Studies of human achondroplasia: oxidative metabolism in tissue culture cells

Mitochondria prepared from the first growth of cells (fibroblasts) from skin biopsies from homozygous (but not heterozygous) achondroplastic human subjects were unable to carry out oxidative phosphorylation. However, successive crops of cells gained the ability to phosphorylate with normal P:O ratios with pyruvate-malate and succinate as substrates. Concentrations of cytochromes a + a3 were markedly and significantly lower in homogenates of homozygous achondroplastic tissue culture cells than in homogenates of normal cells. Levels of cytochromes a + a3 in the heterozygous achondroplastic cells were intermediate between the levels in normal cells and the homozygous achondroplastic cells. Activities of the mitochondrial oxidative systems (NADH, succinic and cytochrome oxidases) were not significantly lower in the achondroplastic cell preparations than in normal cell preparations under standard assay conditions (saturation levels of oxygen).     

Normal somatomedin and somatomedin receptors in achondroplastic dwarfism

It has been proposed that the basic abnormality in achondroplasia may be a quantitative defect in endochondral new bone formation secondary to decreased synthesis of somatomedin (SM) or abnormal binding of SM to specific receptors. To test this hypothesis, we have measured plasma SM levels and SM receptors on circulating mononuclear cells obtained from 5 achondroplastic dwarfs and 5 age-matched controls. Plasma SM levels were 0.82 +/- 0.14 U/ml (mean +/- S.E.M.) for the achondroplastic dwarfs and 0.90 +/- 0.12 U/ml for the controls. The specific binding of 125I-SM to 50 x 10(6) mononuclear cells was 7.66 +/- 1.11% for the dwarf group and 7.66 +/- 1.16% for the controls. Achondroplastic and control cells possessed equal numbers of receptor sites and identical receptor affinity for SM. The data indicate that plasma SM levels and SM binding to circulating mononuclear cells are normal in achondroplastic dwarfs and suggest a primary intrace-lular defect.     

Cytokinetic basis for the impaired activation of lymphocytes from patients with primary intracranial tumors

Patients with malignant brain tumors have a variety of immunologic abnormalities, including the impaired responsiveness of peripheral blood lymphocytes (PBL) to mitogens and alloantigens. We further investigated this impairment of lymphocyte reactivity by employing the techniques of limiting dilution analysis and cytokinetic analysis. PBL preparations from patients have approximately six times fewer phytohemagglutinin (PHA)-responsive cells than PBL from normal subjects. Similar results were obtained with purified T cell preparations. Cytokinetic analysis of PHA-induced [3H]thymidine incorporation employing colchicine blocking of mitosis demonstrated that the number of first generation cells entering the S-phase of mitosis for each 24-hr period was less for PBL from patients than for PBL from normal individuals. First generation responding cells from patients and normal subjects entered DNA synthesis at the same time (48 to 72 hr). Cytokinetic analysis over a period of 168 hr demonstrated that whereas PBL from normal individuals demonstrated second generation responding cells, PBL from the majority of patients did not, thus indicating a defect in their ability to undergo clonal expansion. Measurement of interleukin 2 (IL 2) activity in culture fluids from PHA-activated PBL from normal subjects and patients revealed significantly lower IL 2 levels in culture fluids from PBL from patients. The addition of various concentrations of lectin-free IL 2 to PBL from patients stimulated with PHA did not restore responsiveness to normal values. There was no difference between the levels of interleukin 1 (IL 1) produced by lipopolysaccharide-activated monocytes from normal subjects and patients. Overall, these results suggest that an intrinsic defect exists in T cells obtained from brain tumor patients that renders them unable to enter into normal mitogen-induced blastogenesis.     

In vitro proliferation of achondroplastic and normal mouse chondrocytes, before and after basic fibroblast growth factor stimulation

Achondroplasia in mice is a recessive genetic disorder, characterized by disproportionate dwarfism with reduced bone growth. The cause of this chondrodystrophy is unknown. In this study normal and achondroplastic mouse chondrocytes were cultured in monolayer primary culture, their differentiation was verified by immunofluorescence and their growth was compared. The results showed that achondroplastic cells exhibited a higher proliferative activity than control cells of the same age, confirmed also by a thymidine incorporation assay. Furthermore, basic fibroblast growth factor treatment was found to induce a strong increase in growth of normal mouse chondrocytes, while it did not stimulate statistically significant proliferation of achondroplastic mouse cells. We suppose that this different growth rate could play a role in achondroplastic phenotype development.     

