Subcellular Fractions Of Rat Brain Research Articles

In vitro and ex vivo effects of antidepressants on rat brain membrane-bound phosphatidylinositol synthetase activity.

The in vitro and ex vivo effects of antidepressant drugs on membrane-bound phosphatidylinositol (PI) synthetase and PI: myo-inositol exchange enzyme activities were examined. In rat brain subcellular fractions, PI synthetase occurred exclusively in the microsomes. In comparison, the activity of CDP-diglyceride independent PI:myo-inositol exchange enzyme was low (3%). Of the various CDP-diglycerides tested for the activation of PI synthetase, CDP-dipalmitin was the most active. Addition of 1 mM of desipramine, amitriptyline, imipramine, iprindole, clomipramine and mianserin in vitro significantly inhibited (30-60%) PI synthetase activity, whereas the same concentration of zimelidine and fluoxetine had no effect. At low liponucleotide concentrations, PI synthetase activity was significantly enhanced by imipramine (1 mM), whereas the enzyme activity was inhibited at higher liponucleotide concentrations (greater than 0.3 mM). In contrast, imipramine had no effect on the PI: myo-inositol exchange enzyme activity. No significant alteration in the PI synthetase activity was found following either acute (2 h) or chronic (21 d) treatment of rats with imipramine. The above results indicate that the de novo synthesis of PI is inhibited in vitro but not ex vivo by some antidepressant drugs. However, in view of the high concentration of the drugs required, the pharmacological significance of this inhibitory action with respect to their therapeutic effects is doubtful.

Calcium-activated neutral proteinase in rat brain myelin and subcellular fractions.

The activity of calcium-activated neutral proteinase (mM CANP) was determined in subcellular fractions of rat brain. The CANP activity in whole homogenate and its membrane fractions including myelin was increased ten-fold following treatment with Triton X-100. The majority of the activity (60%) was in the primary particulate fractions P1 (nuclear), P2 (mitochondrial), and P3 (microsomal). Following subfractionation of each particulate fraction, most of the activity (50%) was found in the myelin-enriched fractions (P1A, P2A, and P3A) and separated at the interface of 0.32-0.85 M sucrose. Only 20-30% of the total homogenate activity was in cytosol. The enrichment in the myelin fractions resembled that for 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase) activity. Immunoblotting revealed that the CANP was mainly in myelin and cytosol. In addition to the presence of 72-76 Kd and 80 Kd bands, there were faint high-molecular-weight CANP bands ranging from 110-150 Kd and lower-molecular-weight forms in the region of 30-50 Kd in both purified myelin and cytosol. These studies suggested that CANP is present in myelin and cytosol and that it exists in the brain in membrane-bound and soluble forms.

Phosphatidylinositol kinase, phosphatidylinositol-4-phosphate kinase and diacylglycerol kinase activities in rat brain subcellular fractions

Subcellular fractions isolated and purified from rat brain cerebral cortices were assayed for phosphatidylinositol (PI-), phosphatidylinositol-4-phosphate (PIP-), and diacylglycerol (DG-) kinase activities in the presence of endogenous or exogenously added lipid substrates and [γ- 32P]ATP. Measurable amounts of all three kinase activities were observed in each subcellular fraction, including the cytosol. However, their subcellular profiles were uniquely distinct. In the absence of exogenous lipid substrates, PI-kinase specific activity was greatest in the microsomal and non-synaptic plasma membrane fractions (150–200 pmol/min per mg protein), whereas PIP-kinase was predominantly active in the synaptosomal fraction (136 pmol/min per mg protein). Based on percentage of total protein, total recovered PI-kinase activity was most abundant in the cytosolic, synaptosomal, microsomal and mitochondrial fractions (4–11 nmol/min). With the exception of the microsomal fraction, a similar profile was observed for PIP-kinase activity when assayed in the presence of exogenous PIP (4 nmol/20 mg protein in a final assay volume of 0.1 ml). Exogenous PIP (4 nmol/20 mg protein) inhibited PI-kinase activity in most fractions by 40–70%, while enhancing PIP-kinase activity. PI- and PIP-kinase activities were observed in the cytosolic fraction when assayed in the presence of exogenously added PI or PIP, respectively, but not in heat-inactivated membranes containing these substrates. When subcellular fractions were assayed for DG-kinase activity using heat-inactivated DG-enriched membranes as substrate, DG-kinase specific activity was predominantly present in the cytosol. However, incubation of subcellular fractions in the presence of deoxycholate resulted in a striking enhancement of DG-kinase activities in all membrane fractions. These findings demonstrate a bimodal distribution between particulate and soluble fractions of all three lipid kinases, with each exhibiting its own unique subcellular topography. The preferential expression of PIP-kinase specific activity in the synaptic membranes is suggestive of the involvement of PIP 2 in synaptic function, while the expression of PI-kinase specific activity in the microsomal fraction suggests additional, yet unknown, functions for PIP in these membranes.

