Lumiflavin and Lumichrome Transport in the Central Nervous System

Abstract: The transport of the lipid-soluble sugarless flavins, [¹⁴C]lumiflavin and [¹⁴C]lumichrome, into and from the isolated choroid plexus and brain slices was studied in vitro. The isolated choroid plexus accumulated both [¹⁴C] flavins by a saturable, energy-requiring process that did not depend on binding or intracellular metabolism of the [¹⁴C] flavins. Both sugar-containing and sugarless flavins, as well as cyclic organic acids, significantly inhibited [¹⁴C]lumiflavin and [¹⁴C]Iumichrome uptake by the isolated choroid plexus. Within 2.5 min, 75% of the [¹⁴C]lumiflavin accumulated by the isolated choroid plexus was released into the medium. Brain slices accumulated [¹⁴C]lumiflavin by a saturable process that did not meet all the criteria for active transport. Ninety-five percent of the [¹⁴C]lumiflavin accumulated by brain slices was released into the medium within 7.5 min. In vivo , 2 h after the intraventricular injection of 6.5 nmol [¹⁴C]lumiflavin, almost all of the [¹⁴C]flavin was cleared from the CNS. Addition of 3.5 μmol FMN to the intraventricular injectate significantly decreased the clearance of [¹⁴C]lumiflavin from the CNS. These studies document that the sugarless flavins are transported by the flavin transport systems in the CNS.     

Origin and dynamics of indoleacetic acid under polar transport in Pinus sylvestris

Polarly‐transported IAA is regarded as a long distance correlative signal important in many aspects of plant physiology, including, for example, apical dominance and growth and development of vascular tissues. In this study, we investigated the importance of apical sources in supplying stem tissues with IAA. The current‐yearshoots of 4‐year‐old Pinus sylvestris L. saplings were replaced with a source of [¹³C₆]IAA. Subsequent mass spectrometric analysis showed that most of the IAA present at two positions in the subjacent 1‐year‐old internode consisted of [¹³C₆]IAA, while [¹²C]IAA of endogenous origin formed a minor pool. However, the pool of [¹³C₆]IAA decreased from 90 to 80% of the total free IAA pool [¹³C₆]IAA+[¹²C]IAA) while being transported down the shoot. This dilution with [¹²C]IAA indicates that de novo biosynthesis of IAA occurred. An additional defoliation experiment showed that the synthesis took place in stem tissues rather than in the mature leaves. The results confirm the role of apical shoots as the major source of polarly‐transported IAA, but also indicate that synthesis of IAA takes place in stem tissues. This is important when considering IAA balance at the whole plant level.     

Characterization of l-Arginine and Aminoguanidine Uptake into Isolated Rat Choroid Plexus: Differences in Uptake Mechanisms and Inhibition by Nitric Oxide Synthase Inhibitors

Abstract: Recent reports suggest that nitric oxide (NO) may contribute to several neurodegenerative diseases, e.g., focal cerebral ischemia, N -methyl- d -aspartate-mediated neurotoxicity, and experimental autoimmune encephalomyelitis. Accordingly, an understanding of the CNS transport processes of NO synthase (NOS) inhibitors has important therapeutic implications. The objective of the present study was to characterize the in vitro transport processes governing the uptake of l -[¹⁴C]arginine and the NOS inhibitor [¹⁴C]aminoguanidine in rat choroid plexus tissue. Consistent with previous reports, the uptake of l -[¹⁴C]arginine was mediated by both saturable and nonsaturable processes and was inhibited by the NOS inhibitors N ^G-methyl- l -arginine, N ^G-amino- l -arginine, and N ⁵-imidoethyl- l -ornithine. l -[¹⁴C]Arginine uptake was not inhibited by aminoguanidine or N ^G-nitro- l -arginine. Because aminoguanidine is an organic cation that bears some structural similarity to l -arginine, aminoguanidine might be transported by either an organic cation transporter or by the basic amino acid transporter governing arginine uptake. However, there was no evidence of a saturable uptake process for [¹⁴C]aminoguanidine in isolated rat choroid plexus, in contrast to that observed for l -[¹⁴C]arginine.     

