Mitochondrial-synaptosomal Fraction Research Articles

Localization of rat striatal monoamine oxidase activities towards dopamine, serotonin and kynuramine by gradient centrifugation and nigro-striatal lesions

Subfractionation of the crude synaptosomal-mitochondrial fraction of rat striatum in a continuous sucrose gradient in a zonal rotor led to the following results. The distribution pattern of monoamine oxidase (MAO) activity towards dopamine (DA) was very similar to the pattern of MAO activity towards serotonin (5HT), but differed from the pattern of MAO activity towards kynuramine (KYN). As 5HT is specifically deaminated by MAO-A while KYN is a common MAO substrate, this supports earlier suggestions that in rat striatal preparations DA is deaminated preferentially by MAO-A. The patterns of the MAO activities towards DA and 5HT were clearly dissimilar, despite considerable overlap, to the patterns of tyrosine hydroxylase (TH) and DOPA decarboxylase (DD) activity, both marking the presence of striatal dopaminergic synaptosomes. The peak activities were separated and all patterns were symmetrical without showing a shoulder. This indicates that rat striatal MAO activity towards DA and 5HT is not specifically or for the greater part localized in dopaminergic terminals. We also investigated the effects of electrolytic and 6-hydroxydopamine lesions of the substantia nigra, both causing extensive degeneration of striatal dopaminergic terminals as appeared from the large decrease of striatal TH and DD activity. However, neither type of lesion induced a reduction of the MAO activity towards any of the substrates used. It is concluded towards DA and 5HT (probably MAO-A activity) present in dopaminergic terminals is very low compared with the total activity of this enzyme in rat striatal tissue.

Action of oxotremorine on the sub-cellular distribution of glycine in the rat spinal cord

Intraperitoneal (i.p.) injection of 250 μg kg of oxotremorine (OT) caused a 50% decrease in the glycine content of the synaptosomal-mitochondrial fraction of spinal cord homogenates prepared from rats killed 15 min after treatment. The glycine content of the supernatant fraction was correspondingly raised. In synaptosomes isolated from the spinal cord of OT-treated rats, the decrease in glycine content was 30%. Prior administration of atropine, but not of methylatropine, abolished this effect of OT on synaptosomal glycine content. Eserine exerted a potentiating effect on the action of OT in lowering the glycine content of spinal synaptosomes. Prior administration of l-dopa, apomorphine, haloperidol, muscarine or mecamylamine had no significant effect on the action of OT on synaptosomal glycine content. OT alone or in combination with eserine, and acetylcholine (ACh) in combination with eserine, did not alter the rate of release of glycine from spinal synaptosomes of untreated rats incubated under appropriate conditions. OT was also without effect on the rate of release of glycine from normal spinal synaptosomes subjected to electrical stimulation, as well as on the eventual glycine content of the synaptosomes. On the basis of the present findings it has been suggested that (i) glycine may be released from Renshaw cells at their synapses with motoneurones in response to the muscarinic action of OT; (ii) dopaminergic modulation may not be involved in the OT-induced glycine release from Renshaw cells; and (iii) excessive release of glycine onto motoneurones may be the causative factor of the akinesia observed in OT-induced experimental Parkinsonism.

H-Pro-[3H]Leu-Gly-NH2: uptake and metabolism in rat brain.

The uptake and metabolism of H-Pro-[3H]Leu-Gly-NH2 ([3H]PLG) in rat brain was investigated by reverse-phase paired-ion high pressure liquid chromatography. Following in vitro incubation of [3H]PLG with rat brain subcellular preparations, the microsomal-cytosol fraction was about twice as active in degrading PLG as the crude mitochondrial-synaptosomal fraction. For both enzyme preparations the pH optimum was found at pH 7-7.5. The major labeled metabolite was [3H]leucine, whereas 3H]labeled Leu-Gly-NH2 as the only labeled peptide intermediate was found in trace amounts. After intravenous injection of [3H]PLG the uptake of unmetabolized peptide in the brain appeared to be very low: 0.008% and 0.001% of the administered dose/g tissue at 2 and 5 min after injection respectively, while at longer survival times intact peptide was below the detection limit. Compared with the intravenous route of administration, intracerebroventricular injection of [3H]PLG yielded much higher brain concentrations of unmetabolized PLG. Following both routes of administration, the metabolite profile was in agreement with that obtained after in vitro incubation. However, the in vivo experiments also showed considerable incorporation of [3H]leucine liberated from [3H]PLG into proteins. Both the in vitro and in vivo results indicate that the initial cleavage of PLG in rat brain occurs at the NH2-terminus and that the dipeptide intermediate H-Leu-Gly-NH2 is subsequently hydrolyzed to its constituent amino acids very rapidly.

