Effect of prolonged and intermittent hypoxia on some cerebral enzymatic activities related to energy transduction.

The adaptation to repeated, alternate normobaric hypoxic and normoxic exposures (12 h/day, for 5 days) and to pharmacological treatment was evaluated by studying the specific activities of some enzymes related to cerebral energy metabolism. Measurements were carried out on (a) the homogenate in toto, (b) the purified mitochondrial fraction, and (c) the crude synaptosomal fraction in different areas of rat brain--cerebral cortex, hippocampus, corpus striatum, hypothalamus, cerebellum, and medulla oblongata. The adaptation to intermittent normobaric hypoxic-normoxic exposures was characterized by significant modifications of some enzyme activities in synaptosomes (decrease of cytochrome oxidase activity in the hippocampus, corpus striatum, and cerebellum; decrease of malate dehydrogenase activity in the cerebellum) and in the purified mitochondrial fraction (increase of succinate dehydrogenase activity in the corpus striatum). Daily treatment with three doses of naftidrofuryl (10, 15, and 22.5 mg/kg i.m.) modified some enzyme activities affected or unaffected by intermittent hypoxia and, particularly, decreased acetylcholinesterase activity.

Influence of aging on the acute depletion of reduced glutathione induced by electrophilic agents.

A severe age-dependent depletion of reduced glutathione (GSH) occurs in rat forebrain at 1-3 h from intraperitoneal injection of the electrophilic agents cyclohexene-1-one and cycloheptene-1-one. Chronic pretreatment with central dopamine agonists (i.e., ergot alkaloids; particularly, dihydroergocriptine) partially counteracts the GSH depletion induced in 15-month-old forebrains by the prooxidants tested. In contrast, chronic pretreatment with a vasodilator agent (i.e., papaverine) magnifies the GSH depletion.

The mitochondrial electron transfer alteration as a factor involved in the brain aging.

The tissutal concentrations of reduced glutathione (GSH) and the contents of some key components in the electron transfer chain (namely ubiquinone, cytochromes b, c1, c, and aa3) of the intraterminal mitochondria are measured in the forebrains from 20-, 60-, or 100-week-old Wistar rats. Moreover, in 60-week-old rats, the biochemical analyses are performed also 18 h after the induction of a peroxidative stress by cyclohexene-1-one. The rats have been i.p. pretreated for 8 weeks (7 days/week) with agents acting on macrocirculation (papaverine), carbohydrate metabolism (hopanthenate), lipid metabolism (phosphatidylcholine), energy transduction (theniloxazine), and dopaminergic system (dihydroergocriptine). Brain aging is characterized by the decrease in both GSH and mitochondrial cytochrome aa3, without changes in ubiquinone and cytochrome b populations. In the same way, the peroxidative stress induced by cyclohexene-1-one causes both a GSH depletion and an imbalance among the concentrations of the mitochondrial electron transfer carriers. Only cytochrome aa3 retains all the partially-reduced oxygen intermediates tightly bound to its active sites. Therefore, it is possible to hypothesize that an electron leakage at the level of the auto-oxidizing chain components (i.e., cytochrome b and ubiquinone populations) increases the release of activated oxygen species (superoxide radical, hydroxyl radical). The treatment with the quoted pharmacological tools suggests that GSH and mitochondrial electron transfer carriers are functionally linked, but not interdependent one another.

Effect of aging and acetyl-L-carnitine on energetic and cholinergic metabolism in rat brain regions.

The effect of aging and subchronic treatment with acetyl-L-carnitine (50 mg/kg per day) was studied on mitochondrial bioenergetics and cholinergic metabolism in non-synaptic mitochondria and synaptosomes isolated from cerebral cortex, hippocampus and striatum of rats aged 4, 11 and 18 months. Respiratory activity and cytochrome oxidase specific activity were unaffected by aging in non-synaptic mitochondria. In synaptosomes, pyruvate dehydrogenase, choline acetyltransferase and acetylcholinesterase specific activity remained unchanged, but the high-affinity choline uptake decreased in cerebral cortex and striatum of 18-month-old rats. Acetyl-L-carnitine treatment increased the high-affinity choline uptake in cerebral cortex of 18-month-old rats. The treatment caused also an increase in cytochrome oxidase activity in all the three cerebral regions and in choline uptake in the hippocampus, parameters that were not directly affected by aging processes.

