Inhibition by neomycin of polyphosphoinositide turnover in subcellular fractions of guinea-pig cerebral cortex in vitro

The addition of 10(-5) M to 10(-3) M neomycin to incubations of subcellular fractions of guineapig cerebral cortex increased the labelling of phosphatidylinositol phosphate and decreased the labelling of phosphatidylinositol diphosphate by [gamma-32P]ATP. The effect was observed in all subcellular fractions tested and depended on the cationic form of the antibiotic. Similar effects on lipid labelling were exerted by related aminoglycosidic antibiotics, by neamine, spermine and poly-L-lysine. Other neomycin fragments, antibiotics, local anesthetics or small polyamines were ineffective. Neomycin also inhibited the enzymatic hydrolysis of 32P-polyphosphoinositides. The addition of the drug to aqueous dispersions of these lipids increased the turbidity and lowered the pH of the suspensions. It is suggested that the effects of neomycin on polyphosphoinositide metabolism result from the formation of an ionic complex between the lipids and the antibiotic.     

Studies of blocking mechanisms of carbachol-induced polyphosphoinositide turnover in rat cortical synaptosomes by neuroactive drugs

1. The incorporation of 32Pi into 4 phospholipids of rat cortical synaptosomes was altered in the presence of carbachol (1 mM), viz. a decrease of phosphatidylinositol-4,5-bisphosphate and phosphatidyl-4-phosphate by 34 and 21%, and an increase of phosphatidylinositol and phosphatidic acid by 52 and 96% of basal controls respectively. 2. The IC30 values calculated from the dose-response curves for drugs affecting carbachol-induced 32Pi incorporation into these phospholipids, and [3H]QNB binding to the cortical synaptosomes were similar for the typical antimuscarinic agents (i.e. atropine, pirenzepine and trihexyphenidyl), and tricyclic antidepressants (i.e. amitriptyline, doxepin and imipramine) studied. 3. The IC30 values obtained for drugs affecting carbachol-induced 32Pi incorporation into these phospholipids, and high potassium-induced 45Ca2+-uptake by this preparation were similar for neuroselective calcium channel blockers (i.e. cinnarizine and flunarizine) studied. 4. Our results suggest that the neuroactive drugs studied can either act at, or beyond the receptor level, perhaps on the availability of calcium ion, to block carbachol-induced polyphosphoinositide turnover in rat cortical synaptosomes.     

Inhibition of mitochondrial creatine kinase activity from rat cerebral cortex by methylmalonic acid

Accumulation of methylmalonic acid (MMA) in tissues and biological fluids is the biochemical hallmark of patients affected by the neurometabolic disorder known as methylmalonic acidemia (MMAemia). Although this disease is predominantly characterized by severe neurological findings, the underlying mechanisms of brain injury are not totally established. In the present study, we investigated the effect of MMA, as well as propionic (PA) and tiglic (TA) acids, whose concentrations are also increased but to a lesser extend in MMAemia, on total (tCK), cytosolic (Cy-CK) and mitochondrial (Mi-CK) creatine kinase (CK) activities from cerebral cortex of 30-day-old Wistar rats. Total CK activity (tCK) was measured in whole cell homogenates, whereas Cy-CK and Mi-CK were determined, respectively, in cytosolic and mitochondrial preparations from rat cerebral cortex. We verified that tCK and Mi-CK activities were significantly inhibited by MMA at concentrations as low as 1 mM, in contrast to Cy-CK which was not affected by the presence of the acid in the incubation medium. Furthermore, PA and TA, at concentrations as high as 5 mM, did not alter CK activity. We also observed that the inhibitions provoked by MMA were fully prevented by pre-incubation of the homogenates with reduced glutathione, suggesting that the inhibitory effect of MMA was possibly mediated by oxidation of essential thiol groups of the enzyme. Considering the importance of CK for brain metabolism homeostasis, our results suggest that inhibition of this enzyme by increased levels of MMA may contribute to the neurodegeneration of patients affected by MMAemia and explain previous reports showing an impairment of brain energy metabolism and a reduction of brain phosphocreatine levels caused by MMA.     