NEUROTRANSMITTER UPTAKE INTO SLICES OF RAT CEREBRAL CORTEX IN VITRO: EFFECT OF SLICE SIZE

Abstract— The uptake of [¹⁴C]GABA, [¹⁴C]taurine, [³H] β -alanine and [¹⁴C]dopamine was compared in slices of rat cerebral cortex of three different sizes (0.1 × 0.1 × 2 mm, 0.2 × 0.2 × 2 mm and 0.4 × 0.4 × 2 mm prepared with a mechanical tissue chopper). [¹⁴C]Taurine and [³H] β -alanine uptake increased whereas [¹⁴C]GABA uptake decreased with increasing slice size. [¹⁴C]Dopamine uptake was optimal in 0.2 × 0.2 × 2 mm slices. Increasing slice size was shown to decrease inhibition of [³H] β -alanine and [¹⁴C]GABA uptake by l -2,4-diaminobutyric acid. Lactate dehydrogenase activity increased with increasing slice size indicating decreased tissue damage or increased cellular integrity. The possibility that varying slice size can be used to distinguish between neuronal and glial uptake is discussed. It is suggested that taurine uptake in the cerebral cortex is predominantly glial.     

SYNTHESIS AND RELEASE OF [14C]ACETYLCH0LINE IN SYNAPTOSOMES

Abstract— Synaptosomes took up [¹⁴C]choline, about half or more of which was converted to [^I4C]acetylcholine when incubated in an appropriate medium containing 1 to 5 μ M-[¹⁴C] choline and neostigmine. The amount of [¹⁴C]acetylcholine synthesized in synaptosomes increased in parallel with the increase of Na⁺ concentration in the incubation medium. The effect of Na⁺ on the uptake of [^I4C]choline into synaptosomes was dependent on the concentration of choline in the incubation medium.
About 25 per cent of [¹⁴C]acetylcholine synthesized in synaptosomes was released rapidly into the medium by increasing the K⁺ concentration in the medium from 5 m m to 35 m m . The change of Na⁺ concentration hardly affected the release of [¹⁴C]acetylcholine. The effect of K⁺ on the release of [¹⁴C]choline was rather small compared to that on [¹⁴C] acetylcholine. Ouabain promoted the release of [¹⁴C]acetylcholine.     

Ascorbate Transport and Intracellular Concentration in Cerebral Astrocytes

Abstract: Regulation of the initial rate of uptake and steady-state concentration of ascorbate (reduced vitamin C) was investigated in rat cerebral astrocytes. Although these cells did not synthesize vitamin C, they accumulated millimolar concentrations of ascorbate when incubated with medium containing the vitamin at a level (200 µ M ) typical of brain extracellular fluid. Initial rate of [¹⁴C]-ascorbate uptake and intracellular ascorbate concentration were dependent on extracellular Na⁺ and sensitive to the anion transport inhibitor sulfinpyrazone. Comparison of the efflux profiles of ascorbate and 2',7'-bis(carboxyethyl)-5 (or -6)-carboxyfluorescein from astrocytes permeabilized with digitonin localized most intracellular ascorbate to the cytosol. Pretreatment of astrocytes with dibutyryl cyclic AMP (dBcAMP) doubled their initial rate of sulfinpyrazone-sensitive [¹⁴C]ascorbate uptake compared with cells treated with either n -butyric acid or vehicle. dBcAMP also increased steady-state intracellular ascorbate concentration by 39%. The relatively small size of the change in astrocytic ascorbate concentration was explained by the finding that dBcAMP increased the rate of efflux of the vitamin from ascorbate-loaded cells. These results indicate that uptake and efflux pathways are stimulated by cyclic AMP-dependent mechanisms and that they regulate the cytosolic concentration of ascorbate in astrocytes.     