Age-related modifications of cytochrome C oxidase activity in discrete brain regions.

The apparent Km for cytochrome c of cytochrome oxidase does not change but the Vmax decreases in synaptosomes and non-synaptic mitochondria isolated from the cerebral cortex as a whole of 30-month-old rats compared with 4-month-old ones. When the subcellular organelles are submitted to stressful conditions, namely incubation in media of altered osmolality, the percentage of cytochrome oxidase activity released is much higher in senescent rats. The activity of cytochrome oxidase evaluated in non-synaptic mitochondria and synaptosomes isolated from cortical and subcortical regions and cerebellum of rats aged 4 and 30 months shows a highly significant decrease (P less than 0.001) in the parietotemporal cortex of senescent rats (both in non-synaptic mitochondria and synaptosomes) and in the cerebellum (in synaptosomes).

Amyotrophic lateral sclerosis: oxidative energy metabolism and calcium homeostasis in peripheral blood lymphocytes.

There is evidence of oxidative injury in postmortem brain, spinal cord, and CSF of patients with sporadic amyotrophic lateral sclerosis (SALS patients). We investigated the oxidative metabolism and calcium homeostasis in peripheral blood lymphocytes from such patients and did not find statistical differences in the basal oxygen consumption rate (QO2), cytochrome c oxidase activity, catalase activity, and lactate production. However the increase in QO2, induced by an uncoupler of oxidative phosphorylation, was depressed and the basal (resting) level of free cytosolic calcium ([Ca2+]in) was higher in lymphocytes from SALS patients (p < 0.01). Further increase in free [Ca2+]in challenged by a K+ channel blocker or by an uncoupler of oxidative phosphorylation was similar in SALS and control lymphocytes. The results show that systemic changes consistent with the presence of mitochondrial and of calcium metabolism dysfunction are present in SALS.

Clustering of ALS patients in central Italy due to the occurrence of the L84F SOD1 gene mutation.

OBJECTIVE: To study three new apparently unrelated Italian families with ALS and several sporadic ALS patients living in the same rural area. BACKGROUND: One Italian family with ALS carrying a superoxide dismutase 1 (SOD1) gene mutation (G41S) and no regional ALS clustering has been reported in Italy. METHODS: Genetic analysis was performed by automated and manual sequencing of the SOD1 gene in 13 family members and in 6 of 10 unrelated patients with sporadic cases of ALS living in the same area. The authors also determined SOD1 activity in erythrocytes and lymphocytes. RESULTS: The three families included a total of 28 affected members distributed over six generations. Despite a wide variability in age at onset and disease duration, the clinical pattern is uniform, with onset in the lower limbs, ascending progression, and predominant lower motor neuron involvement in all subjects. Generational anticipation is evident in the last two generations. All familial ALS patients and one of the six sporadic patients carry the same L84F missense point mutation in exon 4 of the SOD1 gene. SOD1 enzyme activity and SOD1 protein levels were not decreased significantly in the L84F patients. CONCLUSION: The ALS patients carrying the L84F mutation derive from a common ancestor. This mutation is responsible for ALS clustering in the area. The L84F mutation does not modify SOD1-specific activity.

Influence of age upon the cerebral metabolic changes induced by acute hypoxia on the synaptosomes from dog brain.

