Control of intracellular glutathione and its effect on ultraviolet radiation-induced K+ efflux in cultured rose cells

Abstract Modification of the ‘intracellular concentration of reduced glutathione’ (IC-GSH) affected the response of cultured rose cells (Rosa damascena) to ultraviolet radiation (UV)-induced leakage of K⁺. High IC-GSH induced by incubation of cells in 10 mol m^?3 GSH (IC-GSH increased linearly with time from 20 to about 600 μmol g^?1 in 61.2 ks) caused cells to become significantly less sensitive to UV. Low IC-GSH induced by treatment with 1 mol m^?3 buthionine sulphoximine (BSO) plus 1 mol m^?3 diethylmaleate (DEM) (IC-GSH decreased from 20 to about 3 μg g^?1 in 61.2 ks) reduced, rather than increased, the UV-sensitivity of the cells. However, treatment with DEM also induced a large transient K⁺ leakage; and treatment with BSO induced a slight leakage. The K⁺ leaked was recovered by 3.24 ks. Following K⁺ recovery, the DEM-treated cells showed almost complete insensitivity to UV, and BSO-treated cells showed a slightly reduced sensitivity to UV. These results are in agreement with our previous findings that other treatments (heat, cycloheximide, UV), which also cause a transient leakage of K⁺, also reduce the induction of K⁺ leakage by a subsequent UV treatment. We conclude that high IC-GSH may play a role in protecting plant cells from UV-induced K⁺ leakage. Increased UV-sensitivity with low ICGSH was not observed, we believe, because of the transient K⁺ leakage, though the mechanism of reduced sensitivity to UV induced by transient leakage of K⁺ is not known at this time. Treatment with UV did not reduce the IC-GSH, showing that this is not the mechanism by which UV induces K⁺ leakage.     

Ca2+ dependence and La3+ interference of ultraviolet radiationinduced K+ efflux from rose cells

A low fluence of ultraviolet radiation (UV) causes cultured cells of Rosa damascena Mill cv. Gloire de Guilan to lose intracellular K⁺. This effect required the presence of Ca²⁺ in the medium. A reduction in the concentration of free Ca²⁺ to 10⁻⁵ M with ethyleneglycol-bis-(β-aminoethyl-ether)-N.N.N',N'-tetraacetic acid (EGTA) buffer inhibited the UV-stimulated efflux; this was correlated with a discharge of the membrane potential and a stimulation of the leakage of K⁺ from unirradiated cells. All the same effects were seen with La³⁺ at 0.2 m M. At 0.02 m M La³⁺, the UV-stimulated efflux of K⁺ was blocked without concomitant effects on the membrane potential or K⁺ efflux from control cells. It is suggested that removal of Ca²⁺ blocks or masks the UV-induced leakage of K⁺ by destabilizing the plasma membrane. In addition, La³⁺ may specifically inhibit the UV-stimulated opening of K⁺ or anion channels.     

Kinetics of efflux of K+ from cultured rose cells treated with ultraviolet light

Irradiation of cultured rose ( Rosa damascena Mill. cv. Gloire de Guilan) cells with ultraviolet light caused of loss of K⁺, which occurred with sigmoid kinetics. The kinetics of loss of K⁺ were not changed when the extracellular concentration of K⁺ was held constant during the period of efflux. Furthermore, the rate of loss of K⁺ was approximately the same even though the K⁺ concentration in the medium was increased from 0.1 to 10 m M . The kinetics of uptake of the lipophilic methyltriphenylphosphonium cation, an indicator of the plasma membrane potential, were linear throughout the period of K⁺ efflux, suggesting that the starting and stopping of K⁺ efflux do not reflect a passive response to changes in the membrane potential of the cells. The results are interpreted in terms of activation and inactivation of an efflux channel or pump for K⁺.     

Compartmentation of [14C]Glutamate and [14C]Glutamine Oxidative Metabolism in the Rat Hippocampus as Determined by Microdialysis

