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Biomedical subjects

R F Villa

Publications and source records attributed to R F Villa.

At least 19 recordsLinked to original sources
PubMed2003

Affective disorders, antidepressant drugs and brain metabolism.

There is increasing evidence that affective disorders are associated with dysfunction of neurotransmitter postsynaptic transduction pathways and that chronic treatment with clinically active drugs results in adaptive modification of these pathways. Despite the close dependence of signal transduction on adenosine triphosphate (ATP) availability, the changes in energy metabolism in affective disorders are largely unknown. This question has been indirectly dealt with through functional imaging studies (PET, SPECT, MRS). Despite some inconsistencies, PET and SPECT studies suggest low activity in cortical (especially frontal) regions in depressed patients, both unipolar and bipolar, and normal or increased activity in the manic pole. Preliminary MRS studies indicate some alterations in brain metabolism, with reduced creatine phosphate and ATP levels in the brain of patients with affective disorders. However, the involvement of the energy metabolism in affective disorders is still debated. We propose direct neurochemical investigations on mitochondrial functional parameters of energy transduction, such as the activities of (a) the enzymatic systems of oxidative metabolic cycle (Kreb's cycle); (b) the electron transfer chain; (c) oxidative phosphorylation, and (d) the enzyme activities of ATP-requiring ATPases. These processes should be studied in affective disorders and in animals treated with antidepressant drugs or lithium.

Adaptation, Physiological↗
PubMed2002

ATPases of synaptic plasma membranes from hippocampus after ischemia and recovery during ageing.

Plasticity and relationships between individual ATPases linked to energy-utilizing systems of hippocampus, a very sensitive functional area to both age and ischemia, were studied during ageing on synaptic plasma membranes of 1-year-old "adult" and 2-year-old "aged" rats after 15 min of complete cerebral ischemia and different reperfusion times (01, 24, 48, 72, and 96 h). Activities of Na+, K+, Mg(2+)-ATPase, Mg(2+)-ATPase ouabain insensitive, Na+, K(+)-ATPase, "direct" or "basal" Mg(2+)-ATPase, and acetylcholinesterase (AChE) were evaluated in synaptic plasma membranes, where they play the major role in the regulation of presynaptic nerve ending homeostasis. This in vivo study of recovery time-course from 15 mins of cerebral ischemia indicated specific biochemical assessments of functional meaning: (a) Na+K(+)-ATPase of synaptic plasma membranes in adult and aged animals is stimulated by ischemia; (b) this "hyperactivity" is more markedly related to adult than to aged animals; (c) these abnormalities still persist after 72 and 96 h during the recirculation times, indicating the delayed postischemic suffering of the brain; (d) specific Mg(2+)-ATPase enzyme system possess a lower catalytic power in aged animals than in adult ones, but remained unaltered in adult animals by ischemia and reperfusion; (e) Mg(2+)-ATPase is stimulated in aged animals by ischemia, further increasing during reperfusion up to 72-96 h, indicating the delayed hyperactivity of hippocampus; (f) the increased metabolic activity of hippocampus is indicated by the increased activity of cholinergic system; (g) integrity of synaptic plasma membranes seems not to be altered by 15 min ischemia to a critical extent to compromise their catalytic functionality during reperfusion; (h) AChE activity increases in both adult and aged at some survival times. There are logical reasons for the hypothesis that the modifications in ATPase's catalytic activities in synaptic plasma membranes, which have been modified by ischemia in presynaptic terminals, may play important functional role during recovery time in cerebral tissue in vivo, especially as regards its responsiveness to noxious stimuli, particularly during the recirculation period from acute (or chronic) brain injury.

Aging↗
PubMed2001

Effect of in vivo treatment of clonidine on ATP-ase's enzyme systems of synaptic plasma membranes from rat cerebral cortex.

The effects on energy-consuming ATP-ases were studied in two types of synaptic plasma membranes from rat cerebral cortex after in vivo injection of clonidine. To study the mechanism of action of clonidine at subcellular level, the enzyme activities of Na+, K+-ATP-ase, Ca2+, Mg2+-ATP-ase, Low- and High-affinity Ca2+-ATP-ase, and Mg2+-ATP-ase were evaluated on synaptic plasma membranes of control and treated animals with clonidine (5 microg x kg(-1); i.p. 30 minutes). Acute treatment with clonidine decreased the catalytic activity of Ca2+, Mg2+-ATP-ase and of low-affinity Ca2+-ATP-ase only in synaptic plasma membranes of II type, that is the fraction enriched in synaptic plasma membranes. The decreases of these enzymatic activities are related to the interference of the drug on Ca2+ homeostasis in synaptoplasm. The reductions of these enzyme-consuming ATP-ases give further evidence that clonidine has not only neuroreceptorial effects, but that the drug also affects the energy metabolism of cerebral tissue, improving the knowledges about the pharmacology of clonidine. Because the elevation of [Ca2+]i, during ischemia/hypoxia contributes to cellular injury, these findings may suggest that the prevention of calcium overload may be the key mechanism of protection by alpha2-agonist.

