Methylammonium transport in Anacystis nidulans R-2

Methylammonium was taken up rapidly by illuminated cells of Anacystis nidulans R-2, leading to internal concentrations of 1.3 +/- 0.1 mM within 1 min, and a gradient of up to 200 between the cells and medium. Accumulation of 14CH3NH3+ required at least 5 mM NaCl, but the uptake rate was independent of medium pH between 6.5 and 9. The kinetics of uptake could be resolved into an initial fast phase lasting less than 1 min (approximate Km, 7.2 microM; Vmax, 12.5 nmol min-1 mg of protein-1 at 15 degrees C). A second, slower phase associated with product formation was eliminated by preincubation with methionine sulfoximine, a specific inhibitor of glutamine synthetase; the rapid phase was unaffected by this treatment. Ammonium ions competed with 14CH3NH3+ for entry, and addition of 5 microM NH4+ or 100 microM CH3NH3+ released 14CH3NH3+ accumulated during the rapid phase of entry. Small additions of NH4+ made at the same time as additions of 14CH3NH3+ delayed the start of radioactivity uptake by a time which corresponded accurately with the period needed for the complete removal of the added NH4+. The effects of inhibitors on accumulation and carbocyanine dye fluorescence suggest that ATP-dependent membrane potential was needed to drive 14CH3NH3+ transport. Spheroplasts were as active as whole cells in accumulating NH4+ and 14CH3NH3+, indicating that soluble periplasmic components are not involved in the translocation. Some significant differences between the translocation of 14CH3NH3 and that of NH4+ were observed: growth with NH4+ in place of NO3- repressed 14CH3NH3+ accumulation ability without affecting the NH4+ uptake rate Na+ was not required for NH4+ uptake, and concentration of KCl inhibitory with 14C3NH3+ did not reduce NH4+ uptake.     

Methylammonium as a Transport Analog for Ammonium in Tomato (Lycopersicon esculentum L.)

Methylammonium (CH3NH3+) has been widely used as an analog of ammonium (NH4+) for examining transport in bacteria and fungi. We compared the kinetics of root CH3NH3+ and NH4+ uptake from solution culture in intact tomato (Lycopersicon esculentum cv T5) plants. Efflux of NH4+ and CH3NH3+ was negligible. The apparent maximum rate of absorption (apparent Vmax) was similar for NH4+ and CH3NH3+, but the apparent affinity (apparent Km) was about 10-fold greater for NH4+ than for CH3NH3+. In characterizing the interaction between NH4+ and CH3NH3+ transport, we used [15N]NH4+ and [14C]CH3NH3+ as well as improved methods for analysis of nonisotopic CH3NH3+ and NH4+. CH3NH3+ acted as an inhibitor of NH4+ influx. Relatively low concentrations of NH4+ strongly inhibited CH3NH3+ influx. Treatments with 1 mM methionine sulfoximine that blocked NH4+ assimilation had little influence on NH4+ inhibition of CH3NH3+ influx. These results suggest that the two ions share a common transport system in tomato, but because this transport system has a much greater affinity for NH4+, CH3NH3+ may be used as a transport analog only when ambient concentrations of NH4+ are very low.     

Characteristics of the methylammonium/ammonium transport systems of the nitrogen-fixing cyanobacterium Anabaena 7120 (ATCC 27893)

NH4(+)-transport in Anabaena 7120 was studied using the NH4+ analogue, 14CH3NH3+. At pH 7, two energy-dependent NH4(+)-transport systems were detected in both N2- and NO3(-)-grown cells, but none in NH4(+)-grown cells. Both transport systems showed a low and a high affinity mode of operation depending on the substrate concentration. One of the transport systems showed Km values of 8 microM (Vmax = 1 nmole min-1mg-1protein) and 80 microM (Vmax = 7 nmole min-1mg-1protein), and was insensitive to L-methionine-DL-sulphoximine, a glutamate analogue and irreversible inhibitor of glutamine synthetase. The other transport system showed Km values of 2.5 microM (Vmax = 0.1 nmole min-1mg-1protein) and 70 microM (Vmax = 0.7 nmole min-1mg-1protein), and was sensitive to L-methionine-DL-sulphoximine. Intracellular accumulation of free 14CH3NH3+ showed a biphasic pattern in response to variation in external 14CH3NH3+ concentrations. A maximum intracellular concentration of 2.5 mM and 7.5 mM was reached in the external 14CH3NH3+ concentration range of 1-50 microM and 1-500 microM, respectively. At pH 9, an energy-independent diffusion of 14CH3NH2 leading to a higher intracellular accumulation and assimilation rate, than that at pH 7, was observed.     