The rat retina is a useful in vivo model to study membrane lipid synthesis: rates of biosynthesis of neutral glycerides and phospholipids.

The phospholipid composition was studied in the whole rat retina, as well as in its subcellular fractions. A relative enrichment of phosphatidic acid, phosphatidylethanolamine, and phosphatidylserine was observed in rod outer segments (ROS) in comparison with entire retina: nuclear-photoreceptor inner segments-synaptic bodies (P1) and synaptosomal-mitochondrial (P2) fractions. Phosphatidylcholine was the predominant phospholipid class found in all subcellular fractions analyzed. The microsomal fraction was relatively enriched in phosphatidic acid and in phosphatidylinositol. In addition, the rat eye has been used as an in vivo system to study membrane lipid synthesis. After intravitreal injections of [2-3H]glycerol a rapid labeling of retinal glycerolipids took place. Up to 120 min after injection only the glycerol backbone of lipids was labeled. Phosphatidic acid and diacylglycerol displayed rapid rates of synthesis and breakdown. Fastest rates of labeling were attained by phosphatidylcholine followed by phosphatidylinositol. Differences were found when in vitro labeling by [2-3H]glycerol was compared with intravitreal injections. Labeling of phospholipids of subcellular fractions by intravitreally injected [2-3H]glycol showed that most of the label accumulated in microsomal phosphatidylcholine and phosphatidylinositol. Diacylglycerols and phosphatidylethanolamine also took up 10 and 20% respectively of the precursor. It is concluded that the rat eye is a useful experimental model to study synthesis and metabolism of membrane lipids in the retina.

Newly synthesized cholecystokinin in subcellular fractions of the rat brain.

The subcellular localization of in vivo synthesized cholecystokinin (CCK) in different parts of the rat brain was studied after intracisternal pulse injections of [35S]methionine. The rats were decapitated 1 h after the injection, and the brain was divided into cortex, hippocampus and remainder. Subcellular fractions were obtained according to Whittaker's method. De novo synthesized CCK in the crude mitochondrial-synaptosomal fraction, P2, and in the purified synaptosomal fraction was demonstrated by affinity chromatography, using antibodies specific for the COOH-terminal sequence of CCK. By subsequent gel chromatography two molecular forms of labelled CCK occurred, with elution constants, Kav, of 1.1 (corresponding to the COOH-terminal octapeptide) and of 1.40 (a component which may correspond to the COOH-terminal tetrapeptide amide, CCK-4). The findings support the idea that the small molecular forms are the transmitter forms of CCK.

Noncholinergic, Saturable Binding of (±)-[3H]Nicotine to Mouse Brain

(+/-)-[3H]Nicotine was bound saturably to crude particulate, and synaptosomal-mitochondrial fraction from mouse brain. Scatchard and Hill plots of the binding data are in agreement with the existence of two independent classes of binding sites with high (Kd of 0.1-0.4 microM) and low(Kd is 20 microM) affinities, although negative cooperativity or a two-step model of ligand-receptor interaction cannot be ruled out. Nicotinic or muscarinic agonists and antagonists had little or no affinity for the nicotine binding sites, suggesting that nicotine binds in brain to noncholinergic sites. The binding did not display stereospecificity; this is consistent with the similarity in the pharmacological effects of (-)- and (+)-nicotine. Our results indicate that binding studies with [3H]nicotine should be interpreted with extreme caution.

Effects of estrogen and progesterone on LHRH release from a hypothalamic synaptosomal fraction of ovariectomized rats.