REDISTRIBUTION OF ADENINE DERIVATIVES AMONG SUBCELLULAR FRACTIONS FROM GUINEA PIG NEOCORTICAL TISSUES, ON INCUBATION IN VITRO

Abstract— After the brief in vitro exposure of guinea-pig neocortical tissue to [¹⁴C]adenine, synaptosomal fractions prepared from the incubated tissue contained about 6% of its retained ¹⁴C. On continued incubation and superfusion with or without stimulation, the synaptosomal proportion of the ¹⁴C increased, while the protein and K content of the fraction underwent smaller changes only. Colchicine, 0.5 m m , diminished the synaptosomal enrichment in [¹⁴C]adenine derivatives and also in some cases increased the ¹⁴C effluent from tissues to superfusates. Colchicine also diminished the uptake of adenosine, but not of adenine, to the neocortical tissues. It is concluded that nerve terminal regions receive adenine derivatives from other tissue components as part of their normal metabolism, and that much of this can arrive by extracellular fluids; transport cytoplasmically is not excluded.     

Cyanate is transported by the nitrate permease in Azotobacter chroococcum

Abstract Azotobacter chroococcum cells exhibiting the capacity to take up nitrate actively could transport [¹⁴C]cyanate. This activity was dependent on the nitrogen source present in the culture medium, ammonium acting as a repressor and nitrate as an inducer. The uptake of cyanate required metabolic energy and was absent from A. Chroococcum TR1, a mutant strain lacking the nitrate transport system, but was present at wild-type levels in A. chroococcum E4, a mutant strain deficient in nitrate reductase. These results show that cyanate is transported by the nitrate permease in A. chroococcum and therefore [¹⁴C]cyanate may be useful as a nitrate analogue for studies on nitrate transport.     

Phosphatidylcholine molecular species involved in Γ-linolenic acid biosynthesis in microsomes from borage seeds

Boraginaceae seeds are particularly rich in Γ -linolenic acid (6,9,12-octadecatrienoic acid, Γ -18:3). In microsomes, the analysis of phosphatidylcholine (PC) molecular species by HPLC led to identification of 15 different molecular species; among them 4 contained Γ -18:3, mostly at position 2 of sn -glycerol. Time courses of acylation and desaturation in PC molecular species were examined when [¹⁴C]oleoyl-CoA or [¹⁴C]linoleoyl-CoA was provided as substrates to isolated microsomes. With [¹⁴C]oleoyl-CoA or [¹⁴C]linoleoyl-CoA and in the absence of NADH, 3 main labelled PC molecular species were found: 18:2/[¹⁴C]18:1, 16:0/[¹⁴C]18:1 and 18:1/[¹⁴C]18:1. When NADH was present in the incubation medium, the fatty acids were progressively desaturated by the Δ12- and Δ6-desaturases successively (with [¹⁴C]oleoyl-CoA as precursor) or by the Δ6-desaturase alone (with [¹⁴C]linoleoyl-CoA as precursor). In both types of experiments, 7 final desaturation products in microsomes were evidenced; among them, 3 contained radioactive Γ -18:3, i.e . 18:2/[¹⁴C] Γ -18:3, 18:1/[¹⁴C] Γ -18:3 and 16:0/[¹⁴C] Γ -18:3. While the Δ12-desaturase had no specificity for position on the glycerol backbone, labelled Γ -linolenic acid was recovered exclusively in the sn -2 position.     

Comparison of DABA and GABA Transport into Plasma Membrane Vesicles Derived from Synaptosomes