The synaptosomal fraction obtained from the motor area of the cerebral cortex of normocapnic, normoxic or hypoxic "young adult," "mature" and "senescent" beagle dogs is incubated and analyzed for : ATP, ADP, AMP, creatine phosphate, pyruvate and lactate. The data are compared with those obtained from the whole controlateral cortical motor area, by the surface freezing technique. After hypoxic hypoxia /15 min; PaO2 = 17-19 mm Hg), the metabolite contents and ratios are differently affected by ageing when the evaluations are performed in the incubated synaptosomal preparation or in the controlateral whole cerebral tissue. In fact, ageing does not affect so much the cerebral changes that occur in the overall energetic state during the hypoxic assault in vivo, but rather those that the synaptosomes remember the tend to reverse during the subsequent incubation in vitro. The protective action of several drugs on the synaptosomal phosphorylation state is tested. Phenobarbital shows a quite broad, age-independent spectrum of action. (-)Eburnamonine and dihydroergocristine exhibits a more limited, age-dependent effectiveness, but are devoid of anesthetic action. Papaverine proves unable to affect the tested biochemical parameters.

The effect of ischemia and pharmacological treatment evaluated on synaptosomes and purified mitochondria from rat cerebral cortex.

Changes in the maximal rate of some cerebral enzymatic activities related to energy transduction (lactate dehydrogenase; citrate synthase and malate dehydrogenase; total NADH-cytochrome c reductase and cytochrome oxidase) as well as both glutamate dehydrogenase and acetylcholine esterase were assayed in the purified mitochondrial fraction or in crude synaptosomal fraction from cerebral cortex. The evaluations were performed in rats before and after a postdecapitative normothermic ischemia of 5, 10, 20 and 40 min duration. The ischemic damage resulted in a decrease in the activity of mitochondrial malate dehydrogenase and total NADH-cytochrome c reductase, and of synaptosomal acetylcholine esterase. The biochemical evaluations were performed also after an i.p. pretreatment with vincamine, trimetazidine and suloctidil (50 mg/kg). The drugs induced different changes in enzyme activities as a function of the ischemia duration. These various interferences are discussed with regard to the possible drugs mode of action.

Early effects of AZT on mitochondrial functions in the absence of mitochondrial DNA depletion in rat myotubes.

Zidovudine (AZT) is a potent inhibitor of human immunodeficiency virus (HIV) replication. In humans, as well as in animal models, long-term treatment with AZT induces a severe myopathy characterised by structural and functional alterations of mitochondria associated with depletion of mitochondrial DNA (mtDNA). In the present work, we compared the effects induced by AZT on mitochondria upon short- or long-term treatments of cultured rat myotubes. Morphological alterations were investigated by electron microscopy, and mtDNA depletion and deletions were analysed by Southern blot. Mitochondrial membrane potential was determined after JC-1 staining by laser-scanning confocal microscopy in whole cells, and by flow cytometry in isolated muscle mitochondria. We found that the early effects of AZT on mitochondrial functions were a marked, yet reversible reduction in mitochondrial membrane potential, in the absence of any effect on mtDNA. The long-term treatment, in addition to mitochondrial membrane potential alterations, induced morphological changes in mitochondria, and a remarkable reduction in the amount of mtDNA, without any significant evidence of mtDNA deletions. In both treatments, a block of the spontaneous contraction of myotubes was observed. To study in more detail the early effects induced by AZT, the ability of the drug to interact with cardiolipin, an important component of internal mitochondrial membrane, was investigated by atomic force microscopy (AFM) in an artificial membrane model system. The results suggest that the primary effects of AZT may be related to a physical interference with the membrane structure leading to a consequent modification of its physical characteristics.

Alteration of pallidal cholinergic activity in MPTP-treated monkeys: effect of dihydro-alpha-ergocryptine (DEK).

Monkeys, intravenously administered with MPTP at the dose of 0.3 mg/kg for 5 consecutive days, develop a severe Parkinson-like syndrome. Cholinergic enzyme activities are increased in the internal segment of the globus pallidus (GPi) and into a lesser extent in the external globus pallidus (GPe). Cholinergic activities are not significantly affected in the caudate and putamen nor in the frontal, parietotemporal, occipital cortices and in the cerebellum. The treatment of the animals twice daily for 2 weeks with dihydro-alpha-ergocryptine (DEK) starting 5 days before the first MPTP administration counteracts the neurotoxin-induced alteration in the internal pallidum and ameliorates some motor related parkinsonian symptoms.