Carbon and ammonia metabolism of Spirillum lipoferum.

Intact cells and extracts from Spirillum lipoferum rapidly oxidized malate, succinate, lactate, and pyruvate. Glucose, galactose, fructose, acetate, and citrate did not increase the rate of O2 uptake by cells above the endogenous rate. Cells grown on NH+/4 oxidized the various substrates at about the same rate as did cells grown on N2. Added oxidized nicotinamide adenine dinucleotide generally enhanced O2 uptake by extracts supplied organic acids, whereas oxidized nicotinamide adenine dinucleotide phosphate had little effect. Nitrogenase synthesis repressed by growth of cells in the presence of NH+/4 was derepressed by methionine sulfoximine or methionine sulfone. The total glutamine synthetase activity from N2-grown cells was about eight times that from NH+/4-grown S. lipoferum; the response of glutamate dehydrogenase was the opposite. The total glutamate synthetase activity from N2-grown S. lipoferum was 1.4 to 2.6 times that from NH+/4-grown cells. The levels of poly-beta-hydroxybutyrate and beta-hydroxybutyrate dehydrogenase were elevated in cells grown on N2 as compared with those grown on NH+/4. Cell-free extracts capable of reducing C2H2 have been prepared; both Mg2+ and Mn2+ are required for good activity.     

Assimilation of 13NH4+ by Azospirillum brasilense grown under nitrogen limitation and excess.

The specific activities of glutamine synthetase (GS) and glutamate synthase (GOGAT) were 4.2- and 2.2-fold higher, respectively, in cells of Azospirillum brasilense grown with N2 than with 43 mM NH4+ as the source of nitrogen. Conversely, the specific activity of glutamate dehydrogenase (GDH) was 2.7-fold higher in 43 mM NH4+-grown cells than in N2-grown cells. These results indicate that NH4+ could be assimilated and that glutamate could be formed by either the GS-GOGAT or GDH pathway or both, depending on the cellular concentration of NH4+. The routes of in vivo synthesis of glutamate were identified by using 13N as a metabolic tracer. The products of assimilation of 13NH4+ were, in order of decreasing radioactivity, glutamine, glutamate, and alanine. The formation of [13N]glutamine and [13N]glutamate by NH4+-grown cells was inhibited in the additional presence of methionine sulfoximine (an inhibitor of GS) and diazooxonorleucine (an inhibitor of GOGAT). Incorporation of 13N into glutamine, glutamate, and alanine decreased in parallel in the presence of carrier NH4+. These results imply that the GS-GOGAT pathway is the primary route of NH4+ assimilation by A. brasilense grown with excess or limiting nitrogen and that GDH has, at best, a minor role in the synthesis of glutamate.     

Purification and characterization of a novel 4-methyleneglutamine synthetase from germinated peanut cotyledons (Arachis hypogaea)

A newly detected amide synthetase, designated 4-methyleneglutamine synthetase, has been partially purified from extracts of 5- to 7-day germinated peanut cotyledons (Arachis hypogaea). Purification steps include fractionation with protamine sulfate and ammonium sulfate followed by column chromatography on Bio-Gel and DEAE-cellulose; synthetase purified over 300-fold is obtained. The enzyme has a molecular weight estimated to be approximately 250,000 and a broad pH optimum with maximal activity at approximately pH 7.5. Maximal rates of activity are obtained with NH+4 (Km = 3.7 mM) as the amide donor and the enzyme is highly specific for 4-methylene-L-glutamic acid (Km = 2.7 mM) as the amide acceptor. Product identification and stoichiometric studies establish the reaction catalyzed to be: 4-methyleneglutamic acid + NH4+ + ATP Mg2+----4-methyleneglutamine + AMP + PPi. PPi accumulates only when F- is added to inhibit pyrophosphatase activity present in synthetase preparations. This enzymatic activity is completely insensitive to the glutamine synthetase inhibitors, tabtoxinine-beta-lactam and F-, and is only partially inhibited by methionine sulfoximine. It is, however, inhibited by added pyrophosphate in the presence of F- as well as by certain divalent metal ions (other than Mg2+) including Hg2+, Ni2+, Mn2+, and Ca2+. All data obtained indicate that this newly detected synthetase is distinct from the well-known glutamine and asparagine synthetases.     

