Biomedical subjects

Frank Norbert Gellerich

Publications and source records attributed to Frank Norbert Gellerich.

8 recordsLinked to original sources

PubMed2005

Effects of extramitochondrial ADP on permeability transition of mouse liver mitochondria.

Carboxyatractylate (CAT) and atractylate inhibit the mitochondrial adenine nucleotide translocator (ANT) and stimulate the opening of permeability transition pore (PTP). Following pretreatment of mouse liver mitochondria with 5 microM CAT and 75 microM Ca2+, the activity of PTP increased, but addition of 2 mM ADP inhibited the swelling of mitochondria. Extramitochondrial Ca2+ concentration measured with Calcium-Green 5N evidenced that 2 mM ADP did not remarkably decrease the free Ca2+ but the release of Ca2+ from loaded mitochondria was stopped effectively after addition of 2 mM ADP. CAT caused a remarkable decrease of the maximum amount of calcium ions, which can be accumulated by mitochondria. Addition of 2 mM ADP after 5 microM CAT did not change the respiration, but increased the mitochondrial capacity for Ca2+ at more than five times. Bongkrekic acid (BA) had a biphasic effect on PT. In the first minutes 5 microM BA increased the stability of mitochondrial membrane followed by a pronounced opening of PTP too. BA abolished the action about of 1 mM ADP, but was not able to induce swelling of mitochondria in the presence of 2 mM ADP. We conclude that the outer side of inner mitochondrial membrane has a low affinity sensor for ADP, modifying the activity of PTP. The pathophysiological importance of this process could be an endogenous prevention of PT at conditions of energetic depression.

Adenine Nucleotide Translocator 3↗

PubMed2004

The problem of interlab variation in methods for mitochondrial disease diagnosis: enzymatic measurement of respiratory chain complexes.

Due to the large importance of mitochondrial function for numerous diseases the detection of respiratory chain defects for diagnostic purposes is an important task of mitochondrial medicine. For comparing the methods, standard mitochondrial homogenate prepared from bovine skeletal muscle was sent on dry ice to 14 labs of 8 countries. Activities of complexes I, I + III, II, II + III, IV (cytochrome c oxidase) and V (F0F1ATPase) as well as of citrate synthase were measured. For all enzymes the results differed at more than one order of magnitude. From eight labs we were able to compare the results with their control values for human skeletal muscle. Four labs found normal activity of cytochrome-c-oxidase whereas three labs found higher and one lab found lower activities compared to the own controls. Since all labs used different temperatures (25, 30 and 37 degrees C) in one lab the temperature dependencies were measured experimentally. The temperature correction did not much reduce the divergence of the results. It is concluded that differences in the lab protocols are the reason for the large variation of results. Since the experimental results strongly depend on the used method a strict standardization is necessary.

Journal Article↗

PubMed2004

Different sensitivities of CPT I and CPT II for inhibition by l-aminocarnitine in human skeletal muscle.

l-Aminocarnitine (l-AC) has been shown to inhibit carnitine palmitoyltransferases (CPT) in rat muscle and in rat liver. However, there are no reports on interactions of l-AC with CPT II and CPT I of human muscle. Therefore, the aim of the present work was to characterize the inhibition of human muscle CPT I and CPT II by l-AC in muscle mitochondria, skinned fibers and muscle homogenates in comparison to the established action of malonyl-CoA. Both isoenzymes were inhibited by l-AC, but sensitivity was different (CPT I, K(d)=3.8 mM l-AC; CPT II, K(d)=21.3 microM l-AC). A mixed inhibition type in respect to carnitine was detected (K(i)=3.5 microM l-AC). At 0.5 mM l-AC, CPT II was completely inhibited without affection of CPT I. In contrast, CPT I was completely inhibited by 0.4 mM malonyl-CoA (K(d)=0.5 microM), whereas CPT II was nearly not affected by this inhibitor. Using these inhibitors in muscle homogenates, activities of CPT II and CPT I were detected to be 38+/-10% and 63+/-10% of total, respectively (n=21). In intact mitochondria and different fractions of muscle homogenates after selective solubilization of CPT II by Tween 20, the extent of specific CPT inhibition changed in relation to the accessible isoenzyme pattern. Palmitoyl-carnitine-dependent respiration in skinned fibers was inhibited by high concentrations of l-AC, indicating that the inhibitor can be transported via the acyl-carnitine transporter, too. The combined use of both inhibitors (l-AC and malonyl-CoA) allows the kinetic characterization of CPT I and CPT II in human muscle homogenates. In addition, it has been shown that l-AC can be used for the study of metabolic consequences of CPT II deficiency on function of intact mitochondria.