Inhibition of polyphosphoinositide phosphodiesterase by aminoglycoside antibiotics

The calcium-activated phosphodiesteratic hydrolysis of³²P-labeled phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-phosphate in prelabeled nerve ending membranes is inhibited by the aminoglycosides neomycin and gentamicin, and to a lesser extent, by streptomycin. The inhibition is overcome by increasing concentrations of Ca²⁺, indicating that the aminoglycosides exert their effect by displacing Ca²⁺ from lipid.Dedicated to Professor Yasuzo Tsukada.     

Inhibition of the mitochondrial respiratory chain complex activities in rat cerebral cortex by methylmalonic acid

Propionic and methylmalonic acidemic patients have severe neurologic symptoms whose etiopathogeny is still obscure. Since increase of lactic acid is detected in the urine of these patients, especially during metabolic decompensation when high concentrations of methylmalonate (MMA) and propionate (PA) are produced, it is possible that cellular respiration may be impaired in these individuals. Therefore, we investigated the effects of MMA and PA (1, 2.5 and 5 mM), the principal metabolites which accumulate in these conditions, on the mitochondrial respiratory chain complex activities succinate: 2,6-dichloroindophenol (DCIP) oxireductase (complex II); succinate: cytochrome c oxireductase (complexII+CoQ+III); NADH: cytochrome c oxireductase (complex I+CoQ+complex III); and cytochrome c oxidase (COX) (complex IV) from cerebral cortex homogenates of young rats. The effect of MMA on ubiquinol: cytochrome c oxireductase (complex III) and NADH: ubiquinone oxireductase (complex I) activities was also tested. Control groups did not contain MMA and PA in the incubation medium. MMA significantly inhibited complex I+III (32–46%), complex I (61–72%), and complex II+III (15–26%), without affecting significantly the activities of complexes II, III and IV. However, by using 1 mM succinate in the assay instead of the usual 16 mM concentration, MMA was able to significantly inhibit complex II activity in the brain homogenates. In contrast, PA did not affect any of these mitochondrial enzyme activities. The effect of MMA and PA on succinate: phenazine oxireductase (soluble succinate dehydrogenase (SDH)) was also measured in mitochondrial preparations. The results showed significant inhibition of the soluble SDH activity by MMA (11–27%) in purified mitochondrial fractions. Thus, if the in vitro inhibition of the oxidative phosphorylation system is also expressed under in vivo conditions, a deficit of brain energy production might explain some of the neurological abnormalities found in patients with methylmalonic acidemia (MMAemia) and be responsible for the lactic acidemia/aciduria identified in some of them.     

Inhibition by N-acetyl-aspartate of aspartate binding to a proteolipid fraction from rat cerebral cortex

Many roles have been suggested for N-acetyl-aspartate in brain function because of it being located almost exclusively in that organ. However, its true role remains to be demonstrated. We show here that N-acetyl-aspartate: 1) binds to a hydrophobic protein fraction from the cerebral cortex of the rat, which specifically bindsl-aspartate,l-glutamate, and -amino-butyric acid; and 2) has a marked inhibitory effect on the aspartate binding sites of this proteolipid fraction. Structural analogs of N-acetyl-asparate, i.e. N-carbamyl-aspartate and N-methyl-aspartate also inhibit thel-aspartate binding by the brain protein fraction used.     