Inhibition of Lipid Peroxidation by Dihydroquinoline-Type Antioxidant (CH 402)

The in vitro effect of a non-toxic, water soluble, low molecular weight, stable dihydroquinoline-type antioxidant, CH 402 (Na (2,2-dimethyl-1,2-dihydroquinoline-4-yl)-methane sulphonic acid) was studied on free radical reactions in brain subcellular fractions. Experiments were performed using rat and mouse brain homogenate and microsomal fractions. Non-enzymatically induced lipid peroxidation by ascorbic acid was studied in correlation with ascorbic acid and CH 402 concentrations and incubation time. Malondialdehyde production during lipid peroxidation was measured by the thiobarbituric acid test. In a concentration range of 10(-2)-10(-5) M CH 402 dose-dependently inhibited the ascorbic acid induced in vitro lipid peroxidation in mouse and rat brain subcellular fractions.

Different localization of receptors for ω-conotoxin and nitrendipine in rat brain

The bindings of radioiodinated ω-conotoxin GVIA and [ 3H]-nitrendipine to subcellular fractions of rat brain were examined. The results indicated that ω-conotoxin binding site was mainly present in the mitochondrial fraction, whereas nitrendipine binding site was rich in the mitochondrial but also present in the post-mitchondrial fraction. Fractionation of the mitochondrial fraction on a sucrose density gradient centrifugation showed that the both binding sites were localized in the heavy synaptosomal fraction. These results strongly suggest that the N- and L-type voltage-sensitive calcium channels have different localizations.

Distribution of physostigmine and metabolites in brain subcellular fractions of the rat

The distribution of 3H-physostigmine (Phy) has been studied in the rat brain subcellular fractions at various time intervals following i.v. injection. 3H-Phy or its metabolites rapidly accumulate into the cytoplasm of cells and penetrates the intracellular compartments. Kinetic studies of the subcellular distribution of radioactivity (RA) per gm of rat brain following i.v. injection of 3H-Phy show peak concentrations at 30 min in all subcellular fractions with the exception of mitochondria. In the mitochondrial fraction the RA levels continue to rise from 4682 ± 875 DPM/gm at 5 min to 27474 ± 2825 DPM/gm at 60 min (P<.05). The cytosol contains the highest RA: 223341 ± 21044 DPM/gm at 30 min which declined to 53475 ± 3756 DPM/gm at 60 min. RA in synaptosome, microsomes and myelin increases from 5 to 30 min, and declines at 60 min. In vitro studies did not show a greater uptake of RA by the mitochondrial or synaptosomal fractions. The finding of relatively high concentrations of RA in the mitochondrial fraction at 60 min increases the likelihood that Phy or its metabolites could interfere with the physiological function of this organelle.

Effect of bicuculline-induced status epilepticus on prostaglandins and hydroxyeicosatetraenoic acids in rat brain subcellular fractions.

Rat cerebrum, prelabeled in vivo by intraventricular injection of [1-14C]arachidonic acid, was used to assess cyclooxygenase and lipoxygenase reaction products in total homogenates, cytosol, synaptosomes, and microsomes. Effects of bicuculline-induced status epilepticus on arachidonic acid metabolism in synaptosomes and microsomes were also measured. Lipoxygenase activity, resulting in the synthesis of hydroxyeicosatetraenoic acids (HETEs), and cyclooxygenase activity, resulting in the synthesis of prostaglandins (PGs), were measured by reverse-phase and normal-phase HPLC with flow scintillation detection. Endogenous lipoxygenase products in synaptosomes were identified by capillary gas chromatography-mass spectrometry. PGs and HETEs were detected in all subcellular fractions. The synaptosomal fraction showed the highest lipoxygenase activity, with 5-HETE, 12-HETE, and leukotriene B4 as the major products. Following bicuculline-induced status epilepticus, endogenous free arachidonic acid and other fatty acids accumulated in synaptosomes, but not in microsomes. Incorporation of [1-14C]arachidonic acid into synaptosomal and microsomal phospholipids was decreased after bicuculline treatment. Bicuculline-induced status epilepticus resulted in increased synthesis of HETEs in synaptosomes. PG synthesis increased in the microsomal fraction. When [1-14C]arachidonic acid-labeled synaptosomes and microsomes were incubated for 1 h at 37 degrees C the synthesis of eicosanoids, particularly PGD2, was increased significantly in bicuculline-treated rats, as compared with untreated rats. Depolarization (45 mM K+) of synaptosomes induced a loss of [1-14C]arachidonic acid from phosphatidylinositol, and increased the synthesis of PGD2 and HETEs, an effect that was enhanced in bicuculline-treated rats. This study localizes changes in arachidonic acid metabolism and lipoxygenase activity resulting from bicuculline-induced status epilepticus in the brain subcellular fraction enriched in nerve endings.

