Utilization of adenine and guanine as nitrogen sources by Chlamydomonas reinhardtii cells

Chlamydomonas reinhardtü Dangeard, adenine or guanine can be used as the sole nitrogen source for growth by means of an inducible system which is repressed by ammonia. Cells grown on either adenine or guanine were able to take up both purines, although the adenine uptake rate was always about 40% of the guanine uptake rate. Both adenine and guanine were taken up by an inducible system(s) exhibiting hyperbolic kinetics with identical apparent A, values of 3-2 mmol m^?3 for adenine and 3-2mmol m^?3 for guanine. Adenine and guanine utilization depended on pH, with similar optimal pH values of 7·3 and 7·4, respectively. Adenine and guanine each acted as a competitive inhibitor of the other's uptake, and their utilization was also inhibited by hypoxanthine, xanthine and urate. Inhibition of adenine uptake by guanine and hypoxanthine was competitive, with A′, values of 5·5 and 1. 6 mmol m^?3 respectively. Guanine uptake was also inhibited competitively by adenine (K₁= 1·3mmol m^?3) and hypoxanthine (K₁= 3. 3 mmol m^?3). Utilization of both adenine and guanine was inhibited by cyanide, azide, 3-(3,4-dichlorophenyl)-1,1-dimethyl urea, 2,4-dinitrophenol and carbonylcyanide m-chlorophenylhydrazone, and was also sensitive to p-hydroxymercuribenzoate and N-ethyl-maleimide. On the basis of these results, taken together, the possibility that adenine and guanine are translocated into Chlamydomonas by a common system is discussed.     

Kinetics of chloride transport in the cyanobacterium Synechococcus R-2 (Anacystis nidulans, S. leopoliensis) PCC 7942

The kinetics of the light-driven Cl^? uptake pump of Synechococcus R-2 (PCC 7942) were investigated. The kinetics of Cl^? uptake were measured in BG-11 medium (pH_o, 7·5; [K⁺]_o, 0·35 mol m^?3; [Na⁺]_o, 18 mol m^?3; [Cl^?]_o, 0·508 mol m^?3) or modified media based on the above. Net³⁶Cl^? fluxes (?Cl^?_o,i) followed Michaelis-Menten kinetics and were stimulated by Na⁺ [18 mol m^?3 Na⁺ BG-11 ?Cl^?_max= 3·29±0·60 (49) nmol m^?2 s^?1 versus Na⁺-free BG-11 ?Cl^?_max= 1·02±0·13 (54) nmol m^?2 s^?1] but the Km was not significantly different in the presence or absence of Na⁺ at pH_o 10; the Km was lower, but not affected by the presence or absence of Na⁺ [Km = 22·3±3·54 (20) mmol m^?3]. Na⁺ is a non-competitive activator of net ?Cl^?_o,i. High [K⁺]_o (18 mol m^?3) did not stimulate net ?Cl^?_o,i or change the Km in Na⁺-free medium. High [K⁺]_o (18 mol m^?3) added to Na⁺ BG-11 medium decreased net ?Cl^?_o,i [18 mol m^?3K⁺ BG-11; ?Cl^?_max= 2·50±0·32 (20) nmol m^?2 s^?1 versus BG-11 medium; ?Cl^?_max= 3·35±0·56 (20) nmol m^?2 s^?1] but did not affect the Km 55·8±8·100 (40) mmol m^?3]. Na⁺-stimulation of net ?Cl^?_o,i followed Michaelis-Menten kinetics up to 2–5 mol m^?3 [Na⁺]_o but higher concentrations were inhibitory. The Km for Na⁺-stimulation of net ?Cl^?_o,i [K_1/2(Na⁺)] was different at 47 mmol m^?3 [Cl^?]_o (K_1/2[Na⁺] = 123±27 (37) mmol m^?3]. Li⁺ was only about one-third as effective as Na⁺ in stimulating Cl^? uptake but the activation constant was similar [K_1/2(Li⁺) = 88±46 (16) mmol m^?3]. Br^? was a competitive inhibitor of Cl^? uptake. The inhibition constant (Ki) was not significantly different in the presence and absence of Na⁺. The overall Ki was 297±23 (45) mmol m^?3. The discrimination ratio of Cl^? over Br^? (δCl^?/δBr^?) was 6·38±0·92 (df = 147). Synechococcus has a single Na⁺-stimulated Cl^? pump because the Km of the Cl^? transporter and its discrimination between Cl^? and Br^? are not significantly different in the presence and absence of Na⁺. The Cl^? pump is probably driven by ATP.     

