Sodium-Dependent Azotobacter chroococcum Strains Are Aeroadaptive, Microaerophilic, Nitrogen-Fixing Bacteria

Na⁺ -dependent strains of Azotobacter chroococcum were observed to have very low reactivities with the H₂O₂ spot test for catalase. The cell extract of the representative Na⁺ -dependent strain 184 contained a catalase specific activity that was 10-to 600-fold lower than those found in Na⁺ -independent strains of A. chroococcum. Peroxidase and superoxide dismutase activities existed in all strains, although only certain Na⁺ -dependent strains contained a peroxidase reactive with p-phenylenediamine. The activities of catalase and peroxidase in the Na⁺ -dependent strain 184 were dependent on iron availability, which helped to explain the iron-dependent growth characteristic of this strain. The activities of these enzymes were not increased by subjecting the cells to increased aeration, nitrogen-fixing conditions, or paraquat. Strain 184 was found to be very sensitive to H₂O₂ or paraquat, even under iron-sufficient conditions, and was difficult to recover quantitatively on solid plating media. Strain 184 was more susceptible to H₂O₂ when grown under low-aeration, nitrogen-fixing conditions than when it was grown in the presence of NH₄⁺. Low population densities of strain 184 grew in nitrogen-free medium under microaerophilic conditions, while more dense populations were able to fix nitrogen under aerobic conditions. Therefore, these bacteria appeared to be aeroadaptive, microaerophilic, nitrogen-fixing bacteria.     

Coupled Metabolic and Photolytic Pathway for Degradation of Pyridinedicarboxylic Acids, Especially Dipicolinic Acid

Three isomers of pyridinedicarboxylic acid (PDCA) (2,3-, 2,5-, and 2,6-PDCA) were partially oxidized by marine bacteria when grown aerobically on the corresponding phthalate analogs. The metabolites, unlike the parent PDCAs, absorbed light in the solar actinic range (wavelengths greater than 300 nm) and were readily degraded in sunlight. The principal product from 2,6-PDCA (dipicolinic acid) metabolism was extracted from a culture fluid, purified by column chromatography, and analyzed by UV-visible, infrared, and ¹³C nuclear magnetic resonance spectroscopy. The compound was identified as 2,3-dihydroxypicolinic acid (2,3-DHPA). 2,3-DHPA was photolyzed in aqueous solution (pH 8.0) with a half-life of 100 min. Eight photoproducts, three of which were photolabile, were detected by high-performance liquid chromatography. Ammonia was also photoproduced from 2,3-DHPA. Analysis of the photoproducts by UV-visible spectroscopy and by high-performance liquid chromatography of 2,4-dinitrophenylhydrazones indicated that the products were conjugated carbonyls and carboxylic acids. Six of the photoproducts were readily consumed by bacterial strain CC9M. In illuminated aquatic environments, coupled bio- and photodegradative mechanisms probably contribute to the degradation of PDCAs.     

Potential for Biodegradation of Phthalic Acid Esters in Marine Regions

Di-(2-ethylhexyl) phthalate was the major phthalic acid ester in the Mississippi River estuary, with mean levels of 0.1 μg/g (dry weight) in surface sediments, 1.0 μg/liter in river water, and 0.7 μg/liter in delta water. Bacteria that grew aerobically on dibutyl phthalate and o-phthalic acid were readily detected in the sediments and water. Pure cultures of bacteria were isolated on seven different phthalic acid esters from freshwater and marine sources. The marine isolates were taxonomically diverse and grew on a variety of phthalic acid esters. Dibutyl phthalate and o-phthalic acid supported growth in full-strength synthetic sea-water medium, but Na⁺ -dependent catabolism was demonstrable only for o-phthalic acid.     

