Presence of gamma-aminobutyric acid in embryonic palates of AJ and SWV mouse strains

The presence of gamma-aminobutyric acid (GABA) in the embryonic palate was sought as a criterion for its role in regulating palate development. GABA was measured by a gas chromatographic-mass spectrometric (GC-MS) method using the heptafluorobutyryl (HFB)-cyclohexyl-GABA derivative, which gave the necessary sensitivity and specificity to measure low levels of GABA in the presence of contaminating substances. GABA was measured in dissected embryonic palates at various times of development in the AJ mouse strain. GABA levels were lower in day 14 AJ palates (0.19 +/- 0.01 nmol/mg protein) than at days 13 (0.28 +/- 0.03) and 15 (0.30 +/- 0.04). Comparable levels were observed in fore- and hindlimbs at day 14, whereas levels were lower in embryonic tongue and higher, as was expected, in embryonic brain. To confirm the presence of GABA in the palate, it was analyzed in growing palate mesenchymal cells in primary and secondary cultures as well as in serum-free medium. In addition, GABA levels were compared in the SWV mouse strain; this strain exhibits a more efficient active uptake mechanism and diazepam produces a higher frequency of cleft palate in this strain than in AJ. SWV contained one and one-half to three times higher concentrations of GABA in excised palates and cultured palate cells than the AJ strain. Furthermore, when GABA levels in skin fibroblasts of the two strains were measured, SWV cells contained 2.7-fold greater GABA than AJ cells. The present results provide additional evidence for the role of GABA in palate development.     

Involvement of GABA in palate morphogenesis and its relation to diazepam teratogenesis in two mouse strains

Previous studies have indicated that serotonin and acetylcholine stimulate palate shelf reorientation. The present studies were undertaken to determine whether gamma-aminobutyric acid (GABA) functions as an inhibitory neurotransmitter in the palate and whether diazepam mimics GABA to inhibit shelf reorientation and cause cleft palate. First, it was shown that 10(-4) M GABA inhibits palate shelf reorientation in day 14.5 AJ embryos cultured for 2 hours. Anterior palate reorientation stimulated by 10(-5) M serotonin was decreased by GABA; 10(-5) M picrotoxin (GABA antagonist) stimulated anterior shelf reorientation and reversed the effect of GABA. Diazepam (10(-4) M) partially inhibited palate shelf reorientation and that stimulated by 10(-5) M serotonin. Diazepam (400 mg/kg) was administered to AJ mice at day 13.5 of gestation and embryos were cultured at day 14.5. The inhibition produced by diazepam was significantly reduced by 10(-5) M picrotoxin. The teratogenic effect of diazepam was compared with AJ and Swiss-Webster Vancouver (SWV) inbred strains. Diazepam produced greater clefting in SWV mice (57% net) than in the AJ (18% net) when compared to their water- and food-starved controls. The greater sensitivity of the SWV strain than the AJ strain to diazepam, as well as to GABA, was also observed in embryo culture. GABA (10(-5) M) markedly inhibited posterior palate reorientation and reversed the stimulation produced by bethanechol in SWV mice. The inhibitory effects of GABA on the posterior palate were partially reversed by picrotoxin. Furthermore, diazepam inhibited palate reorientation either when administered to the pregnant dam or added in embryo culture.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nystatin affects zinc uptake in human fibroblasts

