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.     

CONVERSION OF [1-14C]PALMITIC ACID TO [1-14C]HEXADECANOL BY DEVELOPING RAT BRAIN CELL-FREE PREPARATIONS

—The conversion of [l-¹⁴C]palmitic acid to [1-¹⁴C]hexadecanol has been demonstrated with a cell-free system from developing rat brain. ATP, Coenzyme A and Mg²⁺ were required for the activity. Fatty aldehyde was found to be an intermediate in this reaction. The conversion of fatty acid to fatty alcohol was mainly localized in the microsomal fraction and the formation of hexadecanol showed absolute specificity towards NADPH while fatty aldehyde was formed even in the absence of exogenous reduced pyridine nucleotides. The brain microsomes showed maximal activity with stearic acid and the activities with palmitic and oleic acids were 65% and 38% respectively of that with stearic acid. This enzymic reduction increased with age and showed a maximum in the 15-day old rat brain.     

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.     

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.     

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.

     

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.     

dl-[2-14C]p-CHLOROPHENYLALANINE AS AN INHIBITOR OF TRYPTOPHAN 5-HYDROXYLASE

The distribution in vivo of dl -[2-¹⁴C]p-chlorophenylalanine (p-CP) in regions and subcellular fractions of the rat brain was determined. The half-lives of p-CP and its metabolite p-chlorophenylpyruvic acid (p-CPPA) in plasma and brain were correlated with the development of inhibition of cerebral tryptophan 5-hydroxylase (EC 1.99.1.4). There was active transamination in vivo of p-CP and p-CPPA in the brain. Transport of indolealkylamino acids into brain was impaired by p-CP. Inhibition of tryptophan 5-hydroxylase could not be reversed by administration of large doses of l -tryptophan, l -tyrosine, or l -phenylalanine. After administration of [2-¹⁴C]p-CP in vivo, appreciable radioactivity was bound to cerebral proteins, including those with tryptophan 5-hydroxylase activity, as well as to phenylalanine 4-hydroxylase (EC 1.99.1.2) purified from liver. Amino acid analysis of the acid hydrolysate of purified, radioactive hepatic phenylalanine 4-hydroxylase showed over 80 per cent of the radioactivity to be present as p-CP. Neither the inhibition in vivo nor in vitro of tryptophan 5-hydroxylase could be reversed by dialysis; in controls, dialysis resulted in marked loss of enzyme activity. After incubation for 5 min with p-CP in vitro, enzymic activity was inhibited 60 per cent. In vitro, p-CPPA labelled protein much more extensively than p-CP, yet inhibited the enzyme less. Some of the label from p-CPPA was removable by dialysis.     

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.     

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.     

Comparative Basipetal Transport of 6-Benzylaminopurine-8-14C, Gibberellin A3-3H, IAA-2-14C, and Sucrose-14C in the Root of Intact Citrus aurantium Seedlings

The transport and distribution of IAA-2-¹⁴C, gibberellin A₃-³H, 6-benzylaminopurine-8-¹⁴C and sucrose-¹⁴C (U) were studied in whole seedlings of Citrus aurantium L. after “replacing” the root tip with the solution of radiochemicals. All four substances were transported basipetally in the root and were distributed to the stem and leaves. The pattern of distribution of the label from 6-benzylaminopurine was similar to that of sucrose, while a considerably larger amount of gibberellin A₃ was transported to basal regions of the root, away from the tip, and into the shoot. Contrary to these three substances, the basipetal transport of IAA in the root was very low, and the majority of the label was retained in the terminal sections of the root. It is suggested that the different efficiencies at which various hormones move in the transpiration stream in the root may be an important factor in the attainment of a certain balance of hormones in the shoot.