The α1 Na+−K+ pump of the Dahl salt-sensitive rat exhibits altered Na+ modulation of K+ transport in red blood cells

The properties of the α1 Na⁺-K⁺ pump were compared in Dahl salt-sensitive (DS) and salt-resistant (DR) strains by measuring ouabain-sensitive luxes (mmol/liter cell x hr = FU, Mean ± se) in red blood cells (RBCs) and varying internal ( _i ) and external ( _o ) Na⁺ and K⁺ concentrations. Kinetic parameters of several modes of operation, i.e., Na⁺/ K⁺, K⁺/K⁺, Na⁺/Na⁺ exchanges, were characterized and analyzed for curve-fitting using the Enzfitter computer program. In unidirectional flux studies (n=12 rats of each strain) into fresh cells incubated in 140 mm Na⁺ + 5 mm K⁺, ouabain-sensitive K⁺ influx was substantially lower in the DS than in DR RBCs, while ouabain-sensitive Na⁺ efflux and Na _i were similar in both strains. Thus, the coupling ratio between unidirectional Na⁺∶K⁺ fluxes was significantly higher in DS than in DR cells at similar RBC Na⁺ content. In the presence of 140 mm Na _o , activation of ouabain-sensitive K⁺ influx by K _o had a lower K _m and V _max in DS as estimated by the Garay equation (N=2.70 ± 0.33, K _m 0.74 ± 0.09 mm; V _max 2.87 ± 0.09 FU) than in DR rats (N=1.23 ± 0.36, K _m 2.31 ± 0.16 mm; v _max 5.70 ± 0.52 FU). However, the two kinetic parameters were similar following Na _o removal. The activation of ouabain-sensitive K⁺ influx by Na _i had significantly lower V _max in DS (9.3 ± 0.4 FU) than in DR (14.5 ± 0.6 FU) RBCs but similar K _m. These data suggest that the low K⁺ influx in DS cells is caused by a defect in modulation by Na _o and Na _i . Na⁺ efflux showed no differences in Na _i activation or trans effects by Na _o and K _o , thus accounting for the different Na⁺∶K⁺ coupling ratio in the Dahl strains. Further evidence for the differences in the coupling of ouabain-sensitive fluxes was found in studies of net Na⁺ and K⁺ fluxes, where the net ouabain-sensitive Na⁺ losses showed similar magnitudes in the two Dahl strains while the net ouabainsensitive K⁺ gains were significantly greater in the DR than the DS RBCs. Ouabain-sensitive Na⁺ influx and K⁺ efflux were also measured in these rat RBCs. The inhibition of ouabain-sensitive Na⁺ influx by K _o was fully competitive for the DS but not for the DR pumps. Thus, for DR pumps, K _o could activate higher K⁺ influx in DR pumps without a complete inhibition of ouabain-sensitive Na⁺ influx. This behavior is consistent with K _o interaction with distinct Na⁺ and K⁺ transport sites. In addition, the inhibition of K⁺ efflux by Na, was different between Dahl strains. Ouabain-sensitive K⁺ efflux at Na _i level of 4.6 mmol/liter cell, was significantly higher in DS (3.86 ± 0.67 FU) than in DR (0.86 ± 0.14 FU) due to a threefold higher K₅₀ for Na _i -inhibition 9.66 ± 0.41 vs. 3.09 ± 0.11 mmol/liter cell. This finding indicates that Na⁺ modulation of K⁺ transport is altered at both sides of the membrane. The dissociation of Na⁺ modulatory sites of K⁺ transport from Na⁺ transport sites observed in RBCs of Dahl strains suggests that K⁺ transport by the Na⁺-K⁺ pump is controlled by Na⁺ allosteric sites different from the Na⁺ transport sites. The alterations in K⁺ transport may be related to the amino acid substitution (Leu/Gln₂₇₆) reported for the cDNA of the α1 subunit of the Na⁺-K⁺ pump in the DS strain or to post-translational modifications during RBC maturation. These studies were supported by the following grants: NIH (HL-35664, HL-42120, HL-18318, HL-39267, HL-01967). J.R.R. is a Ford Foundation Predoctoral Fellow. A preliminary report of this work was presented at the International Conference on the Na⁺-K⁺ pump and 44th Annual Meeting of the Society of General Physiologists held at Woods Hole, MA, September 5–9, 1990, and published as an abstract in the J. Gen. Physiol. 96:70a, 1990.     

