M. Cheung

Modification of mitochondrial respiration by aging and dietary restriction.

Effects of aging and of dietary restriction on mitochondrial recovery and respiratory capacities have been assessed in mice. Old mice (23-26 months) did not differ from adult mice (9-12 months) in amounts of protein recovered in mitochondrial fractions of liver, brain and spleen, but did show a decline in specific activity of cytochrome c oxidase (cyt. c ox.) in liver and spleen. Age effects on in vitro respiration by mitochondria occurred in liver and spleen. In liver, only one substrate (beta-hydroxybutyrate) of four tested was respired at a different rate by old than by young mitochondria. Depression of state 3 respiration and 2,4-dinitrophenol (DNP)-uncoupled rates was observed for this substrate; however, this effect depended on expressing respiration on the basis of mitochondrial protein and was less overt if data were expressed per unit of cyt. c ox. activity. Old spleen mitochondria exhibited a grosser defect, showing a 40% decrease in the respiratory control index (RCI) for (succinate + rotenone)- supported respiration (the only substrate tested) due to a possible increase in state 4 rates. Effects of dietary restriction were assessed in liver and brain of 3-7-month-old mice underfed since weaning. Dietary restriction reduced recovery of total liver mitochondrial protein and liver cyt. c ox. specific activity. Liver mitochondria from restricted mice generally showed increased state 3 rates with no differences from controls in state 4 rates for respiration supported by glutamate or pyruvate + malate, resulting in an increased RCI for these substrates. DNP-uncoupled rates were also raised by dietary restriction. Unlike effects observed in old versus young mice, these differences obtained whether the data were expressed on the basis of mitochondrial protein or on cyt. c ox. activity. Electron microscopy of liver mitochondrial preparations revealed more non-mitochondrial contaminants in old mice and larger mitochondria in dietarily restricted mice. These findings are compatible with reports of age-dependent losses of liver mitochondria and suggest that dietary restriction may retard this loss.

ORGANIC MERCURIAL ENCEPHALOPATHY: IN VIVO AND IN VITRO EFFECTS OF METHYL MERCURY ON SYNAPTOSOMAL RESPIRATION

—A reproducible model of subacute methyl mercury (MeHg) intoxication was developed in the adult rat following the daily intragastric administration of 10 mg methyl mercury/kg body wt. Synaptosomes isolated from animals during the latent phase of mercury neurotoxicity (6‐10 days) demonstrated no significant change in respiratory control, State 3, State 4, or 2,4‐dinitrophenol stimulated respiration with succinate, glutamate or pyruvate plus malate. During the neurotoxic phase, a significant decline in respiratory control was evident with all substrates. Cerebellar synaptosomes revealed qualitatively similar but quantitatively greater inhibition of 2,4‐dinitrophenol stimulated respiration during the latent and neurotoxic phases with glutamate.

METHYL MERCURY INHIBITION OF SYNAPTOSOME AND BRAIN SLICE PROTEIN SYNTHESIS: IN VIVO AND IN VITRO STUDIES

Subacute methyl mercury (MeHg) intoxication was induced in adult rats following the daily intragastric administration of 1 mg MeHg/100 g body weight. Decreased [14C]leucine incorporation into cerebral and cerebellar slice protein was found. Weight loss occurred during the latent and neurotoxic phases but pair feeding did not reveal a significant defect in amino acid incorporation into slice protein. There was no decline in synaptosome protein synthesis in vitro during the latent phase but a significant decline in cerebellar and cerebral synaptosome synthesis was found during the neurotoxic phase. MeHg in vitro inhibited cerebral slice and synaptosome protein synthesis at half maximal concentrations of 7.5 and 12.5 μM respectively. Inhibition of synthesis in synaptosomes was non‐competitive with K1 of 4 × 10−6M. MeHg had no effect on [14C]leucine or [14C]proline uptake into synaptosomes. There was no significant inhibition of synaptosome basal ATPase or Na + K ATPase at concentrations of MeHg (12 μM) giving half maximal inhibition of protein synthesis. No preferential inhibition of the chloramphenicol (55S) or cycloheximide sensitive components of synaptosome fraction protein synthesis was found, suggesting that the inhibition is common to both mitochondrial and extramitochondrial protein synthesizing systems. Addition of nucleotides and/or atractylate failed to influence protein synthesis and did not reverse the MeHg inhibition. Mannitol, as a replacement for the predominant cation species of the incubation medium, gave 40% inhibition of protein synthesis in the control but protected against further inhibition by MeHg.