Abstract: Metabolic compartmentation of amino acid metabolism in brain is exemplified by the differential synthesis of glutamate and glutamine from the identical precursor and by the localization of the enzyme glutamine synthetase in glial cells. In the current study, we determined if the oxidative metabolism of glutamate and glutamine was also compartmentalized. The relative oxidation rates of glutamate and glutamine in the hippocampus of free-moving rats was determined by using microdialysis both to infuse the radioactive substrate and to collect ¹⁴CO₂ generated during their oxidation. At the end of the oxidation experiment, the radioactive substrate was replaced by artificial CSF, 2 min-fractions were collected, and the specific activities of glutamate and glutamine were determined. Extrapolation of the specific activity back to the time that artificial CSF replaced ¹⁴C-amino acids in the microdialysis probe yielded an approximation of the interstitial specific activity during the oxidation. The extrapolated interstitial specific activities for [¹⁴C]glutamate and [¹⁴C]glutamine were 59 ± 18 and 2.1 ± 0.5 dpm/pmol, respectively. The initial infused specific activities for [U-¹⁴C]glutamate and [U-¹⁴C]glutamine were 408 ± 8 and 387 ± 1 dpm/pmol, respectively. The dilution of glutamine was greater than that of glutamate, consistent with the difference in concentrations of these amino acids in the interstitial space. Based on the extrapolated interstitial specific activities, the rate of glutamine oxidation exceeds that of glutamate oxidation by a factor of 5.3. These data indicate compartmentation of either uptake and/or oxidative metabolism of these two amino acids. The presence of [¹⁴C]glutamine in the interstitial space when [¹⁴C]glutamate was perfused into the brain provided further evidence for the glutamate/glutamine cycle in brain.     

In Vivo Release from Cerebral Cortex of [14C]Glutamate Synthesized from [U-14C]Glutamine

Awake, unrestrained, and behaviourally normal animals with superfusion cannulae implanted over the sensorimotor cortex were used in a study of the capacity of infused [U-14C]glutamine for labelling glutamate and other amino acids released by depolarising stimuli. A spontaneous background release of [14C]glutamate was detected. This was increased by tityustoxin (1 microM). The specific radioactivity of glutamate increased eightfold during the evoked-release period. [14C]Aspartate was also detected and showed increased release, but not increased specific labelling, in response to depolarisation. Evoked gamma-aminobutyric acid (GABA) release occurred but only small amounts of [14C]GABA were detected. Glutamine showed increased rates of uptake to the sensorimotor cortex during stimulation periods, suggesting an accelerated breakdown via glutaminase.     

Enhanced [3H]DOPA and [3H]Dopamine Turnover in Striatum and Frontal Cortex In Vivo Linked to Glutamate Receptor Antagonism

Abstract: We tested the hypothesis that blockade of NMDA glutamate receptors in brain enhances dopamine turnover. We blocked this class of glutamate receptors in the rat brain in vivo with dizocilpine (MK-801) and measured the accumulation of radiolabeled DOPA and its metabolites as functions of time after intravenous bolus injection. Using the time courses of the accumulated metabolites, we calculated the turnover constants of enzymes mediating dopamine synthesis and catabolism. Dizocilpine treatment for 8 days enhanced the rates of DOPA decarboxylation and dopamine oxidation (monoamine oxidation) 4- and 16-fold, respectively, in neostriatum and 10- and 3-fold, respectively, in frontal cortex. The findings are not inconsistent with the hypothesis that the psychotomimetic properties of dizocilpine may be the manifestation of denervation hypersensitivity linked to activation of key enzymes of dopamine turnover in striatum.     

L-[3H]Glutamate Binding to a Membrane Preparation from Crayfish Muscle

The binding of L-[3H]glutamate to an isolated membrane preparation from crayfish tail muscle has been studied. The muscle homogenate was osmotically shocked, frozen and thawed, and thoroughly washed before incubation with L-[3H]glutamate. The preparation showed high specific binding of L-glutamate with a KD of 0.12 microM and Bmax of 4.7 pmol/mg protein measured in Tris/HCl pH 7.3 and at 4 degrees C. Nonspecific binding was 5-10% of total binding. The glutamate binding was highly stereospecific [K0.5 (D-glutamate), 270 microM] and showed a high degree of discrimination between L-glutamate and L-aspartate [K0.5 (L-aspartate), 54 microM]. In mammalian CNS preparations potent agonists of L-glutamate such as kainate and N-methyl-D-aspartate had no effect at 1 mM, and quisqualate was a weak inhibitor of L-glutamate binding [K0.5 (quisqualate), 162 microM]. Ibotenate was the most potent inhibitor [K0.5 (ibotenate), 0.27 microM], and various esters of L-glutamate were of intermediate potency as displacers of L-[3H]glutamate binding (K0.5 values from 6 to 60 microM). The glutamate binding site from crayfish muscle is clearly different from any of the subclasses of glutamate receptors in mammalian CNS. A possible physiological function of the binding site is a postsynaptic receptor for glutamate, either an extra-junctional or a junctional receptor.     