Lysophospholipase activity in rat brain subcellular fractions.

Lysophospholipase activity in brain subcellular fractions was measured by the release of myristic acid from 1-myristoylglycerophosphocholine or through the formation of [32P]glycerophosphocholine from [32P]lysophosphatidylcholine. Although the lysophospholipase activity was highest in microsomes, considerable enzyme activity was also found in other subcellular membrane fractions. The pH optimum for the microsomal enzyme was around 7, whereas the synaptosomes and non-synaptic plasma membranes exhibited a pH maximum around 8. Although the enzyme did not require divalent cations for activity, divalent cations (1 mM) such as Hg2+, Cu2+, and Zn2+ inhibited potently the enzyme activity. Enzyme activity was also partially inhibited by both saturated and polyunsaturated fatty acids (25-200 microM), and the inhibition seemed to be greater in the membrane than in the cytosolic fractions. Ionic detergents such as deoxycholate and taurocholate inhibited the lysophospholipase. On the other hand, the effect of Triton X-100 was biphasic, i.e., stimulation at concentrations below 100 micrograms/mg protein and inhibition at higher concentrations. Addition of cholesterol (50-250 micrograms/ml), but not cholesteryl esters, also potently inhibited enzyme activity. The presence of active lysophospholipase(s) in brain is probably an important mechanism for preventing unnecessary accumulation of lysophospholipids which may exert a deleterious effect on the membranes because of their detergent properties.

Effects of triethyltin bromide on protein phosphorylation in subcellular fractions from rat and rabbit brain

Phosphorylation of specific proteins in subcellular fractions of rat brain is affected by the presence of low concentrations (1–50 μM) of triethyltin bromide (Et 3SnBr), in vitro. SDS-PAGE and autoradiography showed that Et 3SnBr increased phosphorylation of an M r = 42,000 protein and an M r = 76,000–80,000 doublet band, but decreased phosphorylation of an M r = 52,000 component. The M r = 42,000 and 76,000–80,000 phosphoproteins have been identified as the α-subunit of pyruvate dehydrogenase (PDH) and synapsin, respectively. Et 3SnBr-induced phosphorylation of rabbit brain PDH results in partial inactivation of the PDH complex.

Ethoxyresorufin O-deethylase activity in rat brain subcellular fractions.

Non-polar, lipid-soluble drugs or xenobiotics are able to cross the blood-brain barrier. The brain cytochrome P-450-dependent monooxygenases may oxidize these molecules to more polar and somewhat hazardous metabolites responsible for neurotoxicity. In order to characterize the cytochrome P-450 dependent aryl hydrocarbon hydroxylase activity in brain subcellular fractions, we used 7-ethoxyresorufin as a substrate, as its O-deethylation reflects specifically the activity of the cytochrome P-450 isoform which metabolizes and is induced by polycyclic aromatic hydrocarbons. The results reported here show that this enzymatic activity occurs in both microsomal and mitochondrial fractions, and that the induction after 3-methylcholanthrene treatment remains limited, thus preventing the formation of high levels of harmful metabolites.

Protein turnover in subcellular fractions of brain from the ethanol-fed rat

The effect of ethanol consumption (27% of energy intake) for 21 days, on protein synthesis and degradation in subcellular fractions of rat brain was investigated using the Na 2 14CO 3 labelling procedure. Ethanol administration elicited an increase in the rate of protein synthesis in all fractions which was accompanied by an increase of similar magnitude in the rate of breakdown. These results imply that whilst ethanol increases the rate of protein turnover it is without effect on the rate of protein accretion.

Gamma-Hydroxybutyric acid in subcellular fractions of rat brain.

The presence of gamma-hydroxybutyric acid (GHB) in synaptosome-enriched fractions of rat brain was ascertained using a GLC technique. The stability of GHB in synaptosomes was evaluated by addition of various gamma-aminobutyric acid (GABA) transaminase (GABA-T) inhibitors, GHB, or ethosuximide to the homogenizing medium. Furthermore, changes in whole brain GHB levels were compared with those in the synaptosomal fraction in animals treated with GABA-T inhibitors, GABA, or ethosuximide. GHB was present in synaptosome-enriched fractions in concentrations ranging from 40 to 70 pmol/mg of protein. There was no evidence for redistribution, leakage, or metabolism of GHB during the preparation of synaptosomes. The elevations of whole brain GHB level associated with GABA-T or ethosuximide treatment were reflected by a parallel increase in synaptosomal GHB content. These data add to the growing evidence that GHB may have neurotransmitter or neuromodulator function.