Mitochondrial-synaptosomal fractions (P2) from the basomedial hypothalamus of adult ovariectomized rats were employed to study the effects of estradiol benzoate (EB) and progesterone (P) on the release of luteinizing hormone-releasing hormone (LHRH). Treatment of ovariectomized rats with 5 or 50 micrograms of EB significantly reduced the total LHRH released from P2 under both control and K+-stimulated conditions. Furthermore, rats given 50 microgram EB demonstrated cyclic variations in the magnitude of inhibition of LHRH release. Comparison of LHRH release from P2 of rats sacrificed at 0900 hr with that from those sacrificed at 1500 hr revealed a small but persistent facilitation of LHRH release each afternoon. This facilitation, associated with an increase in the soluble component of LHRH release, was absent when rats also received 5 mg of P. No effects on LHRH release were observed when 17 beta-estradiol alone or when P was applied to P2 in vitro. The data show that the regulatory effects of estrogen and progesterone given in vivo on LHRH secretion can be observed in a subcellular fraction of the hypothalamus containing neurosecretory cell terminals.

Properties of LHRH release from a hypothalamic synaptosomal fraction of estrogen-primed ovariectomized rats

The release of luteinizing hormone-releasing hormone (LHRH) from mitochondrial-synaptosomal fractions (P2) of basomedial hypothalamus was examined under various conditions. Less than 3% of the LHRH in P2 suspensions was released under control conditions while the addition of 60 mM KCl or NaCl effected an 8-fold increase in LHRH as measured by radioimmunoassay. Equiosmolar sucrose effected only a 1.8-fold increase in LHRH release. The stimulatory effects of both Na+ and K+ were significantly inhibited by Mn2+ or La3+. Two forms of released LHRH were observed, one soluble and the other particulate. Soluble LHRH release was effected by hypertonic sucrose or 60 mM KCl and was not inhibited by Ca2+ antagonists. The release of particulate LHRH was unaffected by hpertonic sucrose, was stimulated 10-fold by 60 mM KCl, and was abolished with Ca2+ antagonists. These results suggest that the released soluble LHRH results from nonspecific leakage while the release of particulate LHRH reflects a Ca2+-dependent secretory process.

Isolation of synaptic junction-enriched fraction from the forebrain of day-old chickens. Preparation and characterization of chick forebrain subcellular fractions.

Synaptosomal plasma membrane (SPM) and other subcellular fractions were isolated from the forebrain of 1-day-old chickens by a procedure based on that of Davis and Bloom (16) and Cotman and Taylor (13). The procedure involves the centrifugation through a discontinuous sucrose gradient of a crude synaptosomal-mitochondrial fraction which has been lysed and weighed with iodonitrotetrazolium. SPM isolated by this method contains only small amounts of lysosomal or mitochondrial membranes and is practically devoid of contaminating microsomal membranes, as estimated by enzyme marker assays. The purity of chick-brain SPM prepared by this method is compared to the purity of chick-brain fractions obtained by two other laboratories, using different methods (4, 59). The SPM were extracted with Triton X-100 and all fractions solubilized in sodium dodecyl sulfate (sds). The delipidated proteins of all fractions were subjected to SDS-polyacrylamide electrophoresis on slab gels and stained for protein. A distinct difference was observed between the patterns given by the Triton-soluble and -insoluble fractions. Electron microscopy of the synaptic junction fraction showed numerous junctional complexes.

'High-affinity' binding sites for glycine in synaptosomal-mitochondrial fractions of rat CNS-regions.

'High-affinity' binding mechanisms for glycine exist in synaptosome-enriched preparations of various regions of rat CNS. Such mechanisms may represent interactions of glycine with its synaptic receptors.

High-affinity binding of 3H-glycine and 14C-β-alanine to synaptosomal-mitochondrial fractions of rat CNS regions; Effects of strychnine

Abstract “High-affinity” binding of glycine and β-alanine occurred to synaptosomal-mitochondrial fractions of five regions of rat CNS. Strychnine inhibited the binding of β-alanine to a greater extent than that of glycine to particles of cerebral cortex, midbrain, pons and medulla, but not to those of spinal cord. Both glycine- and β-alanine-receptors may be involved in the convulsant action of strychnine.