Assay and regulation of S-adenosylmethionine synthetase in Saccharomyces cerevisiae and Candida utilis.

A simple and sensitive assay for S-adenosylmethionine (SAM) synthetase is described which depends on the quantitative separation of the product, [14CH3]S-adenosylmethionine, from the substrate, L-[14CH3]methionine, on a Bio-Rex 70 column. L-Methionine protects the enzyme during preparation of cell extracts by sonic treatment but causes repression of enzyme activity during growth of Candida utilis. The presence of 5 mM methionine in the growth medium repressed SAM synthetase specific activity threefold compared to the specific acitivity of the enzyme isolated from cells grown in unsupplemented medium. Conversely, the presence of methionine in the growth medium resulted in an 80-fold increase in the intracellular concentration of SAM as compared to the Sam accumulated intracellularly in unsupplemented cultures.     

Characterization of Salmonella typhimurium Strains Sensitive and Resistant to Methionine Sulfoximine

Methionine sulfoximine inhibits the growth of Salmonella typhimurium at a concentration of 50 muM, and the addition of glutamine, but not glutamate, is sufficient to overcome this inhibition. The analogue causes 50% inhibition of glutamine synthetase activity at 2 to 4 muM and of glutamate synthase at 2 to 3 mM when these enzymes are assayed in vitro. No inhibition of glutamate dehydrogenase activity is observed at analogue concentrations as high as 50 mM. Two mutants selected for their resistance to methionine sulfoximine inhibition have a partial growth requirement for glutamine and a reduction in the glutamine synthetase and glutamate synthase activities. The sensitivity of the remaining glutamine synthetase activity in these mutants to methionine sulfoximine inhibition appears unaltered, and the lesions conferring the analogue resistance may not affect glutamine synthetase directly.     

Benzoate stimulates glutamate release from perfused rat liver.

In isolated perfused rat liver, benzoate addition to the influent perfusate led to a dose-dependent, rapid and reversible stimulation of glutamate output from the liver. This was accompanied by a decrease in glutamate and 2-oxoglutarate tissue levels and a net K+ release from the liver; withdrawal of benzoate was followed by re-uptake of K+. Benzoate-induced glutamate efflux from the liver was not dependent on the concentration (0-1 mM) of ammonia (NH3 + NH4+) in the influent perfusate, but was significantly increased after inhibition of glutamine synthetase by methionine sulphoximine or during the metabolism of added glutamine (5 mM). Maximal rates of benzoate-stimulated glutamate efflux were 0.8-0.9 mumol/min per g, and the effect of benzoate was half-maximal (K0.5) at 0.8 mM. Similar Vmax. values of glutamate efflux were obtained with 4-methyl-2-oxopentanoate, ketomethionine (4-methylthio-2-oxobutyrate) and phenylpyruvate; their respective K0.5 values were 1.2 mM, 3.0 mM and 3.8 mM. Benzoate decreased hepatic net ammonia uptake and synthesis of both urea and glutamine from added NH4Cl. Accordingly, the benzoate-induced shift of detoxication from urea and glutamine synthesis to glutamate formation and release was accompanied by a decreased hepatic ammonia uptake. The data show that benzoate exerts profound effects on hepatic glutamate and ammonia metabolism, providing a new insight into benzoate action in the treatment of hyperammonaemic syndromes.     