Correlation of glutamate binding activity with glutamate-binding protein immunoreactivity in the brain of control and alcohol-treated rats

Specific antibodies raised against a glutamate binding protein purified from bovine brain were used to trace the immunoreactivity of this protein in rat brain subcellular fractions. In the subcellular fractions obtained from whole brain homogenates, the synaptic membranes had the highest immunochemical reactivity towards the anti-glutamate-binding protein antibodies. The combination of measurements of glutamate binding activity and glutamate-binding protein immunoreactivity indicated that in brain synaptic membranes from control animals the highest activity in these two measures was associated with a synaptic plasma membrane subfraction that was enriched with synaptic junctions. In animals treated with ethanol for 14 days, there was a significant increase in the density of synaptic membrane glutamate binding sites. This increase in glutamate binding capacity was correlated with a greater than two-fold increase in the glutamate binding activity and binding protein immunoreactivity of the light synaptic membrane subfraction, a subfraction which does not contain many recognizable synaptic junctions. Acute administration of ethanol to rats produced a moderate but non-significant decrease in glutamate binding capacity of synaptic membranes. The increase in the number of glutamate binding protein subunits in brain plasma membranes may be an adaptive response of central nervous system neurons to the acute effects of ethanol on glutamate synaptic transmission.

The brain 68-kilodalton microtubule-associated protein is a cognate form of the 70-kilodalton mammalian heat-shock protein and is present as a specific isoform in synaptosomal membranes.

The relationship between the 68-kilodalton microtubule-associated protein (68KMAP) and the major heat-induced protein (HSP70) in rat and human cells was investigated by comparison of their heat induction properties and by tryptic and Cleveland peptide mapping procedures. HSP70 synthesis was induced by heat shock of rat and human cells, whereas 68KMAP was a major synthesised protein in the absence of heat shock, with its synthesis being only slightly increased on heat shock. Tryptic peptide mapping, however, indicated strong peptide homology between the two proteins. These data, therefore, confirm that 68KMAP represents a constitutively expressed, heat-shock cognate gene. Two-dimensional gel electrophoretic analysis of subcellular fractions of rat brain, combined with peptide mapping procedures, indicated that 68KMAP exists as at least two isoforms separable by isofocussing, the more acidic of which (alpha 68KMAP) is present in fractions enriched in microtubules, cytosol, microsomes, synaptosomal plasma membranes, and synaptic vesicles, and the more basic of which (beta 68KMAP) is present predominantly in fractions enriched in synaptic vesicles and synaptosomal plasma membranes. These two forms are distinguishable in terms of changes in Cleveland peptide maps, and we conclude that alpha- and beta 68KMAP, therefore, represent distinct forms. The significance of these findings to the molecular pathogenesis of Down's syndrome in the human brain is discussed.

Characterization and cellular localization of a developmentally regulated rat neural sialidase.

A developmentally regulated neural sialidase has been identified in particulate, subcellular fractions of rat brain. Enzyme activity, measured using a [3H]sialoganglioside substrate, was linear with time and had a pH optimum of 4.0-4.5. Protein linearity was only observed at low protein concentrations. This appeared to be caused by enzyme access to a lipophilic substrate, as activity was significantly stimulated by membrane-fluidizing agents. Enzyme activity was developmentally expressed in P2 pellets coincident with in vivo synaptogenesis. It was located on the synaptosome and was particularly high in myelin-containing fractions. Its cellular distribution was confined to neuronal cells and centrally derived oligodendrocytes.

Kinetics of the 5'-nucleotidase and the adenosine deaminase in subcellular fractions of rat brain.

Suspensions of rat brain microsomes, synaptosomes, and synaptic vesicles were able to convert adenosine to inosine by means of adenosine deaminase. Isosbestic points of this transformation, at 222, 250 and 281 nm, remained unchanged with time-course. This fact suggests that adenosine deaminase (ADA, E.C. 3.5.4.4) is located on the surface of the vesicles whereas purine nucleoside phosphorylase (PNP, E.C. 2.1.2.4) is located inside the vesicles. Kinetic parameters of the particulate 5'-nucleotidase (5'N, E.C. 3.1.3.5) and adenosine deaminase were analogous to those of the cytosolic enzymes. These results suggest that soluble and particulate enzymes represent different pools of the same molecular species.