Distribution and properties of NADPH-linked aldehyde reductases from rat brain synaptosomes

Studies on the subcellular distribution of NADPH-linked aldehyde reductase from rat brain showed that 10% of the total reductase activity is located in the mitochondrial-synaptosomal fraction. There are differences in the percentages of reductase activity found in the synaptosomes compared to cytosol in various regions of the brain. The NADPH-linked aldehyde reductase from the synaptosomal fraction exhibited a nonlinear Lineweaver-Burk plot. This nonlinearity is due to the presence of two distinct aldehyde reductases, which can be distinguished by Michealis constants forp-nitrobenzaldehyde of 4.1×10(-5) M and 2.6×10(-6) M. The two NADPH-linked aldehyde reductases isolated from synaptosomes were further characterized according to pH optima, andK i values for inhibition by barbiturates. In addition regional distributions for the two enzymes were determined. TheK i values for pentobarbital for the "highK m " enzyme and the "lowK m " enzyme were estimated to be 2×10(-5) M and 6×10(-5) M, respectively. It was concluded from the above studies that the lowK m reductase is probably responsible for 3,4-dihydroxyphenylglycoaldehyde (derived from norepinephrine) reduction in brain and a role of the highK m enzyme for protection of neurons from high concentrations of chemically reactive aldehydes was proposed.

Subcellular and regional localization of?-glutamyl transpeptidase in sheep brain

Studies of the subcellular distribution ofγ-glutamyl transpeptidase from sheep brain by discontinuous sucrose density gradient centrifugation showed that 40% of the transpeptidase activity associated with the mitochondrial-synaptosomal fraction was localized with the synaptosomal-enriched fraction. The microsomal fraction was found to have the highest specific activity whenγ-glutamylp-nitroanalide was used as substrate. This activity, however, represented only 5% of the totalγ-glutamyl transpeptidase activity. Approximately 90% of the total enzyme activity was apparently associated with the fraction containing cell debris and membrane fragments.The 160,000g supernatant fluid (soluble supernatant fraction) represented the least total activity, with only 1.2% recovery; however, this fraction contained two apparent forms of the enzyme. One form had a highK mand the other a lowK m for the substrate,γ-glutamylp-nitroanilide.It was observed that the enzymeγ-glutamyl transpeptidase was not evenly distributed in all areas of brain when the homogenate was used as the enzyme source. The brain region with the highest enzyme activity was the thalamus, which was able to form 1.10 μmolp-nitroanaline/min/g wet brain tissue. The cortex was found to have the lowest activity. The 40,000g supernatant fluid from each region, however, exhibited only slight distribution differences.

Some properties of acetylcholinesterase from rat retina.

Acetylcholinesterase (AChE, EC 3.1.1.7) of rat retina was studied with respect to its kinetic and other properties, and a comparison was made with the enzyme from brain. The subcellular distribution of the retinal AChE showed that the enzyme was concentrated in the synaptosomal-mitochondrial fraction although in the brain the AChE was distributed more evenly between the fractions studied. The enzyme from both retina and brain was easily solubilised and exhibited a Km of the order of 10(-4) M. The pH optimum was 8.3-8.6 for the AChE from both tissues for both the soluble and particulate enzyme.

Axonal transport in the efferent pathways of the hippocampus: Labeling of projection areas after [ 3Hvaline injections

[ 3HValine was injected into the medial and lateral CAI of the dorsal hippocampus and the radioactivity accumulated in areas of some of its efferent projections was measured by scintillation counting. The labeling of the septum, thalamus, hypothalamus, corpus mammillare, preoptic area and basal telencephalon significantly exceeded the corresponding background activities. Fimbria transection before [ 3Hvaline injection reduced the labeling of these areas to background levels. Differences were observed between medial and more lateral CAI injection sites in the distribution of 3H label among projection areas. The dynamics of axonal transport were studied in 4 of these projection areas: septum, thalamus, hypothalamus and corpus mammillare. A rapidly moving wave of radioactivity began to arrive in all areas 1–3 h after injection. The rate was roughly estimated to be around 100 mm/day in the fibers projecting to the corpus mammillare after injections into the medial CAI. When the brain areas were fractionated into crude nuclear, mitochondrial-synaptosomal and microsomal fractions, projection areas were found to contain more label per milligram of protein in the mitochondrial-synaptosomal fraction than control areas.

