Age-related changes in glutathione and lipid peroxide content in mouse synaptic mitochondria: Relationship to cytochrome c oxidase decline

To elucidate the contribution of lipid peroxides and glutathione to brain aging, we have carried out a comparative study of their contents in synaptic mitochondria from young (10-week-old), adult (24-week-old), and senescent (72-week-old) mice. In synaptic mitochondria, there is a significant decline in lipid peroxide content (P < 0.01) and cytochrome c oxidase activity (P < 0.001) in senescent as compared to adult and young mice. By contrast, glutathione concentration showed an increase in senescent (P < 0.05) in comparison to the other age groups. Moreover, there was a significant inverse correlation between age and lipid peroxide content (r = -0.5, P < 0.001) or cytochrome c oxidase-specific activity (r = -0.58, P < 0.001). We suggest that this age-dependent decrease in lipid peroxide content in synaptic mitochondria may be the result of an age-related decline in the activity of the electron transport chain, with concomitant decrease in oxyradical production, in the senescent organelles.     

Aberration of heme and hemoprotein in aged female rats

The magnitude and duration of drug action is determined partially by the activity of the drug metabolizing enzyme systems in the liver. The pharmacological effectiveness of many drugs is altered during the aging process. In this study, the regulation of heme metabolism and hemoprotein content was examined in livers of aged female rats. The activities of hexobarbital hydroxylase and aniline hydroxylase, indicators of mono-oxygenase function, were decreased in aged rats by 31% and 24%, respectively, as compared to values in young rats. This was accompanied by a proportional decrease in the level of cytochrome P-450 (26%). Additionally, the activity of delta-aminolevulinic acid synthetase (ALA-S), the rate-limiting enzyme in heme synthesis, and the microsomal concentration of heme were also decreased by 33% and 26%, respectively, in these animals. In contrast, the basal activity of microsomal heme oxygenase (MHO), the rate-limiting enzyme in heme degradation, and the percent heme saturation of tryptophan pyrrolase (TPO), a sensitive indicator of changes in the availability of heme in the "regulatory" heme pool, were increased by (87%) and (31%), respectively, in the aged rats. The serum concentration of bilirubin, an indicator of erythrocyte breakdown and/or liver function was likewise increased in these animals. In view of these findings, we suggest that the high activity of MHO and the low level of ALA-S may be a significant causative factor for the decreased microsomal concentration of heme, cytochrome-P-450 and its dependent monooxygenase activities in senescent female rats.     

Heme oxygenase activity and some indices of antioxidant protection in rat liver and kidney in glycerol model of rhabdomyolysis

Activity of heme oxygenase, superoxide dismutase, and catalase, the content of reduced glutathione and total heme in the liver and kidneys, and serum absorption spectrum in the Soret band were studied in rats with glycerol-induced rhabdomyolysis. Glycerol increased the content of heme-containing metabolites in the serum and the total heme content in the liver and kidneys, and decreased the content of reduced glutathione and catalase activity in the examined organs. Superoxide dismutase activity increased in the liver and decreased in the kidneys. Heme oxygenase activity increased in the liver and kidneys 2 and 6 h postinjection, respectively. The effects of heme delivered to the liver and kidneys from the vascular bed on the antioxidant defense and heme oxygenase activity were studied.     

Effect of Hypoxia on Mitochondrial Mass and Cytochrome Concentrations in Rat Heart and Liver during Postnatal Development