The CO2‐concentrating mechanism in the bloom‐forming cyanobacterium Microcystis aeruginosa (Cyanophyceae) and effects of UVB radiation on its operation1

The bloom‐forming cyanobacterium Microcystis aeruginosa (Kütz.) Kütz. 854 was cultured with 1.05 W · m^?2 ultraviolet‐B radiation (UVBR) for 3 h every day, and the CO₂‐concentrating mechanism (CCM) within this species as well as effects of UVBR on its operation were investigated. Microcystis aeruginosa 854 possessed at least three inorganic carbon transport systems and could utilize external HCO₃^? and CO₂ for its photosynthesis. The maximum photosynthetic rate was approximately the same, but the apparent affinity for dissolved inorganic carbon was significantly decreased from 74.7 μmol · L^?1 in the control to 34.7 μmol · L^?1 in UVBR‐treated cells. At 150 μmol · L^?1 KHCO₃ and pH 8.0, Na⁺‐dependent HCO₃^? transport contributed 43.4%–40.2% to the photosynthesis in the control and 34.5%–31.9% in UVBR‐treated cells. However, the contribution of Na⁺‐independent HCO₃^? transport increased from 8.7% in the control to 18.3% in UVBR‐treated cells. The contribution of CO₂‐uptake systems showed little difference: 47.9%–51.0% in the control and 49.8%–47.2% in UVBR‐treated cells. Thus, the rate of total inorganic carbon uptake was only marginally affected, although UVBR had a differential effect on various inorganic carbon transporters. However, the number of carboxysomes in UVBR‐treated cells was significantly decreased compared to that in the control.     

Adaptation to phosphate deprivation in osteoblast-like cells

The rat osteosarcoma cell line UMR-106–01 has an osteoblast-like phenotype. When grown in monolyer culture these cells transport inroganic phosphate and L-alanine via Na⁺-dependent transport systems. Exposure of these cells to a low phosphate medium for 4 h produced a 60–70 per cent increase in Na⁺-dependent phosphate uptake compared to control cells maintained in medium with a normal phosphate concentration. In contrast, Na⁺-dependent alanine uptake and Na⁺-independent phosphate uptake were not changed during phosphate deprivation. The increased phosphate uptake was due, in part, to an increased V_max and was blocked completely by pretreatment with cycloheximide (70 μM). In these cells recovery of intracellular pH after acidification with NH₄Cl is due primarily to the Na⁺/H⁺ exchange system. The rate of this recovery process, monitored with a pH sensitive indicator (BCECF), was decreased by more than 50 per cent in phosphate-deprived cells compared to controls indicating that Na⁺/H⁺ exchange was inhibited during phosphate deprivation.     

DIATOM MINERALIZATION OK SILICIC ACID. I Si(OH)4 TRANSPORT CHARACTERISTICS IN NAVICULA PELLICULOSA

Silicic acid transport was studied in the photosynthetic diatom Navicula pelliculosa (Bréb.) Hilse using [⁶⁸Ge] germanic acid (⁶⁸Ge(OH)₄) as a tracer of silicic acid (Si(OH)₄). The initial uptake rate of Si(OH)₄ was dependent on cell number, pH, temperature, light and was promoted by certain monovalent cations in the medium. Na⁺ was more effective than K⁺, whereas Li⁺ and NH⁺₄ were ineffective at promoting uptake. Uncouplers and inhibitors of oxidative phosphorylation and of photophosphorylation reduced uptake by 40–99% of control values. Uptake was also especially sensitive to the sulfhydryl blocking agents at 10^?5 M and to the ionophorous compound valinomycin (10^?7 M) which inhibited uptake by 82%. The Si(OH)₄ transport system displayed Michaelis-Menten-type saturation kinetics with kinetic parameters of K_S= 4.4 p. mol Si(OH)₄· 1^?1, V_max= 334 pmol Si(OH)₄· 10⁶ cells^?1· min^?1. Calculations of the acid soluble silicic acid pool size based on 60 s uptake at 20 μM Si(OH)₄ suggested that intracellular levels of Si could reach 20 mM and as much as 5 mM could exist as free silicic acid, representing maintenance of a 250-fold concentration gradient compared with the medium. Efflux from preloaded cells was dependent on temperature and the Si(OH)₄ concentration of the external medium. In the presence of 100 μMM “cold” Si(OH)₄, approximately 30% of the Si(OH)₄ in preloaded cells was exchanged in 20 min. The initial uptake rate of Si(OH)₄ in logarithmic phase cells was constant, but the uptake rate increased in a linear fashion for 6 h in stationary phase cells. These results suggest that the first step in silica mineralization by diatoms is the active transmembrane transport of Si(OH)₄ by an energy dependent, saturable, membrane-carrier mechanism which requires the monovalent cations Na⁺ and K⁺ and is sensitive to sulfhydryl blocking agents. Silicic acid transport activity also appears to be regulated during different growth stages of the diatom.     

