The effect of electrical stimulation and ouabain on the uptake and efflux of l-[U-14C]valine in chopped tissue from guinea-pig cerebral cortex

1. Chopped tissue from guinea-pig cerebral cortex carried out an energy-dependent accumulation of l-[(14)C]valine. 2. The uptake was dependent on the extracellular concentration of Na(+) and was markedly inhibited by ouabain (20mum). The extent of the inhibition of uptake by ouabain was also Na(+)-dependent. 3. The accumulation of labelled valine was not directly dependent on the ATP and creatine phosphate contents of the slices. 4. Electrical stimulation increased the rate of [(14)C]valine uptake at first but ultimately led to a net loss of the label so that the amount of label present in the tissue was lower than in the controls. 5. The rate of loss of label during prolonged stimulation was dependent on the extracellular concentration of Na(+). 6. The efflux of labelled valine from slices preloaded at 164mm-Na(+) was studied at 164, 80 and 40mm-Na(+) with and without electrical stimulation or ouabain. 7. Lowering the Na(+) concentration or adding ouabain increased the rate of efflux. 8. Electrical stimulation had little effect on the rate of efflux at first but ultimately led to a more complete loss of label from the tissue than occurred in the control. A kinetic analysis of the efflux curves was attempted.     

The influence of the intracellular concentration on the uptake of l-[14C]valine by chopped tissue from cerebral cortex

1. The intracellular concentrations of sodium, potassium, ATP and creatine phosphate in chopped tissue from guinea-pig cerebral cortex were altered by a 10min period of electrical stimulation. 2. The ability of the tissue to take up l-[(14)C]valine and incorporate it into protein was tested at various times after the cessation of electrical stimulation. 3. The restoration of the ability of the tissue to accumulate l-[(14)C]valine correlated closely with the restoration of the resting intracellular concentrations of sodium and ATP.     

The effect of preganglionic stimulation on the incorporation of l-[U-14C]valine into the protein of the superior cervical ganglion of the guinea pig

The acid glycosaminoglycans were extracted from the skins of young rats less than 1 day post partum. The isolated products were fractionated by a cetylpyridinium chloride-cellulose column technique and identified by chemical analysis, electrophoretic mobility and susceptibility to testicular hyaluronidase digestion. Hyaluronic acid (56%) dermatan sulphate (15.6%) and chondroitin 6-sulphate (9.1%) were the major components, but chondroitin 4-sulphate, heparan sulphate and heparin were also present, together with two further fractions tentatively suggested to be a heparan sulphate-like fraction and a dermatan sulphate fraction, both of short chain length or low degree of sulphation.     

Inhibitory effect of salicylate on the incorporation of l-[U-14C]leucine into the protein of rat tissue preparations in vitro

1. The inhibitory effect of salicylate, in concentrations ranging from 0.1 to 20mm, on the incorporation of radioactivity from l-[U-(14)C]leucine into the protein of isolated rat diaphragm muscle and of cell-free systems from rat liver was studied. 2. The lowest salicylate concentrations producing significant inhibitions of amino acid incorporation were as follows: isolated rat diaphragm, 0.1mm; rat-liver mitochondrial-microsomal system, 0.1mm; rat-liver microsomal system, 0.3mm. 3. Salicylate concentrations of 2.5mm and above were found to inhibit creatine-kinase activity in vitro.     

The effect of methyl palmoxirate on incorporation of [U-14C]palmitate into rat brain

We examined the dose response, time course and reversibility of the effect of methyl 2-tetradecylglycidate (McN-3716, methyl palmoxirate or MEP), an inhibitor of -oxidation of fatty acids, on incorporation of radiolabeled palmitic acid ([U-¹⁴C]PA) from plasma into brain lipids of awake rats. MEP (0.1, 1 and 10 mg/kg) or vehicle was administered intravenously from 10 min to 72 hr prior to infusion of [U-¹⁴C]PA. Two hr pretreatment with MEP (0.1 to 10 mg/kg) increased brain organic radioactivity 1.2 to 1.8 fold and decreased brain aqueous radioactivity by 1.2 to 3.0 fold when compared to control values. At 10 mg/kg, MEP significantly increased brain organic fraction from 40% in controls to 85%, 30 min to 6 hr pretreatment, and resulted in a redistribution of the radiolabeled fatty acid toward triacylglycerol. MEP changed the lipid/aqueous brain ratio of incorporated [U-¹⁴C]PA from 0.67 to 5.7. The incorporation rate coefficient, k^*, was significantly increased by MEP (10 mg/kg) at 2 hr (31%), 4 hr (59%) and 6 hr (34%). All effects were reversed by 72 hr, consistent with a half-life of 2 days for carnitine palmitoyl transferase I. These results indicate that intravenous MEP may be used with [1-¹¹C]palmitic acid for studying brain lipid metabolism in vivo by positron emission tomography, as it significantly reduces the large unincorporated aqueous fraction that would result in high background radioactivity.     

