THE SUBCELLULAR DISTRIBUTION OF [14C]GABA AND [3H]DOPAMINE IN THE RETINA

Abstract— Rabbit retinae were homogenized in isotonic sucrose and subjected to differential and density gradient centrifugation. Preliminary electron microscopic examination of some of the fractions indicated that in addition to the subcellular particles usually observed in brain homogenates, the photoreceptor cells gave rise to several characteristic fragments. These included fragmented outer limbs, aggregations of mitochondria from the inner segments, and photoreceptor terminals. Unlike the synaptosomes formed from the conventional type of synapses in the retina, these photoreceptor terminals appeared to sediment mainly in the low speed crude nuclear pellet (P1).
Retinae were incubated with low concentrations of [¹⁴C]GABA and/or [³H]dopamine prior to subcellular fractionation and in these experiments the P2 pellet was further fractionated on sucrose density gradients. Analysis of the radioactivity in the fractions showed that labelled GABA was accumulated by osmotically sensitive particles which had the sedimentation characteristics of synaptosomes. The panicles accumulating [³H]dopamine appeared to belong to a different, slightly lighter, population than those accumulating [¹⁴C]GABA. It is tentatively suggested that the particles accumulating labelled GABA were synaptosomes because the fractions containing these particles also possessed most of the GAD activity of the gradient. In contrast, GABA-T and MAO activity was found in the dense fractions of the gradients usually associated with mitochondria.
When retinae were incubated with a high concentration of labelled GABA a'lighter'population of particles seemed to accumulate the amino acid than when a low external GABA concentration was used. These results suggest that the high and low affinity uptake processes for GABA in the retina may have different cellular sites.     

INCORPORATION OF [1-14C]OLEIC ACID AND [1-14C]ARACHIDONIC ACID INTO LIPIDS IN THE SUBCELLULAR FRACTIONS OF MOUSE BRAIN

Abstract— The distribution of radioactivity among lipids of subcellular membrane fractions was examined after intracerebral injections of [1-¹⁴C]oleic and [1-¹⁴C]arachidonic acids. Labelled free fatty acids were distributed among the synaptosomal-rich, microsomal, myelin and cytosol fractions at 1 min after injection. However, incorporation of the fatty acids into phospholipids and trïacylglycerols after pulse labelling occurred mainly in the microsomal and synaptosomal-rich fractions. With both types of labelled precursors, there was a higher percentage of radioactivity of diacyl-glycerophosphoryl-inositols in the synaptosomal-rich fraction as compared to the microsomal fraction. Radioactivity of [1-¹⁴C]oleic acid was effectively incorporated into the triacylglycerols in the microsomal fraction whereas radioactivity of the [1-¹⁴C]arachidonic acid was preferentially incorporated into the diacyl-glycerophosphorylinositols in the synaptosomal-rich fraction. Result of the study indicates that synaptosomal-rich fraction in brain is able to metabolize long chain free fatty acids in vivo and to incorporate these precursors into the membrane phosphoglycerides.     

SUBCELLULAR DISTRIBUTION OF RADIOACTIVITY IN NEURONAL AND GLIAL-ENRICHED FRACTIONS AFTER INCORPORATION OF [3H]LEUCINE IN VIVO AND IN VITRO

Abstract— The distribution of protein-bound radioactivity among subcellular organelles of cerebral cortex was followed after intravenous administration of [3H]leucine and after incubation of brain slices in the presence of [3H]leucine. Neuronal and glial cell-enriched fractions were prepared by discontinuous sucroseFicol1 gradient centrifugation of cerebral cortex cell suspensions. Subcellular fractions were obtained from each of the cell prepara- tions and the protein-bound radioactivity determined after in uiuo and in vitro incorporation of [3H]leucine. The unfractionated neuronal material had a considerably higher level of protein-bound radioactivity than the glial material. The most marked neuronal-glial dif- ferences were observed in microsomes and soluble proteins, while the radioactive labelling of the nuclear and mitochondria1 fractions was similar for the two cell types.     