Dopamine-sensitive adenylate cyclase of the caudate nucleus of rats treated with morphine or haloperidol

The effects of morphine and haloperidol were compared on dopamine-sensitive adenylate cyclase activity of the rat caudate nucleus, an enzyme activity which has been related to the “dopamine receptor.” The response of adenylate cyclase activity to dopamine was measured in a hypo-osmotically shocked mitochondrial-synaptosomal fraction. The addition of 3–300 μM morphine, levorphanol, dextrorphan, d- and l-methadone, nalorphine and naloxone in vitro caused no significant changes in the response to dopamine, while addition of haloperidol in vitro completely inhibited the dopamine response at a concentration of 3 μM or higher. When morphine was administered subcutaneously at a dose of 60 mg/kg, significant increases were found in basal and dopamine-sensitive adenylate cyclase activity from the rat caudate from 15 to 120 min after the injection. Twenty to sixty min after the subcutaneous injection of 20 mg/kg of haloperidol, 1.7 to 2.0-fold increases were found in the dopamine-stimulated activity. The increased dopamine-sensitive cyclase activity found after acute administration of haloperidol o r morphine returned to control values 4 hr after the injection. Implantation of two morphine pellets for 2 or 3 days produced a significant increase in the dopamine sensitivity of the cyclase activity without altering basal activity. These results from experiments in vitro and in vivo suggest that, while haloperidol has a direct effect on the dopamine receptor-associated cyclase activity, morphine must act by another mechanism, and that chronic use of either drug produces enhanced dopamine sensitivity.

Sodium dependency of γ-aminobutyric acid binding to particulate fractions of mouse brain

The Na +-dependency of γ-aminobutyric acid (GABA) binding to particulate fractions of mouse brain was studied, by increasing the concentration of Na + in the homogenizing fluid the retention of both [ 3H] GABA and [ 14C] sucrose was increased in the crude nuclear fractions. But, at Na + concentrations greater than 10 meq/l, the retention of both substances was decreased in the synaptosomal-mitochondrial fractions. Although the total [ 3H] GABA bound to all fractions was increased by increasing the Na + concentration of the homogenizing medium, the total [ 14C] sucrose entrapped in these fractions remained rather constant. The changes in GABA binding caused by Na + thus could not be accounted for by the presence of entrapped homogenizing fluid in pellets. The decreased binding of GABA to synaptosome fractions found in the presence of 40 to 100 meq/l Na + was due to “clumping” effects caused by the excess salt. With high salt concentrations many of the particles which bind GABA and which are normally associated with the synaptosome fraction were sedimented in the crude nuclear fraction. In sum, these results indicated that the binding of GABA occurs maximally at a Na + concentration of about 40 meq/l.

Subcellular fractionation of hypothalamus and pituitary stalk of the bull. Morphologic study and serotonin-norepinephrine distribution.

Hypothalamus and pituitary stalk of the bull were fractionated in a sucrose density gradient according to the Hebb and Whittaker (1958) procedure. The crude mitochondrial-synaptosomal fraction was layered on a discontinuous sucrose density gradient (0.6; 0.8; 1; 1.2 M) and, after 63,000 g (average) centrifugation for 2 hours, 4 bands and one pellet were obtained. The fraction layered on 0.8 M sucrose contained mainly myelin and some rare synaptosomes. The fractions layered on 0.8 M, 1.0 M, and 1.2 M sucrose were very rich in synaptosomes. Different populations of the latter were present: one type contained only clear synaptic vesicles; the second type, in addition to clear synaptic vesicles, had a population of vesicles with homogeneous dense core (about 1000 A diameter). There was no significant difference between the light fractions of hypothalamus and of pituitary stalk. However, in the heavier fractions derived from the pituitary stalk a considerable number of large profiles containing dense core granules of 2000 A diameter were present.