Transport Pathways for Therapeutic Concentrations of Lithium in Rat Liver

Although both amiloride- and phloretin-sensitive Na⁺/Li⁺ exchange activities have been reported in mammalian red blood cells, it is still unclear whether or not the two are mediated by the same pathway. Also, little is known about the relative contribution of these transport mechanisms to the entry of therapeutic concentrations of Li⁺ (0.2–2 mm) into cells other than erythrocytes. Here, we describe characteristics of these transport systems in rat isolated hepatocytes in suspension. Uptake of Li⁺ by hepatocytes, preloaded with Na⁺ and incubated in the presence of ouabain and bumetanide, comprised three components. (a) An amiloride-sensitive component, with apparent K _m 1.2 mm Li⁺, V _max 40 μmol · (kg dry wt · min)⁻¹, showed increased activity at low intracellular pH. The relationship of this component to the concentration of intracellular H⁺ was curvilinear suggesting a modifier role of [H⁺] _i . This system persisted in Na⁺-depleted cells, although with apparent K _m 3.8 mm. (b) A phloretin-sensitive component, with K _m 1.2 mm, V _max 21 μmol · (kg · min)⁻¹, was unaffected by pH but was inactive in Na⁺-depleted cells. Phloretin inhibited Li⁺ uptake and Na⁺ efflux in parallel. (c) A residual uptake increased linearly with the external Li⁺ concentration and represented an increasing proportion of the total uptake. The results strongly suggest that the amiloride-sensitive and the phloretin-sensitive Li⁺ uptake in rat liver are mediated by two separate pathways which can be distinguished by their sensitivity to inhibitors and intracellular [H⁺]. Received: 8 April 1999/Revised: 19 July 1999     

The cyanobacterium Synechococcus R-2 (Anacystis nidulans, S. leopoliensis) PCC 7942 has a sodium-dependent chloride transporter

Synechococcus R-2 (PCC 7942) actively accumulated Cl^? in the light and dark, under control conditions (BG-11 media: pH_o, 7·5; [Na⁺]_o, 18 mol m^?3; [Cl^?]_o, 0·508 molm^?3). In BG-11 medium [Cl^?], was 17·2±0·848 mol m^?3 (light), electrochemical potential of Cl^? (ΔμCl^?_i,o) =+211±2mV; [Cl^?]_i= 1·24±0·11 mol m^?3(dark), ΔμCl^?_i,o=+133±4mV. Cl^? fluxes, but not permeabilities, were much higher in the light: ?Cl^?_i,o= 4·01±5·4 nmol m^?2 s^?1, ^PCl^?_i,o= 47±5pm s^?1 (light); ?Cl^?_i,o= 0·395±0·071 nmol m^?2 s^?1, _PCl^?_i,o= 69±14 pm s^?1 (dark). Chloride fluxes are inhibited by acid pH_o (pH_o 5; ?Cl^?_i,o= 0·14±0·04 nmol m^?2 s^?1); optimal at pH_o 7·5 and not strongly inhibited by alkaline pH_o (pH_o 10; ?Cl^?1_i,o= 1·7±0·14 nmol m^?2 s^?1). A Cl^?_in/2H⁺_in coporter could not account for the accumulation of Cl^? alkaline pH_o. Permeability of Cl^? is very low, below 100pm s^?1 under all conditions used, and appears to be maximal at pH_o 7·5 (50–70 pm s^?1) and minimal in acid pH_o (20pm s^?1). DCCD (dicyclohexyl-carbodiimide) inhibited ?Cl^?_i,o in the light about 75% and [Cl^?]_i fell to 2·2±0·26 (4) mol m^?3. Valinomycin had no effect but monensin severely inhibited Cl^? uptake ([Cl^?]_i= 1·02±0·32 mol m^?3; ?Cl^?_i,o= 0·20±0·1 nmol m^?2 s^?1). Vanadate (200 mmol m^?3) accelerated the Cl^? flux (?Cl^?_i,o= 5·28±0·64 nmol m^?2 s^?1) but slightly decreased accumulation of Cl^? ([Cl^?], = 13·9±1·3 mol m^?3) in BG-11 medium but had no significant effect in Na⁺-free media. DCMU (dichlorophenyldimethylurea) did not reduce [Cl^?], or ?Cl^?_i,o to that found in the dark ([Cl^?]_i= 8·41±0·76 mol m^?3; ?Cl^?_i,o= 2·06±0·36 nmol m^?2 s^?1). Synechococcus also actively accumulated Cl^? in Na⁺-free media, [Cl^?]_i was lower but ΔΨ_i,o hyperpolarized in Na⁺-free media and so the ΔμCl^?_i,o was little changed ([Cl^?]_i= 7·98±0·698 mol m^?3; ΔμCl^?_i,o=+203±3 mV). Net Cl^? uptake was stimulated by Na⁺; Li⁺ acted as a partial analogue for Na⁺. Synechococcus has a Na⁺ activated Cl^? transporter which is probably a primary 2Cl^?/ATP pump. The Cl^? pump is voltage sensitive. ΔμCl^?_i,o is directly proportional to ΔΨ_i,o(P»0·01%): ΔμCl^?_i,o= -1·487 (±0·102) ×ΔΨ_i,o, r= -0·983, n= 31. The ΔμCl^?_i,o increased (more positive) as the Δμ_i,o became more negative. The ΔμCl^?_i,o has no known function, but might provide a driving force for the uptake of micronutrients.     