The mechanism(s) by which zinc is transported into cells has not been identified. Since zinc uptake is inhibited by reducing the temperature, zinc uptake may depend on the movement of plasma membrane micoenvironments, such as endocytosis or potocytosis. We investigated the potential role of potocytosis in cellular zinc uptake by incubating normal and acrodermatitis enteropathica fibroblasts with nystatin, a sterol-binding drug previously shown to inhibit potocytosis. Zinc uptake was determined during initial rates of uptake (10 min) following incubation of the fibroblasts in 50 μg nystatin/mL or 0.1% dimethyl-sulfoxide for 10 min at 37°C. The cells were then incubated with 1 to 30 μM ⁶⁵zinc. Michaelis-Menten kinetics were observed for zinc uptake. Nystatin inhibited zinc uptake in both the normal and AE fibroblasts. Reduced cellular uptake of zinc was associated with its internalization, not its external binding. In normal fibroblasts, nystatin significantly reduced theK _m 56% and theV _max 69%. In the AE fibroblasts, nystatin treatment significantly reduced theV _max 59%, but did not significantly affect theK _m. The AE mutation alone affected theV _max for cellular zinc uptake. The control AE fibroblasts exhibited a 40% reduction inV _max compared to control normal fibroblasts. We conclude that nystatin exerts its effect on zinc uptake by reducing the velocity at which zinc traverses the cell membrane, possibly through potocytosis. Furthermore, the AE mutation also effects zinc transport by reducing zinc transport.     

Uptake of GABA by neuronal and nonneuronal cells in dispersed cell cultures of postnatal rat cerebellum

A study was made of the time course and kinetics of [³H]GABA uptake by dispersed cell cultures of postnatal rat cerebellum with and without neuronal cells. The properties of GABA neurons were calculated from the biochemical difference between the two types of cultures. It was found that for any given concentration of [³H]GABA, or any time up to 20 min, GABA neurons in cultures 21 days in vitro had an average velocity of uptake several orders of magnitude greater than that of nonneuronal cells. In addition, the apparent K_m values for GABA neurons for high and low affinity uptake were 0.33 × 10⁻⁶ M and 41.8 × 10⁻⁴ M, respectively. For nonneuronal cells, the apparent K_m for high affinity uptake was 0.29 × 10⁻⁶ M. The apparent V_max values for GABA neurons for high and low affinity uptake were 28.7 × 10⁻⁶ mol/g DNA/min and 151.5 mmol/g DNA/min, respectively. For nonneuronal cells, the apparent V_max for high affinity uptake was 0.06 × 10⁻⁶ mol/g DNA/min. No low affinity uptake system for nonneuronal cells could be detected after correcting the data for binding and diffusion. By substituting the apparent kinetic constants in the Michaelis-Menten equation, it was determined that for GABA concentrations of 5 × 10⁻⁹ M to 1 mM or higher over 99% of the GABA should be accumulated by GABA neurons, given equal access of all cells to the label. In addition, high affinity uptake of [³H]GABA by GABA neurons was completely blocked by treatment with 0.2 mM ouabain, whereas that by nonneuronal cells was only slightly decreased. Most (75–85%) of the [³H]GABA (4.4 × 10⁻⁶ M) uptake by both GABA neurons and nonneuronal cells was sodium and temperature dependent.     

Uptake and metabolism of GABA in astrocytes cultured from dissociated mouse brain hemispheres

Uptake kinetics and contents of GABA in cultured, normal (i.e. nontransformed) glia cells obtained from the brain hemispheres of newborn mice were measured together with the activity of the GABA transaminase. During three weeks of culturing the activity of the transaminase rose from a low neonatal value toward the level in the adult brain. The uptake kinetics indicated an unsaturable component together with an uptake following Michaelis-Menten kinetics. Both theK _m (40 M) and theV _max (0.350 nmol×min^–1×mg^–1 cell protein) were reasonably comparable to the corresponding values in brain slices, and theV _max was much higher than that reported for other glial preparations. The GABA content was low (<5 nmol/mg cell protein), which is in agreement with the high activity of the GABA transaminase.     

Sodium dependency of GABA uptake into glial cells in bullfrog sympathetic ganglia

The kinetics of sodium dependency of GABA uptake by satellite glial cells was studied in bullfrog sympathetic ganglia. GABA uptake followed simple Michaelis-Menten kinetics at all sodium concentrations tested. Increasing external sodium concentration increased bothK _m andV _max for GABA uptake, with an increase in theV _max/K _m ratio. The initial rate of uptake as a function of the sodium concentration exhibited sigmoid shape at 100 M GABA. Hill number was estimated to be 2.0. Removal of external potassium ion or 10 M ouabain reduced GABA uptake time-dependently. The effect of ouabain was potentiated by 100 M veratrine. These results suggest that at least two sodium ions are involved with the transport of one GABA molecule and that sodium concentration gradient across the plasma membrane is the main driving force for the transport of GABA. The essential sodium gradient may be maintained by Na⁺, K⁺-ATPase acting as an ion pump.     

