l-[3H]Nitroarginine and l-[3H]Arginine Uptake into Rat Cerebellar Synaptosomes: Kinetics and Pharmacology

Abstract: Characteristics of the transport of the nitric oxide synthase substrate l -arginine and its inhibitor, N ^G-nitro- l -arginine ( l -NOARG), into rat cerebellar synaptosomes were studied. Uptake of both l -arginine and l -NOARG was linear with increasing amount of protein (up to 40 µg) and time of incubation (up to 5 min) at 37°C. Uptake of both compounds reached a steady state by 20 min. Maximal uptake of l -NOARG (650 pmol/mg of protein) was three to four times higher than that of l -arginine (170 pmol/mg of protein). l -NOARG uptake showed biphasic kinetics ( K _{m 1} = 0.72 m M , V _{max 1} = 0.98 nmol/min/mg of protein; K _{m 2} = 2.57 m M , V _{max 2} = 16.25 nmol/min/mg of protein). l -Arginine uptake was monophasic with a K _m of 106 µ M and a V _max of 0.33 nmol/min/mg of protein. l -NOARG uptake was selectively inhibited by l -NOARG, N ^G-nitro- l -arginine methyl ester, and branched-chain and aromatic amino acids. l -Alanine and l -serine also inhibited l -NOARG uptake but with less potency. Uptake of l -arginine was selectively inhibited by N ^G-monomethyl- l -arginine acetate and basic amino acids. These studies suggest that in rat cerebellar synaptosomes, l -NOARG is transported by the neutral amino acid carrier systems T and L with high affinity, whereas l -arginine is transported by the basic amino acid carrier system y⁺ with high affinity. These data indicate that the concentration of competing amino acids is an important factor in determining the rates of uptake of l -NOARG and l -arginine into synaptosomes and, in this way, may control the activity of nitric oxide synthase.     

Kinetics, Pharmacology, and Autoradiographic Distribution of l-[3H]Nitroarginine Binding Sites in Rat Cerebellum

Abstract: The kinetics and pharmacology of N ^G-nitro- l -[2,3,4,5-³H]arginine ( l -[³H]NOARG) binding to rat cerebellum were investigated using in vitro radioligand binding. Specific l -[³H]NOARG binding in cerebellum was of nanomolar affinity, reversible, saturable, and best fit to a single-site model. Specific binding was Ca²⁺ dependent and sensitive to pH (with an optimum of 5.5–7.0). Added calmodulin (1.5–40 µg/ml) had no influence on specific l -[³H]NOARG binding. However, the calmodulin antagonists W-5, W-13, and calmidazolium inhibited l -[³H]NOARG binding with IC₅₀ values in the micromolar range, and calmodulin (10 µg/ml) competitively reversed this inhibition. Nitric oxide synthase (NOS) inhibitors ( N ^G-nitro- l -arginine methyl ester and N ^G-monomethyl- l -arginine acetate) and l -arginine displaced l -[³H]NOARG binding with IC₅₀ values in the nanomolar range, whereas d -arginine and basic amino acids ( l -lysine and l -histidine) displaced l -[³H]NOARG binding with IC₅₀ values in the millimolar range. A comparison of the NOS functional assay with l -[³H]NOARG binding in rat cerebellum showed similar profiles of Ca²⁺ dependency and inhibitory kinetics. Quantitative autoradiographic distribution of l -[³H]NOARG binding sites was significantly higher in the molecular layer than in the granular layer of cerebellum. These studies confirm the potential use of l -[³H]NOARG binding to study the regional distribution and functional properties of NOS.     