The Extracellular K+ Concentration Dependence of Outward Currents through Kir2.1 Channels Is Regulated by Extracellular Na+ and Ca2+

It has been known for more than three decades that outward Kir currents (I_K1) increase with increasing extracellular K⁺ concentration ([K⁺]_o). Although this increase in I_K1 can have significant impacts under pathophysiological cardiac conditions, where [K⁺]_o can be as high as 18 mm and thus predispose the heart to re-entrant ventricular arrhythmias, the underlying mechanism has remained unclear. Here, we show that the steep [K⁺]_o dependence of Kir2.1-mediated outward I_K1 was due to [K⁺]_o-dependent inhibition of outward I_K1 by extracellular Na⁺ and Ca²⁺. This could be accounted for by Na⁺/Ca²⁺ inhibition of I_K1 through screening of local negative surface charges. Consistent with this, extracellular Na⁺ and Ca²⁺ reduced the outward single-channel current and did not increase open-state noise or decrease the mean open time. In addition, neutralizing negative surface charges with a carboxylate esterifying agent inhibited outward I_K1 in a similar [K⁺]_o-dependent manner as Na⁺/Ca²⁺. Site-directed mutagenesis studies identified Asp¹¹⁴ and Glu¹⁵³ as the source of surface charges. Reducing K⁺ activation and surface electrostatic effects in an R148Y mutant mimicked the action of extracellular Na⁺ and Ca²⁺, suggesting that in addition to exerting a surface electrostatic effect, Na⁺ and Ca²⁺ might inhibit outward I_K1 by inhibiting K⁺ activation. This study identified interactions of K⁺ with Na⁺ and Ca²⁺ that are important for the [K⁺]_o dependence of Kir2.1-mediated outward I_K1.     

Salt tolerance in suspension cultures of sugar beet : induction of na/h antiport activity at the tonoplast by growth in salt

Cell suspension cultures of sugar beet were grown at various salinities (0-200 millimolar NaCl). Their tolerance to Na⁺ was comparable to that of the intact plant. Tonoplast vesicles were prepared by sucrose density gradient centrifugation of microsomal membranes and shown to be highly purified. The vesicles were subjected to a pH jump in the presence of acridine orange and the rate of recovery of fluorescence after addition of Na⁺ was used as a measure of Na⁺-dependent H⁺ efflux. In the presence of K⁺ and valinomycin, the Na⁺/H⁺ antiport showed saturation kinetics. Increasing Na⁺ in the growth medium did not change the apparent K_m for Na⁺, but increased V_max to about twice the control value, suggesting a specific induction of antiport synthesis by salt.     

Calcium Efflux from Barnacle Muscle Fibers : Dependence on External Cations

Calcium-45 was injected into single giant barnacle muscle fibers, and the rate of efflux was measured under a variety of conditions. The rate constant (k) for ⁴⁵Ca efflux into standard seawater averaged 17 x 10^–4 min^–1 which corresponds to an efflux of about 1–2 pmol/cm²·s. Removal of external Ca (Ca_o) reduced the efflux by 50%. In most fibers about 40% of the ⁴⁵Ca efflux into Ca-free seawater was dependent on external Na (Na_o); treatment with 3.5 mM caffeine increased the magnitude of the Na_o-dependent efflux. In a few fibers removal of Na_o, in the absence of Ca_o, either had no effect or increased k; caffeine (2–3.5 mM) unmasked an Na_o-dependent efflux in these fibers. The Na_o-dependent Ca efflux had a Q₁₀ of about 3.7. The data are consistent with the idea that a large fraction of the Ca efflux may be carrier-mediated, and may involve both Ca-Ca and Na-Ca counterflow. The relation between the Na_o-dependent Ca efflux and the external Na concentration is sigmoid, and suggests that two, or more likely three, external Na⁺ ions may activate the efflux of one Ca⁺². With a three-for-one Na-Ca exchange, the Na electrochemical gradient may be able to supply sufficient energy to maintain the Ca gradient in these fibers. Other, more complex models are not excluded, however, and may be required to explain some puzzling features of the Ca efflux such as the variable Na_o-dependence.     