Endothelin 1 Stimulates Na+,K+-ATPase and Na+-K+-Cl− Cotransport Through ETA Receptors and Protein Kinase C-Dependent Pathway in Cerebral Capillary Endothelium

Abstract: The effect of endothelins (ET-1 and ET-3) on ⁸⁶Rb⁺ uptake as a measure of K⁺ uptake was investigated in cultured rat brain capillary endothelium. ET-1 or ET-3 dose-dependently enhanced K⁺ uptake (EC₅₀ = 0.60 ± 0.15 and 21.5 ± 4.1 nM, respectively), which was inhibited by the selective ET_A receptor antagonist BQ 123 (cyclo-d -Trp-d -Asp-Pro-d -Val-Leu). Neither the selective ET_B agonists IRL 1620 [N-succinyl-(Glu⁹,-Ala^11,15)-ET-1] and sarafotoxin S6c, nor the ET_B receptor antagonist IRL 1038 [(Cys¹¹,Cys¹⁵)-ET-1] had any effect on K⁺ uptake. Ouabain (inhibitor of Na⁺,K⁺-ATPase) and bumetanide (inhibitor of Na⁺-K⁺-Cl^? cotransport) reduced (up to 40% and up to 70%, respectively) the ET-1-stimulated K⁺ uptake. Complete inhibition was seen with both agents. Phorbol 12-myristate 13-acetate (PMA), activator of protein kinase C (PKC), stimulated Na⁺,K⁺-ATPase and Na⁺-K⁺-Cl^? cotransport. ET-1-but not PMA-stimulated K⁺ uptake was inhibited by 5-(N-ethyl-N-isopropyl)amiloride (inhibitor of Na⁺/H⁺ exchange system), suggesting a linkage of Na⁺/H⁺ exchange with ET-1-stimulated Na⁺,K⁺-ATPase and Na⁺-K⁺-Cl^? cotransport activity that is not mediated by PKC.     

Some features of hydrogen (ion) secretion by the frog skin

We have studied the movements of H⁺ from the in vitro frog skin into the outside solution because it has been suggested that the movement of sodium from the outside solution into the skin may result from the forced exchange of Na⁺ by H⁺.Our main observations can be summarized as follows: (a) Hydrogen moves from the skin into the outside solution at a rate of 0.04 μequiv · cm^?2 · h^?1 while Na⁺ influx had a value of 0.49 μequiv · cm^?2 · h^?1. (b) The rate of H⁺ secretion is not significantly affected by substituting the Na⁺ in the outside solution by K⁺ nor by inhibiting Na⁺ influx with amiloride (5 · 10^?5 M). (c) Acetazolamide (5 · 10^?3 M) blocked H⁺ secretion without altering the potential difference across the skin. (d) The rate of H⁺ production is not underestimated because it may have been neutralized by HCO₃^? secreted into the outside solution in exchange for Cl^?. Substituting all the Cl^? by SO₄^2? in the outside solutions does not result in an increase in the rate of H⁺ production. (e) The steady-state rate of H⁺ secretion is not affected by large changes in electrochemical potential gradients for H⁺. Neither abolishing the potential difference across the skin nor a 10-fold change in H⁺ concentration in the outside solution affected significantly the steady-state rate of H⁺ secretion. (f) The H⁺ secretion was abolished by the metabolic inhibitors dinitrophenol (1 · 10^?4 M) and Antimycin A (1.5 · 10^?6 M) which also markedly reduced the potential difference across the skin.Observations (a), (b), and (c) suggest that H⁺ and Na⁺ movements across the outer border of the isolated frog skin are not coupled. The ratio of Na⁺ to H⁺ movements is very different from unity and Na⁺ movements can be abolished without any effects on H⁺ secretion and conversely H⁺ movements can be abolished without interruption of Na⁺ uptake.A second conclusion suggested by these results is that the H⁺ secretion does not result from movement of H⁺ following its electrochemical potential gradient since that rate of secretion is not affected by marked changes in either potential or [H⁺]. Furthermore, the effects of metabolic inhibitors suggest that H⁺ secretion requires the expenditure of energy by the cell.     