Simultaneous Measurement of Nitrite and Nitrate Levels as Indices of Nitric Oxide Release in the Cerebellum of Conscious Rats

Abstract: We examined the modulation of nitric oxide production in vivo by measuring levels of nitrite (NO₂⁻) and nitrate (NO₃⁻) in the dialysate of the cerebellum in conscious rats, by using an in vivo brain microdialysis technique. The levels of both NO₂⁻ and NO₃⁻ were decreased by the intraperitoneal injection of N ^G-nitro- l -arginine methyl ester, an inhibitor of nitric oxide synthase, whereas N ^G-nitro- d -arginine methyl ester had no effect. l -Arginine by itself increased NO₂⁻ and NO₃⁻ levels and diminished the reduction of their levels caused by N ^G-nitro- l -arginine methyl ester. Direct infusion of l -glutamate, N -methyl- d -aspartate, or KCl into the cerebellum through a dialysis probe resulted in an increase in NO₂⁻ and/or NO₃⁻ levels. The effects of N -methyl- d -aspartate and KCl were dependent on extracellular calcium. Furthermore, the stimulatory effects of l -glutamate and N -methyl- d -aspartate were inhibited by N ^G-nitro- l -arginine methyl ester and (±)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP), an N -methyl- d -aspartate receptor antagonist. These results suggest that NO₂⁻ and NO₃⁻ levels may be related to nitric oxide production in vivo.     

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.     

Phase I and phase II enzymes produced by Cunninghamella elegans for the metabolism of xenobiotics

Abstract The filamentous fungus Cunninghamella elegans has the ability to metabolize xenobiotics, including polycyclic aromatic hydrocarbons and pharmaceutical drugs, by both phase I and II biotransformations. Cytosolic and microsomal fractions were assayed for activities of cytochrome P450 monooxygenase, aryl sulfotransferase, glutathione S -transferase, UDP-glucuronosyltransferase, UDP-glucosyltransferase, and N -acetyltransferase. The cytosolic preparations contained activities of an aryl sulfotransferase (15.0 nmol min⁻¹ mg⁻¹), UDP-glucosyltransferase (0.27 nmol min⁻¹ mg⁻¹) and glutathione 5-transferase (20.8 nmol min⁻¹ mg⁻¹). In contrast, the microsomal preparations contained cytochrome P450 monooxygenase activities for aromatic hydroxylation (0.15 nmol min⁻¹ mg⁻¹) and N -demethylation (0.17 nmol min⁻¹~' mg⁻¹) of cyclobenzaprine. UDP-glucuronosyltransferase activity was detected in both the cytosol (0.09 nmol min⁻¹ mg⁻¹) and the microsomes (0.13 nmol min⁻¹ mg⁻¹). N -Acetyltransferase was not detected. The results from these experiments provide enzymatic mechanism data to support earlier studies and further indicate that C. elegans has a broad physiological versatility in the metabolism of xenobiotics.     

UPTAKE AND METABOLISM OF GLUTAMATE IN ASTROCYTES CULTURED FROM DISSOCIATED MOUSE BRAIN HEMISPHERES

Abstract— Uptake kinetics of l -glutamate in cultured, normal glia cells obtained from the brain hemispheres of newborn mice were measured together with the activities of the glutamate metabolizing enzymes, glutamic-oxaloacetate-transaminase, glutamate dehydrogenase and glutamine synthetase. During 3 weeks of culturing, the activities of the enzymes rose from low neonatal values toward the levels in the adult brain (206, 12.3 and 25.9 nmol. min⁻¹. mg⁻¹ cell protein for the three enzymes, respectively). The uptake kinetics indicated an unsaturable component together with an uptake following Michaelis-Menten kinetics with a K_m of 220 μ m and a V _max of 7.9 nmol. min⁻¹. mg⁻¹ cell protein. The saturable glutamate uptake was inhibited by d -glutamate, l -aspartate and α-aminoadipate whereas l -glutamine, GABA and glutarate had no effect. The uptake which was Ca²⁺-independent had a K_m for sodium of 18m m and it was stimulated by an increase in the external potassium concentration from 5 to 10 and 25 m m. The results suggest that glia cells are important for the uptake of glutamate from synaptic clefts and for the subsequent metabolism of glutamate.     