Adenosine Triphosphatases↗
PubMed2001

Coenzymes Q9 and Q10, vitamin E and peroxidation in rat synaptic and non-synaptic occipital cerebral cortex mitochondria during ageing.

Great attention has been devoted both to ageing phenomena at the mitochondrial level and to the antioxidant status of membrane structures. These kinds of investigations are difficult to perform in the brain because of its heterogeneity. It is known that synaptic heavy mitochondria (HM) may represent an aged mitochondrial population characterized by a partial impairment of their typical mitochondrial function. We arranged a novel system requiring no extraction procedure, very limited handling of the samples and their direct injection into the HPLC apparatus, to carry out, for the first time, a systematic and concomitant determination of vitamin E, Coenzyme Q9 (CoQ9) and Coenzyme Q10 (CoQ10) contents in rat brain mitochondria. The trends found for CoQ9 and CoQ10 levels in synaptic and non-synaptic occipital cerebral cortex mitochondria during rat ageing are consistent with previous data. Hydroperoxides (HP) differed with age and it was confirmed that in the HM fraction the summation of contributions results in an oxidatively jeopardized subpopulation. We found that vitamin E seems to increase with age, at least in non-synaptic free (FM) and synaptic light (LM) mitochondria, while it was inclined to remain substantially constant in HM.

Age Factors↗
PubMed2000

Cerebral cortex synaptic heavy mitochondria may represent the oldest synaptic mitochondrial population: biochemical heterogeneity and effects of L-acetylcarnitine.

The microheterogeneous nature of intrasynaptic mitochondria has been demonstrated and is widely accepted. However, evidence is still lacking about the role played by the different intrasynaptic mitochondrial subpopulations. The data obtained support the hypothesis that "heavy" mitochondria could represent old mitochondrial populations: in fact, in addition to the well known impairment of typical mitochondrial functions, they possess the highest levels of hydroperoxides and their fatty acids pattern is completely modified. The qualitative and quantitative fatty acid modifications suffered by these organelles deeply altered their protein/lipid ratio, thus modifying their mode of action. The present work also collects a large body of evidence that a subchronic L-acetylcarnitine treatment in 28 days does not structurally affect both nonsynaptic and intrasynaptic mitochondria of normal rat in a "steady-state" metabolic condition.

Acetylcarnitine↗
PubMed2000

Effect of in vivo administration of naloxone on ATP-ase's enzyme systems of synaptic plasma membranes from rat cerebral cortex.

Naloxone is a specific competitive antagonist of morphine, acting on opiate receptors, located on neuronal membranes. The effects of in vivo administration of naloxone on energy-consuming non-mitochondrial ATP-ases were studied in two different types of synaptic plasma membranes from rat cerebral cortex, known to contain a high density of opiate receptors. The enzyme activities of Na+, K(+)-ATP-ase, Ca(2+), Mg(2+)-ATP-ase and Mg(2+)-ATP-ase and of acetylcholinesterase (AChE) were evaluated on synaptic plasma membranes obtained from control and treated animals with effective dose of naloxone (12microg x kg(-1) i.m. 30 minutes). In control (vehicle-treated) animals specific enzyme activities assayed on these two types of synaptic plasma membranes are different, being higher on synaptic plasma membranes of II type than of I type, because the first fraction is more enriched in synaptic plasma membranes. The acute treatment with naloxone produced a significant decrease in Ca(2+),Mg(2+)-ATP-ase activity and an increase in AChE activity, only in synaptic plasma membranes of II type. The decrease of Ca(2+), Mg(2+)-ATP-ase enzymatic activity and the increased AChE activity are related to the interference of the drug on Ca(2+) homeostasis in synaptosoplasm, that leads to the activation of calcium-dependent processes, i.e. the extrusion of neurotransmitter. These findings give further evidence that pharmacodynamic characteristics of naloxone are also related to increase [Ca(2+)]i, interfering with enzyme systems (Ca(2+), Mg(2+)-ATP-ase) and that this drug increases acetylcholine catabolism in synaptic plasma membranes of cerebral cortex.

Acetylcholinesterase↗
PubMed1999

Energy metabolism of synaptosomal subpopulations from different neuronal systems of rat hippocampus: effect of L-acetylcarnitine administration in vivo.