14C]methylammonium transport by Frankia sp. strain CpI1

We describe an NH4+-specific transport system in the N2-fixing symbiotic actinomycete Frankia sp. strain CpI1. [14C]methylammonium was used as an NH4+ analog. No specific transport process was detected when cells were grown on high concentrations of NH4+. A transport system with a high affinity for CH3NH3+ was synthesized after 3 to 4 h of nitrogen starvation. Methylammonium transport was not significantly inhibited by a variety of amino acids, primary amines, and polyamines. Ammonium completely eliminated CH3NH3+ transport. The Km for CH3NH3+ transport was around 2 +/- 1.8 microM with a Vmax of 4 to 5 nmol/min per mg of protein. The electron transport inhibitors cyanide and azide eliminated uptake, as did the uncoupler carbonyl cyanide-m-chlorophenylhydrazone. The sulfydryl reagent p-chloromercuribenzoic acid and the heavy metal thallium also inhibited uptake, suggesting the presence of an NH4+-specific permease. Concentration of CH3NH3+ across the membrane was demonstrated by conducting uptakes at low temperature to slow the metabolism of CH3NH3+ by glutamine synthetase. At 7 degrees C most of the label was concentrated inside the cells in a form that could be chased from the cells by adding excess NH4+ to the medium. At 30 degrees C most of the label was present as an impermeant metabolite. Thin-layer chromatography of cell extracts confirmed that the radioactivity inside the cells was mainly in the form of CH3NH3+ at 7 degrees C but was present as an unidentified metabolite at 30 degrees C. These studies demonstrate that Frankia sp. strain CpI1 has a high-affinity NH4+ transport system that is synthesized in response to NH4+ starvation.     

Characterization of ammonium (methylammonium)/potassium antiport in Escherichia coli

The energetics of ammonium ion transport by Escherichia coli have been studied using [14C]methylammonium as a substrate. Rapid assays for uptake allowed kinetic parameters (CH3NH3+ Km = 36 microM; Vmax = 4 nmol X s-1 X mg-1 to be determined in the absence of CH3NH3+ metabolism. Cells cultured in media containing 1 mM NH4+ failed to express CH3NH3+ transport activity. Methylammonium accumulated at levels which were 100-fold higher than those of the medium. This accumulation was dependent upon the addition of glucose or pyruvate. The entry of CH3NH3+ supported by glucose oxidation in an F1F0-ATPase-deficient mutant was blocked by uncoupler. Transport by wild-type cells under similar conditions was significantly inhibited by arsenate. Thus, CH3NH3+ uptake requires both ATP and an electrochemical H+ gradient. This transport activity was lost upon exposure of E. coli to osmotic shock, but could be recovered by incubation of shocked cells with boiled shock fluid or with glucose plus K+ in the presence of chloramphenicol. Similar reconstitution was observed in K+-depleted parental strains, but not in a mutant defective in K+ transport, demonstrating a requirement for internal K+. However, external K+ proved to be a noncompetitive inhibitor (Ki = 1 mM) of CH3NH3+ uptake by K+ -replete bacteria. External Na+ had no effect on transport. The addition of NH4+ or CH3NH3+ induced a rapid exodus of intracellular 86Rb+, an analog which was able to substitute for K+. The molar ratio of CH3NH3+ uptake to Rb+ exit was 1.12 +/- 0.11. These findings support a mechanism for CH3NH3+ (NH4+) accumulation which requires K+ antiport (exchange) and is driven by the electrochemical K+ gradient.     

Photoproduction of ammonium ion from N2 in Rhodospirillum rubrum.

NH+4 excretion was undetectable in N2-fixing cultures of Rhodospirillum rubrum (S-1) and nitrogenase activity in these cultures was repressed by the addition of 10 mM NH+4 to the medium. The glutamate analog, L-methionine-DL-sulfoximine (MSX), derepressed N2 fixation even in the presence of 10 mM extracellular NH+4. When 10 mg MSX/ml was added to cultures just prior to nitrogenase induction they developed nitrogenase activity (20% of the control activities) and excreted most of their fixed N2 as NH+4. Nitrogenase activities and NH+4 production from fixed N2 were increased considerably when a combined nitrogen source, NH+4 (greater than 40 mumoles NH+4/mg cell protein in 6 days) or L-glutamate (greater than 60 mumoles NH+4/ mg cell protein in 6 days) was added to the cultures together with MSX. Biochemical analysis revealed that R. rubrum produced glutamine synthetase and glutamate synthase (NADP-dependent) but no detectable NADP-dependent glutamate dehydrogenase. The specific activity of glutamine synthetase was observed to be maximal when nitrogenase activity was also maximal. Nitrogenase and glutamine synthetase activities were repressed by NH+4 as well as by glutamate. The results demonstrate that utilization of solar energy to photoproduce large quantities of NH+4 from N2 is possible with photosynthetic bacteria by interfering with their regulatory control of N2 fixation.     