Uptake of glutamine and glutamate by the dinitrogen-fixing cyanobacterium Anabaena sp. PCC7120

Abstract Whole cells of the dinitrogen-fixing cyanobacterium Anabaena sp. PCC7120 exhibited K _m values for l -glutamine and l -glutamate of 33 μM and 0.5 mM, respectively. V _max of uptake was ca. 30 nmol mg⁻¹ (chlorophyll) min⁻¹ for both amino acids. The similar pattern of sensitivity to other amino acids exhibited by both transport activities suggests that a common transport system is involved in glutamine and glutamate uptake by this cyanobacterium.     

PHOSPHATE ACTIVATED GLUTAMINASE ACTIVITY AND GLUTAMINE UPTAKE IN PRIMARY CULTURES OF ASTROCYTES

Abstract— Uptake and release of glutamine were measured in primary cultures of astrocytes together with the activity of the phosphate activated glutaminase (EC 3.5.1.2). In contrast to previous findings of an effective, high affinity uptake of other amino acids (e.g. glutamate, GABA) no such uptake of glutamine was observed, though a saturable, concentrative uptake mechanism did exist (K _m = 3.3 ± 0.5 m m ; V _max= 50.2 ± 12.6 nmol ± min⁻¹± mg⁻¹). The phosphate activated glutaminase activity in the astrocytes (6.9 ± 0.9 nmol ± min⁻¹± mg⁻¹) was similar to the activity found in whole brain (5.4 ± 0.7 nmol ± min ^−l± mg⁻¹), which may contrast with previous findings of a higher activity of the glutamine synthetase (EC 6.3.1.2) in astrocytes than in whole brain. The observations are compatible with the hypothesis of an in vivo flow of glutamate (and GABA) from neurons to astrocytes where it is taken up and metabolized, and a compensatory flow of glutamine towards neurons and away from astrocytes although the latter cell type may be more deeply involved in glutamine metabolism than envisaged in the hypothesis.     

Copper uptake by free and immobilized cyanobacterium

Abstract Copper uptake in free and immobilized cells of the cyanobacterium Nostoc calcicola has been examined. The immobilized cells invariably maintained a higher profile of Cu intake rate (12.7 nmol mg⁻¹ protein min⁻¹) over the free cells (6.0 nmol mg⁻¹ protein min⁻¹). The total Cu uptake in immobilized cells was almost two and a half-times more than their free cell counterpart under identical experimental conditions. Also, the immobilized cells showed a stronger positive correlation between Cu adsorption and uptake. The results have been discussed in terms of improved metabolic efficiency of immobilized cells.     

Endothelin-Stimulated Capacitative Calcium Entry in Enteric Glial Cells: Synergistic Effects of Protein Kinase C Activity and Nitric Oxide

Abstract: Depletion of intracellular calcium stores by agonist stimulation is coupled to calcium influx across the plasma membrane, a process termed capacitative calcium entry. Capacitative calcium entry was examined in cultured guinea pig enteric glial cells exposed to endothelin 3. Endothelin 3 (10 n M ) caused mobilization of intracellular calcium stores followed by influx of extracellular calcium. This capacitative calcium influx was inhibited by Ni²⁺ (89 ± 2%) and by La³⁺ (78 ± 2%) but was not affected by L-, N-, or P-type calcium channel blockers. Chelerythrine, a specific antagonist of protein kinase C, dose-dependently inhibited capacitative calcium entry. The nitric oxide synthase inhibitor N ^G-nitro- l -arginine decreased calcium influx in a dose-dependent manner. The combination of chelerythrine and N ^G-nitro- l -arginine produced synergistic inhibitory effects. Capacitative calcium entry occurs in enteric glial cells via lanthanum-inhibitable channels through a process regulated by protein kinase C and nitric oxide.     