Enzymatic production of the lignin precursor trans-[U-14C]cinnamic acid from l-[U-14C]phenylalanine using l-phenylalanine ammonia-lyase

An enzymatic method using phenylalanine ammonia-lyase (l-phenylalanine ammonia-lyase, EC 4.3.1.5) for the rapid conversion of l-[U-¹⁴C]phenylalanine to the deaminated lignin precursor trans-[U-¹⁴C]cinnamic acid is described. The method produces an experimentally useful ¹⁴C-labelled deaminated lignin precursor unavailable from radiochemical supply companies.     

Anomeric specificity of D-[U-14C]glucose incorporation into glycogen in rat hemidiaphragms

The anomeric specificity of D-[U-14C]glucose incorporation into glycogen in rat hemidiaphragms was investigated. For this purpose, the hemidiaphragms were preincubated for 30 min at 37 degrees C and then incubated for 5 min at the same temperature in the presence of alpha- or beta-D-[U-14C]glucose. The concentrations of D-glucose (5.6 or 8.8 mM) and insulin (0 or 10 mU/ml) were identical during the preincubation and incubation periods. The incubation medium was prepared in D2O/H2O (3:1, v/v) in order to delay the interconversion of the D-glucose anomers. In addition to glycogen labelling, the output of radioactive acidic metabolites was also measured. Insulin caused a preferential stimulation of glycogen labelling relative to glycolysis. Such was not the case in response to a rise in D-glucose concentration. At 5.6 mM D-glucose and whether in the presence or absence of insulin, both glycogen labelling and glycolysis were lower with alpha-D-glucose than with beta-D-glucose suggesting a higher rate of beta-D-glucose than alpha-D-glucose transport across the plasma membrane. A mirror image was found at 8.8 mM D-glucose, especially in the absence of insulin. At this close-to-physiological hexose concentration, insulin lowered the alpha/beta ratio for glycogen labelling. On the contrary, the rise in D-glucose concentration increased such a ratio. Since such a rise is probably little affected by any possible anomeric difference in D-glucose transport across the plasma membrane, the present results strongly suggest that the intracellular factors regulating net glycogen synthesis, as well as glycolytic flux, display obvious preference for alpha-D-glucose.     

[14C]acetylcholine synthesis and [14C]carbon dioxide production from [U-14C]glucose by tissue prisms from human neocortex.

1. [14C]Acetylcholine synthesis and 14CO2 production from [U-14C]glucose has been measured in tissue prism preparations from human neocortex. 2. Electron micrographs of prisms from human and rat neocortex show that both contain intact synaptic endings with evenly-distributed vesicles and normal-appearing mitochondria, but only poorly preserved cell body structure. 3. Synthesis of [14C]acetylcholine in prisms from rat neocortex is similar to estimates for turnover in vivo. Synthesis in prisms from human neocortex is 18% of that in rat tissue and 64% of that in tissue from baboon neocortex for incubations performed in 31 mM-K+. 4. Investigations of prisms prepared from rat brains stored at 37 degrees C after death revealed that synthesis of [14C]acetylcholine in the presence of 31 mM-K+ was greatly decreased within 30 min of post-mortem incubation, whereas synthesis at 5 mM-K+ and production of 14CO2 at both K+ concentrations were only significantly affected after longer periods. Changes were similar in neocortex and striatum. Thus human autopsy material is unlikely to be suitable for use with this system. 5. Investigations using animal models suggest that [14C]acetylcholine synthesis and 14CO2 production are not affected by surgical or anaesthetic procedures. 6. Neither [14C]acetylcholine synthesis nor 14CO2 production in human prisms was significantly changed with age between 15 and 68 years. 7. Samples from patients with the dementing condition Alzheimer's disease showed a significant decrease in [14C]acetylcholine synthesis to 47% of normal samples and a significant increase of 39% in production of 14CO2.     