Cytochrome P-450 distribution in rat liver and the effect of sodium phenobarbitone administration

A microspectrophotometric method for assaying cytochrome P-450 in fresh 24 μm unfixed cryostat sections of rat liver has been developed. When used to assay this cytochrome in sections of microsomal preparations it has yielded results equivalent to those obtained by the conventional spectrophotometric assay of the same preparations. Random measurements made throughout sections of liver have given mean values for cytochrome P-450 concentrations which are twice those measured in microsomes prepared from the livers of the same animals (not corrected for the yield in the homogenate).

Measurements of the cytochrome P-450 content of liver cells by the microspectrophotometric method show that in liver from male Wistar rats, cells nearer to the central veins contain up to twice as much cytochrome P-450 as those nearer to the portal tract (mean cell concentrations of 26.4 (±4.4) μmol/l and 17.5 (±3.0) μmol/l respectively). In the livers from similar rats, killed at the same time, but which had received 1 mg/ml sodium phenobarbitone in their drinking water for one week, the cells near the central vein contained up to five times as much cytochrome P-450 as those near the portal tract (mean cell concentrations of 77.3 (±25.0) μmol/l and 28.3 (±9.6) μmol/l respectively).

The results show a selective increase in cytochrome P-450 content by the cells in the centrilobular region after treatment with sodium phenobarbitone and a smaller increase by some of the cells in the periportal region.     

Cellular non-heme iron content is a determinant of nitric oxide-mediated apoptosis, necrosis, and caspase inhibition

In this report, we tested the hypothesis that cellular content of non-heme iron determined whether cytotoxic levels of nitric oxide (NO) resulted in apoptosis versus necrosis. The consequences of NO exposure on cell viability were tested in RAW264.7 cells (a cell type with low non-heme iron levels) and hepatocytes (cells with high non-heme iron content). Whereas micromolar concentrations of the NO donor S-nitroso-N-acetyl-DL-penicillamine induced apoptosis in RAW264.7 cells, millimolar concentrations were required to induce necrosis in hepatocytes. Caspase-3 activation and cytochrome c release were evident in RAW264.7 cells, but only cytochrome c release was detectable in hepatocytes following high dose S-nitroso-N-acetyl-DL-penicillamine exposure. Pretreating RAW264.7 cells with FeSO(4) increased intracellular non-heme iron to levels similar to those measured in hepatocytes and delayed NO-induced cell death, which then occurred in the absence of caspase-3 activation. Iron loading was also associated with the formation of intracellular dinitrosyl-iron complexes (DNIC) upon NO exposure. Cytosolic preparations containing DNIC as well as pure preparations of DNIC suppressed caspase activity. These data suggest that non-heme iron content is a key factor in determining the consequence of NO on cell viability by regulating the chemical fate of NO.     

Age-dependent decay of cytochrome b5 and cytochrome b5 reductase in human erythrocytes.

Age-dependent decrease in cytochrome b5 was observed in erythrocytes from both a normal person and a patient with hereditary methaemoglobinaemia without neurological symptoms. With aging, concentrations of cytochrome b5 in erythrocytes from the patient were almost the same as those in the control. Age-dependent decrease in cytochrome b5 reductase activity in the control erythrocytes was also shown; however, the reductase activity was very low in erythrocytes from the patient over the whole age range. Our studies show that methaemoglobin content of erythrocytes seems to be dependent on the content of cytochrome b5 in the cells, both in the control subject and in the patient.     