Acclimation to humic substances prevents whole body sodium loss and stimulates branchial calcium uptake capacity in cardinal tetras Paracheirodon axelrodi (Schultz) subjected to extremely low pH

Kinetics of sodium (Na⁺) and calcium (Ca²⁺) uptake were studied in cardinal tetras Paracheirodon axelrodi acclimated to humic substances (HS, 35 mg C l^?1) and low pH (pH 3·72), parallel to analysis of whole body Na⁺ and Ca²⁺ content. This species had a high uptake capacity (J_max) for both Na⁺ and Ca²⁺ in soft, ion‐poor water. The affinity constant (K_m) did not vary significantly among treatments for either Na⁺ or Ca²⁺. J_max Na⁺ increased 30% in fish acclimated to HS for 5 weeks. Acclimation to low pH had no effect on J_max Na⁺ but this treatment was associated with a 32% decrease on whole body Na⁺ content, suggesting that fish were unable to compensate for the increased Na⁺ loss induced by extreme acidity. Exposure of fish to HS + low pH, the treatment most closely approximating to the conditions experienced by the species in its native environment, resulted in an increase in whole body Na⁺ by 31% relative to acclimation to low pH alone. J_max Ca²⁺ in cardinal tetras was high relative to that documented in other freshwater species acclimated to soft water (J_max= 30 nmol g^?1 h^?1). Prolonged exposure of fish to pH 3·72 inhibited J_max Ca²⁺ by 53%, although whole body Ca²⁺ content remained unchanged relative to control. Acclimation of fish to HS + low pH resulted in an increase of J_max Ca²⁺ by 166% relative to low pH alone. Collectively, these results suggest that HS protect cardinal tetras acclimated to soft, acidic waters by preventing excessive Na⁺ loss (as indicated by whole body Na⁺ content) and by stimulating Ca²⁺ uptake (as indicated by increased J_max Ca²⁺) to ensure proper homeostasis.     

UPTAKE OF 3-O-METHYL-d-GLUCOSE INTO CULTURED HUMAN GLIOMA CELLS

Human glioma cells (138 MG) were found to take up 3-O-methyl-d -glucose (3-OMG) by a saturable low affinity transport system with a K_m of 20 mm and a V_max of 500 nmol/mg protein/min. About 20 per cent of the total uptake was due to passive diffusion. d -Glucose was a competitive inhibitor with a K_i of 10 mm . Follow-up experiments indicated that the same transport mechanism is involved in the uptake of n-glucose and 3-OMG. Phloretin (0·02 mm ) and cytochalasin B (0·002 mm ) strongly inhibited the uptake of 3-OMG, whereas phlorizin (0·02 mm ), ouabain (0·1 mm ), NaCN (0·5 mm ) and iodoacetic acid (1·0 mm ) had no effect. The data suggest that 3-OMG and d -glucose enter 138 MG cells mainly by a Na⁺-independent passive carrier-mediated transport system. Serum-deprivation doubled the population doubling time (T_d) without affecting the total uptake of 3-OMG. An increase in the non-specific (diffusional) uptake was balanced by a decrease in the specific (carrier-medíated) uptake. After addition of dibutyryl cyclic AMP (dbcAMP, 0·25 mm ) the cells attained a morphology characteristic of differentiated glia cells. T_d was maintained unchanged. The non-specific uptake of 3-OMG was not affected in cells grown in serum-containing medium plus dbcAMP, whereas the specific uptake increased by 40 per cent and there-fore also the total uptake. Similar, but more pronounced, changes were observed if serum-deprived cells were treated with dbcAMP.     