The effect of pretreatment with pentobarbital on the extent of [14C] incorporation from [U-14C]glucose into various rat brain glycolytic intermediates: relevance to regulation at hexokinase and phosphofrucktokinase

In the present investigation we monitored the incorporation of [¹⁴C] from [U-¹⁴C]glucose into various rat brain glycolytic intermediates of conscious and pentobarbital-anesthetized animals. Labeled glucose was delivered to brain by single bolus intracarotid injection and brain tissue was subsequently prepared at 15, 30 and 45 sec by freeze-blowing. Glycolytic intermediates were then separated by column chromatography. Our results showed a gradual decrease with time of¹⁴C-labeled glucose which gave a calculated rate for glucose metabolism of 0.86 mol/min/g and 0.56 mol/min/g in conscious and anesthetized animals, respectively. Compared to the results obtained using conscious animals the administration of pentobarbital not only resulted in a significant attenuation of the rate of glucose metabolism but also caused a similar reduction in the amount of¹⁴C incorporated into several glycolytic intermediates. These intermediates included: glucose 6-phosphate, fructose 6-phosphate, fructose, 1,6 diphosphate, dihydroxyacetone phosphate and post glycolytic compounds. In addition, pretreatment with pentobarbital resulted in a 75% increase in the endogenous concentration of glucose, 10% increase in glucose 6-phosphate, no change in fructose 6-phosphate and 42% decrease in lactate compared to levels in brains obtained from conscious animals. These results are discussed in relation to control of glycolysis through coupled regulation at hexokinase-phosphofructokinase.     

Decreased incorporation of L-[U-14C]serine into phosphatidylserine by polymorphonuclear leukocytes during phagocytosis

A decreased rate of L-[U-14C]serine incoroporation into phosphatidylserine of polymorphonuclear leukocytes exposed to starch granules was observed. L-[U-14C]serine uptake was also depressed under identical conditions. The degree of reduction in specific radioactivity of phosphatidylserine was parallel to that of L-[U-14C]serine uptake. Both uptake and efflux of 45Ca2+ were enhanced in cells with starch granules, but no significant change in cellular calcium levels was detected. These results suggest that the reduced L-[U-14C]serine incorporation into phospholipids may be attributable to decreased availability of this amino acid. The involvement of Ca2+ fluxes in phosphatidylserine synthesis in intact leukocytes cannot, however, be excluded.     

Studies on the incorporation of [U-14C]glucose and [35S]sulphate into the acid glycosaminoglycans of neonatal rat skin

1. The incorporation of [(35)S]sulphate in vivo into the acid-soluble intermediates extracted from young rat skin showed three sulphated hexosamine-containing components. 2. The rates of synthesis of these components were determined in vivo by measuring the incorporation of radioactivity from [U-(14)C]glucose into their isolated hexosamine moieties. 3. The incorporation of radioactivity from [U-(14)C]glucose into the isolated hexosamine and uronic acid moieties of the acid glycosaminoglycans was also measured. These results, combined with those obtained on the intermediary pathways of hexosamine and uronic acid biosynthesis previously determined in this tissue, indicated that the acid-soluble sulphated hexosamine-containing components were not precursors of the sulphated hexosamine found in the acid glycosaminoglycans. 4. The rates of synthesis of the acid glycosaminoglycan fractions were calculated from the incorporation of radioactivity from [U-(14)C]glucose into the hexosamine moiety. The sulphated components containing principally dermatan sulphate, chondroitin 6-sulphate and in smaller amounts, chondroitin 4-sulphate, heparan sulphate and heparin appeared to be turning over about twice as rapidly as hyaluronic acid and about four times as rapidly as the small keratan sulphate fraction. The relative rates of synthesis of the sulphated glycosaminoglycans were calculated from the incorporation of [(35)S]sulphate and were in agreement with those from (14)C-labelling studies.     

The incorporation of [14C] lysine into the protein of the guinea pig central auditory system

The incorporation of [¹⁴C]lysine into various brain proteins was studied. The proteins of different areas of the auditory system and cortical subcellular fractions were analysed using a disc electrophoretic technique that allows both protein and radioactivity assays along the gels. The highest level of incorporation was found in the mid brain nuclei, particularly the inferior colliculus, and was lowest in the auditory cortex proteins. This was true for both saline soluble proteins and proteins solubilized by Triton X-100 treatment. Of the subcellular fractions, the highest level of activity was found in the microsomal fraction. Considerable radioactivity was also found in the proteins isolated from the synaptosome-rich fraction. Of particular interest in this fraction was a slow migrating protein band which was soluble in Triton X-100, had a high specific activity, and appeared to be synaptosome specific. These observations are in concurrence with the hypothesis that the nerve ending contains protein synthesizing machinery.