Cytochrome concentrations of rat heart and liver mitochondria were measured postnatally from newborn animals to young adults. Mitochondrial cytochrome aa₃ concentration increased shortly after birth in both tissues, this concentration being in a newborn animal 0.149 ± 0.027 nmol/mg protein in liver and 0.333 ± 0.082 nmol/mg protein in heart. The respective values from a two week old animal were 0.216±0.031 and 0.583 10.100. Postnatally cytochromes c±c₁ and b increased markedly in the heart, but in the liver of newborn animals the cytochrome content was more close to the adult values. The amount of mitochondrial protein increased after birth, too. In a newborn animal the mitochondrial protein values were 39.7 ± 3.6 mg/g wet weight in liver and 26.6± 6.5 mg/g wet weight in heart. In adult animals the respective values were 71.5 ± 4.8 and 80.7± 13.1. The effect of postnatal hypoxia (10% O₂, 24 h and 48 h) on liver and heart mitochondrial cytochrome concentration and protein values of newborn animals were investigated. During hypoxia the amount of mitochondrial protein remained about at the level of a newborn animal. The postnatal increase in the mitochondrial cytochrome concentration, which was smaller in the liver than in the heart, was also inhibited by hypoxia.     

Mitochondrial alterations in heart,kidney and liver of rats subjected to anemic hypoxia

The purpose of this investigation was to determine the effects of anemic hypoxia on the mitochondrial respiratory components of heart, kidney, and liver. Male Sprague-Dawley rats were injected subcutaneously with phenylhydrazine hydrochloride (60 mg/kg body weight/48 hr) for 28 days. Determinations on body weight, organ protein, liver glycogen, urea excretion, and plasma glucose concentration suggested that the anemic animals underwent a mild nutritional deficiency during the initial phase of the anemia but that they fully recovered by Day 22. However, the rates of systemic oxygen consumption of the animals fell with the onset of anemia and remained significantly below normal during the 4 weeks of observations. Determinations on the specific activity of cytochrome oxidase on Days 0, 10, and 21 of anemia suggested that no significant changes in the ratios of mitochondrial mass to organ mass occurred for heart, kidney, and liver except for a major diminution in liver at Day 10. However, mitochondria isolated on Days 10 and 21 from heart, kidney, and liver of anemic animals were consistently deficient in one or more of the cytochromes in the b to c portion of the electron transport chain when compared to mitochondria isolated from control animals. This suggested a relationship between systemic hypoxia and cytochrome depletions.     

Induction of heme oxygenase in guinea‐pig stomach: roles in contraction and in single muscle cell ionic currents

The role of heme oxygenase reaction products in modulation of stomach fundus excitability was studied. The presence of constitutive heme oxygenase 2 was verified in myenteric ganglia by immunohistochemistry. The role of inducible heme oxygenase isoenzyme was investigated after invivo treatment of animals with CoCl₂ (80 mg kg^?1 b.w) injected subcutaneously 24 h before they were killed. This treatment resulted in increased production of bilirubin and positive staining for the inducible isoform in stomach smooth muscle and vast induction in the liver. In both control and treated animals haemin, applied to the bath as a substrate of heme oxygenase caused significant decrease of prostaglandin F_2α‐induced tone, and ameliorated the relaxatory response of the fundic strips to electrical field stimulation. Both effects were antagonized by Sn‐protoporphyrin IX, competitive heme oxygenase inhibitor, and were found to be neuronally dependent. In single freshly isolated smooth muscle cells from control animals haemin caused a concentration‐dependent increase of the whole cell K⁺ currents, which was not affected by Sn‐protoporphyrin IX, cyclic guanosine monophosphate (cGMP)‐dependent protein kinase or guanylyl cyclase antagonists, but was reversed by various antioxidants and abolished by an NO scavenger. In cells from treated animals the K⁺ current increasing effect of haemin did not depend on the presence of antioxidants, but was abolished by protein kinase G and guanylyl cyclase inhibitors, depletors of intracellular Ca²⁺ pools or Sn‐protoporphyrin IX. Biliverdin did not affect contraction or ionic currents. Thus, this is the first study demonstrating that heme oxygenase is an inducible enzyme in guinea‐pigs, which exerts a modulatory role on gastric smooth muscle excitability via carbon monoxide production.     