Physiological Effects of Menaquinone Deficiency in Bacillus subtilis

Several aspects of the respiratory physiology of a mutant of Bacillus subtilis deficient in menaquinone-7 (MK-7) and in cytochromes were investigated. The mutant, an aromatic amino acid auxotroph blocked at dehydroshikimate reductase, is unable to synthesize MK-7 unless grown in the presence of the common aromatic amino acid intermediate, shikimate. The inability to synthesize MK-7 prevents the mutant from expressing the normal postexponentialphase cytochrome phenotype. When grown in the presence of shikimate, normal levels of these electron transport components are formed. It was found that the intracellular concentration of MK-7 could be predictably regulated by growing the cells with known concentrations of exogenous shikimate. When the mutant was grown under conditions where MK-7 biosynthesis was severely limited, there was a decrease in oxygen uptake and in membrane-associated reduced nicotinamide adenine dinucleotide (NADH) oxidase and succinate oxidase activity. NADH oxidase, but not succinoxidase, could be restored in membrane preparations by the addition of menadione to the reaction mixture. Reduced-minus-oxidized cytochrome difference spectra indicate that an MK-7 deficiency limits electron flow through the cytochrome chain. Furthermore, oxidation-reduction patterns suggest that MK-7 functions between the primary dehydrogenases and the cytochromes. Although the mutant is asporogenous when grown under conditions where MK-7 biosynthesis is limited, the inability to sporulate does not appear to result from lesions in the electron transport system.     

Respiratory metabolism of normal and divisionless strains of Candida albicans

Respiration of a normal strain of Candida albicans was compared with that of a divisionless mutant which has a biochemical lesion such that metabolically generated hydrogen "spills over," during growth, for non-specific dye reduction. This waste is not at expense of growth, since both strains grow at essentially similar rates, nor at expense of respiration, since the mutant reduces oxygen more rapidly than the normal strain. Respiration in both strains is qualitatively similar, and seemingly unique among highly aerobic organisms in that it is not mediated by cytochrome oxidase. In resting cells of both strains, respiration is not only resistant to, but markedly stimulated by, high concentrations of cyanide, carbon monoxide, and azide. In contrast, growth of these yeasts is inhibited by low concentrations of cyanide and azide. Cytochrome oxidase could not be detected in cell-free preparations; reduced cytochrome c was not oxidized by such preparations. Cytochrome bands could not be observed in thick cell suspensions treated with reducing agents. However, incorporation of superoptimal levels of zinc and iron into the culture medium resulted in growth of cells possessing distinct cytochrome bands; respiration of these cells remained insensitive to cyanide, monoxide, and azide, and the bands were maintained in a reduced form on oxygenation. In the divisionless yeast, tetrazolium dyes compete with oxygen for reduction; this is not the case in the normal strain. The firmness with which hydrogen transfer is channeled in the latter for reduction of disulfide bonds (of importance in the division mechanism) and of oxygen, is contrasted with the lack of such control in the mutant.     

Freezing injury in the yeast respiratory system

The effect of freeze-thawing on the yeast respiratory system was studied at rapid rates of cooling. Freezing of whole cells with liquid nitrogen induced decrease of respiratory activity to under 20% of that of original cells. Mitochondria harvested from freeze-thawed cells have markedly decreased succinate oxidizing activity. Activity of succinate cytochrome c reductase was reduced significantly after freeze-thawing of whole cells while activities of succinate dehydrogenase and cytochrome c oxidase were reduced slightly. By spectrophotometric analysis it was found that about one-half the amount of cytochrome c + c1 was eluted from mitochondria to cytosol after freeze-thawing of cells. The activities of succinate oxidation in mitochondria from freeze-thawed cells were restored to normal levels by the addition of cytochrome c. Freeze-thawing of isolated mitochondria did not induce deactivation of succinate oxidizing activities and succinate cytochrome c reductase, and no elution of cytochrome c was observed. It was concluded that the decreased respiratory activities of yeast cells by freezing of cells with liquid nitrogen can be attributed primarily to the elution of cytochrome c from mitochondria.     

Effect of aluminium phosphide exposure on kinetic properties of cytochrome oxidase and mitochondrial energy metabolism in rat brain

This study involves the effect of aluminium phosphide exposure on the kinetic characteristics of cytochrome oxidase and the mitochondrial respiratory chain function in rat brain. Mitochondrial preparations from both control and aluminium phosphide-treated rats demonstrated significant decrease in the maximal activity of cytochrome oxidase (approximately 50%) when expressed per unit membrane protein and on a turnover number basis (nmol/min/nmol haem a). The results indicated that there was a decrease in the catalytic efficiency of the active oxidase molecules on aluminium phosphide treatment. Arrhenius plot characteristics differ for cytochrome oxidase activity in mitochondria isolated from treated and control rats, in the break point of the biphasic plot which was shifted to a higher temperature. The decreased activity of cytochrome oxidase along with altered NADH and succinic dehydrogenase activities might have contributed towards a significant decline in state 3 and state 4 respiration. These alterations in the electron transport chain complexes in turn affected the ATP synthesis rate adversely in the mitochondria, isolated from treated rats. The data reflect the interaction of aluminium phosphide with redox chain components leading to the impairment of the electron transfer along the respiratory chain.     