Portacaval Shunting and Hyperammonemia Stimulate the Uptake of l-[3H]Arginine but Not of l-[3H]Nitroarginine into Rat Brain Synaptosomes

Abstract: Elevated activities of nitric oxide synthase (NOS) have been reported previously in the brains of portacaval-shunted (PCS) rats, a model of chronic hepatic encephalopathy (HE). As l -arginine availability for nitric oxide synthesis depends on a specific uptake mechanism in neurons, we studied the kinetics of l -[³H]-arginine uptake into synaptosomes prepared from the brains of PCS rats. Results demonstrate that l -arginine uptake is significantly increased in cerebellum (60%; p < 0.01), cerebral cortex (42%; p < 0.01), hippocampus (56%; p < 0.01), and striatum (51%; p < 0.01) of PCS rats compared with sham-operated controls. Hyperammonemia in the absence of portacaval shunting also stimulated the transport of l -[³H]arginine; kinetic analysis revealed that the elevated uptake was due to increased uptake capacity ( V _max) without any change in affinity ( K _m). Incubation of cerebellar synaptosomes with ammonium acetate for 10 min caused a dose-dependent stimulation of l -[³H]arginine uptake. Neither portacaval shunting nor hyperammonemia had any significant effect on the synaptosomal uptake of N ^G-nitro- l -[³H]arginine. These studies demonstrate that increased NOS activity observed in experimental HE may result from increased availability of l -arginine resulting from a direct stimulatory effect of ammonia on l -arginine transport.     

Na+-Dependent Uptake and Release of Taurine by Neuroblastoma × Glioma Hybrid Cells

Abstract In neuroblastoma × glioma hybrid cells, a cell line of neuronal character, a saturable uptake system for taurine is found which displays high affinity and high capacity ( K _m= 38 μ m , V = 1.25 nmol mg⁻¹ min⁻¹)- Only the closely related structural analogues hypotaurine and β-alanine are able to inhibit the transport of radioactively labeled taurine. Imipramine or haloperidol at 100 μ m effectively blocks taurine uptake. High-affinity taurine uptake shows an absolute and highly specific requirement for Na⁺. The hybrid cells internalize taurine very slowly and, with 1 m m extracellular taurine, attain a plateau only after more than 20 h, at which time approximately 34 m m labeled taurine has accumulated in the cytosol. Generally there is hardly any spontaneous release of accumulated taurine. Efflux can, however, be induced by increasing the intracellular Na⁺ content and is then accelerated by lowering the extracellular Na⁺ concentration. The hypothesis is forwarded that taurine may exert its function by driving the extrusion of Na⁺ in emergency situations.     

Mycorrhiza formation on Norway spruce (Picea abies) roots affects the pathway of anaplerotic CO2 fixation

Activities of carboxylation enzymes were analyzed in the mycelium of the mycorrhizal fungus Amanita muscaria (L. ex Fr.) Hooker, in non-mycorrhizal short roots of Norway spruce ( Picea abies [L.] Karst.) and in myconhizas of these two partners. While pyruvale carboxylase (PC, EC 6.4.1.1) and phosphoenolpyruvate carboxykinase activities (PEPCK.EC 4.1.1.49) could be detected in the mycelium of A. muscaria , phosphoenolpyruvate carboxyknase (PEPC, EC 4.1.1.31) was only active in root tissue. In A. muscaria , PC activity was generally low (around 10 nmol mg^−tprotein min⁻) but PEPCK activity was above 250 nmol mg⁻¹ protein min⁻¹. Mycorrhizal development on short roots decreased PEPC activity by more than 75%, although dilution by the fungal biomass in mycorrhizas was only 35%. This reduction in activity was paralleled by a decreased content of PEPC protein. By means of micro-analytical methods it was shown that PEPC activity was lowest in the central zones of the mycorrhizas, Whereas PEPC activity was highest in the corresponding central sections in non-mycorrhizal short roots. ¹⁴CO₂ labelling, on the other hand, revealed that in vivo CO₂ fixation was higher in mycorrhizas compared to non-mycorrhizal short roots. It is concluded that fungal carboxylases (probably PEPCK) are important for anaplerotic CO₂ fixation during nitrogen assimilation in mycorrhizas of Norway spruce.     