Sodium transport measured in plasma membrane vesicles isolated from wheat genotypes with differing K+/Na+ discrimination traits

Right-side-out plasma membrane vesicles were isolated from wheat roots using an aqueous polymer two-phase system. The purity and orientation of the vesicles were confirmed by marker enzyme analysis. Membrane potential (Ψ)-dependent ²²Na⁺ influx and sodium/proton (Na⁺/ H⁺) antiport-mediated efflux across the plasma membrane were studied using these vesicles. Membrane potentials were imposed on the vesicles using either K⁺ gradients in the presence of valinomycin or H⁺ gradients. The ΔΨ was quantified by the uptake of the lipophilic cation tetraphenylphosphonium. Uptake of Na⁺ into the vesicles was stimulated by a negative ΔΨ and had a K_m for extrav-esicular Na⁺ of 34.8 ± 5.9 mol m³. The ΔΨ-dependent uptake of Na⁺ was similar in vesicles from roots of hexaploid (cv. Troy) and tetraploid (cv. Langdon) wheat differing in a K⁺/Na⁺ discrimination trait, and was also unaffected by growth in 50 mol m^?3 NaCl. Inhibition of ΔΨ-dependent Na⁺ uptake by Ca²⁺ was greater in the hexaploid than in the tetraploid. Sodium/proton antiport was measured as Na⁺-dependent, amiloride-inhibited pH gradient formation in the vesicles. Acidification of the vesicle interior was measured by the uptake of ¹⁴C-methylamine. The Na⁺/H⁺ antiport had a K_m, for intravesicular Na⁺ of between 13 and 19 mol m^?3. In the hexaploid, Na⁺/H⁺ antiport activity was greater when roots were grown in the presence of 50 mol m^?3NaCl, and was also greater than the activity in salt-grown tetraploid wheat roots. Antiport activity was not increased in a Langdon 4D chromosome substitution line which carries a trait for K⁺/Na⁺ discrimination. It is concluded that neither of the transport processes measured is responsible for the Na⁺/K⁺ discrimination trait located on the 4D chromosome of wheat.     

Na/H Antiport in Isolated Tonoplast Vesicles from Storage Tissue of Beta vulgaris

The pH-dependent fluorescence quenching of acridine orange was used to study the Na⁺- and K⁺-dependent H⁺ fluxes in tonoplast vesicles isolated from storage tissue of red beet and sugar beet (Beta vulgaris L.). The Na⁺-dependent H⁺ flux across the tonoplast membrane could be resolved into two components: (a) a membrane potential-mediated flux through conductive pathways; and (b) an electroneutral flux which showed Michaelis-Menten kinetics relationship to Na⁺ concentration and was competitively inhibited by amiloride (K_i = 0.1 millimolar). The potential-dependent component of H⁺ flux showed an approximately linear dependence on Na⁺ concentration. In contrast, the K⁺-dependent H⁺ flux apparently consisted of a single component which showed an approximately linear dependence on K⁺ concentration, and was insensitive to amiloride. Based on the Na⁺- and K⁺-dependent H⁺ fluxes, the passive permeability of the vesicle preparation to Na⁺ was about half of that to K⁺.

The apparent K_m for Na⁺ of the electroneutral Na⁺/H⁺ exchange varied by more than 3-fold (7.5-26.5 millimolar) when the internal and external pH values were changed in parallel. The results suggest a simple kinetic model for the operation of the Na⁺/H⁺ antiport which can account for the estimated in vivo accumulation ratio for Na⁺ into the vacuole.

     

Evidence for Na-Independent HCO(3) Uptake by the Cyanobacterium Synechococcus leopoliensis

At low levels of dissolved inorganic carbon (DIC) and alkaline pH the rate of photosynthesis by air-grown cells of Synechococcus leopoliensis (UTEX 625) was enhanced 7- to 10-fold by 20 millimolar Na⁺. The rate of photosynthesis greatly exceeded the CO₂ supply rate and indicated that HCO₃⁻ was taken up by a Na⁺-dependent mechanism. In contrast, photosynthesis by Synechococcus grown in standing culture proceeded rapidly in the absence of Na⁺ and exceeded the CO₂ supply rate by 8 to 45 times. The apparent photosynthetic affinity (K_½) for DIC was high (6-40 micromolar) and was not markedly affected by Na⁺ concentration, whereas with air-grown cells K_½ (DIC) decreased by more than an order of magnitude in the presence of Na⁺. Lithium, which inhibited Na⁺-dependent HCO₃⁻ uptake in air-grown cells, had little effect on Na⁺-independent HCO₃⁻ uptake by standing culture cells. A component of total HCO₃⁻ uptake in standing culture cells was also Na⁺-dependent with a K_½ (Na⁺) of 4.8 millimolar and was inhibited by lithium. Analysis of ¹⁴C-fixation during isotopic disequilibrium indicated that standing culture cells also possessed a Na⁺-independent CO₂ transport system. The conversion from Na⁺-independent to Na⁺-dependent HCO₃⁻ uptake was readily accomplished by transferring cells grown in standing to growth in cultures bubbled with air. These results demonstrated that the conditions experienced during growth influenced the mode by which Ssynechococcus acquired HCO₃⁻ for subsequent photosynthetic fixation.     

