Decarboxylation and Transport of Auxin in Segments of Sunflower and Cabbage Roots. II. A Chromatographic Study Using IAA-1-14C and IAA-5-3H

The polar movement of IAA has been examined in 5-mm root segments of Brassica oleracea and Helianthus annum. The movement was studied partly with IAA-1-¹⁴C and partly with IAA-5-³H. In both plants a slight acropetal flux of ¹⁴C and IAA-³H was found through the segments. The recovered radioactivity in the agar receiver blocks and in the receiver end of the segments increased as a function of time. A large portion of the applied IAA was converted on the cut surfaces and in the tissues of the segments. Chromatographic analysis indicated different destruction products when estimated by scintillation counting and by spraying with in-dole reagent (DMCA). Chromatograms run in isopropanol: ammonia: water, 8:1:1, yielded three different substances, one spot near the starting line and one near the front, neither of which has been identified. Finally there was a spot with R_f 0.4–0.6, probably representing IAA.     

Metabolism of DL-Tryptophan-3-14C by the Fruitlets of Citrus aurantifolia

Developing lime fruit [Citrus aurantifolia (Christm.) Swingle] was supplied with dl-tryptophan-3-¹⁴C in a special medium. An incubation period of six hours was sufficient for the radioactivity to reach an equilibrium between the fruit tissue and the incubation medium. Analyses of the fruit tissue and the medium for acidic and neutral metabolites of tryptophan indicated the existence of indolic products. The auxin indole-3-acetic acid (IAA) was identified among the products by dry column chromatography and biological assay. Other acidic metabolites included indolepyruvic acid and an unidentified material. Neutral metabolites included indolealdehyde, indoleacetaldehyde, and two unidentified compounds. Biological activity in the Avena curvature test was obtained from extracted compounds which corresponded to IAA and indolepyruvic acid in the acidic fraction and indoleacetaldehyde in the neutral fraction. Radioactive tryptophan was also found in both the acidic and the neutral fractions due to its amphoteric nature. The experiment demonstrated the conversion of tryptophan to its indolic metabolites, including indole-3-acetic acid, in this Citrus tissue.     

Characteristics of Root-to-Shoot Transport of Cytokinin 6-Benzylaminopurine in Intact Seedlings of Citrus aurantium

Transport of the cytokinin 6-benzylaminopurine-8-¹⁴C in the root and shoot of intact Citrus aurantium L. seedlings was studied by “replacing” the 0.5 cm root tip with the uptake solution. The cytokinin was transported basipetally in the root and was distributed in an acropetal direction in the stem and into the leaves. Kinetic analysis of the transport for periods of up to 96 h revealed a characteristic advancing front of the label along the axis of the seedling. The estimated velocity of transport of 6-benzylamino-purine-8-¹⁴C in various regions of the intact root was 2.6 to 5.1 mm/h. The transport of 6-benzylaminopurine was predominantly in the transpiration stream, in stelar tissues of the root. Conditions of high transpiration favored enhanced transport to the shoot and an overall greater accumulation of the label. The total accumulation of 6-benzylaminopurine in roots of intact seedlings after 48 h of transport was 354% of that in roots of shoot-less seedlings. Root girdling and treatment of the root with KCN did not reduce the basipetal transport of the label in the root and into the shoot. Radiochromalogram scanning of root extracts and analysis of the ethanol insoluble-NaOH soluble fraction revealed considerable metabolic changes in the translocated cytokinin. Only 51% of the radioactivity remained in the original 6-benzylaminopurine peak after 24 h of incubation. Two other, unidentified, metabolites were detected. It is suggested that all the factors that affect the ascent of sap are involved in the long-distance transport of cytokinins, and that the rate and mode of transport of cytokinins from the root system to the shoot may be a major factor in the expression of their physiological activity.     