Sodium/potassium selectivity and pleiotropy in stl2, a highly salt-tolerant mutation of Ceratopteris richardii

The roles of Na⁺ and K⁺ (Rb⁺) uptake were further studied in a NaCl-tolerant strain of Ceratopteris richardii containing the stl2 mutation by direct comparison with the wild-type strain. In addition to Na⁺ tolerance, stl2 also confers tolerance to Mg²⁺ and sensitivity to K⁺. In addition to higher K⁺ (Rb⁺) uptake at concentrations commonly associated with low-affinity K⁺ transport, stl2 maintained higher uptake down to 0·1 mol m^–3 Rb⁺. Up to a 25-fold excess of Na⁺ had little effect in either genotype on K⁺ (Rb⁺) uptake at low concentrations, i.e. 0·2 and 0·5 mol m^–3 RbCl. Pretreatment with K⁺ (20 mol m^–3) inhibited uptake of K⁺ (Rb⁺) in the wild type, whereas concurrent inclusion of K⁺ inhibited uptake of Rb⁺ more in stl2. In the absence of K⁺, Na⁺ uptake (0·01–60 mol m^–3) was nearly identical in the wild type and stl2. K⁺ inhibited Na⁺ uptake more effectively in stl2 than the wild type, especially at 60 mol m^–3 Na⁺. Greater inhibition of K⁺ uptake in stl2 occurred with MgCl₂ or TEA (tetraethylammonium chloride) preincubation or with simultaneous inclusion of Al³⁺ (Al₂SO₄). The higher effective velocity of K⁺ uptake at a wide range of concentrations and the enhanced selectivity for K⁺ and against Na⁺ contribute to the preservation of higher cytosolic K⁺ and lower Na⁺ under salinity stress.     

High-Affinity Uptake of [3H]Norepinephrine by Primary Astrocyte Cultures and Its Inhibition by Tricyclic Antidepressants

Abstract: Primary astrocyte cultures from neonatal rat brains show uptake of [³H]norepinephrine ([³H]NE). This uptake has a high-affinity component with an apparent K_m of approximately 3 × 10^?7 M. At 10^?7 M [³H]NE both the initial rate of uptake and steady-state content of [³H]NE is inhibited by up to 95% by omission of external Na⁺. The Na⁺-dependent component of this uptake is totally inhibited by the tricyclic antidepressants desipramine (DMI) and amitryptyline with IC₅₀ values of 2 × 10^?9 and 4 × 10^?8 M, respectively. Inhibition of [³H]NE uptake by DMI shows competitive kinetics. These characteristics are essentially identical to those found for high-affinity uptake of NE in total membrane or synaptosome fractions from rodent brains and suggests that such uptake in neural tissue is not exclusively neuronal.     

15N MEASUREMENTS OF AMMONIUM AND NITRATE UPTAKE BY ULVA FENESTRATA (CHLOROPHYTA) AND GRACILARIA PACIFICA (RHODOPHYTA): COMPARISON OF NET NUTRIENT DISAPPEARANCE,RELEASE OF AMMONIUM AND NITRATE,AND 15N ACCUMULATION IN ALGAL TISSUE 1

Ammonium and nitrate uptake rates in the macroalgae Ulva fenestrata (Postels and Ruprecht) (Chlorophyta) and Gracilaria pacifica (Abbott) (Rhodophyta) were determined by ¹⁵N accumulation in algal tissue and by disappearance of nutrient from the medium in long‐term (4–13 days) incubations. Nitrogen‐rich algae (total nitrogen> 4% dry weight [dw]) were used to detect isotope dilution by release of inorganic unlabeled N from algal thalli. Uptake of NH₄ ⁺ was similar for the two macroalgae, and the highest rates were observed on the first day of incubation (45 μmol N·g dw ^{? 1}·h ^{? 1} in U. fenestrata and 32 μmol N·g dw ^{? 1}·h ^{? 1} in G. pacifica). A significant isotope dilution (from 10 to 7.9 atom % enrichment) occurred in U. fenestrata cultures during the first day, corresponding to a NH₄ ⁺ release rate of 11 μmol N·g dw ^{? 1}·h ^{? 1}. Little isotope dilution occurred in the other algal cultures. Concurrently to net NH₄ ⁺ uptake, we observed a transient free amino acid (FAA) release on the first day in both macroalgal cultures. The uptake rates estimated by NH₄ ⁺ disappearance and ¹⁵N incorporation in algal tissue compare well (82% agreement, defined as the percentage ratio of the lower to the higher rate) at high NH₄ ⁺ concentrations, provided that isotope dilution is taken into account. On average, 96% of added ¹⁵NH₄ ⁺ was recovered from the medium and algal tissue at the end of the incubation. Negligible uptake of NO₃ ^? was observed during the first 2–3 days in both macroalgae. The lag of uptake may have resulted from the need for either some N deprivation (use of NO₃ ^? pools) or physiological/metabolic changes required before the uptake of NO₃ ^? . During the subsequent days, NO₃ ^? uptake rates were similar for the two macroalgae but much lower than NH₄ ⁺ uptake rates (1.97–3.19 μmol N·g dw ^{? 1}·h ^{? 1}). Very little isotope dilution and FAA release were observed. The agreement between rates calculated with the two different methods averaged 91% in U. fenestrata and 95% in G. pacifica. Recovery of added ¹⁵NO₃ ^? was virtually complete (99%). These tracer incubations show that isotope dilution can be significant in NH₄ ⁺ uptake experiments conducted with N‐rich macroalgae and that determination of ¹⁵N atom % enrichment of the dissolved NH₄ ⁺ is recommended to avoid poor isotope recovery and underestimation of uptake rates.     