Inhibition of creatine kinase activity from rat cerebral cortex by D-2-hydroxyglutaric acid in vitro

D-2-Hydroxyglutaric acid (DGA) is the biochemical hallmark of patients affected by the neurometabolic disorder known as D-2-hydroxyglutaric aciduria (DHGA). Although this disease is predominantly characterized by severe neurological findings, the underlying mechanisms of brain injury are virtually unknown. In the present study, we investigated the effect of DGA on total, cytosolic, and mitochondrial creatine kinase (CK) activities from cerebral cortex of 30-day-old Wistar rats. Total CK activity (tCK) was measured in whole cell homogenates, whereas cytosolic and mitochondrial activities were measured in the cytosolic and mitochondrial preparations from cerebral cortex. We verified that CK activities were significantly inhibited by DGA (11-34% inhibition) at concentrations as low as 0.25 mM, being the mitochondrial fraction the most affected activity. Kinetic studies revealed that the inhibitory effect of DGA was non-competitive in relation to phosphocreatine. We also observed that this inhibition was fully prevented by pre-incubation of the homogenates with reduced glutathione, suggesting that the inhibitory effect of DGA on tCK activity is possibly mediated by oxidation of essential thiol groups of the enzyme. Considering the importance of CK activity for brain metabolism homeostasis, our results suggest that inhibition of this enzyme by increased levels of DGA may be related to the neurodegeneration of patients affected by DHGA.     

Guanine nucleotides have a direct inhibitory effect on polyphosphoinositide turnover in rat cortical synaptosomes

1. The possible involvement of guanosine 5'-triphosphate (GTP)-binding proteins in the receptor mediated polyphosphoinositide (PPI) turnover event was investigated in rat cortical synaptosomes. 2. It was studied under the effects of guanine nucleotides on 32Pi incorporation into synaptosomal phospholipids in the absence or presence of carbachol. 3. The basal 32Pi incorporation into these phospholipids was altered by the presence of 1 mM carbachol: i.e. a decrease in 32Pi incorporation into phosphatidylinositol-4,5-bisphosphate and phosphatidylinositol-4-phosphate and an increase in the incorporation of 32Pi into phosphatidylinositol and phosphatidic acid. 4. In the presence of guanine nucleotides: GTP, Gpp(NH)p and GDP at suitable concentrations, there was a general decreasing effect on 32Pi incorporation into all 4 phospholipids, which are all involved in PPI turnover cycle, either in the basal or carbachol-stimulated levels. 5. There was no selective effect among the guanine nucleotides studied on this PPI turnover event. It is, therefore, likely that these nucleotides have a direct inhibitory effect on PPI turnover, and this action may not act through a GTP-binding protein.     

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 g · 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 ₂-agonist.     

Inhibition by gamma-hexachlorocyclohexane of acetylcholine-stimulated phosphatidylinositol synthesis in cerebral cortex slices and of phosphatidic acid-inositol transferase in cerebral cortex particulate fractions

• 1 γ-Hexachlorocyclohexane inhibits the ACh-stimulated synthesis of phosphatidylinositol in guinea pig cerebral cortex slices, as measured either by the incorporation of [2-³H]inositol or of ³²P. Phosphatidylinositol synthesis in the control slices is not inhibited.
• 2 The synthesis of phosphatidylinositol from CDP-diglyceride in cerebral cortex microsomal preparations is inhibited by γ-hexachlorocyclohexane. The incorporation of [2-³H]inositol into lipid in the absence of added cytidine nucleotide in these preparations is not inhibited.
• 3 δ-Hexachlorocyclohexane profoundly inhibits phosphatide synthesis and phosphate metabolism in cerebral cortex slices both in the presence and absence of ACh. This isomer also inhibits the exchange reaction for the incorporation of [2-³H]inositol into lipid in the microsomal preparations.
• 4 α-, and β-Hexachlorocyclohexanes do not inhibit either ACh-stimulated or control synthesis of phosphatidylinositol in cerebral cortex slices; nor do they inhibit the exchange reaction for [2-³H]inositol incorporation into lipid in the microsomal preparations.
• 5 The specific effects of γ-hexachlorocyclohexane are taken as providing evidence that ACh-stimulated phosphatidylinositol synthesis in cerebral cortex slices probably involves the CDP-diglyceride pathway. The possibility is discussed that the primary action of ACh in this system is to cause an increased activity of diglyceride kinase to provide phosphatidic acid for this pathway.