The maximum rate (Vmax) of some enzyme activities related to glycolysis, Krebs' cycle, acetylcholine catabolism and amino acid metabolism were evaluated in different types of synaptosomes obtained from rat hippocampus. The enzyme characterization was performed on two synaptosomal populations defined as "large" and "small" synaptosomes, supposed to originate mainly from the granule cell glutamatergic mossy fiber endings and small cholinergic nerve endings mainly arising from septohippocampal fiber synapses, involved with cognitive processes. Thus, this is an unique model of pharmacological significance to study the selective action of drugs on energy metabolism of hippocampus and the sub-chronic i.p. treatment with L-acetylcarnitine at two different dose levels (30 and 60 mg x kg(-1), 5 day a week, for 4 weeks) was performed. In control animals, the results indicate that these two hippocampal synaptosomal populations differ for the potential catalytic activities of enzymes of the main metabolic pathways related to energy metabolism. This energetic micro-heterogeneity may cause their different behaviour during both physiopathological events and pharmacological treatment, because of different sensitivity of neurons. Therefore, the micro-heterogeneity of brain synaptosomes must be considered when the effect of a pharmacological treatment is to be evaluated. In fact, the in vivo administration of L-acetylcarnitine affects some specific enzyme activities, suggesting a specific molecular trigger mode of action on citrate synthase (Krebs' cycle) and glutamate-pyruvate-transaminase (glutamate metabolism), but mainly of "small" synaptosomal populations, suggesting a specific synaptic trigger site of action. These observations on various types of hippocampal synaptosomes confirm their different metabolic machinery and their different sensitivity to pharmacological treatment.

Acetylcarnitine↗
PubMed1998

Action of L-acetylcarnitine on different cerebral mitochondrial populations from cerebral cortex.

The maximum rate (Vmax) of some mitochondrial enzymatic activities related to the energy transduction (citrate synthase, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, malate dehydrogenase, cytochrome oxidase) and amino acid metabolism (glutamate dehydrogenase, glutamate-pyruvate-transaminase, glutamate-oxaloacetate-transaminase) was evaluated in non-synaptic (free) and intra-synaptic mitochondria from rat brain cerebral cortex. Three types of mitochondria were isolated from rats subjected to i.p. treatment with L-acetylcarnitine at two different doses (30 and 60 mg.kg-1, 28 days, 5 days/week). In control (vehicle-treated) animals, enzyme activities are differently expressed in non-synaptic mitochondria respect to intra-synaptic "light" and "heavy" ones. In fact, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, malate dehydrogenase, glutamate-pyruvate-transaminase and glutamate-oxaloacetate-transaminase are lower, while citrate synthase, cytochrome oxidase and glutamate dehydrogenase are higher in intra-synaptic mitochondria than in non-synaptic ones. This confirms that in various types of brain mitochondria a different metabolic machinery exists, due to their location in vivo. Treatment with L-acetylcarnitine decreased citrate synthase and glutamate dehydrogenase activities, while increased cytochrome oxidase and alpha-ketoglutarate dehydrogenase activities only in intra-synaptic mitochondria. Therefore in vivo administration of L-acetylcarnitine mainly affects some specific enzyme activities, suggesting a specific molecular trigger mode of action and only of the intra-synaptic mitochondria, suggesting a specific subcellular trigger site of action.

Acetylcarnitine↗
PubMed1998

Regulation of cytochrome c oxidase and FoF1-ATPase subunits expression in rat brain during aging.

In the present study we analyzed the age-dependent changes of mRNA levels for cytochrome c oxidase and FoF1-ATP synthase subunits in rat cerebral cortex and cerebellum. To establish whether the regulation of expression is transcriptional or post-transcriptional, the results were compared to those related to protein subunits levels, of the same enzymatic complexes, previously observed. The different patterns of age-related changes of mRNA subunits, in particular the lower increments, compared with those related to protein subunits, indicate that post-transcriptional mechanisms of regulation might be involved in the coordinated expression of the various subunits of each complex. Northern blotting analyses of RNA from the cerebellum of rats at the various ages, showed also differences in age-dependent patterns of transcription between cerebral cortex and cerebellum. Moreover, the major age-dependent changes of mitochondrial-encoded subunits, compared with the nuclear-encoded ones, previously observed at proteins level, occur also during transcription.

Aging↗
PubMed1997

Decrease of rotenone inhibition is a sensitive parameter of complex I damage in brain non-synaptic mitochondria of aged rats.