Effect of energy shortage and oxidative stress on amyloid precursor protein metabolism in COS cells.

The present study investigates the influence of energy related metabolic stress on amyloid precursor protein (APP) non-amyloidogenic secretory processing in COS cells. The effect of glucose deprivation on soluble APP (sAPP) secretion has been evaluated: incubation of COS cells with 50 mM 2-deoxy-D-glucose (2-DG) in glucose free medium was able to reduce sAPP secretion (-26%). Sodium azide (NaN3), an inhibitor of cytochrome c oxidase (complex IV of the mitochondrial electron transfer chain) decreased sAPP release in a concentration dependent way (maximum -75%). Treatment of COS cells with the antioxidant glutathione (GSH) fully antagonized the inhibitory effect of azide (1 mM) and elicited sAPP release over basal level. These results suggest that the inhibition of energy metabolism can influence APP processing leading to a decreased secretion of non-amyloidogenic fragments of APP.

Energy metabolism inhibition impairs amyloid precursor protein secretion from Alzheimer's fibroblasts.

The present study investigates the influence of aglycemia and sodium azide (a Cytochrome c Oxidase inhibitor) on sAPP secretion from skin fibroblasts derived from sporadic AD patients and control subjects. Aglycemia reduced sAPP release in the medium of both AD and control fibroblasts to a similar extent after 2 h incubation. Treatment for 2 h with increasing azide concentrations (1 microM-100 mM) under glucose deprivation did not significantly affect sAPP secretion from control fibroblasts, but was able to significantly inhibit sAPP secretion from AD fibroblasts (maximal inhibition 51%). The failure of antioxidants like glutathione (GSH) or N-acetylcysteine (NAC) to antagonize the azide effect on AD fibroblasts and lipoperoxidation data seemed to rule out the possibility that oxidative stress could mediate the sodium azide effect on sAPP release from AD fibroblasts.

Oxidative metabolism in cultured fibroblasts derived from sporadic Alzheimer's disease (AD) patients.

Fibroblasts from Alzheimer's disease (AD) patients displayed decreased cytochrome c oxidase (complex IV) activity (P < 0.05). The basal oxygen consumption rate (QO2) and the response to an uncoupler of oxidative phosphorylation did not differ between AD and control fibroblasts. The QO2 of AD fibroblasts was more susceptible (P < 0.05) to inhibition by azide in the range 0.5-5 mM. The basal intracellular pH (pHi) in AD fibroblasts was significantly more acidic than in control ones. The results support the hypothesis that subtle dysfunctions of oxidative energy-producing processes are present in fibroblasts from sporadic AD patients. The alterations observed scantly influence the fibroblasts functioning even in stressful conditions; however in tissues, such as the brain, that rely heavily on oxidative metabolism for their function, similar alterations may trigger molecular mechanisms leading to cell damage.

Heterogeneous distribution of amyotrophic lateral sclerosis patients with SOD-1 gene mutations: preliminary data on an Italian survey.

We report the absence of superoxide dismutase (SOD-1) gene mutations in 30 patients with amyotrophic lateral sclerosis (ALS) including individuals with a confirmed family history of ALS (familial ALS/FALS), ALS with an unclear family history (UFALS) and sporadic ALS (SALS). Single strand conformation polymorphism (SSCP) and sequence analysis of the 5 SOD-1 gene exons were undertaken to improve the accuracy of the mutation detection. Our preliminary data appear to diverge from the results of studies by other groups using different populations. We discuss the possible reasons for this disparity and the apparent heterogeneous distribution of ALS with SOD-1 gene mutations among different ethnic groups.

Role of synaptosomal enzymatic alterations and drug treatment in brain aging.

