UPTAKE OF [14C]NIPECOTIC ACID INTO RAT DORSAL ROOT GANGLIA

Abstract— [¹⁴C]Nipecotic acid was accumulated in isolated desheathed rat dorsal root ganglia by a saturable process with K _m= 48.8 μ m and V _max= 2.2 nmol/g/min. The concentration of l -2.4-diamino-butyric acid required to inhibit the uptake of nipecotic acid by 50% was three times the concentration of β-alanine required to do the same. Light microscopic autoradiography indicated that the sites of uptake of [¹⁴C]nipecotic acid were principally confined to satellite glial cells. It is concluded that nipecotic acid is transported by the GABA uptake system in glia but that it has less affinity for this system than GABA.     

UPTAKE OF [3H-METHYL]CHOLINE BY MICROSOMAL, SYNAPTOSOMAL, MITOCHONDRIAL AND SYNAPTIC VESICLE FRACTIONS OF RAT BRAIN

Abstract— Microsomal, mitochondrial, synaptosomal and synaptic vesicle fractions of rat brain took up [³H-methyl]choline by a similar carrier-mediated transport system. The apparent K_m for the uptake of [³H-methyl]choline in these subcellular fractions was about 5 × 10^?5 M. Choline uptake was also observed in microsomal fractions prepared from liver and skeletal muscle. Virtually identical kinetic properties for [³H-methyl]choline transport were found in the synaptosomal fractions prepared from the whole brain, cerebellum or basal ganglia. Countertransport of [³H-methyl]choline from the synaptosomal fraction was demonstrated against a concentration gradient. HC-3 was a competitive inhibitor of the uptake of [³H-methyl]choline in brain microsomal, synaptosomal and mitochondria] fractions with respective values for K_i of 4.0, 2.1 and 2.3 × 10^?5 M. HC-15 was a competitive inhibitor of the transport of [³H-methyl]choline in the synaptosomal fraction, with a K_i of 1.7 × 10^?4 M. Upon entry into the microsomal fraction, 74 per cent of the radioactivity could be recovered as unaltered choline, 10 per cent as phosphorylcholine, 1.5 per cent as acetylcholine and 2.5 per cent as phospholipid. Choline acetyltransferase (EC 2.3.1.6) was assayed with [¹⁴C]acetylCoA in synaptosomal fractions prepared from basal ganglia and cerebellum, and in the 31,000 g supernatant fraction of a rat brain homogenate. Enzyme activity was 11-fold greater in the synaptosomal fraction from the basal ganglia than in that from the cerebellum. HC-3 did not inhibit choline acetyltransferase and there was no evidence for acetylation of HC-3. Our findings suggest that choline uptake is a ubiquitous property of membranes in the CNS and cannot serve to distinguish cholinergic nerve endings and their synaptic vesicles.     

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.     

MEDIATION BY β-ADRENERGIC RECEPTORS OF EFFECT OF NOREPINEPHRINE ON PINEAL SYNTHESIS OF [14C]SEROTONIN AND [14C]MELATONIN

Rat pineal organs maintained in organ culture converted [¹⁴C]tryptophan to [¹⁴C]serotonin and [¹⁴C]melatonin. The synthesis of both indoles was stimulated by the presence of norepinephrine or dibutyryl adenosine 3′,5′-monophosphate. This effect of norepinephrine could be blocked by the α-adrenergic blocking drug, propranolol, but was not modified by the a-adrenergic blocking agent, phenoxybenzamine. Neither blocking agent modified the pineal response to dibutyryl adenosine 3′,5′-monophosphate. Unlike dibutyryl adenosine 3′,5′-monophosphate, the naturally occurring adenosine phosphates did not stimulate synthesis of [¹⁴C]melatonin in vitro.     