Insulin effect on GABA uptake in astroglial primary cultures

Astroglial cultures from newborn mouse cerebral cortex contain [¹²⁵I]insulin binding sites. Binding was specific reversible, time dependent and reached equilibrium after 45 min. Insulin analogues compete for this [¹²⁵I]Insulin binding. Incubation of cerebral cortex astroglial cultures with insulin induced a time-and dose-dependent inhibition of the [³H]GABA high affinity uptake. A decrease in theV _max rather than, an effect on theK _m was observed. This effect was dose-dependent and effective at 10^–10 M. Autoradiographic observations on the cell monolayer showed the presence of two groups of cells: one which strongly takes up [³H]GABA and consist in smaller GFAP positive process-bearing cells and another group of much flatter and larger GFAP positive cells which uptake was lower. The smaller stellate cells were apparently the most sensitive to insulin effect. These results: 1) confirm the presence of insulin binding sites on astroglial primary cultures, 2) show an effect of insulin of [³H]GABA high affinity uptake of these cells; this effect being optimal on a stellate-like population of astrocytes, and 3) indicate, that insulin may interfere in neuromodulation through astroglial signals.     

KINETICS OF THE SODIUM-DEPENDENT TRANSPORT OF GAMMA-AMINOBUTYRIC ACID BY SYNAPTOSOMES

The kinetics of transport of gamma-aminobutyric acid [2,3-³H] by synaptosomes from rat brain was studied by means of a rapid filtration technique. The rate of uptake was proportional to the protein concentration over the range 0.05—0.2 mg of synaptosomal protein per ml. Although apparent allosteric kinetics were observed with sodium, transport followed simple saturation kinetics with respect to GABA and no heterotropic, cooperative effects of GABA on sodium on kinetics were observed. A minimum of three interacting sodium sites is suggested the basis of Hill plots of the sodium data. Both the apparent K_m and V_max for GABA were functions of the sodium ion concentration but the effect of sodium was considerably greater on V_max than on the apparent K_m The V_max for GABA was 1.1 ± 0.5 nmol.min^?1 mg^?1 of protein at 95 mm sodium and decreased to 12 per Cent of this value at 19 mm sodium. The apparent K^m for GABA increased from 4.0 ± 1.0 μm at 95 mm sodium to 8.4 ± 2.0 μm at 19 mm sodium. Potassium was a noncompetitive inhibitor with respect to GABA and did not affect the apparent cooperativity observed with sodium. These findings are discussed in terms of models of GABA transport.     

Effects of X-irradiation induced loss of cerebellar granule cells on the synaptosomal levels and the high affinity uptake of amino acids.