     

Pipecolic acid receptors in rat cerebral cortex

Identification of [¹⁴C]pipecolic acid (PA) receptors was attempted in the solubilized membrane fraction from rat cerebral cortex. Specific binding proteins for both PA and muscimol, a potent -aminobutyric acid (GABA) agonist, were detected in the same preparation. Separation of labeled PA and GABA binding proteins by glycerol gradient centrifugation has shown labeled protein bands of similar sedimentation rates, suggesting that PA and GABA may be binding to identical proteins. It seems likely that the PA binding receptor either may possess the same sedimentation characteristics as that of the GABA receptor, or both GABA and PA which is an endogenous and weak GABA agonist may bind to the same receptor complex, if not to the same binding site.     

Sensory mapping of the rat cerebral cortex by thermographic methods

Using thermovision and digital image processing, the dynamics of thermomap in the brain cortex has been studied on albino rats through the opened skull before and during sensory stimulation. Photic, sonic and somatosensory stimuli lead to the onset of local heating foci in the corresponding cortical zones, as well as to local multiple thermoresponses in other areas. Some quantitative parameters of these effects are given and their possible mechanisms are discussed.     

Inhibition of cytochrome c oxidase activity in rat cerebral cortex and human skeletal muscle by d-2-hydroxyglutaric acid in vitro

l-2-Hydroxyglutaric (LGA) and d-2-hydroxyglutaric (DGA) acids are the characteristic metabolites accumulating in the neurometabolic disorders known as l-2-hydroxyglutaric aciduria and d-2-hydroxyglutaric aciduria, respectively. Although these disorders are predominantly characterized by severe neurological symptoms, the neurotoxic mechanisms of brain damage are virtually unknown. In this study we have evaluated the role of LGA and DGA at concentrations ranging from 0.01 to 5.0 mM on various parameters of energy metabolism in cerebral cortex slices and homogenates of 30-day-old Wistar rats, namely glucose uptake, CO₂ production and the respiratory chain enzyme activities of complexes I to IV. DGA significantly decreased glucose utilization (2.5 and 5.0 mM) by brain homogenates and CO₂ production (5 mM) by brain homogenates and slices, whereas LGA had no effect on either measurement. Furthermore, DGA significantly inhibited cytochrome c oxidase activity (complex IV) (EC 1.9.3.1) in a dose-dependent manner (35–95%) at doses as low as 0.5 mM, without compromising the other respiratory chain enzyme activities. In contrast, LGA did not interfere with these activities. Our results suggest that the strong inhibition of cytochrome c oxidase activity by increased levels of DGA could be related to the neurodegeneration of patients affected by d-2-hydroxyglutaric aciduria.     

Ultrastructural demonstration of dehydrogenases in rat cerebral cortex

Summary Techniques for the ultrastructural demonstration of dehydrogenases in cerebral cortex are described. The best fixation for good fine structural preservation and retension of LDH and NADH-diaphorase was obtained by perfusion with a mixture of formaldehyde and glutaraldehyde and for SDH by perfusion with formaldehyde. Comparison of incubation conditions showed that consistent results were obtained using enzyme markers NBT and DS-NBT for LDH and NADH-diaphorase; DS-NBT was more satisfactory than NBT and BSPT for SDH. Penetration of incubation media was improved by Triton X-100; DMSO and ultrasonic treatment were less effective. The techniques enabled the first electron cytochemical demonstration of dehydrogenases in different elements of prefixed cerebral cortex. Ultrastructural sites of enzyme activities were localized within cristae and intermembrane spaces of mitochondria in nerve cell cytoplasm and its processes, oligodendrocytes and astrocytes. Authenticity of the ultrastructural sites was confirmed by four different control experiments.