THE EFFECT OF MORPHINE ADMINISTRATION ON THE INCORPORATION OF [14C]LEUCINE INTO PROTEIN IN CELL-FREE SYSTEMS FROM RAT BRAIN AND LIVER

—Ribosomes isolated from the brains of rats treated with morphine in vivo were less active in promoting the incorporation of [¹⁴C]leucine into protein than ribosomes isolated from untreated rats. This inhibitory phenomenon was studied in relation to dose of morphine, time after drug administration and the pharmacological responses of hypothermia and analgesia. The inhibition of [¹⁴C]leucine incorporation into brain proteins in vitro was transient after a single injection of morphine and dose-dependent, and related to the hypothermic response, but not prevented by keeping the rats at an ambient temperature which prevented hypothermia. The incorporation of [¹⁴C]leucine into protein by liver ribosomes was also inhibited in preparations from morphine treated rats.     

INCORPORATION OF 14C FROM [U-14C]GLUCOSE INTO FREE AMINO ACIDS IN MOUSE BRAIN REGIONS UNDER CYANIDE INTOXICATION

—During anoxia induced by the administration of potassium cyanide, [U-¹⁴C]glucose was injected intraperitoneally into adult mice and they were decapitated at 5, 15 and 30 min after the injection. After freeze-drying in vacuo, differences in the uptake of radioactive carbon from [U-¹⁴C]glucose into free amino acids (glutamate + glutamine, aspartate + asparagine, GABA, alanine and glycine) in mouse cerebral neocortex, cerebellar hemisphere, caudate nucleus, thalamus, hypothalamus and medulla oblongata were investigated (by macroautoradiography and GLC separation) and compared with those obtained under normal conditions. (1) During anoxia, autoradiographical densities in the thalamus and medulla oblongata were higher than that in the cerebral neocortex and caudate nucleus. (2) Among specific radioactivities (d.p.m./μmol) of free amino acids, alanine gave the highest value during anoxia, except in the cerebellar hemisphere and hypothalamus at 5 min and the medulla oblongata at 30 min. (3) During anoxia, the specific radioactivities of alanine and glycine in each brain region did not significantly decrease at 15 and 30 min compared with those under normal conditions. During anoxia, the specific radioactivity of glutamate + glutamine in the cerebellar hemisphere and hypothalamus did not significantly decrease compared with the normal conditions, while that of GABA, aspartate + asparagine and glutamate + glutamine in the cerebral neocortex, caudate nucleus, thalamus and medulla oblongata showed an increase. (4) The percentage decrease of glutamate + glutamine and aspartate + asparagine at 5 and 15 min was highly significant in the cerebral neocortex and caudate nucleus.     

INCORPORATION OF 14C FROM [U-14C]GLUCOSE INTO FREE AMINO ACIDS IN MOUSE BRAIN LOCI IN VIVO UNDER NORMAL CONDITIONS

By macroautoradiography and by GLC separation, differences in the uptake of radioactive carbon from [U-¹⁴C]glucose into free amino acids (glutamate + glutamine, aspartate + asparagine, GABA, alanine and glycine) in mouse cerebral neocortex, hippocampus, thalamus and hypothalamus were investigated. (1) The autoradiographical densities in the thalamus, cerebral neocortex and hippocampus measured with a microdensitometer were higher than that in the hypothalamus at 5 min after subcutaneous injection. At 180 min, densities in the cerebral neocortex, hippocampus and hypothalamus were higher than that in thalamus. (2) The free amino acid levels determined by GLC varied with each brain region. (3) The specific radioactivity (d.p.m./μmol) of alanine in each brain region was higher than that of the other amino acids at 5 min after the injection. The specific radioactivity of GABA in the brain regions was clearly higher than that of (glutamate + glutamine), (aspartate + asparagine) and glycine at 5 and 15 min. (4) The autoradiographical data were in good agreement with the chemical data at 5 min but were different at 180 min. (5) Variations in specific radioactivity of each free amino acid among brain regions at 5 min were influenced greatly by existing free amino acid concentrations in each region.     