Abstract— Crude synaptosomal (P₂) preparations were obtained from the cerebella of rats in which the granule cell population had been selectively reduced by X-irradiation treatment and from the cerebella of control animals. In the P₂ fraction from control cerebella, the level of glutamate was greater than any other of the 5 amino acids measured and was 2-fold higher than taurine, which was present at the next highest level. The content of taurine was slightly higher than that found for aspartate and was 3-fold greater than that observed for GABA. Alanine and glycine were present in the lowest amounts. The levels of glutamate and aspartate were significantly (P < 0.05) lower by 25 and 15%, respectively, in the P₂ fraction isolated from the X-irradiated cerebella in comparison to control values. The content of taurine, GABA, glycine, and alanine were not changed by the X-irradiation treatment. The uptake of 1.0 μm -l -[³H]glutamate and l -[³H]aspartate was reduced approx 20% by X-irradiation treatment, whereas the uptake of 1.0 μm -[³H]GABA and [³H]taurine was unchanged. A more detailed kinetic analysis of l -[³H]glutamate uptake revealed there was a 20% decrease in the V_max value with X-irradiation treatment and no change in the apparent K_m value. In a second study, the uptake of l -[³H]glutamate, l -[³H]aspartate and [³H]GABA was measured using P₂ fractions obtained from the cerebella of rats in which the population of granule, stellate and basket cells had been reduced by X-irradiation treatment. The uptake of 1.0μm -l -[³H]glutamate, l -[³H]aspartate and [³H]GABA was significantly (P < 0.05) reduced to 57, 68, and 59%, respectively, of control values. A more detailed kinetic analysis of [³H]GABA uptake revealed no significant change in the apparent K_m and a 35% decrease in the V_max value. The data are discussed in terms of glutamate being the excitatory neurotransmitter released from granule cells and GABA being the inhibitory neurotransmitter released from basket cells.     

Transport of valproate and its effects on GABA uptake in astroglial primary culture

The antiepileptic drug Na⁺-valproate (VPA) is a broadspectrum anticonvulsant. It has been proposed to be involved in the inhibitory mechanisms of GABA-ergic systems. In this study, transport of the drug and possible influence on the GABA uptake were investigated in primary astroglial cell cultures from newborn rat cerebral cortex. The results show a Na⁺ and K⁺ independent high affinity uptake for VPA, withK _m andV _max not significantly different from those observed for the GABA uptake. In the presence of the drug, the K_m-value of the GABA uptake increased. The GABA uptake inhibitors guvacine, (RS)-Cis-4-OH-nipecotic acid and 4,5,6,7-tetrahydroisoxazolo (4,5-c) pyridin-3-ol (THPO) did not influence upon the uptake of VPA, suggesting a transport mechanism for the drug, separated from the GABA uptake carrier.     

Uptake of ethanolamine in neuronal and glial cell cultures

The uptake of radioactive ethanolamine has been studied in exclusively neuronal and glial cell cultures from dissociated cerebral hemispheres of chick embryos. Both cell types show saturable kinetics; neurons have an apparentK _m of 6.7 M,V _max 41.4 pmol mg prot.^–1 min^–1 and glial cells aK _m of 119.6 M,V _max 3,917 pmol mg prot^–1 min^–1. The lower affinity of the transport and the 100 fold increase inV _max observed in glial cells correlated with a more important accumulation of free ethanolamine found in glial cells and with a higher degree of phosphorylation of ethanolamine. The uptake appeared to be temperature and Na⁺ ions dependent but was not affected by CN^– or ouabain. Monomethyl-, dimethylethanolamine and choline were effective in inhibiting the uptake. Little or no effect was observed with serine, methionine, carnitine, alanine or glutamate.     

Changes in regional brain synaptosomal high affinity choline uptake during the development of hypertension in spontaneously hypertensive rats

The high-affinity uptake of choline (HAChU) by freshly prepared crude synaptosomal fractions was employed as relative measure of regional brain cholinergic activity. TheV _max for uptake as determined by the accumulation of a tracer amount of³H-choline in the presence of unlabeled choline (0.2–2 M) varied 6 fold depending upon the region examined (striatum>hypothalamus>medulla-pons). HAChU was hemicholinium-3-sensitive and linear at 37°C from 1 to 8 min in all brain regions. Respective brain synaptosomal fractions derived from adult (12 week old) spontaneously hypertensive (SH) rats and normotensive Wistar Kyoto (WK) rats revealed no difference in theV _max for HAChU from synaptosomes derived from the striatum of either strain. However, there was a significant increase in theV _max for HAChU measured from the medulla-pons of SH rats compared with WK rats. In older (22 weeks) rats, theV _max for HAChU was 78% greater than age-matched WK control rats. In addition, a highly significant correlation was found between resting systolic blood pressure and theV _max for HAChU both in the medulla-pons (r=0.76) and hypothalamus (r=0.48). That the increase in HAChU in SH rats was not a consequence of elevated pressure, was indicated by the lack of effect of prolonged i.v. infusion of pressor agents in normotensive rats on HAChU. These findings are consistent with a role for brain cholinergic neurons in the maintenance of hypertension in SH rats.     

