F. Dagani

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.

Sequential damage in mitochondrial complexes by peroxidative stress

The biochemical characteristics of the electron transfer chain are evaluated in purified non-synaptic (“free”) mitochondria from the forebrain of 60-week-old rats weekly subjected to peroxidative stress (once, twice, or three times) by the electrophilic prooxidant 2-cyclohexene-1-one. The following parameters are evaluated: (a) content of respiratory components, namely ubiquinone, cytochrome b, cytochrome c1, cytochrome c; (b) specific activity of enzymes, namely citrate synthase, succinate dehydrogenase, rotenone-sensitive NADH: cytochrome c reductase, cytochrome oxidase; (c) concentration of reduced glutathione (GSH). Before the first peroxidative stress induction, the rats are administered for 8 weeks by intraperitoneal injection of vehicle, papaverine, δ-yohimbine, almitrine or hopanthenate. The rats are treated also during the week(s) before the second or third peroxidative stress. The cerebral peroxidative stress induces: (a) initially, a decrease in brain GSH concentration concomitant with a decrease in the mitochondrial activity of cytochrome oxidase of aa3-type (complex IV), without changes in ubiquinone and cytochrome b populations; (b) subsequently, an alteration in the transfer molecule cytochrome c and, finally, in rotenone-sensitive NADH-cytochrome c reductase (complex I) and succinate dehydrogenase (complex II). The selective sensitivity of the chain components to peroxidative stress is supported by the effects of the concomitant subchronic treatment with agents acting at different biochemical steps. In fact, almitrine sets limits to its effects at cytochrome c content and aa3-type cytochrome oxidase activity, while δ-yohimbine sets limits to its effects at the level of tricarboxylic acid cycle (citrate synthase) and/or of intermediary between tricarboxylic acid cycle and complex II (succinate dehydrogenase). The effects induced by sequential peroxidative stress and drug treatment are supportive of the hypothesis that leakage of electrons (as a mandatory side-effect of the normal flux of electrons from both NADH and succinate to molecular oxygen) would be due to alteration in both availability of GSH and the content of components in the respiratory chain associated to energy-transducing system. In this field there is a cascade of derangements involving, at the beginning, the complex IV and, subsequently, other chain components, including cytochrome c and, finally, complexes II and I.

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.

No evidence for altered muscle mitochondrial function in Parkinson's disease.

Recent reports indicate that reductions in mitochondrial respiratory chain function occur in substantia nigra, platelets, and muscle from patients with Parkinsons disease. To confirm and further characterise the presence of a generally distributed mitochondrial defect, mitochondrial metabolism was evaluated in muscle obtained from subjects with Parkinsons disease and from normal controls. Oxygen consumption rates in muscle mitochondria represented by complex I, complexes II-III, or complex IV did not differ between the two groups. Likewise, activities of rotenone sensitive NADH cytochrome c reductase, succinate cytochrome c reductase, or cytochrome oxidase in muscle mitochondria were not significantly different between Parkinsonian and control subjects. These findings fail to provide support for a generalised defect in mitochondrial function in Parkinsons disease but do not exclude an abnormality in respiratory function confined to the substantia nigra.

Relations Between Intracellular Ions and Energy Metabolism Under Acidotic Conditions: A Study with Nigericin in Synaptosomes, Neurons, and C6 Glioma Cells

Abstract: Effects of nigericin were investigated in rat brain synaptosomes, cultured neurons, and C6 glioma cells to characterize the relations among ATP synthesis, [Na+]i., [K+]i, and [Ca2+]i, and pH under conditions when [H+]i is substantially increased and transmembrane electrical potential is decreased. Intracellular acidification and loss of K+ were accompanied by enhanced oxygen consumption and lactate production and a decrease in cellular energy level. Changes in the last three parameters were attenuated by addition of 1 mM ouabain. In synaptosomes treated with nigericin, neither respiration nor glycolysis was affected by 0.3 μM tetrodotoxin, whereas 1 mM amiloride reduced lactate production by 20% but did not influence respiration. In C6 cells, amiloride decreased the nigericin‐stimulated rate of lactate generation by about 50%. The enhancement by nigericin of synaptosomal oxygen uptake and glycolytic rate decreased with time. However, there was only a small reduction in respiration and none in glycolysis in C6 cells. Measurements with ion‐selective microelectrodes in neurons and C6 cells showed that nigericin also caused a rise in [Ca2+], and [Na+]., The increase in [Na+], in C6 cells was partially reversed by 1 mM amiloride. It is concluded that nigericin‐induced loss of K+ and subsequent depolarization lead to an increase in Na+ influx and stimulation of the Na+/K+ pump with a consequent rise in energy utilization; that acidosis inhibits mitochondrial ATP production; that a rise in [H+] does not decrease glycolytic rate when the energy state (a fall in [ATP] and rises in [ADP] and [AMP]) is simultaneously reduced; that a fall in [K+], depresses both oxidative phosphorylation and glycolysis; and that the nigericin‐induced alterations in ion levels and activities of energy‐producing pathways can explain some of the deleterious effects of ischemia and hypoxia.