UPTAKE OF [14C]ASPARTIC ACID AND [14C]GLUTAMIC ACID BY RETINAL SYNAPTOSOMAL FRACTIONS

Abstract— Uptake systems for [¹⁴C]aspartate and [¹⁴C]glutamate were characterized in two distinct synaptosomal fractions solated from rabbit retina. The P, synaptosomal fraction was highly enriched in large photoreceptor cell synaptosomes but contained very few conventional sized synaptosomes from amacrine, horizontal or bipolar cells. In contrast, the P₂ synaptosomal fraction contained numerous conventional sized synaptosomes and was virtually free of photoreceptor cell synaptosomes. Both synaptosomal fractions took up [¹⁴C]aspartate and [¹⁴C]glutamate with high affinity [ K _m= 1–2μM). Uptake characteristics were similar to those described for high affinity uptake systems in brain synaptosomes, i.e. saturation kinetics; temperature and Na⁺ dependence. Although the presence of a high affinity uptake system is not a definitive criterion for demonstration of functional neurotransmitter systems, it is an important and necessary prerequisite and can thus be considered as supportive evidence for the involvement of asparate and glutamate in neurotransmission in rabbit retina.     

INCORPORATION OF d-[3H]GLUCOSAMINE INTO THE SIALOGLYCOPROTEIN GP-350 OF ADULT RAT BRAIN

—The incorporation of d -[³H]glucosamine into the nervous specific sialoglycoprotein GP-350 has been studied in adult rat brain. Both the 100,000 g supernatant fluid and the 12,500 g pellet were used for the investigation, since GP-350 could only be detected in the soluble cell fraction (Van Nieuw Amerongen et al., 1972) and in the synaptosomal membranes, sedimenting in the crude mitochondrial fraction (Van Nieuw Amerongen & Roukema , 1973, 1974). GP-350 was separated from the other proteins by polyacrylamide gel electrophoresis at pH 7.5 and the incorporation of radioactivity into GP-350 was measured at different time intervals, ranging from 1 to 96 h after the administration of the radioisotope. The maximal incorporation of radioactivity into the soluble GP-350 was obtained after about 2 h and into the membrane-bound GP-350 after about 3 h. After 2 h there is a very rapid decrease of the radioactivity of GP-350 from the soluble cell fraction (up to 70 per cent within 2 h). Thereafter the disappearance is more gradual and of the same order as that found for the membrane-bound fraction of GP-350. The half-life of the soluble GP-350 was estimated to be 19 h and for the membrane-bound GP-350 a value of 18 h was calculated. Compared to the total pool of brain (glyco) proteins and specific nervous (glyco) proteins GP-350 has a very rapid turnover. The rapid initial decrease of the radioactivity from the soluble GP-350 may be interpreted in terms of a rapid transport of the newly-synthesized GP-350 from the cytoplasma of the perikaryon to the membranes of the synaptic region by an axoplasmic flow.     

SUBCELLULAR LOCALIZATION OF NEWLY-FORMED [3H]ACETYLCHOLINE IN RAT CEREBRAL CORTEX IN VITRO

—Slices from rat brain cortex were incubated for either 5 or 60 min in a medium containing [³H]choline and 4·7 or 25 mm -KCl. Bioassayable ACh and labelled ACh were determined in the incubation medium, in the total tissue homogenate and in subcellular fractions. Raising the KCl concentration from 4·7 to 25 mm stimulated the release and synthesis of total and of labelled ACh. In medium containing 25 mm -KCl the amounts of ACh decreased in the tissue and in the nerve ending cytoplasm, but remained constant in the synaptic vesicles. After incubation in 25 mm -KCl medium the ACh in the vesicles was labelled to the same extent as the cytoplasmic ACh but after incubation in 4·7 mm -KCl medium vesicular ACh was labelled less than cytoplasmic ACh. During 5 min incubation in medium containing 25 mm -KCl the ratio of labelled to total ACh was much higher in the medium than in the homogenate, the vesicles or the cytoplasm. During the last 15 min of the 60 min incubation the ratio of labelled to total ACh in the medium was still higher than that in the tissue fractions, but less so than during the 5 min incubation. It is concluded that the vesicular and cytoplasmic fractions are not identical with the store in the tissue from which newly-synthesized ACh is preferentially released.     