Oxidative Metabolism of 4-Aminobutyrate by Rat Brain Mitochondria: Inhibition by Branched-chain Fatty Acid

Abstract: The oxidation of 4-aminobutyric acid (GABA) by nonsynaptosomal mitochondria isolated from rat forebrain and the inhibition of this metabolism by the branched-chain fatty acids 2-methyl-2-ethyl caproate (MEC) and 2, 2-dimethyl valerate (DMV) were studied. The rate of GABA oxidation, as measured by O₂ uptake, was determined in medium containing either 5 or 100 mM-[K⁺]. The apparent K_m for GABA was 1.16 ± 0.19 mM and the V_max in state 3 was 23.8 ± 5.5 ng-atoms O₂. min^?1. mg protein^?1 in 5 mM-[K⁺]. In a medium with 100 mM-[K⁺] the apparent K_m was 1.11 ± 0.17 mM and V_max was 47.4 ± 5.7 ng-atoms O₂. min^?1. mg protein^?1. The K_m for MEC was determined to be 0.58 ± 0.24 or 0.32 ± 0.08 mM, in 5 or 100 mM-[K⁺], respectively. For DMV, the K_i was 0.28 ± 0.05 or 0.34 ± 0.06 mM, in 5 or 100 mM-[K⁺] medium, respectively. The O₂ uptake of the mitochondria in the presence of GABA was coupled to the formation of glutamate and aspartate; the ratio of oxygen uptake to the rate of amino acid formation was close to the theoretical value of 3. Neither the [K²] nor any of the above inhibitors had any effect on this ratio. The metabolism of exogenous succinic semialdehyde (SSA) by these same mitochondria was also examined. The V_max for utilization of oxygen in the presence of SSA was much greater than that found with exogenously added GABA, indicating that the capacity for GABA oxidation by these mitochondria is not limited by SSA dehydrogenase. In addition, the branched-chain fatty acids did not inhibit the metabolism of exogenously added SSA. Thus, the inhibitors examined apparently act by competitively inhibiting the GABA transaminase system of the mitochondria.     

Developmental changes in high-affinity uptake of GABA by cultured neurons

High-affinity uptake of [³H]-aminobutyric acid (GABA) was studied in cultures of neonatal rat cortical neurons grown on pre-formed monolayers of non-neuronal (glial) cells. Both the maximum rate (V _max) and, to a smaller extent, theK _m of [³H]GABA uptake increased with time. In addition, in parallel with these changes, 2,4-diaminobutyric acid and cis-3-aminocyclohexane-1-carboxylic acid (ACHC), compounds which are considered typical substrate/inhibitors of GABA uptake in neurons, became progressively stronger inhibitors of [³H]GABA uptake. Consequently, the present results may mean that the studies using uptake, of [³H]GABA, [³H]ACHC, or [³H]DABA as a specific marker for GABAergic neurons differentiating during the ontogenetic development of the central nervous system may have to be interpreted with caution.     

Amino acid and monoamine transport in primary astroglial cultures from defined brain regions

