxidative energy transformation and energy consumption at mitochondrial and cellular levels.

The adaptation of oxidative energy transformation in mitochondria to the energy demand of cellular metabolism was investigated in experiments with isolated mitochondria and liver cells and by computer simulation in terms of a mathematical model. Separate draining of different energy pools allowed the determination of the relation between these pools and the elucidation of the importance of the connecting enzyme reactions to the regulation of the whole process. The following conclusions can be drawn from the results: 1. The intramitochondrial adenine nucleotide pool exhibits a homogeneous behaviour, and its changes are the signal for ATP synthesis. 2. The proton-motive force which is in near-equilibrium with the intramitochondrial phosphorylation potential is the immediate signal for the respiratory chain. 3. The intramitochondrial phosphorylation potential is transformed into the external one by a flux-dependent non-equilibrium reaction of the translocator. 4. The rate of respiration-linked ATP formation is regulated by more than one reaction step with varying control strength. 5. In both isolated mitochondria and hepatocytes an activation of respiration is provoked by a decrease in the mitochondrial energy state caused by cellular energy utilization.

Adenosine Triphosphate↗