ESTIMATION OF NORADRENALINE AND ITS MAJOR METABOLITES SYNTHESIZED FROM [3H]TYROSINE IN THE RAT BRAIN

Abstract— A new procedure is described for the estimation of [³H]noradrenaline (NA) and its major metabolites free and conjugated 3-methoxy-4-hydroxyphenylglycol (MOPEG) and free and conjugated 3,4-dihydroxyphenylglycol (DOPEGI in the rat brain. The procedure involves adsorption on to alumina, cation exchange chromatography. enzymatic hydrolysis of conjugates and thin-layer-chromatography after intraventricular (IVT) or intravenous injection of [³H]tyrosine. In a time-course study the formation and accumulation of the metabolites have been measured from 15min to 23h after IVT injection of [³H]tyrosine. [³H]MOPEG and [³H]DOPEG were found in almost equal amounts during the synthesis phase of [³H]NA as well as during the storage and disappearance phase of [³H]NA. The maximum levels of conjugated [³H]MOPEG and conjugated [³H]DOPEG were found 2 h after IVT [³H]tyrosine. At this time interval the levels of free [³H]MOPEG and free [³H]DOPEG amounted to 25% and 11%, respectively of the corresponding conjugates. Increasing doses of IVT injected [³H]tyrosine (10-90 °Ci) revealed that the accumulation of [³H]NA and metabolites was linear up to about 50 °Ci. Following intravenous instead of IVT injection of [³H]tyrosine. much higher doses (325 °Ci) were needed to obtain measurable amounts of total [³H]MOPEG and [³H]DOPEG-SO₄ in the rat brain. The formation of labelled NA metabolites from [³H]NA in the rat brain in vim measured as total [³H]MOPEG and [³H]DOPEG-SO₄ was influenced by drugs affecting [³H]NA synthesis, release and metabolism. Synthesis inhibition with a-methyltyrosine (250mg-kg^?1) or FLA-63 (30mg-kg^?1) and inhibition of monoamine oxidase with pargyline (75mg-kg^?1) or clorgyline (2mg-kg^?1) strongly decreased the accumulation of total [³H]MOPEG and [³H]DOPEG-SO₄. Noradrenaline receptor blockade with phenoxybenzamine (20mg-kg^?1) increased both total [³H]MOPEG and [³H]DOPEG-SO₄ to about 160% of the control values. NA release and uptake inhibition induced by d-amphetamine (10mg-k^?1) or phenylethylamine (two doses of 80mg-kg^?1) decrease strongly the levels of [³H]NA and [³H]DOPEG-SO₄. whereas total [³H]MOPEG was only very slightly decreased or even increased as compared to controls.     

EFFECTS OF SPREADING CORTICAL DEPRESSION ON THE INCORPORATION OF [14C]LEUCINE INTO PROTEINS OF RAT BRAIN

Abstract— Incorporation of dl -[1-¹⁴C]leucine into proteins of the cerebral cortex of the rat was measured during spreading cortical depression (CSD) evoked by a single topical application of 25% (w/v) KCI. Maximal inhibition (42 per cent) of the rate of incorporation occurred 1 hr after application of KCI. Spreading depression of 2–3 hr duration was associated with 22 per cent and 13 per cent decreases, respectively, of incorporation of labelled leucine. Specific activity of the free pool leucine was not decreased during CSD but appeared to be higher than controls at 20 min after initiation of CSD. The specific activity of the total free pool amino acids was also increased at 10, 20, 60 and 120 min after application of KCI.
The inhibitory effect of CSD on incorporation of leucine into proteins was uniformly distributed among the crude mitochondrial, microsomal and soluble subcellular fractions from brains of adult animals, while in fractions from 25-day old animals there appeared to be relatively more inhibition in the crude mitochondrial fraction.     