Cytochromes c-dependent aerobic respiration of Paracoccus denitrificans

Terminal parts of the respiratory chain of Paracoccus denitrificans containing cytochromes c have been investigated through the use of NNN′Nprime;-tetramethyl-p-phenylenediamine (TMPD) as an electron donor. The cells have been shown to possess two major membrane bound TMPD oxidases characterized by their different sensitivities to cyanide. The more sensitive one, identified as cytochrome aa₃, appears to be produced under oxygen-rich growth conditions, whereas a less sensitive and as yet uncharacterized enzyme accompanied with only moderate amounts of cytochrome aa₃ functions when oxygen becomes limiting. The soluble periplasmic cytochrome cd₁ does not contribute significantly to the total oxygen consumption rate, although its maximal catalytic capacity may be high. The fact thap the activity of alternative TMPD oxidase is not proportional to cytochrome o content is discussed in relation to possible occurrence of two distinct cytochrome o-type terminal oxidases.     

Mutations in the structural gene for cytochrome c result in deficiency of both cytochromes aa 3 and c in Neurospora crassa

The cyt-12-12 mutant of Neurospora crassa is characterized by slow growth and a deficiency of spectrophotometrically-detectable cytochromes aa ₃ and c. Using a sib-selection procedure we have isolated the cyt-12 ⁺ allele from a cosmid library of N. crassa genomic DNA. Characterization of the cyt-12 ⁺ allele reveals that it encodes the structural gene for cytochrome c. DNA sequence analysis of the cyt-12-12 allele revealed a mutation in the cytochrome c coding sequence that results in replacement of a glycine residue, which is invariant in the cytochrome c of other species, with an aspartic acid. Genetic analysis confirms that cyt-12-12 is allelic with the previously-characterized cyc-1-1 mutant, which was also shown to affect the single locus encoding cytochrome c in N. crassa. We suggest that the amount of functional cytochrome c present in mitochondria influences the level of cytochrome aa ₃ .     

Heme oxygenase-1, heme oxygenase-2 and biliverdin reductase in peripheral ganglia from rat,expression and plasticity

The expression of inducible and constitutive heme oxygenase and biliverdin reductase was studied in normal and cultured peripheral ganglia from adult rats, using immunocytochemistry and in situ hybridization. Dramatic changes were induced by one to two days' culturing of dorsal root ganglia, nodose ganglia, otic ganglia, sphenopalatine ganglia and superior cervical ganglia. An up-regulation of inducible heme oxygenase was found in satellite cells of the cultured nodose ganglia, dorsal root ganglia, sphenopalatine ganglia and otic ganglia, whereas only a few satellite cells in the superior cervical ganglia responded with an increase in inducible heme oxygenase immunoreactivity. In the superior cervical ganglia inducible heme oxygenase also appeared in a subpopulation of macrophages. During culturing, expression of inducible heme oxygenase immunoreactivity also increased in axons and in nerve cell bodies. In situ hybridization corroborated the immunocytochemical findings, revealing a strong up-regulation of inducible heme oxygenase messenger RNA in satellite cells, and less pronounced up-regulation in nerve cell bodies. Constitutive heme oxygenase immunoreactivity was found in most neurons in all of the ganglia studied. No significant changes in constitutive heme oxygenase immunoreactivity could be observed in cultured ganglia. Biliverdin reductase immunoreactivity was barely detectable in any of the normal ganglia; however, after culturing it appeared in axons, single nerve cell bodies and nerve cell nuclei.The results show that inducible heme oxygenase is up-regulated in peripheral ganglia after axonal injury, and suggest a role for carbon monoxide in cellular signaling and a requirement for the antioxidant (bilirubin) during the regeneration process.     

Role of interleukin-1beta in the modulations of cytochrome P450 and heme metabolism in rat liver.

