THE INCORPORATION OF [1-14C]LINOLENATE INTO LIPIDS OF DEVELOPING RAT BRAIN DURING ESSENTIAL FATTY ACID DEPRIVATION

Abstract— Twenty-one-day old essential fatty acid (EFA) deprived rats incorporated about twice the radioactivity from [1-¹⁴C]linolenate into brain lipid fractions as did controls. At 5 min after injection, 2/3 of the radioactivity was associated with the less polar lipid fraction of both control and EFA deprived animals. By 30 min after injection, 70% of the radioactivity was in the phospholipid fraction. This value increased to 90% at later time points.
The specific activity of brain phospholipids from EFA deprived rats was always greater than that of controls. This held true for the individual phosphatide fractions also. In general, phosphatidylcholine (PC) was labeled most rapidly. With increasing time intervals, radioactivity was transferred to phospha-tidylethanolamine (PE) and phosphatidylserine + phosphatidylinositol (PS + PI).
The transfer of fatty acid radioactivity into phospholipid and the distribution of radioactivity among individual phosphatides did not appear to be affected by the dietary state. However, the total amount of radioactivity incorporated was related to the amount initially retained by brain after injection. Our data suggest that one or more components of the less polar lipid fraction may act as a 'trap' or reservoir for fatty acids which are required for phospholipid synthesis.     

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.     

UPTAKE AND RELEASE OF EXOGENOUS [1-14C]ACETYLCHOLINE BY BRAIN CORTEX SLICES FROM THE RAT

Abstract— Radioactive acetylcholine ([¹⁴C]ACh) that is taken up by rat cerebral cortex slices, incubated aerobically in a physiological saline-glucose paraoxon-[¹⁴C]ACh medium, apparently by a passive diffusion process at concentrations > 1 mm consists essentially of two forms, a readily exchangeable and releaseable or mobile form, and a bound or retained form, poorly (or not) exchangeable. The quantity of retained ACh consists of a considerable fraction of that taken up amounting to 54% with external 0.1 mm -[¹⁴C]ACh and about constant, 27%, for the range 5-50mm -[¹⁴C]ACh. All its ACh is released on homogenization with 0.1 n -perchloric acid or on tissue disintegration in distilled water. The cerebral uptake of ACh differs basically from that of urea as there is no retention of the latter following its uptake. Cerebral cortex slices are superior to those of cerebellar cortex, subcortical white matter, kidney cortex, liver and spleen in taking up and retaining [¹⁴C]ACh. Deprivation in the incubation media of glucose or Na⁺ or Ca²⁺. or the presence of dinitrophenol, whilst causing little change in ACh uptake, induces considerable changes in swelling and ACh retention; the greater the amount of swelling the smaller is that of retention. It seems that the latter is segregated in compartments characterized by a low permeability to exogenous ACh. About half of it is independent of changes in incubation conditions whilst the other half enters the compartment by an Na⁺, Ca²⁺ and energy-dependent process. At least part of the retention is neuronal as it is diminished by protovera-trine, the diminution being blocked by tetrodotoxin. Mobile ACh (i.e. total uptake minus retained ACh) is largely unaffected by protoveratrine, ouabain, etc. It seems that the retained ACh is directly proportional to the amount of mobile ACh minus the amount that enters with swelling. If the latter is largely glial in location, then the retained ACh is simply proportional to the mobile neuronal ACh. Suggestions are made as to the location of the retained ACh in the brain cells and to the processes involved in its segregation there. Release of retained ACh occurs on change of the Na⁺ gradient. Atropine and d-tubocurarine also diminish the amount of retained ACh but the percentage diminution falls with increase of the concentration of exogenous ACh.     

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.     