Metabolic effects of propionate in normal and vitamin B12-deficient rats

1. Administration of propionate caused a twofold increase in the concentrations of lactate and pyruvate in the blood of vitamin B(12)-deficient rats, whereas there was a slight decrease in lactate and a 50% increase in pyruvate in normal rats. 2. Concentrations of total ketone bodies in the blood of normal rats were not significantly altered by propionate administration but the [3-hydroxybutyrate]/[acetoacetate] ratio decreased from 3.0 to 2.0. In the vitamin B(12)-deficient rats there was a 40% decrease in total ketone bodies and a change in the ratio from 3.4 to 1.2. 3. The changes in the concentration of ketone bodies in freeze-clamped liver preparations were similar in pattern to those observed in blood. 4. Propionate administration caused a decrease in the concentration of acetyl-CoA in the livers of both groups of animals, but the absolute decrease was greater in the vitamin B(12)-deficient group. The decrease in the concentration of CoA was similar in both groups. 5. As in blood, there were threefold increases in the concentrations of lactate and pyruvate in the livers of the vitamin B(12)-deficient rats after propionate administration, whereas there was no significant change in the concentrations of these metabolites in the normal rats. 6. There was a 50% inhibition of glucose synthesis in perfused livers from vitamin B(12)-deficient rats when lactate and propionate were substrates as compared with lactate alone. 7. It is concluded that the conversion of lactate into glucose is inhibited in vitamin B(12)-deficient rats after propionate administration, and that this effect is due to inhibition of the pyruvate carboxylase step resulting from a decrease in acetyl-CoA concentration and a postulated increase in methylmalonyl-CoA concentration.     

Rate enhancement of the internal electron transfer in cytochrome c oxidase by the formation of a peroxide complex; its implication on the reaction mechanism of cytochrome c oxidase

The oxidation of reduced cytochrome c oxidase by hydrogen peroxide was investigated with stopped-flow methods. It was reported by us previously (A.C.F. Gorren, H. Dekker and R. Wever (1986) Biochim. Biophys. Acta 852, 81-92) that at low H2O2 concentrations cytochrome a is oxidised simultaneously with cytochrome a3, but that at higher H2O2 concentrations the oxidation of cytochrome a is slower than that of cytochrome a3. We now report that for high peroxide concentrations (10-45 mM) the oxidation rate of cytochrome a increased linearly with the concentration of H2O2 (k = 700 M-1.S-1). Upon extrapolation to zero H2O2 concentration an intercept with a value of 16 s-1 (at 20 degrees C and pH 7.4) was found. A reaction sequence is described to explain these results; according to this model the rate constant (16 S-1) at zero H2O2 concentration represents the true value of the rate of electron transfer from cytochrome a to cytochrome a3 when the a3-CuB site is oxidised and unligated. However, when a complex of hydrogen peroxide with oxidised cytochrome a3 is formed, this rate is strongly enhanced. The slope (700 M-1.S-1) would then represent the rate of cytochrome a3(3+)-H2O2 complex formation. From experiments in which the pH was varied, we conclude that the reaction of H2O2 with cytochrome a3(2+) is independent of pH, whereas the electron-transfer rate from cytochrome a to cytochrome a3 gradually decreases with increasing pH. From the temperature dependence we could calculate values of 23 kJ.mol-1 and 45 kJ.mol-1 for the activation energies of the oxidations by H2O2 of cytochrome a3(2+) and cytochrome a2+, respectively. The similarity of the values that were obtained for cytochrome a oxidation both with H2O2 and with O2 as the electron acceptor suggests that the reactions share the same mechanism. In 2H2O the reactions studied decreased in rate. For the reaction of 2H2O2 with reduced cytochrome a3 in 2H2O, a small effect was found (15% decrease in rate constant). However, the internal electron-transfer rate from cytochrome a to cytochrome a3 decreased by 50%, Our results suggest that the internal electron transfer is associated with proton translocation.     