Potassium activation of Na+-dependent leucine transport in brush-border membrane vesicles from rat jejunum

Na⁺-dependent leucine uptake was greater in potassium loaded brush-border membrane vesicles compared with controls. This effect was not mediated by an electrical potential difference, since it was still present in voltage-clamped conditions. Inhibition experiments indicate the same Na⁺-dependent leucine transport activity in the presence or in the absence of potassium. The affinity of sodium for the cotransporter was identical at 10 or 100 mM potassium. Leucine kinetics at different potassium concentrations showed a maximum 2.4-fold increase in V_max, while K_m was unaffected. The secondary plots of the kinetic results were not linear. This kinetic behaviour suggests that K⁺ acts as a non-essential activator of Na⁺-dependent leucine cotransport. A charge compensation of sodium-leucine influx is most probably a component of the potassium effect in the presence of valinomycin.     

Human SLC4A11 Is a Novel NH3/H+ Co-transporter

SLC4A11 has been proposed to be an electrogenic membrane transporter, permeable to Na⁺, H⁺ (OH⁻), bicarbonate, borate, and NH₄⁺. Recent studies indicate, however, that neither bicarbonate or borate is a substrate. Here, we examined potential NH₄⁺, Na⁺, and H⁺ contributions to electrogenic ion transport through SLC4A11 stably expressed in Na⁺/H⁺ exchanger-deficient PS120 fibroblasts. Inward currents observed during exposure to NH₄Cl were determined by the [NH₃]_o, not [NH₄⁺]_o, and current amplitudes varied with the [H⁺] gradient. These currents were relatively unaffected by removal of Na⁺, K⁺, or Cl⁻ from the bath but could be reduced by inclusion of NH₄Cl in the pipette solution. Bath pH changes alone did not generate significant currents through SLC4A11, except immediately following exposure to NH₄Cl. Reversal potential shifts in response to changing [NH₃]_o and pH_o suggested an NH₃/H⁺-coupled transport mode for SLC4A11. Proton flux through SLC4A11 in the absence of ammonia was relatively small, suggesting that ammonia transport is of more physiological relevance. Methylammonia produced currents similar to NH₃ but with reduced amplitude. Estimated stoichiometry of SLC4A11 transport was 1:2 (NH₃/H⁺). NH₃-dependent currents were insensitive to 10 μm ethyl-isopropyl amiloride or 100 μm 4,4′- diisothiocyanatostilbene-2,2′-disulfonic acid. We propose that SLC4A11 is an NH₃/2H⁺ co-transporter exhibiting unique characteristics.     

Comparative genomic analysis of <Emphasis Type="Italic">Geosporobacter ferrireducens</Emphasis> and its versatility of anaerobic energy metabolism

Members of the family Clostridiaceae within phylum Firmicutes are ubiquitous in various iron-reducing environments. However, genomic data on iron-reducing bacteria of the family Clostridiaceae, particularly regarding their environmental distribution, are limited. Here, we report the analysis and comparison of the genomic properties of Geosporobacter ferrireducens IRF9, a strict anaerobe that ferments sugars and degrades toluene under iron-reducing conditions, with those of the closely related species, Geosporobacter subterraneus DSM 17957. Putative alkyl succinate synthase-encoding genes were observed in the genome of strain IRF9 instead of the typical benzyl succinate synthase-encoding genes. Canonical genes associated with iron reduction were not observed in either genome. The genomes of strains IRF9 and DMS 17957 harbored genes for acetogenesis, that encode two types of Rnf complexes mediating the translocation of H⁺ and Na⁺ ions, respectively. Strain IRF9 harbored two different types of ATPases (Na⁺-dependent F-type ATPase and H⁺-dependent V-type ATPase), which enable full exploitation of ion gradients. The versatile energy conservation potential of strain IRF9 promotes its survival in various environmental conditions.     