The uptake ofl-[³H]glutamate,l-[³H]aspartate, -[³H]aminobutric acid (GABA), [³H]dopamine,dl-[³H]norepinephrine and [³H]5-hydroxytryptamine (5-HT) was studied in astrocytes cultured from the cerebral cortex, striatum and brain stem of newborn rat and grown for 2 weeks in primary cultures. The astrocytes exhibited a high-affinityl-glutamate uptake withK _m values ranging from 11 to 110 M.V _max values were 4.5 in cerebral cortex, 39.1 in striatum, and 0.4 in brain stem, nmol per mg cell protein per min. There was a less prominent high-affinity uptake ofl-aspartate withK _m values from 88 to 187 M.V _max values were 7.4 in cerebral cortex, 37.1 in striatum, and 3.1 in brain stem, nmol per mg cell protein per min. The high-affinity GABA uptake exhibitedK _m values ranging from 5 to 17 M andV _max values were 0.01 for cerebral cortex, 0.04 for striatum, and 0.1 for brain stem, nmol per mg cell protein per min. No high-affinity, high-capacity uptake was found for the monoamines. The results demonstrate a heterogeneity among the astroglial cells cultivated from the different brain regions concerning the uptake capacity of amino acid neurotransmitters. Furthermore, amino acid transmitters and monoamines are taken up by the cells in different ways.     

Characterization of the Lactose Transport System in the Strain Bifidobacterium bifidum DSM 20082

Lactose was fermented but not assimilated by the strain Bifidobacterium bifidum DSM 20082. The sugar uptake was measured with lactose ¹⁴C. K _m and V _max values were respectively 2.6 mM and 12.11 nmol/min/mg of cell protein. The lactose transport system and the β-D-galactosidase were stimulated when the cells were grown with lactose, but isopropyl-β-D-thiogalactopyranoside had no effect. Lactose uptake was inhibited by compounds which interfered with proton and metal ionophore. Na⁺, Li⁺, or K⁺ did not affect incorporation of lactose. Furthermore, the lactose uptake decreased when an inhibitor of ATP synthesis was used. From the results of this study, the strain contained an active lactose transport system, probably a proton symport as described for Escherichia coli but with a different regulation system.     

THE UPTAKE OF [3H]GABA BY SLICES OF RAT CEREBRAL CORTEX

—A rapid accumulation of [³H]GABA occurs in slices of rat cerebral cortex incubated at 25° or 37° in a medium containing [³H]GABA. Tissue medium ratios of almost 100:1 are attained after a 60 min incubation at 25°. At the same temperature no labelled metabolites of GABA were found in the tissue or the medium. The process responsible for [³H]GABA uptake has many of the properties of an active transport mechanism: it is temperature sensitive, requires the presence of sodium ions in the external medium, is inhibited by dinitrophenol and ouabain, and shows saturation kinetics. The estimated K_m value for GABA is 2·2 × 10^?5m , and V_max is 0·115 μmoles/min/g cortex. There is only negligible efflux of the accumulated [³H]GABA when cortical slices are exposed to a GABA-free medium. [³H]GABA uptake was not affected by the presence of large molar excesses of glycine, l -glutamic acid, l -aspartic acid, or β-aminobutyrate, but was inhibited in the presence of l -alanine, l -histidine, β-hydroxy-GABA and β-guanidinopropionate. It is suggested that the GABA uptake system may represent a possible mechanism for the inactivation of GABA or some related substance at inhibitory synapses in the cortex.     

SHORT‐TERM TEMPORAL VARIABILITY OF AMMONIUM AND UREA UPTAKE BY ALEXANDRIUM CATENELLA (DINOPHYTA) IN CULTURES1

In batch cultures of four Mediterranean strains (from France, Italy, and Spain) of Alexandrium catenella (Whedon et Kof.) Balech growing on a daily light cycle, ammonium and urea uptake were estimated by the ¹⁵N tracer technique. Ammonium uptake could be described by Michaelis–Menten kinetics along a substrate gradient of 0.1–10 μgat N · L^?1 for the four strains, while two different patterns were observed for urea uptake with Michaelis–Menten kinetics for one strain and linear kinetics for the others. In all cases, an increase in uptake rates with time was noted over the daylight period. This trend led to a net increase in the maximum uptake rate (V_max; for saturable kinetics) and in the initial slope α. For ammonium, V_max increased by a factor of 2–10 depending on the strain, and, for urea, the maximal uptake rates measured increased by a factor of 2–18. Temporal variations of half‐saturation constants (K_s) for both nutrients did not show a clear trend. Increases in V_max and α showed an acclimation of the cells’ uptake system over time to a N pulse, which may be explained by the light periodicity. For two strains, extensive ammonium release was observed during urea assimilation. This mechanism removes urea from the medium, so it is no longer available to other potential competitors, but supplies N back to the medium in the form of ammonium. From a methodological point of view, the phenomenon leads to considerable underestimates of the contribution of urea to phytoplankton growth.     