CHEMICAL SYNTHESIS OF [3H]CEREBROSIDE [35S]SULFATE AND ITS IN VIVO METABOLISM IN RAT BRAIN

—Double-labeled sulfatide containing [3-³H]lignoceric acid and [³⁵S]sulfate was synthesized and injected intracerebrally into 28-day-old rats. The ³H-labeled sulfatide was synthesized by condensing (RS)-[3-³H]lignoceroyl chloride with lysosulfatide which had been obtained by saponification of sulfatide. The ³⁵S-labeled sulfatide was synthesized by using [³⁵S]sulfuric acid for sulfating 2′, 4′, 6′-tri-benzoyl-galactosyl N-fatty acyl, N-benzoyl-3-0-benzoyl-sphingosine, which had been obtained by per-benzoylation followed by solvolysis of calf brain nonhydroxycerebrosides. The perbenzoylated [³⁵S]sul-fatide was then subjected to mild alkaline saponification. Eight hours following the injection, the brain lipids contained various radioactive sphingolipids in addition to sulfatides. Fourteen per cent of the injected ³H was recovered in total lipids, and 26% of this was found in sulfatide. Nonhydroxy- and hydroxyceramides, nonhydroxy- and hydroxycerebrosides, and polar lipids contained 7, 1, 8, 3, and 22 per cent of the ³H found in total lipids, respectively. On the other hand, only 6% of the ³⁵S injected was recovered in total lipids; 63% of this was found in sulfatide, 5% in a mixture of seminolipid and cholesterol sulfate and 10% in a water-soluble material.     

INCORPORATION OF [14C]N-ACETYL NEURAMINIC ACID INTO BRAIN GLYCOPROTEINS AND GANGLIOSIDES IN VIVO1

—Intracerebrally administered [¹⁴C]N-acetyl neuraminic acid was incorporated into brain glycoproteins and gangliosides. Incorporation into both classes of compounds was markedly inhibited by acetoxycycloheximide but incorporation into the soluble glycoproteins of the nerve-ending fraction was inhibited least of all. In contrast to glucosamine and fucose, a relatively small proportion of the injected [¹⁴C]NANA was incorporated.     

CHARACTERIZATION AND SUBCELLULAR LOCALIZATION OF BRAIN MUSCARINIC RECEPTORS LABELLED IN VIVO BY [3H]DEXETIMIDE

Abstract— [³H]Dexetimide specifically labels brain muscarinic receptors in vivo . After i.v. injection of labelled drug into rats, radioactivity specifically accumulates in brain regions containing muscarinic receptors but not in cerebellum. This accumulation is stereospecific, saturable and displaceable by unhbelled dexetimide. In contrast, [³H]levetimide, the inactive enantiomer, does not show such preferential uptake or stereospecific displacement.
An analytical approach was used to study the subcellular distribution of [³H]dexetimide binding sites. After differential centrifugation the binding sites are mainly recovered in the microsomal fraction from different brain regions but not from the cerebellum. After displacement the radioactivity is found in the supernatant. After equilibration in a density gradient the distribution pattern of [³H]dexetimide is bimodal, like that of 5'-nucleotidase, with a major peak in a region of low density.
When the microsomal fraction was treated with digitcnin, three groups of membrane were characterized by isopycnic centrifugation on the basis of their differential shift to higher densities. Evidence is provided that the postsynaptic membranes bearing muscarinic receptors belong to the class of plasma membranes. Finally, digitonin treatment may represent a useful tool to produce subfractions enriched in postsynaptic membranes which can now be identified biochemically in binding experiments.     

POTASSIUM-INDUCED RELEASE OF [3H]GABA AND OF [3H]NORADRENALINE FROM NORMAL AND RESERPINIZED RAT BRAIN CORTEX SLICES. DIFFERENCES IN CALCIUMDEPENDENCY, AND IN SENSITIVITY TO POTASSIUM IONS

The release of [³H]GABA induced by elevated extracellular potassium (K)_o, from thin rat brain cortex slices, has been compared with that of [³H]noradrenaline ([³H]NA), released by the same procedures, both from normal slices, and from slices pre-treated with reserpine and nialamide, [³H]NA being predominantly a vesicular component in the former situation, and a soluble substance in the latter one. 46 mM-(K)_o released considerably more [³H]NA from normal than from drug-treated slices, while the release of GABA was about two thirds of the latter. When 4min ‘pulses’ of increasing concentrations of potassium were applied, it was observed that the release of GABA and of [³H]NA from drug-treated slices increased in proportion to (K)_o, up to 36-46 mM and then declined considerably with higher (K)_o. The dependency of potassium-induced release on the concentration of calcium in the medium, indicated that release of [³H]NA from normal slices was proportional to calcium up to 1.5-2 mM, while that of [³H]NA from drug-treated slices increased up to 0.5 mM-Calcium, and then declined with higher concentrations. GABA release also increased up to 0.5 mM-calcium, but no further changes were observed at higher concentrations. The calcium antagonist D-600 inhibited high (K)_o-induced release of [³H]NA from normal slices to a greater extent than that of [³H]GABA or of [³H]NA from drug-treated slices. These results, in which elevated (K)_o-induced release of [³H]GABA resembles considerably that of soluble NA, but differs from that of NA present in synaptic vesicles, suggest that release of [³H]GABA also occurs from the soluble cytoplasmic compartment, and that the partial calcium requirement that is found is unrelated to that of transmitter secretion. These findings are also a further indication of the lack of specificity of elevated (K)_o as a stimulus for inducing transmitter secretions.     