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.     

Effect of internal and external K+ on Na+−Ca2+ exchange in dialyzed squid axons under voltage clamp conditions

The effect of external and internal K⁺ on Na_o⁺-dependent Ca²⁺ efflux was studied in dialyzed squid axons under constant membrane potential. With axons clamped at their resting potentials, external K⁺ (up to 70 mM) has no effect on Na⁺?Ca²⁺ exchange. Removal of K_i⁺ causes a marked inhibition in the Na_o⁺-dependent Ca²⁺ efflux component. Internal K⁺ activates the Na⁺?Ca²⁺ exchange with low affinity $\text{(K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 90 mM)}$. Activation by K_i⁺ is similar in the presence or in the absence of Na_i⁺, thus ruling out a displacement of Na_i⁺ from its inhibitory site. Axons dialyzed with ATP also show a dependency of Ca²⁺ efflux on K_i⁺. The present results demonstrate that K_i⁺ is an important cofactor (partially required) for the proper functioning of the forward Na⁺?Ca²⁺ exchange.     

Characterization of Na+-Dependent Phosphate Uptake in Cultured Fetal Rat Cortical Neurons

Abstract: Our laboratory has recently cloned and expressed a brain- and neuron-specific Na⁺-dependent inorganic phosphate (P_i) cotransporter that is constitutively expressed in neurons of the rat cerebral cortex, hippocampus, and cerebellum. We have now characterized Na⁺-dependent ³²P_i cotransport in cultured fetal rat cortical neurons, where >90% of saturable P_i uptake is Na⁺-dependent. Saturable, Na⁺-dependent ³²P_i uptake was first observed in primary cultures of cortical neurons at 7 days in vitro (DIV) and was maximal at 12 DIV. Na⁺-dependent P_i transport was optimal at physiological temperature (37°C) and pH (7.0–7.5), with apparent K_m values for P_i and Na⁺ of 54 ± 12.7 µM and 35 ± 4.2 mM, respectively. A reduction in extracellular Ca²⁺ markedly reduced (>60%) Na⁺-dependent P_i uptake, with a threshold for maximal P_i import of 1–2.5 mM CaCl₂. Primary cultures of fetal cortical neurons incubated in medium where equimolar concentrations of choline were substituted for Na⁺ had lower levels of ATP and ADP and higher levels of AMP than did those incubated in the presence of Na⁺. Furthermore, a substantial fraction of the ³²P_i cotransported with Na⁺ was concentrated in the adenine nucleotides. Inhibitors of oxidative metabolism, such as rotenone, oligomycin, or dinitrophenol, dramatically decreased Na⁺-dependent P_i import rates. These data establish the presence of a Na⁺-dependent P_i cotransport system in neurons of the CNS, demonstrate the Ca²⁺-dependent nature of ³²P_i uptake, and suggest that the neuronal Na⁺-dependent P_i cotransporter may import P_i required for the production of high-energy compounds vital to neuronal metabolism.     

Bumetanide-sensitive Ion Fluxes in Vascular Smooth Muscle Cells: Lack of Functional Na+, K+, 2 Cl− Cotransport