Mitochondrial biogenesis in human fibroblasts

It is not known whether limitation of lifespan represents a programmed genetic event or is a result of environmental factors imposed by the conditions of culture. An investigation of the factors surrounding the limitedin vitro lifespan of human diploid fibroblasts has been undertaken. We have investigated the role of mitochondria in the finite lifespan of WI-38 human lung fibroblasts. Mitochondrial function was depressed in a controlled manner by treating cells with ethidium bromide and chloramphenicol both of which inhibit normal biogenesis. These antibiotics decrease cytochrome oxidase activity, change cell ultrastructure, and inhibit growth at high concentration. At lower concentrations the antibiotics do not affect cell proliferation for several generations. However, their effect is cumulative and after several generations the cells enlarge, stop dividing and die. Removal of antibiotics from the culture media before death restores proliferative capacity. At still lower concentrations cytochrome oxidase activity was decreased but continuous growth in the presence of the antibiotics caused no decrease inin vitro lifespan. Thus, the potential for oxidative metabolism appears to be in excess of that needed for cell proliferation at all stages of thein vitro lifespan of a culture. The importance of cytoplasmic protein synthesis was evaluated using cycloheximide, a specific inhibitor of this process. Cycloheximide was used to try to distinguish between the effects due to general inhibition and that due to specific inhibition of mitochondrial biogenesis. Exposure of cultures to concentrations of cycloheximide which inhibited growth drastically caused no decrease in cytochrome oxidase activity.     

Maintenance of oxidative phosphorylation protects cells from Actinobacillus actinomycetemcomitans leukotoxin-induced apoptosis

Subnanomolar concentrations (3 × 10⁻¹⁰ M) of Actinobacillus actinomycetemcomitans leukotoxin (Ltx) trigger apoptosis of JY cells, as shown by a decrease in mitochondrial transmembrane potential (ΔΨ_m), hyperproduction of reactive oxygen species (ROS) and release of cytochrome c from the intermembrane space. When compared with heat-inactivated leukotoxin (ΔI Ltx) controls, ATP levels in Ltx-treated JY cells continued to decrease during a 24 h experiment while cytoplasmic ADP concentrations were increasing. These results suggest that a blockage occurred in ATP/ADP exchange. To maintain ATP/ADP exchange, JY cells were transfected with bcl -2 and bcl -x_L and incubated with Ltx. ATP levels of the transfected cells decreased to 67% (JY/ bcl -2) and 73% (JY/ bcl-x _L) after the experiment. Furthermore, cytochrome c remained localized to the mitochondrial fraction of Ltx-treated JY/ bcl -2 and JY/ bcl-x _L cells, whereas its presence in the cytoplasmic fraction of JY/ gen cells suggests an uncoupling of electron transport. Expression of bcl -2 and bcl-x _L in cells inhibited downstream apoptotic events such as cleavage of poly(ADP-ribose) polymerase, DNA fragmentation and activation of a family of caspases. The results indicate that Ltx induces apoptosis through a mitochondrial pathway that involves decreased levels of the ADP in the mitochondrial matrix, a lack of substrate for ATP synthetase and arrest of oxidative phosphorylation.     

Alpha7 nicotinic receptor activation inhibits ethanol-induced mitochondrial dysfunction,cytochrome c release and neurotoxicity in primary rat hippocampal neuronal cultures

Primary hippocampal neuronal cultures exhibited a concentration- and time-dependent loss of cells when exposed to ethanol (EtOH). EtOH-induced neurotoxicity was attenuated by 2,4-dimethoxybenzilidene anabaseine (DMXB) which selectively activates alpha7 nicotinic receptors in a concentration-dependent manner. We further investigated the mechanisms of the protective effect of DMXB on EtOH- induced neurotoxicity. We found that EtOH decreased the mitochondrial membrane potential and released cytochrome c from mitochondria at neurotoxic concentrations. DMXB (3 microm) attenuated both of these actions in a manner that was in turn blocked with the nicotinic antagonist methyllyconitine (MLA) 100 nm. Neither DMXB nor MLA alone affected these parameters. These results suggest that the neuroprotection conferred by alpha7 nicotinic receptor activation may be mediated, at least in part, through preventing the decrease in the mitochondrial membrane potential and the increase in the release of cytochrome c caused by EtOH.     