Effect of Methionine Sulfoximine and Methionine Sulfone on Glutamate Synthesis in Klebsiella aerogenes

At least two pathways exist in Klebsiella aerogenes for glutamate synthesis. A mutant blocked in one pathway due to the loss of glutamate dehydrogenase (gltD) does not require glutamate and has the same growth characteristics as the parent strain in most media; however, its growth is inhibited by the analogues methionine sulfoximine and methionine sulfone. Wild-type Klebsiella is resistant to 0.1 M methionine sulfoximine or methionine sulfone, whereas the gltD mutant is sensitive to 1 mM concentrations. Either glutamate or glutamine is effective in overcoming this inhibition. Activities of both glutamine synthetase and glutamate synthetase, two enzymes involved in the second pathway of glutamate synthesis, are inhibited by methionine sulfoximine and methionine sulfone. The primary effect of methionine sulfoximine appears to be the prevention of glutamine production necessary for subsequent glutamate synthesis via glutamate synthetase enzyme.     

Activity of nitrogenase and glutamine synthetase in relation to availability of oxygen in continuous cultures of a strain of cowpea Rhizobium sp. supplied with excess ammonium.

In samples from nitrogen-fixing continuous cultures of strain CB756 of the cowpea type rhizobia (Rhizobium sp.), newly fixed NH+4 is in equiblibrium with the medium, from where it is assimilated by the glutamine synthetase/glutamate synthase pathway. In samples from steady state cultures with different degrees of oxygen-limitation, nitrogenase activity was positively correlated with the biosynthetic of glutamine synthetase in cell free extracts. Also, activities in biosynthetic assays were positively correlated with activities in gamma-glutamyl transferase assays containing 60 mM Mg2+. Relative adenylylation of glutamine synthetase was conveniently measured in cell free extracts as the ratio of gamma-glutamyl transferase activities without and with addition of 60 mM Mg2+. Automatic control of oxygen supply was used to facilitate the study of transitions between steady-state continuous cultures with high and low nitrogenase activities. Adenylylation of glutamine synthetase and repression of nitrogenase activity in the presence of excess NH+4, were masked when oxygen strongly limited culture yield. Partial relief of the limitation in cultures supplied with 10 mM NH+4 produced early decline in nitrogenase activity and increase in relative adenylylation of glutamine synthetase. Decreased oxygen supply produced a rapid decline in relative adenylylation, followed by increased nitrogenase activity, supporting the concept that control of nitrogenase synthesis is modulated by glutamine synthetase adenylylation in these bacteria.     

Ammonia assimilation pathways in nitrogen-fixing Clostridium kluyverii and Clostridium butyricum.

Pathways of ammonia assimilation into glutamic acid were investigated in ammonia-grown and N2-fixing Clostridium kluyverii and Clostridium butyricum by measuring the specific activities of glutamate dehydrogenase, glutamine synthetase, and glutamate synthase. C. kluyverii had NADPH-glutamate dehydrogenase with a Km of 12.0 mM for NH4+. The glutamate dehydrogenase pathway played an important role in ammonia assimilation in ammonia-grown cells but was found to play a minor role relative to that of the glutamine synthetase/NADPH-glutamate synthase pathway in nitrogen-fixing cells when the intracellular NH4+ concentration and the low affinity of the enzyme for NH4+ were taken into account. In C. butyricum grown on glucose-salt medium with ammonia or N2 as the nitrogen source, glutamate dehydrogenase activity was undetectable, and the glutamine synthetase/NADH-glutamate synthase pathway was the predominant pathway of ammonia assimilation. Under these growth conditions, C. butyricum also lacked the activity of glucose-6-phosphate dehydrogenase, which catalyzes the regeneration of NADPH from NADP+. However, high activities of glucose-6-phosphate dehydrogenase as well as of NADPH-glutamate dehydrogenase with a Km of 2.8 mM for NH4+ were present in C. butyricum after growth on complex nitrogen and carbon sources. The ammonia-assimilating pathway of N2-fixing C. butyricum, which differs from that of the previously studied Bacillus polymyxa and Bacillus macerans, is discussed in relation to possible effects of the availability of ATP and of NADPH on ammonia-assimilating pathways.     

Role of metabolism in the chemotactic response of Rhodobacter sphaeroides to ammonia.