Evidence for l-glutamate release in frog vestibular organs

The present study was devised in order to ascertain whether L-glutamate (Glu) is the neurotransmitter at the primary afferent synapse in frog vestibular organs. To this end different groups of frog isolated semicircular canals were stimulated by means of solutions slightly enriched in K+ (5 mM K(+)-rich solutions are sufficient to produce a strong, long-lasting, transmitter release from the basal pole of sensory cells) both in normal conditions and after low-Ca(2+)-high-Mg2+ impairment of the synaptic transmission. The concentration of Glu in the surrounding medium, determined by means of a bioluminescence-enzymatic method, was evaluated in two different experimental conditions: a) when the canals (5 canals placed inside little net bags) were immersed in a 5 mM K(+)-stimulating solution; b) during the superfusion of the canals (25 canals placed into a little perfusion chamber) with a 5 mM K(+)-stimulating solution. The net bag experiments demonstrated that K(+)-rich solutions can provoke an outflow of Glu from canal organs only if the crista ampullaris is present and functioning. Glu fluctuations were in fact suppressed by employing canals deprived of the ampulla or after low-Ca2(+)-high-Mg2+ synaptic blockade. The superfusion experiments demonstrated that the time course of 5 mM K(+)-induced release of Glu from the sensory organ strictly parallels the time course of 5 mM K(+)-induced EPSPs and spike discharge in afferent axons. These results strongly support the hypothesis that Glu is, or is released with, the afferent transmitter in frog inner ear sensory organs.

High-performance liquid chromatographic separation with electrochemical detection of amino acids focusing on neurochemical application

Abstract Twenty-three amino acids and dipeptides, including compounds of neurochemical interest, are measured by high-performance liquid chromatography using electrochemical detection after precolumn derivatization with o -phthalaldehyde/β-mercaptoethanol. The method uses a multistep polarity gradient system and the entire separation is performed in less than 23 min of analysis. The minimum detectable quantity was 0.66 pmol injected, corresponding to 50 n m concentration in the sample; the response was linear in the tested range of 1.33–1333 pmol (0.1–100 μ m ). Relative standard deviations ranged from 0.75 to 6.089% for area measurements (mean, 2.33) and from 0.209 to 0.779% for retention times (mean, 0.546). Examples of application of the method to analysis of biological samples such as rat brain homogenate and human cerebrospinal fluid are shown.

Effect of chronic treatment with some drugs on the enzymatic activities of the rat brain

Abstract In untreated and treated rats, age-dependent changes of some cerebral enzymatic activities (lactate dehydrogenase; citrate synthase and malate dehydrogenase; total NADH-cytocrome c reductase and cytochrome oxidase) were studied in the homogenate in toto and in the crude mitochondrial fraction of the brain from the 16th to the 28th week of age, at 4-week intervals. All the activities studied exhibited a natural peak around the 20th week of life, and subsequently they decreased to lower values. The tested drugs (medibazine. trimetazidine, (−)eburnamonine, papaverine, suloctidil, bamethan, inositol niacinate, and UDP-glucose) were administered daily for periods of 4, 8 or 12 weeks each (16–20, 16–24. 16–28 or 24–28 weeks of life) by intraperitoneal route and at one dose level (1 or 5 mg/kg). The drugs tested exerted different effects in the various administration periods, thus enabling us to differentiate drug action on some important cerebral enzymatic activities after chronic treatment.

Oxidative Metabolism of Nonsynaptic Mitochondria Isolated from Rat Brain Hippocampus: A Comparative Regional Study

Abstract: Nonsynaptic mitochondria isolated from rat brain hippocampus were compared with those obtained by means of the same preparative procedure from cerebral cortex and striatum. Protein recovery, marker enzyme activities (lactate dehydrogenase, citrate synthase, and acid phosphatase), state 4 respiration, and response to hypoosmotic shock showed no difference among the three cerebral regions, suggesting homogeneous behavior during the subfractionation procedure. Cholinergic markers—choline acetyltransferase, acetylcholinesterase activities, and high‐affinity choline uptake—evaluated on synaptosomes showed the classic regional pattern with an enrichment in the striatum (striatum cortex > hippocampus). The coupling state of the mitochondrial fractions was maintained (respiratory control ratios ranging from 3.62 to 5.08 with glutamate + malate as oxidizable substrates), showing a metabolic competence sufficient to perform metabolic studies. Regional differences were found in state 3, uncoupled state of respiration, and cytochrome oxidase activity. Hippocampus showed the lower values (hippocampus < striatum < cortex). A possible role of this lower capacity of mitochondrial energy metabolism in determining the sensitivity of hippocampal neurons to ischemia or epileptic seizures is suggested.

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.