Transport of Kinetin-814C in Petioles

Several differences in the translocation pattern of radioactive kinetin in plant petioles were determined. Radioactivity from kinetin-8-¹⁴C (Kn*) moved from donor agar blocks through petioles of bean and cocklebur but not of cotton. There was no difference in basipetal or acropetal movement of radioactivity from Kn* in cocklebnr petioles, but there was in bean petioles. In bean petioles this movement was preferentially basipetal, but it was influenced by the age of the petiole and by the presence of added indoleactic acid. The combination treatment accelerated the basipetal movement of radioactivity from Kn* in young bean petioles and not in old ones. All data is based on radioactivity translocated into receiver agar blocks which were assayed individually in a liquid scintillation spectrometer. The results show that plant species, direction of transport, age of tissue, and presence of IAA can all influence the translocation of Kn* in petioles.     

Cerebral Glucose Utilization: Comparison of [14C]Deoxyglucose and [6-14C]Glucose Quantitative Autoradiography

The [14C]deoxyglucose [Sokoloff et al., J. Neurochem. 28, 897-916 (1977)] and [6-14C]glucose [Hawkins et al., Am. J. Physiol. 248, C170-C176 (1985)] quantitative autoradiographic methods were used to measure regional brain glucose utilization in awake rats. The spatial resolution and qualitative appearance of the autoradiograms were similar. In resting animals, there was no significant difference between the two methods among 18 gray and three white matter structures over a fourfold range in glucose utilization rates (coefficient of correlation = 0.97). In rats given increasing frequencies of photoflash visual stimulation, the two methods gave different results for glucose utilization within visual pathways. The linearity of the metabolic response was studied in the superior colliculus using an on-off checkerboard stimulus between 0 and 33 Hz. The greatest increment in activity occurred between 0 and 4 Hz stimulation with both methods, probably representing recruitment of neuronal elements into activity. Above 4 Hz, there was a progressive increase in labeling with [14C]deoxyglucose up to 1.7 times control at 33 Hz. With [6-14C]-glucose, there was no further increment in change above a 30% increase seen at 4 Hz. Measurement of tissue glucose revealed no drop in the visually stimulated structures compared to control. We interpret these results to indicate that, with increasing rates of physiological activity, the products of deoxyglucose metabolism accumulate progressively, but the products of glucose metabolism are removed from brain in 10 min.     

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.     

STUDIES WITH MURINE LPC-1 PLASMACYTOMA USING [6-14C]ARGININE

[6-¹⁴C]Arginine ([6-¹⁴C]Arg) was used as an in vivo pulse label to study BALB/c murine LPC-1 plasmacytoma synthesis and secretion of its tumour-associated M component (IgG2a, k). With this isotope, an eight- to ten-fold enhancement in the labelling of the γ globulin region and ten-fold reduction in the albumin labelling were observed. Production and secretion of the M component was detected (within 30 min) after cell transfer. Only mice which received tumour cells showed significant labelling in the γ globulin region 24 hr after isotope injection. The labelling behaviour of the tumour M component correlated with the administered cell dose. The peak heights of radioactivity in the γ region increased with increments in cell number. When the percentage radioactivity diverted into M component was plotted as a function of cell dose, a linear relationship was noted. This study demonstrates the feasibility of using [6-¹⁴C]Arg as a tool to follow the newly synthesized tumour-associated protein, and provides a means of estimating tumour cell number.     

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.     

Comparison of Rates of Local Cerebral Glucose Utilization Determined with Deoxy[l-14C]glucose and Deoxy[6-14C]glucose