Inorganic carbon uptake by phytoplankton in Vineyard Sound,Massachusetts. II. Comparative primary productivity and nutritional status of winter and summer assemblages

Using time-course, natural-light incubations, we assessed the rate of carbon uptake at a range of light intensities, the effect of supplemental additions of nitrogen (as NH₄⁺ or urea) on light and dark carbon uptake, and the rates of uptake of NH₄⁺ and urea by phytoplankton from Vineyard Sound, Massachusetts from February through August 1982. During the winter, photoinhibition was severe, becoming manifested shortly after the start of an incubation, whereas during the summer, there was little to no evidence of photoinhibition during the first several hours after the start of an incubation. At light levels which were neither photoinhibiting nor light limiting, rates of carbon uptake normalized per liter were high and approximately equal during winter and summer (22–23 μg C·l^?1 · h^?1), and low during spring (<10 μgC·l^?1· h^?1). In contrast, on a chlorophyll a basis, rates of carbon fixation were as high during spring (15–20μg C·μg Chl a^?1·h^?1), when concentrations of chlorophyll a were at the yearly minimum (<0.5 μg · l^?1) as during the summer, when chlorophyll a concentrations were substantially higher (0.8–1.3 μg · l^?1). Highest rates of NH₄⁺ and urea uptake were observed during summer, and at no time of the year was there evidence for severe nitrogen deficiency, although moderate nitrogen nutritional stress was apparent during the summer months.     

METHYLAMINE UPTAKE IN THE GREEN ALGA CHLORELLA PYRENOIDOSA1

Methylamine uptake in nitrogen-starved Chlorella pyrenoidosa Beij. follows Michaelis-Menten kinetics: maximum uptake is about 1.6 nmol μl^?1· cells · min^?1, half-saturation occurs at 4 μM methylamine, and the slope in the range where uptake is proportional to concentration is 0.4 nmol μl^?1· min^?1·μM^?1. In cells grown in the presence of a non-limiting nitrogen concentration, methylamine uptake is directly proportional to concentration up to at least 0.5 mM, and the slope is 1/500 that for starved cells. Similar uptake kinetics have been reported for Penicillium chrysogenum and attributed to an inducible “ammonium permease.” Apparently, a similar permease occurs in algae.     

Discrimination of nitrogen isotopes during absorption of ammonium and nitrate at different nitrogen concentrations by rice (Oryza sativa L.) plants

Studies of uptake of ionic sources of N by two hydroponically grown rice (Oryza sativa L.) cultivars (paddy‐field‐adapted Koshihikari and dryland‐adapted Kanto 168) showed that the magnitude of the nitrogen isotope fractionation (?) for uptake of NH₄⁺ depended on the concentrations of NH₄⁺ and cultivar (averaging –6·1‰ for Koshihikari and –12·0‰ for Kanto 168 at concentrations from 40 to 200 mmol m^?3 and, respectively, –13·4 and –28·9‰ for the two cultivars at concentrations from 0·5 to 4 mol m^?3). In contrast, the ? for uptake of NO₃^? in similar experiments was almost insensitive to the N concentration, falling within a much narrower range (+3·2‰ to –0·9‰ for Koshihikari and –0·9‰ to –5·1‰ for Kanto 168 over NO₃^? concentrations from 0·04 to 2 mol m^?3). From longer term experiments in which Norin 8 and its nitrate‐reductase deficient mutant M819 were grown with 2 or 8 mol m^?3 NO₃^? for 30 d, it was concluded that the small concentration‐independent isotopic fractionation during absorption of this ion was not related to nitrate reductase activity.     