High-Affinity Uptake of (RS)-Nipecotic Acid in Astrocytes Cultured from Mouse Brain. Comparison with GABA Transport

Abstract: (RS)-Nipecotic acid is taken up into cultured astrocytes by a saturable high-affinity transport system with a K_m, of 28.8 ± 2.8 μM and a V_max of 0.294 ± 0.022 nmol × min⁻¹× [mg cell protein]⁻¹. The uptake which represents a net inward transport was sodium-dependent, requiring translocation of one sodium ion for each molecule of nipecotic acid taken up. The most potent inhibitors of GABA uptake into astrocytes (GABA, (R)-nipecotic acid, (3RS,4SR)-4-hydroxynipecotic acid, and guvacine) were shown to be potent inhibitors of nipecotic acid uptake (IC₅₀) 20, 25, 25, and 50 μm respectively), GABA being a competitive inhibitor. (S)-2,4-Diaminobutyric acid was a more efficient inhibitor than β-alanine of glial uptake of (RS)-nipecotic acid. It is concluded that astroglial uptake of (RS)-nipecotic acid and GABA is mediated by the same transport system.     

Transport of Zinc-65 at the Blood-Brain Barrier During Short Cerebrovascular Perfusion in the Rat: Its Enhancement by Histidine

Abstract: Zinc-65 transport into different regions of rat brain has been measured during short vascular perfusion of one cerebral hemisphere with an oxygenated HEPES-containing physiological saline at pH 7.40. The [Zn²⁺] was buffered with either bovine serum albumin or histidine. In each case uptake was linear with time up to 90 s. ⁶⁵Zn flux into brain in the presence of albumin followed Michaelis-Menten kinetics and for parietal cortex had a K _m of 16 n M and a V _max of 44 nmol/kg/min. Increasing concentrations of l -histidine enhanced ⁶⁵Zn flux into brain at [Zn²⁺] values between 1 and 1,000 n M . The combined effect of [histidine] and [Zn²⁺] was best accounted for by a function of [ZnHis⁺], i.e., flux = 64.4 · [ZnHis⁺]/(390 + [ZnHis⁺]) + 0.00378 · [ZnHis⁺], with concentrations being nanomolar. d -Histidine had an influence similar to that of l -histidine. ⁶⁵Zn flux in the presence of 100 µ M l -histidine was not affected by either 500 µ M l -arginine or 500 µ M l -phenylalanine. The results indicate specific transport of Zn²⁺ across the plasma membranes of brain endothelium. The enhancement due to histidine has been attributed to diffusion of ZnHis⁺ across unstirred layers "ferrying" zinc to and from transport sites.     

Purification and characterization of a feruloyl esterase from the fungus Penicilliumexpansum

An extracellular phenolic acid esterase produced by the fungus Penicillium expansum in solid state culture released ferulic and ρ-coumaric acid from methyl esters of theacids, and from the phenolic-carbohydrate esters O-[5-O-(trans-feruloyl)-α- l -arabinofuranosyl]-(1 → 3)-O-β- d -xylopyranosyl-(1 → 4)- d -xylopyranose (FAXX) and O-[5-O-((E)-ρ-coumaroyl)-α- l -arabinofuranosyl]-(1 → 3)-O-β- d -xylopyranosyl-(1 → 4)- d -xylopyranose(PAXX). The esterase was purified 360-fold in successive stepsinvolving ultrafiltration and column chromatography by gel filtration, anion exchange andhydrophobic interaction. These chromatographic methods separated the phenolic acid esterasefrom α- l -arabinofuranosidase, pectate and pectin lyase, polygalacturonase,xylanase and β- d -xylosidase activities. The phenolic acid esterase had an apparentmass of 65 kDa under non-denaturing conditions and a mass of 57·5 kDa underdenaturing conditions. Optimal pH and temperature were 5·6 and 37 °C,respectively and the metal ions Cu2+ and Fe³+ atconcentrations of 5 mmol l⁻¹ inhibited feruloyl esterase activity by 95% and44%, respectively, at the optimum pH and temperature. The apparent K_m and V_max of the purified feruloyl esterase for methyl ferulate at pH 5·6 and 37 °Cwere 2·6 mmol l⁻¹ and 27·1 μmol min⁻¹ mg⁻¹. The corresponding constants of ρ-coumaroylesterase for methyl coumarate were 2·9 mmol l⁻¹ and 18·6μmol min−1 mg⁻¹.     