The activity of the pentose phosphate shunt pathway in brain is thought to be linked to neurotransmitter metabolism, glutathione reduction, and synthetic pathways requiring NADPH. There is currently no method available to assess flux of glucose through the pentose phosphate pathway in localized regions of the brain of conscious animals in vivo. Because metabolites of deoxy[1-14C]glucose are lost from brain when the experimental period of the deoxy[14C]glucose method exceeds 45 min, the possibility was considered that the loss reflected activity of this shunt pathway and that this hexose might be used to assay regional pentose phosphate shunt pathway activity in brain. Decarboxylation of deoxy[1-14C]glucose by brain extracts was detected in vitro, and small quantities of 14C were recovered in the 6-phosphodeoxygluconate fraction when deoxy[14C]glucose metabolites were isolated from freeze-blown brains and separated by HPLC. Local rates of glucose utilization determined with deoxy[1-14C]glucose and deoxy[6-14C]glucose were, however, similar in 20 brain structures at 45, 60, 90, and 120 min after the pulse, indicating that the rate of loss of 14CO2 from deoxy[1-14C]glucose-6-phosphate in normal adult rat brain is too low to permit assay pentose phosphate shunt activity in vivo. Further metabolism of deoxy[1-14]glucose-6-phosphate via this pathway does not interfere during routine use of the deoxyglucose method or explain the progressive decrease in calculated metabolic rate when the experimental period exceeds 45 min.     

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.

     

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.     

Incorporation of label from root-applied N6[8-14C]furfiuryIadenine into the guanine nucleotide fraction of pea bud ribonucleic acid

The pattern of incorporation of label into the nucleotides of axillary bud ribonucleic acid was investigated in Pisum sativum L. cv. Meteor following the application of N ⁶[8-^I4C]furfuryladenine or of [8-¹⁴C]adenine to the root system of decapitated plants and to cultured excised buds. When N ⁶[8-¹⁴C]furifaryladenine was applied to the root system label was confined to the guanine nucleotide moiety of the axillary bud ribonucleic acid; label from [8-¹⁴C]adenine was incorporated preferentially into adenine nucleotide in the molar ratio adenine nucleotide/guanine nucleotide = 3.23. When isolated buds were incubated in media containing [8-¹⁴C]adenine or N ⁶[8-¹⁴C]furfuryladenine, label was incorporated into both purine moieties of the ribonucleic acid. However, the relative incorporation into the guanine nucleotide fraction was considerably greater for N ⁶[8-^I4C]furfuryladenine (adenine nucleotide/guanine nucleotide = 2.23) than for [8-¹⁴C]adenine (ratio = 4.67).
It was concluded that the pattern of metabolism of adenine to guanine and its incorporation into the guanine nucleotide moiety of pea axillary bud ribonucleic acid, is influenced by the presence of a substitution in the N ⁶ position of the adenine base.     

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.     

Effect of Vanadium Pentoxide on the Incorporation of (2-14C)-Acetate into Fungal Lipids

Mycelia of Glomerella cingulata grown in the presence of vanadium pentoxide (V₂O₅), incorporated many times less (2-¹⁴C)-acetate into their lipids than mycelia of the same organism grown in the absence of V₂O₅. All neutral and polar lipid fractions investigated were affected. These data suggested that V₂O₅ depressed lipid biosynthesis in G. cingulata.     

Adenosine A2a Receptor-Mediated Modulation of Striatal [3H]GABA and [3H]Acetylcholine Release

Abstract: The ability of adenosine agonists to modulate K⁺-evoked 4D†-[³H]aminobutyric acid ([³H]GABA) and acetylcholine (ACh) release from rat striatal synaptosomes was investigated. The A_2a receptor-selective agonist CGS 21680 inhibited Ca²⁺-dependent [³H]GABA release evoked by 15 m M KCI with a maximal inhibition of 29 ± 4% (IC₅₀ of ∼4 ± 10 ⁻¹² M ). The relative order of potency of three agonists was CGS 21680 ± 5'- N -ethylcarboxamidoadenosine > R-phenylisopropyladenosine (R-PIA), with the inhibition being blocked by A_2a receptor-selective antagonists (CP 66,713 and CGS 15943A) but not by the A₁-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). When release of [³H]GABA was evoked by 30 mM KCI, no significant inhibition was observed. In contrast, CGS 21680 stimulated the release of [³H]ACh evoked by 30 m M KCI, with a maximal stimulation of 26 ± 5% (IC₅₀ of ∼10⁻¹¹ M ). This effect was blocked by CP 66,713 but not by DPCPX. The A₁ agonist R -PIA inhibited [³H]ACh release, an effect blocked by DPCPX. It is concluded that adenosine A_2a receptors are present on both GABAergic and cholinergic striatal nerve terminals where they inhibit and stimulate transmitter release, respectively. Key Words : GABA—Acetylcholine—Adenosine receptors—Striatum.     