Characterization of separated cell types from developing rat cerebellum

The characteristics of [³H]GABA transport were investigated in preparations greatly enriched in different classes of cerebellar cells. In contrast to observations in situ, isolated Purkinje cells readily accumulated [³H]GABA. In comparison with astrocytes, theV _max of the high-affinity uptake process was sixfold higher (0.31 vs. 0.05 nmol/min/10⁶ cells) and the apparentK _t twofold greater (2 vs. 1 M). In contrast to these cell types, uptake was very low in granule cell-enriched preparations.cis-1,3-Aminocyclohexane carboxylic acid was a potent inhibitor of [³H]GABA uptake by the Purkinje cells and a weak blocker in astrocytes, while the converse was the case for -alanine. Diaminobutyric acid strongly inhibited uptake in both cell types. [³H]GABA transport was Na⁺ dependent in both cell classes. However, veratridine and ouabain selectively blocked [³H]GABA accumulation in the Purkinje cells, which were also more sensitive than the astrocytes to the glycolysis inhibitor, NaF. The results indicated, therefore, marked differences between Purkinje cells and astrocytes in the properties of both the [³H]GABA transport systems and the underlying metabolic processes.     

Nutrient uptake and alkaline phosphatase (ec 3:1:3:1) activity of emiliania huxleyi (PRYMNESIOPHYCEAE) during growth under n and p limitation in continuous cultures

Emiliania huxleyi (strain L) expressed an exceptional P assimilation capability. Under P limitation, the minimum cell P content was 2.6 fmol P·cell^?1, and cell N remained constant at all growth rates at 100 fmol N·cell^?1. Both, calcification of cells and the induction of the phosphate uptake system were inversely correlated with growth rate. The highest (cellular P based) maximum phosphate uptake rate (V_max^P) was 1400 times (i.e. 8.9 h^?1) higher than the actual uptake rate. The affinity of the P‐uptake system (dV/dS) was 19.8 L·μmol^?1·h^?1 at μ = 0.14 d^?1. This is the highest value ever reported for a phytoplankton species. V_max and dV/dS for phosphate uptake were 48% and 15% lower in the dark than in the light at the lowest growth rates. The half‐saturation constant for growth was 1.1 nM. The coefficient for luxury phosphate uptake (Q_maxt/Q_min) was 31. Under P limitation, E. huxleyi expressed two different types of alkaline phosphatase (APase) enzyme kinetics. One type was synthesized constitutively and possessed a V_max and half‐saturation constant of 43 fmol MFP·cell^?1·h^?1 and 1.9 μM, respectively. The other, inducible type of APase expressed its highest activity at the lowest growth rates, with a V_max and half‐saturation constant of 190 fmol MFP·cell^?1·h^?1 and 12.2 μM, respectively. Both APase systems were located in a lipid membrane close to the cell wall. Under N‐limiting growth conditions, the minimum N quotum was 43 fmol N·cell^?1. The highest value for the cell N‐specific maximum nitrate uptake rate (V_max^N) was 0.075 h^?1; for the affinity of nitrate uptake, 0.37 L·μmol^?1·h^?1. The uptake rate of nitrate in the dark was 70% lower than in the light. N‐limited cells were smaller than P‐limited cells and contained 50% less organic and inorganic carbon. In comparison with other algae, E. huxleyi is a poor competitor for nitrate under N limitation. As a consequence of its high affinity for inorganic phosphate, and the presence of two different types of APase in terms of kinetics, E. huxleyi is expected to perform well in P‐controlled ecosystems.