Experimental Methyl Mercury Neurotoxicity: Locus of Mercurial Inhibition of Brain Protein Synthesis In Vivo and In Vitro

Brain cell‐free protein synthesis is inhibited by methyl mercury chloride (MeHg) following in vivo or in vitro administration. In this report, we have identified the locus of mercurial inhibition of translation. Intraperitoneal injection of MeHg (40 nmol/g body wt) induced variable inhibition of amino acid incorporation into the postmitochondrial supernatant (PMS) harvested from the brain of young (10‐20‐day‐old) rats. No mercurial‐induced disaggregation of brain polyribosomes nor change in the proportion of 80S monoribosomes was detected on sucrose density gradients. No difference in total RNA was found in the PMS. Initiation complex formation was stimulated by MeHg, as detected by radiolabelled methionine binding to 80S monoribosomes following continuous sucrose density gradient centrifugation. After micrococcal nuclease digestion of endogenous mRNA, both in vivo and in vitro MeHg inhibited polyuridylic acid‐directed incorporation of [3H]phenylalanine. However, the in vivo inhibition was no longer observed when [3H]phenylalanyl‐tRNAPhe replaced free [3H]phenylalanine in the incorporation assay. The formation of peptidyl[3H]puromycin revealed no difference from controls. There was significant mercurial inhibition of phenylalanyl‐tRNAPhe synthetase activity in pH 5 enzyme fractions derived from brain PMS of MeHg‐poisoned rats. These experiments revealed that the apparent MeHg inhibition of brain translation in vivo and in vitro is due primarily to perturbation in the aminoacylation of tRNA and is not associated with defective initiation, elongation, or ribosomal function.

Experimental methyl mercury neurotoxicity: Similar in vivo and in vitro perturbation of brain cell-free protein synthesis☆

Perturbation of brain protein synthesis by methyl mercury chloride (MeHg) was compared in vivo and in vitro. MeHg-stimulated and/or inhibited brain cell-free protein synthesis following in vivo or in vitro administration. Although pretreatment with GSH protected the postmitochondrial supernatant (PMS) from the in vitro inhibition, direct addition of -SH compounds did not reverse the in vivo or in vitro perturbations in synthesis induced by MeHg. Inhibition of synthesis induced by both in vivo and in vitro methyl mercury administration resulted in inactivation of component(s) in brain pH 5 enzymes. Stimulation of amino acid incorporation following in vivo administration of MeHg was apparently associated with the ribosome fraction, but in vitro preincubation of PMS with MeHg produced stimulation associated with the pH 5 enzyme fraction. A model of MeHg neurotoxicity was proposed providing a common molecular locus of interaction in vivo and in vitro.

Delayed phytohemagglutinin-stimulated production of adenosine triphosphate by aged human lymphocytes: possible relation to mitochondrial dysfunction.

The decreased immune response associated with aging may, in part, reflect intrinsic age-related biochemical alterations in lymphocytes from older animals. We measured levels of lymphocyte adenosine triphosphate (ATP) and continuous [3H]thymidine incorporation in phytohemagglutinin-stimulated lymphocytes from young and old humans, and the effects thereon of inhibitors of mitochondrial oxidative phosphorylation and protein synthesis. No difference was found in adenine nucleotide content between young and old subjects. After 24 hours of culture there was a decrease in ATP, with recovery and 2--3-fold increase at 48 hours in young cells after phytohemagglutinin stimulation. We observed a clearcut delay in older lymphocytes of the increase in ATP and [3H]thymidine incorporation following phytohemagglutinin stimulation. We found no evidence for decreased viability or diminished number of responding units in aged cultures. The evidence suggests that mitochondrial dysfunction may play a role in the immunodeficiency of aging.

ON THE MECHANISM OF OUABAIN INHIBITION OF SYNAPTOSOME PROTEIN SYNTHESIS

Abstract— Ouabain (200μm) inhibited incorporation of radiolabelled leucine or glycine into the protein of neonatal synaptosome fractions but had minimal effect on preparations from adult rats. Leucine uptake into synaptosomes was rapid but not influenced by 200μm‐ouabain in contrast to ouabain inhibition of [14C]glycine and [14C]γ‐aminobutyric acid uptake. Ouabain blocked the Na+ ‐dependent (stimulated) component of synaptosome fraction protein synthesis in the presence of 25mm‐K+. Ouabain inhibition was not alleviated by addition of ADP or ATP. 100μm‐atractylate failed to influence [3H]leucine uptake or incorporation. Synergistic inhibition by ouabain was observed with the cycloheximide‐sensitive component of protein synthesis and the chloramphenicol sensitive phase. Increasing the medium Ca2+ concentration stimulated protein synthesis and this stimulated component was inhibited by ouabain. Ouabain inhibition was associated with decreasing intraterminal K+ concentration and [K]i was linearly related to the protein synthesis rate in control and ouabain treated preparations.