The effect of recombinant human interleukin-1beta (IL-1beta) on the modulation of hepatic cytochrome P450 (P450) was investigated by in vivo subcutaneous dosing studies in male Sprague-Dawley rats. To assess the effect of IL-1beta on heme metabolism, we determined the delta-aminolevulinic acid synthetase (delta-ALAS) and heme oxygenase activities in the liver. IL-1beta suppressed the microsomal total P450 and heme contents and delta-ALAS activity in the liver. In contrast, microsomal heme oxygenase activity was significantly increased by the IL-1beta treatments. Western blot analysis and marker enzyme activities for individual P450 isoforms demonstrated that IL-1beta suppressed CYP2C6, 2C13, 2E1, and 3A2, whereas CYP2A, 2B1/2, 2C11, and 4A1 were not influenced by the treatments. IL-1beta inhibited both allylisopropylamide- and phenobarbital-inducible delta-ALAS activities in the liver. These results indicate that IL-1beta has differential effects on the constitutive P450, and also on delta-ALAS and heme oxygenase activities in rat liver. Thus, the modulation of hepatic P450 by IL-1beta is complex, and IL-1beta may be involved in the regulation of both apoprotein synthesis for each P450 isoform and the heme pools in the liver.     

Influence of aging on cerebral derangement by acute severe hypoxia during hypovolemic hypotension

In synaptosomes isolated from the motor area of the cerebral cortex of beagle dogs and incubated in Krebs-Henseleit-Hepes buffer (for 10 min at 24°C), the energetic state was defined by the balance of the labile phosphates (ATP, ADP, AMP, and creatine phosphate), the redox state of the intramitochondrial NAD-couple, and the respiratory rate. By the experimental model utilized in the present research, it is possible to evaluate the potential synaptosomal damage induced by the in vivo hypoxic insult. Aging affects the phosphorylation state of the posthypoxic incubated synaptosomes. Although the oxygen consumption rate is the same in the synaptosomal fractions from the motor area of hypoxic beagle dogs of different ages, the cytochrome c and a contents are lower in the preparations from older brains. This points to higher activity of cytochromes in the synaptosomes from “mature” and “senescent” hypoxic animals. In dogs of different ages, hypoxia lowers the respiration of the synaptosomes but aging affects the oxygen consumption rates only in post-hypoxic synaptosomes incubated with succinate. In synaptosomes isolated from older hypoxic brains, the free energy utilized for the synthesis of two moles of ATP (ΔG_ATP) is progressively lower than that released upon the transfer of electrons from the NADH to cytochrome c (ΔG_{ox red}).     

The damage of the hepatic mixed functional oxygenase system by CCl4: significance of incorporation of 14CCl4 metabolites in vivo

The relation between incorporation of ¹⁴C from ¹⁴CCl₄ into liver lipids and damage to the mixed functional oxygenase system of liver microsomes was investigated in rats of different sex or differently pretreated. Under the respective conditions of pretreatment the ¹⁴C incorporation rate and the relative decrease of the cytochrome P-450 level as the parameters most influenced by CCl₄ were compared with those for untreated female rats. Induction by 3-methylcholanthrene does not after both parameters. Induction by sodium phenobarbital enhances the ¹⁴C incorporation rate as well as the cytochrome P-450 decrease but the latter is more affected. In male rats only the ¹⁴C incorporation rate increased. Vitamin D₃-pretreated rats showed a smaller decrease of cytochrome P-450 but an enhanced ¹⁴C incorporation rate. Therefore, there is no correlation between the cytochrome P-450 decrease and ¹⁴C incorporation rate. Reasons for the lack of that correlation are discussed with regard to the mechanism of liver cell damage by carbon tetrachloride.     

Gingipains from Porphyromonas gingivalis increase the chemotactic and respiratory burst-priming properties of the 77-amino-acid interleukin-8 variant