Translocation of 14C, carbohydrate content and activity of the enzymes of sucrose metabolism in rose petals temperatures

Both export of ¹⁴C from the source leaves of roses (Rosa × hybrida cv. Golden Times) and import of ¹⁴C to the petals were reduced by plant exposure to low night temperature. However, the import was affected to a greater extent than the export. During all stages of flower bud development the concentration of reducing sugars in petals of roses grown at reduced night temperature was lower than in petals of plants grown at higher night temperature. There was no significant difference in starch content in response to the night temperature, and the content of starch decreased toward complete flower bud opening. The concentration of sucrose in flowers at the low night temperature remained low during all stages of flower bud development, while at the high night temperature the concentration of sucrose increased during flower bud development, reaching a peak at the stage when petals start to unfold. At both temperatures the concentration of sucrose declined at complete flower opening. The activity of sucrose synthase (EC 2.4.1.14) was inhibited by low temperature in young rose shoots more than in the petals, while the activity of acid invertase (EC 3.2.1.26) was affected similarly in both tissues by the temperature treatments.     

Glucose and Synaptosomal Glutamate Metabolism: Studies with [15N]Glutamate

The metabolism of [15N]glutamate was studied with gas chromatography-mass spectrometry in rat brain synaptosomes incubated with and without glucose. [15N]Glutamate was taken up rapidly by the preparation, reaching a steady-state level in less than 5 min. 15N was incorporated predominantly into aspartate and, to a much lesser extent, into gamma-aminobutyrate. The amount of [15N]ammonia formed was very small, and the enrichment of 15N in alanine and glutamine was below the level of detection. Omission of glucose substantially increased the rate and amount of [15N]aspartate generated. It is proposed that in synaptosomes (a) the predominant route of glutamate nitrogen disposal is through the aspartate aminotransferase reaction; (b) the aspartate aminotransferase pathway generates 2-oxoglutarate, which then serves as the metabolic fuel needed to produce ATP; (c) utilization of glutamate via transamination to aspartate is greatly accelerated when flux through the tricarboxylic acid cycle is diminished by the omission of glucose; (d) the metabolism of glutamate via glutamate dehydrogenase in intact synaptosomes is slow, most likely reflecting restriction of enzyme activity by some unknown factor(s), which suggests that the glutamate dehydrogenase reaction may not be near equilibrium in neurons; and (e) the activities of alanine aminotransferase and glutamine synthetase in synaptosomes are very low.     

Glutathione Turnover in Cultured Astrocytes: Studies with [15N]Glutamate

The incorporation of [15N]glutamic acid into glutathione was studied in primary cultures of astrocytes. Turnover of the intracellular glutathione pool was rapid, attaining a steady state value of 30.0 atom% excess in 180 min. The intracellular glutathione concentration was high (20-40 nmol/mg protein) and the tripeptide was released rapidly into the incubation medium. Although labeling of glutathione (atom% excess) with [15N]glutamate occurred rapidly, little accumulation of 15N in glutathione was noted during the incubation compared with 15N in aspartate, glutamine, and alanine. Glutathione turnover was stimulated by incubating the astrocytes with diethylmaleate, an electrophile that caused a partial depletion of the glutathione pool(s). Diethylmaleate treatment also was associated with significant reductions of intraastrocytic glutamate, glycine, and cysteine, i.e., the constituents of glutathione. Glutathione synthesis could be stimulated by supplementing the steady-state incubation medium with 0.05 mM L-cysteine, such treatment again partially depleting intraastrocytic glutamate and causing significant reductions of 15N labeling of both alanine and glutamine, suggesting that glutamate had been diverted from the synthesis of these amino acids and toward the formation of glutathione. The current study underscores both the intensity of glutathione turnover in astrocytes and the relationship of this turnover to the metabolism of glutamate and other amino acids.     

Characterisation of Na+-Independent L-[3H]Glutamate Binding Sites in Human Temporal Cortex

The binding of L-[3H]glutamate to membranes from human temporal cortex was studied in the absence of Na+, Ca2+, and Cl- ions. Pharmacological characterisation revealed that approximately 35% of specific binding at 50 nM L-[3H]glutamate was sensitive to a combination of kainate and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid. The remaining approximately 65% of specific binding was to a single population of sites with a KD of 844 nM and a Bmax of 0.92 pmol/mg protein. The pharmacological characteristics were consistent with an interaction at the N-methyl-D-aspartate subclass of excitatory amino acid receptor. The inclusion of Cl- ions revealed additional glutamate binding; this was sensitive to quisqualate and DL-2-amino-4-phosphonobutyrate, but not to kainate, DL-2-amino-7-phosphonoheptanoate, or alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid.     