Pyruvate Carboxylase Activity in Primary Cultures of Astrocytes and Neurons

Abstract: The activity of the pyruvate carboxylase was determined in brains of newborn and adult mice as well as primary cultures of astrocytes, of cerebral cortex neurons, and of cerebellar granule cells. The activity was found to be 0.25 ± 0.14, 1.24 ± 0.07, and 1.75 ± 0.13 nmol · min⁻¹· mg⁻¹ protein in, respectively, neonatal brain, adult brain, and astrocytes. Neither of the two types of neurons showed any detectable enzyme activity (i.e., < 0.05 nmol · min⁻¹· mg⁻¹). It is therefore concluded that pyruvate carboxylase is an astrocytic enzyme.     

Carbon monoxide dehydrogenase and acetate thiokinase in Methanothrix soehngenii

Abstract In Methanothrix soehngenii acetate is first activated by an acetate thiokinase rather than a phosphotransacetylase. The specific activity of the acetate thiokinase was 5.29 μmol acetate activated min⁻¹ mg⁻¹ protein with a half maximum rate at 0.74 mM acetate and at 0.047 mM CoA. In cell-free extracts a CO-dehydrogenase activity was measured of 3.02 μmol min⁻¹ mg⁻¹ protein with a half maximum rate at 0.44 mM CO and at 0.18 mM methylviologen. NADP and NAD could not replace methylviologen. F₄₂₀ showed only low activity as electron acceptor.     

Choline Uptake in Cultured Human Y79 Retinoblastoma Cells: Effect of Polyunsaturated Fatty Acid Compositional Modifications

Abstract: Choline uptake in Y79 human retinoblastoma cells occurs through two kinetically distinguishable processes. The high-affinity system shows little sodium or energy dependence, and it does not appear to be linked to acetyl CoA: choline O -acetyltransferase. When the cells are grown in a culture medium containing 10% fetal bovine serum, the high-affinity system has a K' _m= 2.16 ± 0.13 μ m and V' _max= 27.0 ± 2.9 pmol min⁻¹ mg⁻¹, whereas the low-affinity system has K' _m= 20.4 ± 1.3 μ m and V' _max= 402 ± 49 pmol min⁻¹ mg⁻¹. Under these conditions, the polyunsaturated fatty acid content of the cell membranes is relatively low. When the polyunsaturated fatty acid content of the microsomal membrane fraction was increased by supplementing the culture medium with linolenic or docosahexaenoic acids (n-3 polyunsaturated fatty acids) or arachidonic acid (n-6 polyunsaturated fatty acid), the K' ^m of the high-affinity choline transport system was reduced by 40–60%. The V' _max also was reduced by 20–40%. Supplementation with oleic acid, the most prevalent monounsaturated fatty acid, did not affect either kinetic parameter. The results suggest that one functional effect of the high unsaturated fatty acid content of neural cell membranes is to facilitate the capacity of the high-affinity choline uptake system to transport low concentrations of choline. This effect appears to be specific for polyunsaturated fatty acids but not for a single type, for it is produced by members of both the n-3 and n-6 classes of polyunsaturated fatty acids.