METABOLISM OF γ-HYDROXY-[1-14C] BUTYRATE BY RAT BRAIN: RELATIONSHIP TO THE KREBS CYCLE AND METABOLIC COMPARTMENTATION OF AMINO ACIDS

Abstract— Ninhydrin decarboxylation experiments were carried out on the labelled amino acids produced following intraventricular injection of either γ-hydroxy-[1-¹⁴C]butyric acid (GHB) or [1-¹⁴C] succinate. The loss of isotope (as ¹⁴CO₂) was similar for both substances. The [1-¹⁴C]GHB metabolites lost 75% of the label and the [1-¹⁴C] succinate metabolites lost 68%. This observation gives support to the hypothesis that the rat brain has the enzymatic capacity to metabolize [1-¹⁴C]GHB to succinate and to amino acids that have the isotope in the carboxylic acid group adjacent to the a-amino group. These results also indicate that the label from [1-¹⁴C]GHB does not enter the Krebs cycle as acetate. The specific activity ratio of radiolabelled glutamine to glutamic acid was determined in order to evaluate which of the two major metabolic compartments preferentially metabolize GHB. It was found that for [1-¹⁴C]GHB this ratio was 4.20 ± 0.18 (S.E. for n = 7) and for [l-¹⁴C]succinate this ratio was 7.71 (average of two trials, 7.74 and 7.69). These results suggest that the compartment thought to be associated with glial cells and synaptosomal structures is largely responsible for the metabolism of GHB. Metabolism as it might relate to the neuropharmacological action of GHB is discussed.     

THE EFFECTS OF CARBAMYLCHOLINE ON INCORPORATION IN VIVO OF [1-14C]ARACHIDONIC ACID INTO GLYCEROLIPIDS OF MOUSE BRAIN

Abstract— The effects of carbamylcholine on incorporation of [1-¹⁴C]arachidonate into the glycerolipids in mouse brain synaptosome-rich and microsomal fractions were examined at 1, 3 and 10 min after intracerebral injection of the labeled precursor. When carbamylcholine was included with the labeled arachidonate, there was a decrease in the proportion of labeled fatty acid incorporated into the phospholipids. Among the phospholipids in the synaptosome-rich fraction, a decrease in incorporation of radioactivity into diacyl-glycerophosphoinositols and diacyl-glycerophosphocholines was observed at 1 and 3 min after injection. A decrease in labeling of diacyl-glycerophosphoethanolamines and diacyl-glycerophosphocholines in the microsomal fraction was observed at 3 and 10 min after injection. The decrease in phospholipid labeling was marked by an increase in labeling of diacylglycerols which was observed initially in the synaptosome-rich fraction, but also in the microsomal fraction at later time periods. Other lipid changes included an increase in triacylglycerol labeling which was found in the synaptosome-rich fraction and an increase in phosphatidic acid labeling which was found in the microsomal fraction. Results of the in vivo study have demonstrated changes in brain lipid metabolism during carbamylcholine stimulation. Furthermore, these changes appear to be initiated mainly in the synaptosome-rich fraction.     

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.     

14C-LABELLED AMINO ACIDS AND GLUCOSE IN RAT BRAIN AND LIVER AFTER INJECTION OF [2-14C]PROPIONATE

Abstract— [2-¹⁴C]Propionate injected into rats was metabolized into [¹⁴C]glucose and ¹⁴C-labelled aspartate, glutamate, glutamine and alanine. The results are consistent with the conversion of propionate into succinate and the oxidation of succinate into oxaloacetate, the precursor of labelled amino acids and the substrate for gluconeogenesis.
The ratio of the specific radioactivity of glutamine to glutamate was greater than 1 during the 30 min period in the brain, indicating that propionate taken up by the brain was metabolized mainly in the 'small glutamate compartment' in the brain. The results, therefore, support the previous conclusion (G aitonde , 1975) that the labelling of amino acids by [¹⁴C]propionate formed from [U-¹⁴C>]-threonine in thiamin-deficient rats was metabolized in the 'large glutamate compartment' of the brain.
The specific radioactivity ratio of glutamine to glutamate in the liver was less than 1 during the 10 min period but greater than 1 at 30min. These findings which gave evidence against metabolic compartments of glutamate in the liver, were interpreted as indicative of the entry of blood-borne [¹⁴C]glutamine synthesized in other tissues, e.g. brain. The labelling of amino acids when compared to that after injection of [U-¹⁴C]glucose showed that [2-¹⁴C]propionate was quantitatively a better source of amino acids in the liver. The concentration of some amino acids in the brain and liver was less in the adult than in the young rats, except for alanine and glutathione, where the liver content was more than double that in the adult.     