[14C]GLUTAMATE UPTAKE AND COMPARTMENTATION IN GLIA OF RAT DORSAL SENSORY GANGLION

Abstract— The characteristics of the uptake of l -[U-¹⁴C] glutamate into rat dorsal sensory ganglia were investigated. The uptake was mediated by two distinct kinetic systems, with apparent K_m values of the order of 10⁻³ M (low affinity) and 10⁻⁵ m (high affinity). The high affinity uptake system was strongly dependent upon temperature and sodium ion concn, and was depressed by a number of metabolic inhibitors. Following uptake, [¹⁴C] glutamate was extensively metabolized, primarily to glutamine, although this was not so with cultured ganglia, where in addition to an increased uptake of [¹⁴C] glutamate, the specific radioactivity of glutamate was increased and that of glutamine decreased. The labelled substrates [U-¹⁴C]pyruvate and [U-¹⁴C] acetate were used to investigate this phenomenon and the results are discussed in relation to current knowledge of metabolic compartmentation in nervous tissue.     

MECHANISMS FOR THE EFFLUX OF [14C]DOPA AND [14C]DOPAMINE FROM THE CSF OF RHESUS MONKEYS

—Clearance of [¹⁴C]DOPA and [¹⁴C]dopamine from CSF was investigated in anaesthetized rhesus monkeys (M. Mulatta) subjected to ventriculocisternal perfusion. The efflux coefficients, k_VE, at tracer concentrations (3–5 m ) in the perfusate were 0.0487 ml/min and 0.0325 ml/min for [¹⁴C]DOPA and [¹⁴C]dopamine, respectively. Carrier DOPA (10 mm ) in the perfusate decreased the efflux of [¹⁴C]DOPAsignificantly, but carrier dopamine had no appreciable effect on the clearance of [¹⁴C]dopamine. These findings suggest that DOPA is cleared from CSF in part by a saturable mechanism which may be located in the choroid plexus, whereas dopamine leaves the ventricular system by passive diffusion. Radioactivity in the caudate nucleus immediately adjacent to the perfused ventricle averaged 15.5 % and 12.6% of the radioactivity in the perfusates with [¹⁴C]DOPA or [¹⁴C]dopamine, respectively. These distribution percentages were similar to those found for various extracellular indicators after ventriculocisternal perfusion and may indicate that the efflux of intraventricularly-administered exogenous DOPA and dopamine occurs in part through extracellular channels.     

STUDY OF RNA IN SUBCELLULAR FRACTIONS FROM RAT BRAIN: SIMULTANEOUS INCORPORATION OF [14C]URIDINE AND [3H]METHYL-l-METHIONINE

Abstract— By using a combination of subcutaneous and intraventricular injections of [¹⁴C]uridine and [³H]methyl- l -methionine we have obtained maximum incorporation in about 40 min of both radioactive precursors into nuclear RNA from rat brain. In this nuclear fraction we found at least two different types of RNA that were rapidly labelled. One of them incorporated both [¹⁴C]uridine and [³H]methyl groups and seemed to correspond to species of rRNA and their precursors. The other RNA fraction was less methylated or non-methylated and exhibited sedimentation coefficients distributed along a continuous 8–30 % sucrose density gradient. At least part of the latter type of RNA very probably was mRNA, but much of it must conespond to a different RNA similar to that recently described in HeLa cells by P enman , V esco and P enman (1968).
We also found that labelled 185 and 285 rRNA components began leaving the nucleus for the cytoplasm within 24 to 33 min after the radioactive precursors had been injected, and, in the cytoplasmic fraction, the patterns of incorporation for [¹⁴C]uridine and [³H]-methyl groups were similar for the 18S and 28S rRNA components. We estimate that in this fraction of rat brain the 18S rRNA component was 1·4 times more methylated than the 28S component. We also detected a lower sedimentation coefficient for the non- or slightly methylated, species of soluble RNA found in the cytoplasmic fraction.     