Characterization of Two [3H]Glutamate Binding Sites in Rat Hippocampal Membranes

The specific binding of L-[3H]glutamate was investigated in the presence and the absence of sodium ions in freshly prepared membranes from rat hippocampus. Sodium ions were found to have a biphasic effect; low concentrations induced a marked inhibition of the binding (in the range 0.5-5.0 mM), whereas higher concentrations resulted in a dose-dependent stimulation of binding (in the range 10-150 mM). These results permit the discrimination of two binding sites in hippocampal membranes. Both Na+-independent and Na+-dependent binding sites were saturable, exhibiting dissociation constants at 30 degrees C of 750 nM and 2.4 microM, respectively, with Hill coefficients not significantly different from unity, and maximal number of sites of 6.5 and 75 pmol/mg protein, respectively. [3H]Glutamate binding to both sites reached equilibrium between 5 and 10 min and was reversible. The relative potencies of a wide range of compounds, with known pharmacological activities, to inhibit [3H]glutamate binding were very different for the Na+-independent and Na+-dependent binding and suggested that the former sites were related to post-synaptic glutamate receptors, whereas the latter were related to high-affinity uptake sites. This conclusion was also supported by the considerable variation in the regional distribution of the Na+-dependent binding site, which paralleled that of the high-affinity glutamate uptake; the Na+-independent binding exhibited less regional variation.     

Pharmacology of Sodium-Dependent High-Affinity l-[3H]Glutamate Transport in Glial Cultures

Abstract: Pharmacological and molecular biological studies provide evidence for subtypes of sodium-dependent high-affinity glutamate (Glu) transport in the mammalian CNS. At least some of these transporters appear to be selectively expressed in different brain regions or by different cell types. In the present study, the properties of l -[³H]Glu transport were characterized using astrocyte-enriched cultures prepared from cerebellum and cortex. In both brain regions, the kinetic data for sodium-dependent transport were consistent with a single site with K_m values of 91 ± 17 µM in cortical glial cells and 66 ± 23 µM in cerebellar glial cells. The capacities were 6.1 ± 1.6 nmol/mg of protein/min in cortical glial cells and 8.4 ± 0.9 nmol/mg of protein/min in cerebellar glial cells. The potencies of ～40 excitatory amino acid analogues for inhibition of sodium-dependent transport into glial cells prepared from cortex and cerebellum were examined, including compounds that are selective inhibitors of transport in synaptosomes prepared from either cerebellum or cortex. Of the analogues tested, 14 inhibited transport activity by >50% at 1 mM concentrations. Unlike l -[³H]Glu transport in synaptosomes prepared from cerebellum or cortex, there were no large differences between the potencies of compounds for inhibition of transport measured in glial cells prepared from these two brain regions. With the exception of (2S,1′R,2′R)-2-(carboxycyclopropyl)glycine and l -α-aminoadipate, all of the compounds examined were ～10–200-fold less potent as inhibitors of l -[³H]Glu transport measured in glial cells than as inhibitors of transport measured in synaptosomes prepared from their respective brain regions. The pharmacology of transport measured in these glial cells differs from the reported pharmacology of the cloned Glu transporters, suggesting the existence of additional uncloned Glu transporters or Glu transporter subunits.     

Perinatal Hypoxia-Ischemia Disrupts Striatal High-Affinity [3H]Glutamate Uptake into Synaptosomes

We examined the impact of hypoxia-ischemia on high-affinity [3H]glutamate uptake into a synaptosomal fraction prepared from immature rat corpus striatum. In 7-day-old pups the right carotid artery was ligated, and pups were exposed to 8% oxygen for 0, 0.5, 1, or 2.5 h, and allowed to recover for up to 24 h before they were killed. High-affinity glutamate uptakes in striatal synaptosomes derived from tissue ipsilateral and contralateral to ligation were compared. After 1 h of hypoxia plus ischemia, high-affinity glutamate uptake in the striatum was reduced by 54 +/- 13% compared with values from the opposite (nonischemic) side of the brain (p less than 0.01, t test versus ligates not exposed to hypoxia). There were similar declines after 2.5 h of hypoxia-ischemia. Activity remained low after a 1 h recovery period in room air, but after 24 h of recovery, high-affinity glutamate uptake was equal bilaterally. Kinetic analysis revealed that loss of activity could be attributed primarily to a 40% reduction in the number of uptake sites. Hypoxia alone had no effect on high-affinity glutamate uptake although it reduced synaptosomal uptake of [3H]3,4-dihydroxyphenylethylamine. Addition of 1 mg/ml of bovine serum albumin to the incubation medium preferentially stimulated high-affinity glutamate uptake in hypoxic-ischemic brain compared with its effects in normal tissue. These studies demonstrate that hypoxia-ischemia reversibly inhibits high-affinity glutamate uptake and this occurs earlier than the time required to produce neuronal damage in the model.     