Lysosome-vacuolar system reactivity during early cell regeneration☆

The activities of five selected acid hydrolases have been measured in early regenerating liver, 24 hr after partial hepatectomy. A comparable significant decline in the specific activity of acid phosphohydrolase, β-glucuronidase, β-glucosidase, and β-galactosidase was found. Less significant changes were noted in N-acetylglucosaminidase. No significant decline in the specific activity of cytochrome c oxidase or lactate dehydrogenase was observed. An apparent resistance to thermal and osmotic activation of lysosomal N-acetyl-β-glucosaminidase was noted in the partially hepatectomized animals at 24 hr. A significant increase in the ratio; free/total activity of β-galactosidase and N-acetylglucosaminidase was noted following 50 μg100 g body wt glucagon injection in sham and control animals. Glucagon administration to partially hepatectomized animals failed to produce a corresponding increase in the free/total ratio suggesting that regenerating liver is insensitive to the action of large doses of glucagon. Electron microscopy failed to reveal autophagic vacuole formation at 24 hr after partial hepatectomy. Quantitative analysis of organelle populations after partial hepatectomy revealed a significant decline in the number of lysosomes per 100 μm2. Small decreases in mitochondria and uricosomes were evident when expressed per unit area but the lysosome/mitochondria index was significantly lowered after partial hepatectomy. These observations suggest a potential decrease in the reactivity of the lysosome-vacuolar apparatus after partial hepatectomy and may contribute evidence suggesting a decrease in the catabolic potential of the cell at a time of increased macromolecular synthesis.

The specificity of glutamate inhibition of protein synthesis in synaptosomal fractions from rat cerebral cortex.

In vitro addition of glutamate (GLU) resulted in a dose-dependent inhibition of protein synthesis in synaptosomes from adult rat brain cortex. There was significant (20-25%) inhibition at 25 ?M GLU and a maximum (30-50%) inhibition was observed at [GLU] ? 200 ?M. The excitatory amino acids, N- methyl- d -aspartic acid (NMDA), kainic acid (KA), quinolinic acid (QUIN) and selected non-excitatory amino acids did not markedly inhibit protein synthesis at all concentrations tested. On the other hand, aspartic acid (ASP), ibotenic acid (IBO), quisqualic acid (QA) and ?-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) produced a significant but apparently less potent inhibition than GLU. In the presence of AMPA plus ASP or GLU (500 ?M each), protein synthesis inhibition was equivalent to the maximum effect of GLU alone. However, only partial additive effects were observed with high concentrations of AMPA + IBO or AMPA + QA. In the presence of 200 ?M ouabain, synergistic GLU inhibition was not observed suggesting that GLU specifically inhibited the sodium-dependent, ouabain-sensitive component of synaptosomal protein synthesis. The inhibitory action of GLU is not dependent on externally added Ca(2+) or Cl(?). Supplementation with 5 mM MgCl(2) or 1.0 mM GLU antagonists, 2-amino-5-phosphonovaleric acid (AP5), 2-amino-7-phosphonoheptanoic acid (AP7), ?-glutamylglycine (?-DGG), kynurenic acid (KYN), MK-801, l-glutamic acid diethyl ester (GDEE) and 6-cyano-7-nitro quinoxaline-2,3-dione (CNQX) did not protect the synaptosomes from 50 ?M GLU. Inhibiting glutamate uptake or phosphoinositide metabolism with 1 mM dihydrokainic acid (DHK) and 5 mM LiCl(2), respectively, was also non-protective. These data suggest that GLU and other excitatory amino acids (EAA) specifically inhibit synaptosomal protein synthesis. The inhibitory effect is partially associated with activation of QA-preferring receptors which may not be coupled to phosphoinositide metabolism. A possible candidate might be the AMPA specific QA receptor located in the postsynaptic density which is in close proximity to rosettes of polyribosomes. In addition, there is a GLU-sensitive, QA-resistant component of synaptosome protein synthesis which does not appear to be dependent on activation of any of the known pharmacologically defined GLU receptor subtypes.

Failure of atractyloside to inhibit synaptosomal mitochondrial energy transduction.

Studies on synaptosome mitochondrial respiration are complicated by “free” mitochondria. Veratridine stimulation of synaptosomal respiration was due to increased Na+ cycling at the synaptosome membrane associated with increased oxidative phosphorylation of intraterminal ADP and was inhibited by oligomycin, ouabain or Na+ free medium. Atractylate or carboxyatractyloside failed to block veratridine-stimulated respiration but inhibited exogenous-ADP-stimulated respiration. Protein synthesis in the synaptosome fraction was inhibited by oligomycin, valinomycin or 2,4-dinitrophenol but was unaffected by excess atractylate. No change in synaptosomal adenine nucleotide content was found in the presence of atractylate, although a significant decrease in the [ATP]/[ADP] was found with oligomycin, veratridine or valinomycin. These findings show that atractylate does not modify intraterminal mitochondrial energy transduction and indirectly suggest an impermeability of the synaptosome membrane to atractylate.