We investigated NADH oxidation in non-synaptic and synaptic mitochondria from brain cortex of 4- and 24-month-old rats. The NADH oxidase activity was significantly lower in non-synaptic mitochondria from aged rats; we also found a significant decrease of sensitivity of NADH oxidation to the specific Complex I inhibitor, rotenone. Since the rotenone-binding site encompasses Complex I subunits encoded by mtDNA, these results are in accordance with the mitochondrial theory of aging, whereby somatic mtDNA mutations are at the basis of cellular senescence. Accordingly, a 5 kb deletion was detected only in the cortex of the aged animals.

Aging↗
PubMed1997

Coenzyme Q homologs and vitamin E in synaptic and non-synaptic occipital cerebral cortex mitochondria in the ageing rat.

The coenzyme Q8 (CoQ8) and alpha-tocopherol contents of different mitochondrial fractions were investigated from occipital cerebral cortices of different ages. The highest CoQ8 and vitamin E concentrations were found in non-synaptic free mitochondria (FM) fractions. In several cases heavy mitochondria (HM) fractions displayed the lowest values. Occipital cerebral cortex mitochondria contained higher CoQ9 and lower CoQ10 amounts than those typical for other brain regions.

Aging↗
PubMed1996

Is the Mg(2+)-ATP-dependent proton pumping activity of the synaptic vesicles a factor involved in the cerebral hypoxia?

The changes in the Mg(2+)-dependent V-type ATPase activity and the Mg(2+)-ATP-dependent H+ pumping activity of the synaptic vesicles from the cerebral cortex of rats submitted to intermittent chronic (4 weeks) mild or severe hypoxia were evaluated. The adaptation to the chronic severe hypoxia increases both the ATPase and the H+ pumping activities which are inhibited by NEM with an exponential relationship between the IC(50) values and the in vivo O2 concentration. The Mg(2+)-dependent increase in H+ pumping activity of synaptic vesicles from the rats subjected to in vivo chronic hypoxia may be antagonized by nigericin (dissipating delta pH) and by FCCP (dissipating delta pH and delta psi SV). In contrast, valinomycin (dissipating the delta psi SV) and facilitating an enhancement in delta pH) increases in vitro the H+ pumping activity that is inhibited by the addition of high concentration of K gluconate (reducing the rate of K+ efflux). The preincubation of vesicles from hypoxic rats with FCCP, but not with nigericin, inhibits the valinomycin-increased H+ pumping activity. L-glutamate increases the H+ pumping activity in synaptic vesicles from the cerebral cortex of chronic hypoxic rats, whereas other amino acids (i.e., L-aspartate and L-homocysteate) and glutamate analogs (i.e., quisqualate and ibotenate) are ineffective. The adaptation to both chronic intermittent severe hypoxia and in vivo treatment with posatireline causes a decrease in the Mg(2+)-ATPase activity consistent with the decrease in the H+ pumping one of the synaptic vesicles. The addition of nigericin into incubation medium magnifies the decrease in the H+ pumping activity, while the addition of FCCP is ineffective, suggesting that the treatment with posatireline interferes with the delta psi SV component in the delta mu H+ of the synaptic vesicles from rats submitted to chronic hypoxia. The results of the in vivo and in vitro experiments suggest that in the synaptic vesicles from hypoxic rats the delta psi SV component in delta mu H+ may be most effective in increasing the Mg(2+)-ATP-dependent H+ pumping activity.

Adaptation, Physiological↗
PubMed1996

Coenzyme Q, peroxidation and cytochrome oxidase features after parkinson's-like disease by MPTP toxicity in intra-synaptic and non-synaptic mitochondria from Macaca fascicularis cerebral cortex and hippocampus: action of dihydroergocriptine.

The effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration on respiratory chain features were studied in synaptic and non-synaptic mitochondrial populations from cerebral cortex and hippocampus of Macaca Fascicularis (Cynomolgus monkey). Enzymatic activity, cytochrome a + a3 content and turnover numbers of Complex IV, contents of Coenzyme Q10, of hydroperoxides and membrane fluidity were assessed in non-synaptic "perikaryal" and intra-synaptic "light" and "heavy" mitochondria isolated: (a) from the dopaminergic ascending terminal areas of cerebral cortex of monkeys treated p.o. with dihydroergocriptine at the dose of 2, 6 or 20 mg/kg/day for 52 weeks; (b) from the dopaminergic terminal areas of hippocampus of monkeys treated p.o. with dihydroergocriptine at the dose of 12 mg/kg/day before and during the induction of a Parkinson's-like syndrome by MPTP administration (i.v., 0.3 mg/kg/day for 5 days). Dihydroergocriptine administration moderately increased both cytochrome oxidase activity and cytochrome a + a3 content in "light" intra-synaptic mitochondria and hydroperoxides/CoQ10 ratio in all the types of mitochondria, as a consequence of the enhanced energy metabolism. The Parkinson's-like syndrome by MPTP changed the biochemical investigated parameters, affecting both directly the respiratory chain structures, i.e. by respiratory chain complexes inhibition and indirectly, i.e. by free radical mediated damages. MPTP administration negatively influenced Complex IV activity and Turnover Number of intra-synaptic mitochondria, without affecting the total cytochrome a + a3 amount. In all types of mitochondria and particularly on the "light" intra-synaptic ones, MPTP-induced lesion enhanced hydroperoxides/Coenzyme Q10 molar ratio due to the fall in Coenzyme Q10 levels and the concomitant increase in hydroperoxides. Dihydroergocriptine treatment appeared to be effective in MPTP-treated animals in improving those mitochondrial features that probably suffered free radical insults.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed1995

Coenzyme Q content in synaptic and non-synaptic mitochondria from different brain regions in the ageing rat.

We investigated the Coenzyme Q (CoQ) content of different mitochondrial fractions [free mitochondria (FM), synaptic heavy (HM) and light mitochondria (LM)] from three brain areas (cortex, striatum, hippocampus) of rats at different ages. In rats from 2 to 26 months of age, we observed only small differences in total CoQ content (CoQ9 + CoQ10). In FM and LM fractions, values are very similar and appear to be much higher than in HM fractions. The CoQ10/CoQ9 ratios are much higher in brain mitochondria than in other organs, suggesting possible modifications of CoQ biosynthetic pathways in brain; nevertheless they appear to remain constant during ageing. CoQ9 and CoQ10 contents slowly decrease reaching their minimum in rats of 18 months of age, then increase in the older ages. Considering ageing as partially driven by a summation of free radical-mediated processes, we can hypothesize that damage occurring to biological structures in the first half of life might be followed by induction phenomena tending to re-establish the primitive levels of antioxidant molecules.

Aging↗
PubMed1995

Modifications by chronic intermittent hypoxia and drug treatment on skeletal muscle metabolism.

The energy metabolism was evaluated in gastrocnemius muscle from 3-month-old rats subjected to either mild or severe 4-week intermittent normobaric hypoxia. Furthermore, 4-week treatment with CNS-acting drugs, namely, alpha-adrenergic (delta-yohimbine), vasodilator (papaverine, pinacidil), or oxygen-increasing (almitrine) agents was performed. The muscular concentration of the following metabolites was evaluated: glycogen, glucose, glucose 6-phosphate, pyruvate, lactate, lactate-to-pyruvate ratio; citrate, alpha-ketoglutarate, succinate, malate; aspartate, glutamate, alanine; ammonia; ATP, ADP, AMP, creatine phosphate. Furthermore the Vmax of the following muscular enzymes was evaluated: hexokinase, phosphofructokinase, pyruvate kinase, lactate dehydrogenase; citrate synthase, malate dehydrogenase; total NADH cytochrome c reductase; cytochrome oxidase. The adaptation to chronic intermittent normobaric mild or severe hypoxia induced alterations of the components in the anaerobic glycolytic pathway [as supported by the increased activity of lactate dehydrogenase and/or hexokinase, resulting in the decreased glycolytic substrate concentration consistent with the increased lactate production and lactate-to-pyruvate ratio] and in the mitochondrial mechanism [as supported by the decreased activity of malate dehydrogenase and/or citrate synthase resulting in the decreased concentration of some key components in the tricarboxylic acid cycle]. The effect of the concomitant pharmacological treatment suggests that the action of CNS-acting drugs could be also related to their direct influence on the muscular biochemical mechanisms linked to energy transduction.

Adenine Nucleotides↗
PubMed1995

Changes of mitochondrial cytochrome c oxidase and FoF1 ATP synthase subunits in rat cerebral cortex during aging.

The contents of subunits I, II/III, and IV of cytochrome c oxidase and of subunits alpha, beta and gamma of FoF1 ATP synthase in inner mitochondrial membrane proteins purified from cerebral cortex of rat at 2, 6, 12, 18, 24, and 26 months of age were analyzed by western blot. Age-related changes in the content of subunits, either of mitochondrial or nuclear origin, were observed. All the cytochrome c oxidase (COX) subunits examined showed an age-related increase from 2-month-old rats up to 24 months with a decrease at the oldest age (26 months). The same pattern of age-dependent changes was observed for gamma ATP synthase, while the alpha and beta subunits increased progressively up to 26 months.

Aging↗