Betaine↗

PubMed2002

Mitochondrial respiratory rates and activities of respiratory chain complexes correlate linearly with heteroplasmy of deleted mtDNA without threshold and independently of deletion size.

To clarify the importance of deleted protein and tRNA genes on the impairment of mitochondrial function, we performed a quantitative analysis of biochemical, genetic and morphological findings in skeletal muscles of 16 patients with single deletions and 5 patients with multiple deletions of mtDNA. Clinically, all patients showed chronic progressive external ophthalmoplegia (CPEO). The size of deletions varied between 2.5 and 9 kb, and heteroplasmy between 31% and 94%. In patients with single deletions, the citrate synthase (CS) activity was nearly doubled. Decreased ratios of pyruvate- and succinate-dependent respiration were detected in fibers of all patients in comparison to controls. Inverse and linear correlations without thresholds were established between heteroplasmy and (i) CS referenced activities of the complexes of respiratory chain, (ii) CS referenced maximal respiratory rates, (iii) and cytochrome-c-oxidase (COX) negative fibers. In patients with single and multiple deletions, all respiratory chain complexes as well as the respiratory rates were decreased to a similar extent. All changes detected in patients with single deletions were independent of deletion size. In one patient, only genes of ND5, ND4L as well as tRNA(Leu(CUN)), tRNA(Ser(AGY)), and tRNA(His) were deleted. The pronounced decrease in COX activity in this patient points to the high pathological impact of these missing tRNA genes. The activity of nuclear encoded SDH was also significantly decreased in patients, but to a lesser extent. This is an indication of secondary disturbances of mitochondria at CPEO. In conclusion, we have shown that different deletions cause mitochondrial impairments of the same phenotype correlating with heteroplasmy. The missing threshold at the level of mitochondrial function seems to be characteristic for large-scale deletions were tRNA and protein genes are deleted.

Citrate (si)-Synthase↗

PubMed2002

The quantitation of ADP diffusion gradients across the outer membrane of heart mitochondria in the presence of macromolecules.

We have previously provided evidence that diffusion of metabolites across the porin pores of mitochondrial outer membrane is hindered. A functional consequence of this diffusion limitation is the dynamic compartmentation of ADP in the intermembrane space. These earlier studies were done on isolated mitochondria suspended in isotonic media without macromolecules, in which intermembrane space of mitochondria is enlarged. The present study was undertaken to assess the diffusion limitation of outer membrane in the presence of 10% (w/v) dextran M20, in order to mimic the action of cytosolic macromolecules on mitochondria. Under these conditions, mitochondria have a more native, condensed configuration.Flux-dependent concentration gradients of ADP were estimated by measuring the ADP diffusion fluxes across the porin pores of isolated rat heart mitochondria incubated together with pyruvate kinase (PK), both of which compete for ADP regenerated by mitochondrial creatine kinase (mtCK) within the intermembrane space or by yeast hexokinase (HK) extramitochondrially. From diffusion fluxes and bulk phase concentrations of ADP, its concentrations in the intermembrane space were calculated using Fick's law of diffusion. Flux-dependent gradients up to 23 microM ADP (for a diffusion rate of J(Dif)=1.9 micromol ADP/min/mg mitochondrial protein) were observed. These gradients are about twice those estimated in the absence of dextran and in the same order of magnitude as the cytosolic ADP concentration (30 microM), but they are negligibly low for cytosolic ATP (5 mM). Therefore, it is concluded that the dynamic ADP compartmentation is of biological importance for intact heart cells. If mtCK generates ADP within the intermembrane space, the local ADP concentration can be clearly higher than in the cytosol resulting in higher extramitochondrial phosphorylation potentials. In this way, mtCK contributes to ensure optimal kinetic conditions for ATP-splitting reactions in the extramitochondrial compartment.