Abstract: Transport of GABA by a high-affinity transport system ( K _m≃ 10⁻⁵ M) is thought to terminate the action of this postulated neurotransmitter. 2,4-Diaminobutyric acid (DABA), a structural analogue, is taken up by neuronal elements and inhibits GABA uptake. Localization of [³H]DABA by auto-radiography has been used to identify neurons with the GABA high-affinity transport system. After reconstitution of lysed synaptosomal fractions in potassium salts, transfer of these membrane vesicles to sodium salts produces sodium and potassium ion gradients which drive [³H]GABA and [³H]DABA transport. For each, transport requires external sodium, is abolished by ionophores that dissipate the Na⁺ gradient, and is enhanced by conditions which make the intravesicular electromotive force more negative. Some characteristics of the transport of these substances, however, differ. For example, external chloride is required for GABA, but not DABA, transport. Internal potassium is required for DABA, but not GABA, transport. DABA is a competitive inhibitor ( K _i≃ 0.6 MM) of GABA transport into membrane vesicle and synaptosomes. GABA, however, is a feeble inhibitor of DABA uptake into the membrane vesicles. These differences suggest that the two substances are transported by different mechanisms and possibly by different carriers. In addition to these experiments, using enzymatic-fluorometric techniques, it was shown that the artificially imposed ion gradients drive net chemical transport of GABA into the vesicles.     

Effects of Internal pH on the Acetylcholine Transporter of Synaptic Vesicles

Abstract: Uptake of acetylcholine (ACh) by synaptic vesicles isolated from the electric organ of Torpedo was induced with an artificially imposed proton gradient. The gradient was formed by hyposmotic lysis and resealing of vesicles in a low pH buffer to form vesicular ghosts followed by sudden elevation of the pH of the ghost suspension. [³H]ACh accumulated rapidly, the proton gradient collapsed spontaneously within 5 min as monitored by [¹⁴C]methylamine uptake, and the accumulated ACh leaked out of the ghosts after 5 min. Vesamicol blocked both uptake and efflux of the [³H]ACh, demonstrating that both processes are mediated by the ACh transporter. The protonophore nigericin also blocked uptake very potently. Specific uptake was titrated with variable concentrations of [³H]ACh. It exhibited K _m and V _max values of ∼200–500 µ M and 7–30 nmol [³H]ACh/mg at 5 min, respectively, which are values close to those commonly observed for ATP-dependent uptake by intact vesicles. Specific uptake by ghosts was titrated with variable internal pH and constant external pH. It exhibited maximal uptake between internal pH 4.5 and 5.5. The dependence was very steep and could be fit best by assuming that the active form of the transporter requires protonation of two internal sites of apparent pK value of 5.3 ± 0.2. A similar result was obtained when the uptake was titrated with variable internal pH with a constant thermodynamic driving force maintained by keeping the external pH ∼2.6 units higher. The origin of the transport inhibition that sets in at very low internal pH values is not clear. In vivo, the steep dependence of transport on the transmembrane pH gradient might serve to minimize leakage of ACh from the cytoplasm due to ACh transporter in the plasma membrane.     

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.     

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.     

Pyrimidine deoxyribonucleotide metabolism during maturation and germination of white spruce (Picea glauca) somatic embryos: metabolic fate of C-labelled cytidine, deoxycytidine and thymidine

Pyrimidine deoxyribonucleotide metabolism was investigated during maturation and germination of white spruce somatic embryos by following the metabolic fate of [2‐¹⁴C]cytidine, [2‐¹⁴C]deoxycytidine and [2‐¹⁴C]thymidine. The de-novo pathway of deoxyribonucleotides was estimated indirectly, by the ability of the tissue to incorporate cytidine into DNA after conversion to dCTP. The salvage pathway was estimated by the utilization of labelled cytidine, deoxycytidine and thymidine for synthesis of deoxyribonucleotides and nucleic acids. Utilization of cytidine for DNA synthesis, via the de novo pathway, was always lower than that observed for RNA throughout the course of the experiment. Incorporation of cytidine into RNA was found to occur either directly, after conversion to CTP, mediated by the enzymes cytidine kinase, nucleoside monophosphate kinase and nucleoside diphosphate kinase, or indirectly, after conversion to UTP via uridine and UMP. Active incorporation of uridine into RNA of white spruce-cultured cells was demonstrated previously. Salvage of deoxycytidine and thymidine was operative in maturing and germinating white spruce somatic embryos, as label from both compounds was recovered in nucleotides and DNA. However, the utilization of these precursors by the cells was different. Salvage of deoxycytidine was always higher than that observed for thymidine, which was extensively catabolized to CO₂ at all stages of embryo development.     