To examine the involvement of Na⁺,K⁺,2Cl⁻ cotransport in monovalent ion fluxes in vascular smooth muscle cells (VSMC), we compared the effect of bumetanide on ⁸⁶Rb, ³⁶Cl and ²²Na uptake by quiescent cultures of VSMC from rat aorta. Under basal conditions, the values of bumetanide-sensitive (BS) inward and outward ⁸⁶Rb fluxes were not different. Bumetanide decreased basal ⁸⁶Rb uptake by 70–75% with a K _i of ∼0.2–0.3 μm. At concentrations ranging up to 1 μm, bumetanide did not affect ³⁶Cl influx and reduced it by 20–30% in the range from 3 to 100 μm. In contrast to ⁸⁶Rb and ³⁶Cl influx, bumetanide did not inhibit ²²Na uptake by VSMC. BS ⁸⁶Rb uptake was completely abolished in Na⁺- or Cl⁻-free media. In contrast to ⁸⁶Rb, basal BS ³⁶Cl influx was not affected by Na⁺ _o and K⁺ _o . Hyperosmotic and isosmotic shrinkage of VSMC increased ⁸⁶Rb and ³⁶Cl influx to the same extent. Shrinkage-induced increments of ⁸⁶Rb and ³⁶Cl uptake were completely abolished by bumetanide with a K _i or ∼0.3 μm. Shrinkage did not induce BS ⁸⁶Rb and ³⁶Cl influx in (Na⁺ or Cl⁻)- and (Na⁺ or K⁺)-depleted media, respectively. In the presence of an inhibitor of Na⁺/H⁺ exchange (EIPA), neither hyperosmotic nor isosmotic shrinkage activated ²²Na influx. Bumetanide (1 μm) did not modify basal VSMC volume and intracellular content of sodium, potassium and chloride but abolished the regulatory volume increase in isosmotically-shrunken VSMC. These data demonstrate the absence of the functional Na⁺,K⁺,2Cl⁻ cotransporter in VSMC and suggest that in these cells basal and shrinkage-induced BS K⁺ influx is mediated by (Na⁺ _o + Cl⁻ _o )-dependent K⁺/K⁺ exchange and Na⁺ _o -dependent K⁺,Cl⁻ cotransport, respectively. Received: 30 January 1996/Revised: 20 May 1996     

Degradation of <Emphasis Type="Italic">o</Emphasis>-xylene and <Emphasis Type="Italic">m</Emphasis>-xylene by a novel sulfate-reducer belonging to the genus <Emphasis Type="Italic">Desulfotomaculum</Emphasis>

A strictly anaerobic bacterium, strain OX39, was isolated with o-xylene as organic substrate and sulfate as electron acceptor from an aquifer at a former gasworks plant contaminated with aromatic hydrocarbons. Apart from o-xylene, strain OX39 grew on m-xylene and toluene and all three substrates were oxidized completely to CO₂. Induction experiments indicated that o-xylene, m-xylene, and toluene degradation were initiated by different specific enzymes. Methylbenzylsuccinate was identified in supernatants of cultures grown on o-xylene and m-xylene, and benzylsuccinate was detected in supernatants of toluene-grown cells, thus indicating that degradation was initiated in all three cases by fumarate addition to the methyl group. Strain OX39 was sensitive towards sulfide and depended on Fe(II) in the medium as a scavenger of the produced sulfide. Analysis of the PCR-amplified 16S rRNA gene revealed that strain OX39 affiliates with the gram-positive endospore-forming sulfate reducers of the genus Desulfotomaculum and is the first hydrocarbon-oxidizing bacterium in this genus.     

The form of sodium-calcium competition at the frog myoneural junction

The times required for a steady rate of miniature end-plate potential discharge to be reached in response to changes in extracellular [K⁺], [Na⁺], and [Ca⁺⁺] have been measured. In the presence of 15 mM KCl, Ca⁺⁺ raises and Na⁺ lowers the steady-state mepp frequency; but the depressive effect on Na⁺ is not specific: Li⁺ can replace Na⁺ to a large extent. Mepp frequency has been found to depend on the ratio of [Ca_o ⁺⁺]/[Na_o ⁺]. It is assumed that in the steady state, intracellular sodium will change when extracellular sodium is changed. Because both intracellular and extracellular sodium at motor nerve endings affect acetylcholine release, it is proposed that mepp frequency depends on the ratio [Ca_o] [Na_i]²·/[Na_o]² Two models are proposed. Firstly, to account for the action of sodium and calcium a carrier is postulated for which Ca⁺⁺ and Na⁺ compete. The carrier determines a maximum level of intracellular Ca⁺⁺ far lower than predicted by the Nernst equation for Ca. Secondly, to account for activation of acetylcholine release by a small influx of Ca⁺⁺, the ions are presumed to enter the nerve ending in a two stage process through a small intermediate compartment and to act on the acetylcholine release site in this region rather than after entering directly into the cell.