The activity patterns of enzyme linked to energy transduction are measured as an estimate of the energy potential capacity of the brain during aging. Early investigations provided information on age-related modifications in the apparent activity of these enzymes in the brain as a whole without taking into account the anatomical, morphological, and functional heterogeneity of the discrete brain regions, the metabolic compartments, and their different time course of aging processes. These considerations prompted the investigators to focus their efforts on subcellular organelles, representative of metabolic compartments, isolated from selected brain regions. In the present study, to better elucidate the role of the synaptic compartment during aging, the maximum rate (Vmax) of enzymes involved in energy metabolic pathways is evaluated in synaptosomes isolated from the cerebral cortex of rats aged 4, 12, and 24 months. The potential catalytic activity of phosphofructokinase and citrate synthase is not affected by aging. In contrast, the Vmax of pyruvate dehydrogenase and particularly of cytochrome oxidase decreases in aged rats. A marked increase is found in the Vmax of glucose-6-phosphate dehydrogenase in 24-month-old rats and could support the availability of nicotinamide adenine dinucleotide phosphate (NADPH) for antiperoxidative processes. Pretreatments of the animals with certain drugs are performed in order to check the responsiveness of the tissue and the plasticity of enzyme proteins during aging. Papaverine (acting on macrocirculation) is ineffective, but raubasine (acting on microcirculation and metabolism) and almitrine (acting on oxygen availability) both interfere with the potential activity of some of the enzymes tested. Their influence differs with the age of the animal and are in agreement with their action on brain carbohydrate and phospholipid metabolism.

Enzymes related to energy metabolism in human gliomas.

The evaluation of the specific activity of some enzymes related to energy transduction was performed in 7 fresh samples of malignant gliomas and in 4 samples of normal brain tissue. Compared with normal brain tissue, the hexokinase, phosphofructokinase and citrate synthase activities are lower; the lactate dehydrogenase and succinate dehydrogenase are unchanged, while glucose-6-phosphate dehydrogenase and NADP+-isocitrate dehydrogenase activities are higher in gliomas.

Alteration of complex IV and acetylcholine-related enzymes in experimental spinal cord injury.

Acute, severe injury of the rabbit spinal cord, induced by the weight-drop method, causes alterations of the enzyme activities related to cholinergic and energy metabolism. Morphological examinations at the trauma site show degenerative processes in neurons 0.5 hr posttrauma and a marked decrease in the number of living cells 24 hrs later. Both biochemical and cytochemical findings show that the tissue metabolic and morphologic derangement, caused by severe spinal cord injury, is mostly confined to the gray matter at an early stage (0.5 hr), whereas 24 hrs later the white matter is also involved. The decrease in choline acetyl-transferase and acetylcholinesterase activities in the gray matter parallels the impairment of complex IV (cytochrome c oxidase) of the respiratory chain and the presence of morphological alteration in neurons. The dramatic drop in the enzyme activities, observed 24 hrs after the induction of the severe trauma is clearly associated with the loss of cells.

Extrusion-induced changes to the chemical profile and viscosity generating properties of citrus fiber.

Each of 8 variants in extrusion conditions was applied to a commercially available citrus fiber. Extrusion under conditions where the specific mechanical energy (SME) exceeded 400 kJ·kg(-1) was able to solubilize up to 30% of the fibers. Where the SME was ∼200 kJ·kg(-1) the degree of fiber solubilization was between 8 and 12%. All extruded fibers showed a loss of water-retaining capacity compared to the reference fiber, and this was attributed to the disruption of the integrated cell wall structure during the extrusion process. Nevertheless, within the 8 extruded variants there was a wide range of viscosity generating capacity which depended on the level of SME to which the fibers were subjected. The SME also had a pronounced effect on the nature of the solubilized fibers in terms of both their monosaccharide composition and their molecular weight profile. Both pectic and hemicellulosic polysaccharides were solubilized. It is concluded that extrusion has promise as a physical process for manipulating both the technological functionality and the health promoting properties of dietary fibers.