METABOLISM OF [14C]LEUCINE AND [14C]ACETATE IN SENSORIMOTOR CORTEX, THALAMUS, CAUDATE NUCLEUS AND CEREBELLUM OF THE CAT

Abstract— —In the head of the caudate nucleus, the relative specific activity of glutamine (glutamic acid specific activity = 1) was less than 1 with intravenous [¹⁴C]leucine as the tracer metabolite. This is in contrast to observations made in other brain areas (cortex, hippocampus, thalamus, pons, and medulla) where the relative specific activity of glutamine was greater than 1. This is also in contrast to findings when [l-¹⁴C]acetate was utilized as the tracer; under these conditions, in all brain areas, including the head of the caudate nucleus, the relative specific activity of glutamine was greater than 1. It is inferred that the differences in metabolism of [¹⁴C]leucine and [¹⁴C]acetate in the head of the caudate from that in other brain areas reflect differences in compartmentation of the glutamate-glutamine system.     

Production of [14C]Acetylcholine and [14C]Carbon Dioxide from [U–14C]Glucose in Tissue Prisms from Aging Rat Brain

Abstract: Production of [¹⁴C]acetylcholine and ¹⁴CO₂ was examined by using tissue prisms from neocortex, hippocampus, and striatum from rats aged approximately 5 months, 13 months, and 27 months. [¹⁴C]Acetylcholine synthesis in the striatum showed highly significant decreases with age for measurements in the presence of both 5 m m - and 31 m m -K⁺, contrasting with the lack of significant change in ¹⁴CO₂ production in this region. The neocortex and hippocampus showed only small changes, especially when comparison was made between 13-month and senescent animals. Measurements of the release of [¹⁴C]acetylcholine and influence of atropine on this release confirmed the relative stability with age of the cholinergic system in the neocortex.     

THE BIOSYNTHESIS OF CHOLESTEROL AND OTHER STEROLS BY BRAIN TISSUE: DISTRIBUTION IN SUBCELLULAR FRACTIONS AS A FUNCTION OF TIME AFTER INJECTION OF [2-14C]MEVALONIC ACID, SODIUM [2-14C]ACETATE AND [U-14C]GLUCOSE INTO 15-DAY-OLD RATS

The distribution of [¹⁴C]-labelled material into subcellular fractions of 15-day-old rat brain was studied at 2 and 24 h following intraperitoneal and intracerebral injection of [2-¹⁴C]sodium acetate, [U-¹⁴C]glucose and [2-¹⁴C]mevalonic acid respectively. The total quantity of labelled isoprenoids in the brain was, except for glucose, greater when the precursor was administered intracerebrally. The intraperitoneal route was more advantageous in the case of [U-¹⁴C]glucose. The subcellular distribution of both labelled total isoprenoid material and sterol was distinct for each labelled precursor. Intracerebrally injected [U-¹⁴C]glucose at both time periods studied suggested no dominance of labelling in any fraction. After intraperitoneal injection of [U-¹⁴C]glucose the microsomes were more prominently labelled. Both methods of administration of sodium [2-¹⁴C]acetate resulted in heavy labelling of the myelin fraction after 24 h. The total labelled isoprenoids resided mainly in the microsomes 24 h after injection of [2-¹⁴C]mevalonic acid. Labelled sterol was found to be localized more in the myelin and microsomal fractions for all three precursors than was the labelled total isoprenoids. Depending on the type of experiment to be conducted, each of these precursors can give different results, which must be interpreted accordingly.     

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.     

Metabolism of [14C]ss-glucose and [14C]-acetate by lettuce embryos during early germination

The metabolism of ['⁴C]-labelled glucose and acetate has been investigated during the early germination - before radicle emergence - of lettuce ( Lactuca sativa L., cv. Val d'Orge) embryos. Similar amounts of radioactivity from both substrates were evolved as C., or incorporated into organic acids, amino acids and proteins. A large part of the [¹⁴C]-glucose was also incorporated into sucrose and polysaecharides, and a small part into the glycerol moiety of lipids. Acetate was massively incorporated into lipids, and only slightly into neutral compounds. These results show that both glucose and acetate can be utilized as respiratory substrates during early germination of lettuce embryos. Various biosynthetic pathways leading to amino acids, proteins, polysaecharides and lipids are active during this period.     