Effects of drought on [1-14C]-oleic and [1-14C]-linoleic acid desaturation in cotton leaves

The effects of water stress on [1-¹⁴C]-oleic and [1-¹⁴C]-linoleic acid desaturations were studied in leaves of two varieties of cotton ( Gossypium hirsutum L.), one drought-sensitive (Reba) and the other more resistant (Mocosinho). After 24 h incorporation, [1-¹⁴C]-oleate led to the appearance of linoleate in phospholipids and, additionally, of linolenate in galactolipids. [1-¹⁴C]-Linoleate was desaturated to linolenate only in galactolipid fractions. Water stress markedly inhibited the incorporation of the precursors into the leaf lipids. The two desaturation steps were affected, particularly the transformation of linoleate to linolenate in monogalactosyldiacylglycerol in the drought-sensitive variety of cotton. The metabolic implications of the inhibition of the biosynthesis of C₁₈-polyunsaturated fatty acids are discussed.     

SIMULTANEOUS INCORPORATION OF ORALLY ADMINISTERED [9, 10-3H2]OLEIC AND [1-14C]LINOLEIC ACIDS INTO LIPIDS OF THE ADULT RAT BRAIN

—The incorporation of an orally administered mixture of [9,10-³H₂joleic acid and [1-¹⁴C]linoleic acid into the brain and spinal cord lipids was maximal after 24 h compared with 4 h for extraneural tissue. In the latter, both acids were utilized equally well for triglyceride biosynthesis, but linoleate entered phosphatidylcholine more rapidly than oleate. Oleic acid was preferentially incorporated into newly synthesized cholesterol esters although 4 h after dosing most cholesterol esters present in serum were formed preferentially from linoleate presumably by the action of lecithin-cholesterol acyl transferase. In neural tissue, a considerable amount of [1-¹⁴C]linoleate was metabolized to higher polyunsaturated fatty acids, whereas in the case of oleate, 90 per cent of the tritium activity remained in monoenic acids at all time periods studied. Both acids were initially incorporated most rapidly into the lecithin fraction of brain and spinal cord, but after 7 days diacyl phosphatidylethanolamine had the highest specific activity. These data are consistent with the view that the uptake of labelled fatty acids by the brain takes place principally as free acids but that some uptake of esterified forms, probably largely as phosphatidylcholine, also occurs. The low linoleate content of the brain and probably also of cerebrospinal fluid cannot be explained on the basis of a selective restriction on the uptake of this lipid from plasma.     

DECARBOXYLATION STUDIES OF GLUTAMATE, GLUTAMINE, AND ASPARTATE FROM BRAIN LABELLED WITH [1-14C] ACETATE, l-[U-14C]-ASPARTATE, AND l-[U-14C]GLUTAMATE

Studies in vivo and in vitro of the distribution of label in C-1 of glutamate and glutamine and C-4 of aspartate in the free amino acids of brain were carried out. [1-¹⁴C]-Acetate was used both in vivo and in vitro and l -[U-¹⁴C]aspartate and l -[U-¹⁴C]glutamate were used in vitro.