Porphyromonas gingivalis, a gram-negative anaerobe which is implicated in the etiology of active periodontitis, secretes degradative enzymes (gingipains) and sheds proinflammatory mediators (e.g., lipopolysaccharides [LPS]). LPS triggers the secretion of interleukin-8 (IL-8) from immune (72-amino-acid [aa] variant [IL-8_72aa]) and nonimmune (IL-8_77aa) cells. IL-8_77aa has low chemotactic and respiratory burst-inducing activity but is susceptible to cleavage by gingipains. This study shows that both R- and K-gingipain treatments of IL-8_77aa significantly enhance burst activation by fMLP and chemotactic activity (P < 0.05) but decrease burst activation and chemotactic activity of IL-8_72aa toward neutrophil-like HL60 cells and primary neutrophils (P < 0.05). Using tandem mass spectrometry, we have demonstrated that R-gingipain cleaves 5- and 11-aa peptides from the N-terminal portion of IL-8_77aa and the resultant peptides are biologically active, while K-gingipain removes an 8-aa N-terminal peptide yielding a 69-aa isoform of IL-8 that shows enhanced biological activity. During periodontitis, secreted gingipains may differentially affect neutrophil chemotaxis and activation in response to IL-8 according to the cellular source of the chemokine.     

Status of hepatic heme and heme oxygenase during Plasmodium yoelii nigeriensis infection in mice

Plasmodium yoelii nigeriensis infection in albino mice caused a significant increase in hepatic heme level, the increase being concomitant with a rise in parasitemia. This elevated heme was found to be associated with all the subcellular fractions except the cytosol, where its content remained unaltered. Activity of heme oxygenase, the key enzyme responsible for catabolism of heme, also increased progressively with rise in parasitemia. Treatment of normal mice with cobalt chloride [60 mg (kg body wt)-1; subcutaneously] brought about a 150% increase in the level of heme oxygenase; similar treatment of infected mice at low parasitemia could induce the enzyme activity while at high parasitemia the enzymic activity remained unaltered as compared to untreated infected mice. In spite of an increased level of heme oxygenase in the cobalt-treated mice, the level of heme did not show any noticeable change. Oral administration of chloroquine [64 mg (kg body wt)-1 x 4 days] brought about a 56% reduction in the level of heme oxygenase of normal animals but there was no change in infected animals when compared with the corresponding untreated infected mice. However, the amount of chloroquine present in livers of normal and infected animals was not significantly different.     

Age-dependent changes in rat brain mitochondria of synaptic and non-synaptic origins

Synaptic and non-synaptic mitochondria were isolated from the brains of 3- 12-and 28-30-month-old female Fisher 344 rats. Total oxygen consumption and oxygen consumption due to mitochondrial respiration decreases 83% with increasing age in synaptic mitochondria using malate plus glutamate as substrate, but only 33% in non-synaptic mitochondria; succinate-driven activity is not affected. Succinate-driven superoxide generation decreases over 90% in both fractions; malate plus glutamate-driven superoxide generation decreases 50% in synaptic mitochondria only. The amount of c- and a-type cytochromes decreases approximately 50% in synaptic mitochondria. The absorbance wavelength maximum of cytochrome b decreases 2.6 nm in synaptic mitochondria from senescent brains but only 1.6 nm in non-synaptic mitochondria.     

Hepatic heme metabolism in rats with fever induced by interleukin 1beta

We have recently reported that the content of hepatic cytochrome P450 (CYP) apparently decreased in fever model rats, which were created by repeated injection of recombinant human interleukin-1beta (rhIL-1beta) into the cerebroventricle. To make clear the biochemical mechanism of the decreased CYP content, we examined the effect of fever on the activities of hepatic enzymes involved in the biosynthetic and degradative pathways of heme. The activities of delta-aminolevulinic acid synthase, a rate-limiting enzyme in the heme biosynthesis, and porphobilinogen synthase in the liver of rhIL-1beta-induced fevered rat were significantly lower than those in the control, whereas the activity of heme oxygenase, a key enzyme in the heme-degradative pathway, markedly increased in the fevered rat. Moreover, the heme saturation of tryptophan 2,3-dioxygenase in the fevered rat liver was decreased to 43% of the control. These results indicate that fever diminishes the hepatic heme content by decreasing the heme biosynthesis and by accelerating the heme degradation. The deficiency of hepatic heme pool may be one of the main mechanisms that cause the impairment of CYP synthesis.     