THE EFFECT OF PHOSPHOLIPASE C, PHOSPHOLIPASE A2 AND NEURAMINIDASE ON THE UPTAKE OF [3H]NOREPINEPHRINE AND [3H]SEROTONIN

Abstract— The uptake of [³H]norepinephrine ([³H]NE) and [³H]serotonin ([³H]5-HT) by rat brain synaptosomes is reduced as a result of pretreatment of the synaptosomes with phospholipase C (EC 3.1.4.3) or phospholipase A₂ (EC 3.1.1.4). This effect is not due to inhibition of the Na⁺-K⁺-ATPase but rather is caused by hydrolysis of neuronal membrane phospholipids, mainly phosphatidylcholine, which seem to be important to the uptake.     

Metabolic Fluxes Between [14C]2-Deoxy-D-Glucose and [14C]2-Deoxy-D-Glucose-6-Phosphate in Brain In Vivo

The rates of the phosphorylation and dephosphorylation of 2-deoxyglucose were measured in rat brain in vivo using tracer kinetic techniques. The rate constant for each reaction was estimated from two separate experiments with different protocols for tracer administration. Tracer amounts of [1-14C]2-deoxyglucose (1 microCi) were injected through the internal carotid artery (intraarterial experiment), or through the atrium (intravenous experiment). Brains were sampled by freeze-blowing at various times after the injection. In the intraarterial experiment, the rate constant for the forward reaction from 2-deoxyglucose to 2-deoxyglucose phosphate was calculated by dividing the initial rate of 2-deoxyglucose phosphate production by the 2-deoxyglucose content in brain. The rate constant for the reverse reaction from 2-deoxyglucose phosphate to 2-deoxyglucose was calculated from the decay constant of 2-deoxyglucose phosphate. The rate constants estimated were 10.1 +/- 1.4%/min (SD) and 3.00 +/- 0.01%/min (SD), respectively, for the forward and reverse reactions. In the intravenous experiment, rate constants for both reactions were estimated by compartmental analysis. By fitting data to program SAAM-27, the rate constants for the forward and reverse reactions were estimated as 11.4 +/- 0.4%/min (SD) and 5.1 +/- 0.4%/min (SD), respectively. The rate constants determined were compared to those for the reactions between glucose and glucose-6-phosphate, estimated previously from labeled glucoses. It is concluded that the rate of glucose utilization measured by the 2-deoxyglucose method reflects the rate of the hexokinase reaction and not the rate of glucose utilization or brain energy utilization.     

Effect of Denervation and Reinnervation on Oxidation of [6-14C]Gucose by Rat Skeletal Muscle Homogenates

Abstract: We studied the effects of denervation and reinnervation of the rat extensor digitorum longus muscle (EDL) on the oxidation of [6-¹⁴C]glucose to ¹⁴CO₂. The rate of ¹⁴CO₂ production decreased dramatically following denervation, and the decrease became significant 20 days after nerve section. Prior to day 20, changes apparently reflected the decline of muscle mass. Decreased ¹⁴CO₂ production was due to reduced capacity of the enzymatic system (apparent V_max); there was no change in apparent affinity for glucose (apparent K _m). Mixing experiments revealed that the loss of oxidative capacity following denervation is not caused by production of soluble inhibitors by degenerating muscle. Oxidative metabolism, as measured by ¹⁴CO₂ evolution, recovered during reinnervation. Surprisingly, the specific activity in reinnervated muscles displayed an "overshoot" of approximately 50%, which returned to control by day 60, possibly reflecting increased energy demand by the growing muscle. The time-course of the denervation-mediated change indicates that altered oxidative capacity is secondary to events that initiate denervation changes in muscle. Nevertheless, diminished oxidative capacity may be of considerable metabolic significance in denervated muscle.