THE RELEASE OF AMINO ACIDS FROM THE HEMISECTED SPINAL CORD DURING STIMULATION

Abstract— Isolated frog or toad hemicords were incubated for 40 min with either [¹⁴C]glycine, [³H]GABA, l -[¹⁴C]glutamate. l -[¹⁴C]aspartate, l -[¹⁴C]serine, l [¹⁴C]threonine or l -[³H]leucine, and the release of these compounds from the cord was measured under resting conditions and during electrical stimulation. Stimulation of spinal roots produced no significant change in the efflux of any of the compounds tested. Direct stimulation of the rostral cord however, produced a large increase in the efflux of [¹⁴C]glycine, [³H]GABA, l -[¹⁴C]glutamate and l -[¹⁴C]aspartate. These increased effluxes were calcium dependent, the effects of stimulation being reduced in a calcium-free, or magnesium-supplemented (10 mM) medium. Stimulation failed to produce an increase in the efflux of l -[¹⁴C]serine, l -[¹⁴C]threonine, l -[¹⁴H]leucine, [¹⁴C]mannitol or [¹⁴C]urea. These results are consistent with the suggestions that glycine, GABA, glutamate and aspartate may be synaptic transmitters in the spinal cord.     

Synthesis and proof-of-function of a [14C]-labelled form of the plant iron chelator nicotianamine using recombinant nicotianamine synthase from barley

The amino acid nicotianamine (NA) is essential for micronutrient metabolism in plants. Lack of NA results in a chlorotic phenotype and oxidative stress, since NA is a chelator of iron and other metal nutrients. To investigate the precise cellular function of NA in micronutrient transport and homeostasis, a protocol for the production of [¹⁴C]-labelled NA was developed. Recombinant NA synthase was used to generate [¹⁴C]-NA from [¹⁴C]- S -adenosylmethionine. After purification by solid-phase ion exchange about 66% yield was achieved. The identity of the [¹⁴C]-NA with chemically synthesized NA was demonstrated by several independent methods, including two TLC systems, two HPLC systems and immuno-detection. Moreover, biological function was shown by complementation of the Lycopersicon esculentum mutant chloronerva that is free of NA due to a defect in NA synthase. Proof-of-function for the produced [¹⁴C]-NA as a suitable tool for transport studies was provided monitoring the distribution of [¹⁴C]-NA after feeding to tomato and Ricinus communis seedlings.     

Quantitative aspects of triacylglycerol metabolism and sterol synthesis from 14C-acetate in etiolated seedlings of Euphorbia lambii

Triacylglycerols occur in both the endosperm and embryo of Euphorbia lambii seeds. Upon germination, the amount of these neutral lipids in the endosperm decreased with 1.06 mg fatty acid day^-1. The embryo contained 1.4 mg fatty acids in the triacylglycerols and this value declined slowly to 0.4 mg seedling^-1 during the 8 day period of endosperm depletion. Radioactive acetate was rapidly taken up by the cotyledons of intact seedlings, translocated throughout the entire seedling, and up to 10.5% of the ¹⁴C proceeded to the sterols and latex triterpenols. Maximum uptake values of 1.4 μmol seedling^-1 day^-1 of acetate were measured. Acetate uptake and subsequent incorporation into sterols and triterpenols decreased substantially in the presence of increasing amounts of sucrose (up to 0.3 M). Traces of acetate did not effect [¹⁴C]-sucrose uptake and corresponding synthesis of [¹⁴C]-sterols and triterpenols, but increased concentrations of acetate (0.05 M and up) reduced both uptake of sucrose and its conversion into unsaponifiable lipids.
The uptake capacity of the cotyledons for [¹⁴C]-glycerol exceeded the daily production in the endosperm, but only a small amount of label proceeded to the sterols and triterpenols. [¹⁴C]-Triacylglycerols were never detected in the seedling, regardless of the labeled substrate used. Although acetate is an efficient precursor in triterpenol and sterol synthesis, the uptake capacity of the cotyledons for this metabolite is too small in relation to the daily production of water soluble substrates in the endosperm. If acetate is released by the endosperm, only a marginal contribution towards triterpenol and sterol synthesis in the seedling is to be anticipated from this substrate.     