Heterogeneous localization of some purine enzymes in subcellular fractions of rat brain and cerebellum.

The activity of guanine deaminase (GAH, E.C.3.5.4.3) was lower in rat cerebellum soluble and microsomal fractions than in rat brain subfractions. Adenosine deaminase (ADA, E.C.3.5.4.4) activity was released in higher proportion than guanine deaminase, purine nucleoside phosphorylase (PNP, E.C.2.1.2.4), 5'-nucleotidase (5'N, E.C.3.1.3.5), and lactate (LDH, E.C. 1.1.1.27) and malate (MDH, E.C. 1.1.1.37) dehydrogenase in press-juices of rat brain. Furthermore, nerve ending-derived fractions (synaptosomes and synaptic vesicles) showed an enrichment of adenosine deaminase and also of 5'-nucleotidase. The action of deoxycholate over the subfractions did not increase the activity of either enzyme. The contrary occurred with the remaining enzymes studied. Thus, it is possible that one set of enzymes are located on the surface of the particulate vesicles, whereas another set are located inside these vesicles, suggesting a compartmentation of purine catabolic enzymes in different areas of the central nervous system.

Peroxisomal oxidation of lignoceric acid in rat brain.

The oxidation of [1-14C]lignoceric acid was studied in different subcellular fractions of rat brain. The highest specific activity for oxidation of [1-14C]lignoceric acid to acetate was observed in the light mitochondrial fraction. The oxidation of [1-14C]lignoceric acid had an absolute requirement for CoASH and ATP. It was stimulated by NAD and FAD by 400 and 280 percent, respectively, whereas addition of carnitine and KCN had no effect. These properties suggest that in brain [1-14C]lignoceric acid is oxidized in peroxisomes.

Diacylglycerol kinase and lipase activities in rat brain subcellular fractions

Phosphatidic acid synthesis via diacylglycerol kinase and free fatty acid release via diacylglycerol lipase were investigated in rat brain subcellular fractions using membrane-bound [I-(14)C]arachidonoyl-diacylglycerol as substrate. Labeled diacylglycerol was generated by incubating brain membranes containing [I-(14)C]arachidonoyl-phosphatidylinositols in the presence of deoxycholate and Ca(2+). Incubation of the prelabeled synaptosomes enriched in [1-(14)C]arachidonoyl-diacylglycerols or incubation of brain subcellular fractions with heat-treated prelabeled membranes resulted in the release of free fatty acids from the diacylglycerols. When incubations were carried out in the presence of ATP, MgCl(2) and NaF, both free fatty acid release and conversion of diacylglycerols to phosphatidic acids were observed. The conversion of diacylglycerols to phosphatidate or their hydrolysis to free fatty acids were linear with time for at least 15 min. In three brain subcellular fractions examined, diacylglycerol kinase activity indicated a pH maximum of 7.4. The free fatty acid release was enhanced slightly by Ca(2+) (1 mM), but Ca(2+) (0.5-4 mM) in the presence of Mg(2+) (10 mM) was inhibitory to the diacylglycerol kinase reaction. Phosphatidate formation was also inhibited by an excessive amount of deoxycholate added to the incubation mixture. Among the brain subcellular fractions, diacylglycerol kinase was more active in synaptic vesicles and cytosol than in the microsomal fraction, whereas diacylglycerol lipase activity was higher in the cytosol fraction than in the membrane fractions. Upon washing the membranes by centrifugation, a substantial portion of the diacylglycerol kinase activity was removed after the first washing, whereas the diacylglycerol lipase activity remained essentially unchanged. The metabolic role of arachidonoyl-diacylglycerols in brain membranes in relation to the biosynthesis of phosphatidate and the release of arachidomic acid is discussed.

Subcellular localization of "peripheral-type" binding sites for benzodiazepines in rat brain.

The binding of [3H]Ro 5-4864, a specific ligand for "peripheral-type" benzodiazepine binding sites and [3H]Ro 15-1788, a specific ligand for the central benzodiazepine receptors, was determined in subcellular fractions of rat brain. As previously reported, the highest levels of "peripheral-type" benzodiazepine binding sites and benzodiazepine receptors were found in the crude P1 and P2 fractions, respectively. Purification of these crude fractions revealed that high levels of both [3H]Ro 5-4864 and [3H]Ro 15-1788 binding were present in the mitochondrial and synaptosomal fractions. In contrast, the purified nuclei and myelin contained low levels of both [3H]Ro 5-4864 and [3H]Ro 15-1788 binding.