Dementia of the Alzheimer's Type: Changes in Hippocampal L-[3H]Glutamate Binding

Abstract: Glutamate or a related excitatory amino acid is thought to be the major excitatory neurotransmitter of hippocampal afferents, intrinsic neurons, and efferents. We have used an autoradiographic technique to investigate the status of excitatory amino acid receptors in the hippocampal formation of patients dying with dementia of the Alzheimer type (DAT). We examined l-[³H]glutamate binding to sections from the hippocampal formation of six patients dying of DAT and six patients without DAT and found marked reductions in total [³H]glutamate binding in all regions of hippocampus and adjacent parahippocampal cortex in DAT brains as compared to controls. When subtypes of excitatory amino acid receptors were assayed, it was found that binding to the N -methyl-d-aspartate (NMDA)-sensitive receptor was reduced by 75–87%, with the greatest loss found in stratum moleculare and stratum pyramidale of CA1. Binding to quisqualate (QA)-sensitive receptors was reduced by 45–69%. There were smaller reductions (21–46%) in GABA_A receptors in DAT cases. Muscarinic cholinergic receptors assayed in adjacent sections of hippocampal formation were unchanged in DAT. Benzodiazepine receptors were reduced significantly only in parahippocampal cortex by 44%. These results suggest that glutamatergic neurotransmission within the hippocampal formation is likely to be severely impaired in Alzheimer's disease. Such impairment may account for some of the cognitive decline and memory deficits that characterize DAT.     

Heterogeneity of [3H]Dipyridamole Binding to CNS Membranes: Correlation with [3H]Nitrobenzylthioinosine Binding and [3H]Uridine Influx Studies

The relationship between the nucleoside transport system and the nitrobenzylthioinosine-sensitive and -resistant [3H]dipyridamole binding sites was examined by comparing the characteristics of [3H]dipyridamole binding with those of [3H]nitrobenzylthioinosine binding and [3H]-uridine influx in rabbit and guinea pig cerebral cortical synaptosomes. Two distinct high-affinity synaptosomal membrane-associated [3H]dipyridamole binding sites, with different sensitivities to inhibition by nitrobenzylthioinosine, were characterized in the presence of 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS, 0.01%) to prevent [3H]dipyridamole binding to glass tubes and filters. The nitrobenzylthioinosine-resistant [3H]-dipyridamole binding sites represented a greater proportion of the total membrane sites in guinea pig than in rabbit (40 vs. 10% based on inhibition studies). In rabbit, nitrobenzylthioinosine-sensitive [3H]dipyridamole binding (KD = 1.4 +/- 0.2 nM) and [3H]nitrobenzylthioinosine binding (KD = 0.30 +/- 0.01 nM) appeared to involve the same membrane site associated with the nitrobenzylthioinosine-sensitive nucleoside transporter. By mass law analysis, [3H]-dipyridamole binding in guinea pig could be resolved into two components based on sensitivity to inhibition by 1 microM nitrobenzylthioinosine. The nitrobenzylthioinosine-resistant [3H]dipyridamole binding sites were relatively insensitive to inhibition by all of the nucleoside transport substrates and inhibitors tested, with the exception of dipyridamole itself. In guinea pig synaptosomes, 100 microM dilazep blocked nitrobenzylthioinosine-resistant [3H]uridine transport completely but inhibited the nitrobenzylthioinosine-resistant [3H]dipyridamole binding component by only 20%. Furthermore, a greater percentage of the [3H]dipyridamole binding was nitrobenzylthioinosine resistant in guinea pig compared with rabbit, yet both species had a similar percentage of nitrobenzylthioinosine-resistant [3H]uridine transport.(ABSTRACT TRUNCATED AT 250 WORDS)     