IN VIVO FORMATION AND CATABOLISM OF [14C]HISTAMINE IN MOUSE BRAIN

Abstract— The formation of histamine in brain was studied in mice injected with l -[¹⁴C]-histidine (ring 2-¹⁴C) intravenously (i.v.) or intracerebrally; [¹⁴C]histamine appeared rapidly and exhibited a rapid rate of turnover. Drugs known to block various pathways of histamine catabolism were tested for effects on brain–[¹⁴C]histamine and [¹⁴C]-methyl-histamine in mice given (1) [¹⁴C]histamine i.v., (2) [¹⁴C]histamine intracerebrally, and (3) l -[¹⁴C]histidine i.v. Blood-borne histamine did not enter brain; brain histamine was formed locally by decarboxylation of histidine Methylhistamine did cross the blood-brain barrier. Methylation was the major route of histamine catabolism in mouse brain and some of the methylhistamine formed was destroyed by monoamine oxidase. No evidence for catabolism by the action of diamine oxidase was found.     

CATECHOLAMINE METABOLISM IN THE DOG: COMPARISON OF INTRAVENOUSLY AND INTRAVENTRICULARLY ADMINISTERED [14C]DOPAMINE AND [3H]NOREPINEPHRINE

—The urinary excretion of labelled metabolites was measured in dogs which had been injected intravenously or intraventricularly with [³H]norepinephrine or [¹⁴C]dopamine. [³H]Norepinephrine injected by either route produced more labelled 3-methoxy-4-hydroxy-phenylglycol than 3-methoxy-4-hydroxymandelic acid, as did [¹⁴C]dopamine after intravenous administration. In contrast, following the intraventricular injection of [¹⁴C]dopamine, more [¹⁴C]3-methoxy-4-hydroxymandelic acid was formed than [¹⁴C]3-methoxy-4-hydroxyphenylglycol. These observations suggest that the metabolism of exogenously-administered and endogenously-formed norepinephrine may proceed through different routes and that the predominant metabolite of norepinephrine in canine brain may be 3-methoxy-4-hydroxymandelic acid rather than 3-methoxy-4-hydroxyphenylglycol.     

DEVELOPMENT OF THE UPTAKE AND STORAGE OF L-[3H]NOREPINEPHRINE IN THE RAT BRAIN

The uptake and storage of L-[³H]norepinephrine at various stages of development was examined in homogenates of rat brain. For the adult animal, active uptake accounted for 80 per cent of the total uptake. At 14 days of gestation, no active uptake was demonstrable At 18 days of gestation, saturable uptake of L-[³H]norepinephrine with a K_m of 3 × 10 ^?7m was first demonstrable; the K_m value did not vary during subsequent development. The V_max. of uptake increased five-fold between 18 days of gestation and 28 days postnatally, at which stage it was the same as the adult value. The development of saturable uptake paralleled but preceded the increase in endogenous norepinephrine. When homogenates were incubated with l -[³H]norepinephrine and subjected to centrifugation on linear sucrose gradients, there was a peak of tritium in the synaptosomal fractions; the magnitude of the peak increased with maturation of the brain. The increase in the peak of tritium paralleled the increase in particulate LDH activity and was distinct from the peak of MAO activity. Desipramine, a compound that blocks the initial uptake of norepinephrine, first exhibited inhibition of uptake at 19 days of gestation; the degree of inhibition did not vary during subsequent development. In contrast, reserpine, a compound which inhibits the intra-neuronal storage of norepinephrine, exhibited a progressive increase of inhibition with maturation of the brain at and subsequent to 19 days of gestation.