Bacterial Terminal Oxidases

Abstract

Oxidases, as such, regardless of their source, represent a diverse and complex series of enzymes. What they have in common is the ability to react with molecular oxygen, activate it chemically (in a manner which is still not understood), and utilize the “activated atoms of oxygen” primarily as electron acceptors. Should the “activated oxygen atoms” be used directly for oxygenating substrate molecules, such as hydrocarbons, then according to the conventions used today, the enzyme is termed an oxygenase rather than an oxidase. The subject of oxygenases is far too complex to be reviewed in any detailed treatment of oxidases. All oxidases serve as electron acceptors for specific dehydrogenation reactions that are carried out by the multitude of dehydrogenases that are found in tissues as well as in bacteria. The major end product that results from the oxidase reaction is either H₂O or H₂O₂. The oxidases can be (1) simple flavoprotein-containing enzymes, such as the glucose oxidase or the D- and L-amino acid oxidases, (2) metalloflavoprotein-containing enzymes, such as the xanthine oxidase which in addition to flavin contains both molybdenum and nonheme iron, (3) the heme-containing oxidases which essentially are free of flavoprotein yet carry out a peroxidase-oxidase type of reaction, and finally (4) the heme-containing and flavoprotein-free cytochrome (or terminal) oxidase molecule which may contain as many as two heme “a” components (multiple heme iron), copper protein, and the active form of which requires an enzyme complex containing some phospholipid.^{1 4 5} It is this latter group, the cytochrome or terminal oxidases, that will be considered in this review, particularly what is known about them in bacterial systems. The cytochrome or terminal oxidases are usually membrane-bound entities, play a major role in electron transport, and are very important in the bioenergetic mechanism of aerobic cells that allow for respiration.     

Age-related changes of long-chain fatty acid metabolism in rat liver

Palmitate oxidation activity and the activities of several enzymes involved in long-chain fatty acid metabolism were examined in the liver of young adult (2-month-old) and senescent (32-month-old) female rats. Palmitate oxidation activity in rat liver mitochondria showed age-related decrease, as judged by the rates of both 14CO2 production and formation of radioactive acid-soluble products from [1-14C]palmitate. In addition, long-chain acyl-coenzyme A synthetase activity was found to be decreased in liver mitochondria and increased in liver microsomes in senescent rats. These results suggest that, in the rat liver, preferential channeling of long-chain fatty acids through the triacylglycerol synthetic pathway may increase with age, and as a result, energy production by their oxidation may decrease.     

Bioenergetics and permeability transition pore opening in heart subsarcolemmal and interfibrillar mitochondria: Effects of aging and lifelong calorie restriction

Loss of cardiac mitochondrial function with age may cause increased cardiomyocyte death through mitochondria-mediated release of apoptogenic factors. We investigated ventricular subsarcolemmal (SSM) and interfibrillar (IFM) mitochondrial bioenergetics and susceptibility towards Ca²⁺-induced permeability transition pore (mPTP) opening with aging and lifelong calorie restriction (CR). Cardiac mitochondria were isolated from 8-, 18-, 29- and 37-month-old male Fischer 344 × Brown Norway rats fed either ad libitum (AL) or 40% calorie restricted diets. With age, H₂O₂ generation did not increase and oxygen consumption did not significantly decrease in either SSM or IFM. Strikingly, IFM displayed an increased susceptibility towards mPTP opening during senescence. In contrast, Ca²⁺ retention capacity of SSM was not affected by age, but SSM tolerated much less Ca²⁺ than IFM. Only modest age-dependent increases in cytosolic caspase activities and cytochrome c levels were observed and were not affected by CR. Levels of putative mPTP-modulating components: cyclophilin-D, the adenine nucleotide translocase (ANT), and the voltage-dependent ion channel (VDAC) were not affected by aging or CR. In summary, the age-related reduction of Ca²⁺ retention capacity in IFM may explain the increased susceptibility to stress-induced cell death in the aged myocardium.