A Novel Chloride-Dependent L-[3H]Glutamate Binding Site in Astrocyte Membranes

Membrane fractions prepared from astrocytes grown in culture exhibit a specific binding site for L-[3H]glutamate that is Cl--dependent and Na+-independent. The binding site is a single saturable site with a KD of about 0.5 microM, is inhibited by L-aspartate, L-cysteate, and quisqualate, and is insensitive to kainate, N-methyl-D-aspartate, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate, and 2-amino-4-phosphonobutyrate. The pharmacological characteristics of the binding site indicate that it is distinct from any site previously described in synaptic membrane preparations. Comparisons of ionic requirements, ligand specificity, and inhibitor sensitivities, however, suggest the described binding is the first step in a Cl--dependent high-affinity glutamate uptake system. Such binding studies provide a useful model system in which to investigate the close association between excitatory amino acids, astrocytes, the termination of glutamate's excitatory action by high-affinity uptake, and the excitotoxic action of acidic amino acids in membranes of a single cell type.     

[3H]Aniracetam Binds to Specific Recognition Sites in Brain Membranes

Abstract: [³H]Aniracetam bound to specific and saturable recognition sites in membranes prepared from discrete regions of rat brain. In crude membrane preparation from rat cerebral cortex, specific binding was Na⁺ independent, was still largely detectable at low temperature (4°C), and underwent rapid dissociation. Scatchard analysis of [³H]aniracetam binding revealed a single population of sites with an apparent K_D value of ～70 nM and a maximal density of 3.5 pmol/mg of protein. Specifically bound [³H]aniracetam was not displaced by various metabolites of aniracetam, nor by other pyrrolidinone-containing nootropic drugs such as piracetam or oxiracetam. Subcellular distribution studies showed that a high percentage of specific [³H]aniracetam binding was present in purified synaptosomes or mitochondria, whereas specific binding was low in the myelin fraction. The possibility that at least some [³H]aniracetam binding sites are associated with glutamate receptors is supported by the evidence that specific binding was abolished when membranes were preincubated at 37°C under fast shaking (a procedure that substantially reduced the amount of glutamate trapped in the membranes) and could be restored after addition of either glutamate or α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) but not kainate. The action of AMPA was antagonized by DNQX, which also reduced specific [³H]aniracetam binding in unwashed membranes. High levels of [³H]aniracetam binding were detected in hippocampal, cortical, or cerebellar membranes, which contain a high density of excitatory amino acid receptors. Although synaptosomal aniracetam binding sites may well be associated with AMPA-sensitive glutamate receptors, specifically bound [³H]aniracetam could not be displaced by cyclothiazide or GYKI 52466, which act as a positive and negative modulator of AMPA receptors, respectively.     

Effects of genetic modification of nitrate reductase expression on 15NO3– uptake and reduction in Nicotiana plants

The physiological consequences for NO₃^– utilization by the plant of underexpression and overexpression of nitrate reductase (NR) were investigated in nine transformants of Nicotiana tabacum and Nicotiana plumbaginifolia. The in vitro NR activities (NRAs) in both roots and leaves of low- and high-NR tobacco transformants ranged from 5–10% to 150–200%, respectively, of those measured in wild-type plants. The level of NR expression markedly affected the NO₃^– reduction efficiency in detached leaves and intact plants. In both species, ¹⁵NO₃^– reduction ranged from 15–45% of ¹⁵NO₃^– uptake in the low-NR plants, to 40–80% in the wild-type, and up to 95% in high-NR plants. In the high-NR genotypes, however, total ¹⁵NO₃^– assimilation was not significantly increased when compared with that in wild-type plants, because the higher ¹⁵NO₃^– reduction efficiency was offset by lower ¹⁵NO₃^– uptake by the roots. The inhibition of NO₃^– uptake appeared to be the result of negative feedback regulation of NO₃^– influx, and is interpreted as an adjustment of NO₃^– uptake to prevent excessive amino acid synthesis. In genotypes underexpressing NR, the low ¹⁵NO₃^– reduction efficiency also was generally associated with a decrease in net ¹⁵NO₃^– uptake as compared with the wild type. Thus, underexpression of NR resulted in an inhibition of reduced ¹⁵N synthesis in the plant, although the effect was much less pronounced than that expected from the very low NRAs. The restricted NO₃^– uptake in low-NR plants emphasizes the point that the products of NO₃^– assimilation are not the only factors responsible for down-regulation of the NO₃^– uptake system.