Characterization of Na+/H+ Exchanger Isoform (NHE1, NHE2 and NHE3) Expression in Prairie Dog Gallbladder

Gallbladder Na⁺ absorption is linked to gallstone formation in prairie dogs. Na⁺/H⁺ exchange (NHE) is one of the major Na⁺ absorptive pathways in gallbladder. In this study, we measured gallbladder Na⁺/H⁺ exchange and characterized the NHE isoforms expressed in prairie dogs. Na⁺/H⁺ exchange activity was assessed by measuring amiloride-inhibitable transepithelial Na⁺ flux and apical ²²Na⁺ uptake using dimethylamiloride (DMA). HOE-694 was used to determine NHE2 and NHE3 contributions. Basal J ^Na _ms was higher than J ^Na _sm with J ^Na _net absorption. Mucosal DMA inhibited transepithelial Na⁺ flux in a dose-dependent fashion, causing J ^Na _ms equal to J ^Na _sm and blocking J ^Na _net absorption at 100 μm. Basal ²²Na⁺ uptake rate was 10.9 ± 1.0 μmol · cm⁻²· hr⁻¹ which was inhibited by ∼43% by mucosal DMA and ∼30% by mucosal HOE-694 at 100 μm. RT-PCR and Northern blot analysis demonstrated expression of mRNAs encoding NHE1, NHE2 and NHE3 in the gallbladder. Expression of NHE1, NHE2 and NHE3 polypeptides was confirmed using isoform-specific anti-NHE antibodies. These data suggest that Na⁺/H⁺ exchange accounts for a substantial fraction of gallbladder apical Na⁺ entry and most of net Na⁺ absorption in prairie dogs. The NHE2 and NHE3 isoforms, but not NHE1, are involved in gallbladder apical Na⁺ uptake and transepithelial Na⁺ absorption. Received: 9 February 2001/Revised: 11 April 2001     

Induction of the Na+/Pi cotransport system in the plasma membrane of Chara corallina requires external Na+ and low levels of Pi

It was shown in previous studies that the giant freshwater alga Chara corallina does not control its Na⁺‐dependent Pi uptake by monitoring the internal Pi concentration and it was hypothesized that Chara may instead detect changes in Pi supply from the environment. The present work investigated the conditions that control the induction and inactivation of high affinity Na⁺/Pi influx in Chara. Withdrawal of Pi from the external medium resulted in a gradual increase in the rate of uptake measured immediately after Pi was resupplied. The increase continued for at least 7 d of starvation. In the initial stages, 0·5 or 1 µm Pi were more effective at inducing transport activity than no Pi, suggesting that low levels of Pi are actually required for induction. The high Na⁺‐dependent Pi uptake observed in Pi‐starved cells was inactivated by treatment with as little as 1 µm Pi over 6 d. External Na⁺ plays a major role in controlling the capacity for Na⁺/Pi cotransport activity, and in the absence of Na⁺, both induction and inactivation were either delayed or abolished. Na⁺ starvation stimulated Na⁺ uptake even though there were no measurable changes in the concentrations of Na⁺, or of K⁺ or Pi in either the vacuole or cytoplasm. It was concluded that both substrate (Pi) and driver ion (Na⁺) are required at adequate concentrations for the induction of the cotransporter. In the case of Pi, it was suggested that passive leakage of Pi from the cell into the apoplast is sufficient for this purpose but that supplementation by up to 1 µm Pi is more effective at the earlier stage. A mechanism for sensing the external supply of Pi is proposed.     

CHOLINE UPTAKE BY CHOLINERGIC NEURON CELL SOMAS

The cellular compartments of ciliary ganglia take up choline by a single, saturable process with K_m=7.1 × 10^?5 M and V_max= 4.66 pmol/min per ganglion: Denervation of the ganglia and the resultant degeneration of nerve terminals caused no significant decrease of the rate of accumulation of choline by the ganglia. This indicates that the measured uptake is by the postganglionic ncurons and nonneural elements (NNE: glial and connective tissue cells) in the ganglia. This uptakc is not dependent on metabolic energy and is not affectcd by lowcring Na⁺ or raising K⁺ concentrations in the incubating mcdia but is depressed in the presence of ouabain and hemicholinium-3. The presence or Na⁺-dependent. rapidly saturable uptake in the preganglionic nerve terminals which is not detectablc kinetically is, however, inferred from a decrease in ACh synthesis in dcncrvatcd prcparations and a similar decrcasc in intact ganglia incubated in low Na+ solution.