ISOLATION OF HYDROPHOBIC PROTEINS BINDING AMINO ACIDS. STEREOSELECTIVITY OF THE BINDING OF L-[14C]GLUTAMIC ACID IN CEREBRAL CORTEX

From the total lipid extract of ncrve-ending membranes or the homogenate of cerebral cortex a hydrophobic protein fraction binding L-[¹⁴C]glutamic acid was separated by chromatography on Sephadex LH20. This protein could only be partially separated from the [¹⁴C]GABA-binding protein and from the lipids that are present in the fraction; however, it was demonstrated that both amino acids bind to different sites. The saturation of the binding showed a high (Kd₁= 0.3μM), a medium (Kd, = 5 μM) and a low (Kd, = 55 μM) affinity binding site. The high affinity binding site had a binding capacity of 0.53 nmol/mg of protein and was highly stereoselective for the L-enantiomer. The binding of L-[¹⁴C]glutamic acid was not inhibited by GABA, was slightly inhibited by glycine and glutamine and was strongly inhibited in a progressive order by DL-a-methylglutamic acid, L-nuciferine, L-aspartic acid and L-glutamic acid diethyl ester. These results are compared with those previously obtained with the L-glutamic acid-binding protein isolated from crustacean muscle. The stereoselectivity of the binding and the possible role of this protein in synaptic transmission are discussed.     

A SOLUBLE PREPARATION FROM RAT BRAIN THAT INCORPORATES INTO ITS OWN PROTEINS [14C]ARGININE BY A RIBONUCLEASE-SENSITIVE SYSTEM AND [14C]TYROSINE BY A RIBONUCLEASE-INSENSITIVE SYSTEM

Abstract— A 100,000 g supernatant fraction from rat brain that was passed through a column of Sephadex G-25-40 was able, after addition of some factors, to incorporate [^I4C]arginine (apparent K_m= 5 μM) and [¹⁴C]tyrosine (apparent K_m= 20 μM) into its own proteins. The factors required for the incorporation of [¹⁴C]arginine were: ATP (optimal concentration = 0-25-2 μM) and Mg²⁺ (optimal concentration 5 mM). For the incorporation of [^I4C]tyrosine the required factors were: ATP (apparent K_m= 0-75 μM), Mg²⁺ (optimalconcentration 8-16 mM) and K⁺ (apparent K_m= 16 mM). Addition of 19 amino acids did not enhance these incorporations. Optimal pHs were: for [¹⁴C]arginine and [¹⁴C]tyrosine, respectively, 7-4 and 7-0 in phosphate buffer and 7–9 and 7-3-8-1 in tris-HCl buffer. Pancreatic ribonuclease abolished the incorporation of [¹⁴C]arginine but had practically no effect in the incorporation of [¹⁴C]tyrosine. Furthermore, [¹⁴C]arginyl-tRNA was a more effective donor of arginyl groups than [¹⁴C]arginine, whereas [¹⁴C]tyrosyl-tRNA was considerably less effective than [¹⁴C]tyrosine. The incorporations of [¹⁴C]arginine and [¹⁴C]tyrosine into brain proteins were from 25- to 2000-fold higher than for any other amino acid tested (12 in total). In brain [¹⁴C]arginine incorporation was higher than in liver and thyroid but somewhat lower than in kidney. In comparison to brain, the incorporation of [¹⁴C]tyrosine was negligible in liver, thyroid or kidney. Kinetic studies showed that the macromolecular factor in the brain preparation was complex. The protein nature of the products was inferred from their insolubilities in hot TCA and from the action of pronase that rendered them soluble. [¹⁴C]Arginine was bound so that its a-amino group remained free. Maximal incorporation of [¹⁴C]tyrosine in brain of 30-day-old rats was about one-third of that in the 5-day-old rat. The changes with postnatal age in the incorporation of [¹⁴C]arginine were not statistically significant.