• 1 The results obtained with labelled acetate and aspartate suggest that CO₂ and a 3-carbon acid may exchange at different rates on a CO_a-fixing enzyme.
• 2 The apparent cycling times of both glutamate and glutamine show fast components measured in minutes and slow components measured in hours.
• 3 With [1-¹⁴C]acetate in vitro glutamine is more rapidly labelled in C-1 than is glutamate at early time points; the curves cross over at about 7 min.
• 4 The results support and extend the concept of metabolic compartmentation of amino acid metabolism in brain.

     

ACETYLCHOLINE SYNTHESIS FROM [2-14C]PYRUVATE IN RAT STRIATAL SLICES

Abstract— Rat striatal slices were incubated with [2-¹⁴C]pyruvate or [6-¹⁴C]glucose as sole carbon source. The method devised to study the accumulation of labelled ACh in tissues and incubating medium in the presence or absence of eserine 200 μM derived from the previous studies of FONNUM (1969) and H emsworth and M orris (1964). Total ACh was estimated by biological assay.
The specific activity of newly synthesized ACh was found to be equal to that of the precursors, even for short incubation times and low substrates concentrations. When slices were incubated with [2-¹⁴C]pyruvate and eserine, the spontaneous release of ACh occurred at a constant rate, was not modified by the addition of 2 mM-choline in the medium, and consisted only of newly synthesized transmitter.
The initial rate of ACh synthesis was found to be independent of choline concentration, but dependent on the [2-¹⁴C]pyruvate concentration, and reached a maximal value corresponding to about 5 per cent of the measured striatal choline acetyltransferase activity.
The appearance of the so called 'surplus ACh' pool, obtained in the presence of eserine, could be detected only after 30 min and represented 26 per cent of the total tissue ACh content after 180 min of incubation.
In the absence of eserine, tissue ACh levels increased six-fold in 80 min and then remained stable until the end of the incubation period (180 min), if sufficient substrate was provided. The maximal ACh accumulation in slices was independent of both excess of choline and [2-¹⁴C]pyruvate.
The 'ACh plateau' represented the attainment of a new dynamic equilibrium, since ACh synthesis could still be stimulated by 30 mM-K⁺. From these results, it was concluded that ACh synthesis is controlled by a negative feed-back regulation.     

COMPARTMENTATION AND LABELLING OF AMINO ACIDS IN THE DEVELOPING RAT BRAIN AFTER INJECTION OF [U-14C]RIBOSE

Abstract— [U-¹⁴C]Ribose was given by subcutaneous injection to young rats aged 2–56 days. During the first week after birth ¹⁴C in the brain was found mainly combined in glucose, fructose and sedoheptulose which contained 46–57 per cent of the ¹⁴C in the acid soluble metabolites in the rat brain. In contrast, during the critical period (10–15 days after birth) the ¹⁴C in the free sugars decreased from 24 to 3 per cent, while the ¹⁴C content of amino acids in the brain increased from 11 to 44 per cent of the total perchloric acid-soluble ¹⁴C. The increase in labelling of amino acids during the critical period was attributed to increased glycolysis and increased oxidation of pyruvate. The relative specific radioactivity of y -aminobutyrate and aspartate in the rat brain at 28 days after birth was equal to or greater than the relative specific radioactivity of glutamate. Assuming that the increase in amino acid content following the cessation of cell proliferation in the brain is located mainly in cell processes (cytoplasm of axons, dendrites, glial processes and nerve terminals), tentative values were estimated for the pool sizes of glutamate, glutamine, aspartate and y -amino butyrate.     

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.     

THE INCORPORATION OF [1-14C]ACETATE INTO UNESTERIFIED FATTY ACIDS IN RAT CEREBRAL CORTEX IN VIVO

—The incorporation of [1-¹⁴C]acetate into unesterified fatty acids and into the fatty acids of neutral glycerides and of phospholipids has been measured in rat cerebral cortex in vivo. The most rapid incorporation is seen in the unesterified fatty acids which have a turnover time of 5-6 min. It is suggested that unesterified fatty acids are precursors to neutral glycerides and phospholipids rather than being derived from them by lipase activity.     

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.