Adenosine Diphosphate↗

PubMed2002

Mitochondrial dysfunction in sepsis: evidence from bacteraemic baboons and endotoxaemic rabbits.

Mitochondria, that provide most of the ATP needed for cell work, and that play numerous specific functions in biosyntheses and degradations, as well as contributing to Ca2+ signaling, also play a key role in the pathway to cell death. Impairment of mitochondrial functions caused by mutations of mt-genome, and by acute processes, are responsible for numerous diseases. The involvement of impaired mitochondria in the pathogenesis of sepsis is discussed. By means of the skinned fiber technique and high resolution respirometry, we have detected significantly reduced rates of mitochondrial respiration in heart and skeletal muscle of endotoxaemic rabbits. Mitochondria from heart were more affected than those from skeletal muscle. Decreased respiration rates were accompanied by reduced activities of complex I + III of the respiratory chain. Endotoxin-caused impairment was also detectable at the level of the Langendorff perfused heart, where the coronary vascular resistance was significantly increased. For an investigation of the influence of bacteraemia on the mitochondrial respiratory chain, baboons were made septic by infusion of high and low amounts of E. coli. For complex I + III and II + III, a clear dose-dependent decrease was detectable and in animals which died in septic shock, a further decrease of enzyme activities in comparison to the controls were found. These results are discussed in the light of current knowledge on the role of mitochondria in cell pathology in respect to sepsis. In conclusion, we present evidence that mitochondrial function is disturbed during sepsis. Besides ischaemic and poison-induced disturbances of mitochondrial function, sepsis is a further example of an acute disease where impaired mitochondria have to be taken into account.

Animals↗

PubMedDate not supplied

Energetic depression caused by mitochondrial dysfunction.

Mitochondria, providing most of ATP needed for cell work, realizing numerous specific functions as biosyntheses or degradations, contributing to Ca2+ signalling also play a key role in the pathways to cell death. Impairment of mitochondrial functions caused by mutations of mt-genome and by acute processes are responsible for numerous diseases. The relations between changes on the level of molecules and the clinical state are rather complex, and the prediction of thresholds is difficult. Therefore investigations on different levels of an organismus (genome, metabolites, enzymes, mitochondrial function in vivo and in vitro) are necessary (multi level approach). Metabolic control theory is a valuable tool for understanding the different effects of mutations on the level of enzyme activities and mitochondrial function. Decreased concentrations of adenine nucleotides, leaky outer and inner mitochondrial membranes, decreased rates of mitochondrial linked pathways and decreased activities of respiratory chain enzymes contribute to depression of cellular energy metabolism characterized by decreased cytosolic phosphorylation potentials as one of the most important consequences of mitochondrial impairments. This review regards classical bioenergetic mechanisms of mitochondrial impairment which contribute to energetic depression.

Animals↗

PubMedDate not supplied

Energetic depression caused by mitochondrial dysfunction.

Mitochondria not only provide most of the ATP needed for cell work and numerous specific anabolic and catabolic functions, they also contribute to Ca++ signalling and play a key role in the pathway to cell death. Impairment of mitochondrial functions caused by mutations of the mt-genome or by acute processes, is responsible for numerous diseases. Decreased concentrations of adenine nucleotides, leaky outer and inner mitochondrial membranes, and decreased activities of respiratory chain enzymes contribute to depression of cellular energy metabolism, one of the most important consequences of mitochondrial impairment as characterized by decreased cytosolic phosphorylation potentials.

Adenine Nucleotides↗