Does light-promoted export from Commelina benghalensis leaves result from differential light-sensitivity of the cells in the mesophyll-to-sieve tube path?

In contrast to the light-promoted uptake by mesophyll cells, light slightly inhibited sucrose uptake by stripped leaf disks of Commelina benghalensis L. This phenomenon appeared to result from a light-promoted vein-associated release, as light stimulated photosynthate release from stripped disks and inhibited that from mesophyll cells. In the -presence of the resorption-blocker p -chloromercuriphenylsulfonic acid, (PCMBS) the release of preloaded [¹⁴C]-sugars (sucrose, glucose) and [¹⁴C]-amino acids (alanine, asparagine, proline, valine, α-aminoisobutyric acid) from stripped disks was doubled in the light. Illumination enhanced by 20 to 60% the release of endogenous leaf cell compounds (sucrose, H₂PO-₄, K⁺, Mg²⁺, Ca²⁺) from stripped disks in the presence of PCMBS. Light also increased the export of [¹⁴C]-assimilates from intact leaves by 20% after pulse-labelling with ¹⁴CO₂. A model for loading is proposed, based on the differential light sensitivities of the plasma membranes in the mesophyll-to-sieve tube path.     

Effect of the crown on uptake and transport in embryonic shoots of Picea abies

Embryonic shoots of Picea abies (L.) Karst, isolated from 10-year-old trees, were excised either with or without the crown. Various short-term uptake experiments (3, 6 and 24 h) and one long-term uptake experiment (4 weeks) were performed with these shoots to obtain information about the physiological role of the crown as translocation barrier for different substances. Transport through the embryonic shoots was followed in both acropetal and basipetal directions using radiolabelled substances supplied in an agarified Schenk and Hildebrandt medium. The medium was labelled with [¹⁴C]-IAA and/or [³²P]-phosphate, or with [³⁵S]-sulphate and ⁸⁶Rb (as a tracer for K⁺). The experiments were conducted in light at 20°C, with the exception of one of the short-term experiments, which was carried out at 5°C to evaluate the connection between transport and metabolism. The main observation is that the crown in its collenchymatous stage of development acts as a selective barrier both acropetally and basipetally for transport of substances such as [¹⁴C]-IAA and [³²P]-phosphate or their metabolized forms. This could explain why the embryonic shoot when cultured plus or minus its crown shows different growth and developmental patterns in vitro.     

ON THE MECHANISM OF OUABAIN INHIBITION OF SYNAPTOSOME PROTEIN SYNTHESIS

Abstract— Ouabain (200μ m ) inhibited incorporation of radiolabelled leucine or glycine into the protein of neonatal synaptosome fractions but had minimal effect on preparations from adult rats. Leucine uptake into synaptosomes was rapid but not influenced by 200μ m -ouabain in contrast to ouabain inhibition of [¹⁴C]glycine and [¹⁴C]γ-aminobutyric acid uptake. Ouabain blocked the Na⁺ -dependent (stimulated) component of synaptosome fraction protein synthesis in the presence of 25m m -K⁺. Ouabain inhibition was not alleviated by addition of ADP or ATP. 100μ m -atractylate failed to influence [³H]leucine uptake or incorporation. Synergistic inhibition by ouabain was observed with the cycloheximide-sensitive component of protein synthesis and the chloramphenicol sensitive phase. Increasing the medium Ca²⁺ concentration stimulated protein synthesis and this stimulated component was inhibited by ouabain. Ouabain inhibition was associated with decreasing intraterminal K⁺ concentration and [K]_i was linearly related to the protein synthesis rate in control and ouabain treated preparations.