Differential effects of adrenocorticotropic hormone on steroid hydroxylase activities in the inner and outer zones of the guinea pig adrenal cortex.

We have studied the effects of ACTH treatment on steroid hydroxylase activities in the inner (zona reticularis) and outer (zona fasciculata plus zona glomerulosa) zones of the guinea pig adrenal cortex. Animals received 5 or 10 U of ACTH daily for 6 days and enzyme activities were then assessed in isolated microsomal or mitochondrial preparations. In control animals, microsomal cytochrome P-450 concentrations were greater in the inner than outer zone, but mitochondrial P-450 levels were similar in the two zones. Microsomal 17 alpha-hydroxylase and mitochondrial 11 beta-hydroxylase activities were greater in the outer than inner zone, but microsomal 21-hydroxylase activity was greater in the inner zone. ACTH treatment decreased cytochrome P-450 concentrations in inner but not outer zone microsomes; mitochondrial P-450 levels were unaffected in both zones. ACTH caused a dose-dependent increase in inner zone 17 alpha-hydroxylase activity and decrease in 21-hydroxylase activity without affecting the activity of either enzyme in outer zone microsomes. ACTH also decreased 11 beta-hydroxylase activity in outer but not inner zone mitochondrial preparations. The net effect of ACTH treatment was to diminish the differences in steroid metabolism between the two zones. The results indicate that the effects of ACTH on steroid hydroxylase activities are both zone- and enzyme-dependent, suggesting the existence of multiple and independent regulatory mechanisms.     

Cytochrome P-450-dependent metabolism in alveolar type II epithelial cells: modulation by platelet-activating factor

Platelet-activating factor (PAF), an ether lipid mediator released from activated pulmonary phagocytes, was evaluated for its ability to affect cytochrome P-450-dependent activities in isolated rat alveolar type II cells. The data indicate that at non-toxic doses, PAF caused an increase in beta-naphthoflavone (BNF) inducible/alpha-naphthoflavone (ANF) sensitive ethoxyphenoxazone deethylase (EtOPx'ase) activity. At high concentrations of PAF, inhibition of both EtOPx'ase and metyrapone (MP) sensitive benzyloxyphenoxazone debenzylase (BzOPx'ase) activities and aggregation of type II cells were observed. The PAF analogs, lyso-PAF and enantio-PAF, exhibited actions similar to those observed with PAF. PAF-induced enhancement of EtOPx'ase activity required the presence of intact cells, whereas at high PAF concentrations decreased enzyme activities were observed in both intact cell and sonicated cell preparations. The data thus suggest that xenobiotic metabolism in alveolar type II cells can be modified by an inflammatory mediator, such as PAF, produced by alveolar phagocytes.     

Enhanced cytochrome oxidase activity and modification of lipids in heart mitochondria from hyperthyroid rats

In order to further investigate the mechanism regulating the control of mitochondrial respiration by thyroid hormones, the effect of the hyperthyroidism on the kinetic characteristics of cytocrome c oxidase in rat heart mitochondria was studied. Mitochondrial preparations from both control and hyperthyroid rats had equivalent K_m values for cytochrome c, while the maximal activity of cytochrome oxidase was significantly increased (by around 30%) in mitochondrial rats. This enhanced activity of cytochrome oxidase was associated to a parallel increases in mitochondrial State 3 respiration. The hormone treatment resulted in a decrease in the flux control coefficient of the oxidase. The enhanced activity of cytochrome oxidase in hyperthyroid rats does not appear to be dependent on an increases in the mass of this enzyme complex in that the heme aa₃ content was equivalent in both hyperthyroid and control preparations. The Arrhenius plot characteristics differ for cytochrome oxidase activity in mitochondria from hyperthyroid rats as compared with control rats in the breakpoint of the biphasic plot is shifted to a lower temperature. Cardiolipin content was significantly increased in mitochondrial preparations from hyperthyroid rats, while there were no significant alterations in the fatty acid composition of cardiolipin of control and hyperthyroid preparations. The results support the conclusion that the enhanced cytochrome oxidase activity in heart mitochondrial preparations from hyperthyroid rats is due to a specific increase in the content of cardiolipin.