Rhodobacter sphaeroides only showed chemotaxis towards ammonia if grown under nitrogen-limited conditions. This chemotactic response was completely inhibited by the addition of methionine sulfoximine. There was no effect of methionine sulfoximine treatment on motility or taxis towards propionate, demonstrating that the effect is specific to ammonia taxis. It is known that methionine sulfoximine inhibits glutamine synthetase and hence blocks ammonia assimilation. Methionine sulfoximine does not inhibit ammonia transport in R. sphaeroides; therefore, these results suggest that limited metabolism via a specific pathway is required subsequent to transport to elicit a chemotactic response to ammonia. Bacteria grown on high ammonia show transport but no chemotactic response to ammonia, suggesting that the pathway of assimilation is important in eliciting a chemotactic response.     

Interactions between glutamine metabolism and cell-volume regulation in perfused rat liver

1. In the presence of near-physiological glutamine concentrations, exposure of perfused rat liver to hypotonic perfusion media switched glutamine balance across the liver from net release to net uptake. This was due to both stimulation of flux through glutaminase and inhibition of flux through glutamine synthetase. Conversely, during exposure to hypertonic media, net glutamine release from the liver increased due to inhibition of glutaminase flux and slight stimulation of flux through glutamine synthetase. The effect of perfusate osmolarity on glutaminase flux was observed at an NH4Cl concentration (0.5 mM) sufficient for near-maximal ammonia stimulation of glutaminase. This indicates the involvement of different mechanisms of glutaminase flux control by extracellular osmolarity changes and ammonia. The effects of anisotonicity on flux through glutamine-metabolizing enzymes were fully reversible. Glutamine (0.6 mM) stimulated urea synthesis from NH4Cl (0.5 mM) during hypotonic and normotonic conditions. 2. Exposure to hypotonic and hypertonic media led, after initial liver-cell swelling and shrinkage, respectively to volume-regulatory K+ fluxes which largely restored the initial liver-cell volume despite the continuing osmotic challenge. Even after completion of cell-volume regulatory K+ fluxes, the effects of perfusate osmolarity on hepatic glutamine metabolism persisted. This indicates that in anisotonicity the liver cell is left in an altered metabolic state, even after completion of volume-regulatory responses. 3. During perfusion with isotonic media, addition of glutamine (3 mM) led to an increase of liver mass by about 4% within 2 min, which was accompanied by a net K+ uptake by the liver. Thereafter, the new steady state of increased liver mass was maintained throughout glutamine infusion. When the liver mass had reached this new steady state, a net release of K+ from the liver of about 3 mumol/g liver was observed during the following 10 min. Withdrawal of glutamine was followed by a slow reuptake of K+ and the liver mass returned to its initial value. Following exposure to glutamine (3 mM), the intracellular glutamine concentration (as calculated from glutamine tissue levels, taking into account the extracellular space determined with the [3H]inulin technique) rose from about 1 mM to 30-35 mM within about 12 min, indicating a 10-12-fold concentrative uptake of glutamine into the liver cells and an osmotic challenge for the hepatocyte. When intracellular glutamine had reached its steady-state concentration, net K+ efflux from the liver was also terminated.(ABSTRACT TRUNCATED AT 400 WORDS)     

Regulation of glutamine metabolism in Chlorella pyrenoidosa. Mechanisms of regulating the activity of glutamine synthetase during ammonia assimilation]

Glutamine synthetase (GS) (E.C.6.3.1.2) activity in Chlorella cells decreased when NH4+ was added to nitrogen-free growth medium. This GS inactivation had such a rate, that it could not be due to the repression of enzyme synthesis: the GS activity decreased by 20% within 5 minutes of NH4+ assimilation. Glutamine content in cell increased in 2.5 times for this period. In vitro experiments have shown that glutamine is a strong inhibitor of GS from Chlorella grown in the presence of NO3-, and in a less degree--an inhibitor of GS from cells grown in ammonium-containing medium. The data obtained are negative with respect to possible mechanisms of GS activity regulation via adenylation and ATP-dependent destruction of glutamine synthetase.     

Effects of L-glutamate/D-aspartate and monensin on lactic acid production in retina and cultured retinal Müller cells

We have investigated the dependence of the rate of lactic acid production on the rate of Na(+) entry in cultured transformed rat Müller cells and in normal and dystrophic (RCS) rat retinas that lack photoreceptors. To modulate the rate of Na(+) entry, two approaches were employed: (i) the addition of L-glutamate (D-aspartate) to stimulate coupled uptake of Na(+) and the amino acid; and (ii) the addition of monensin to enhance Na(+) exchange. Müller cells produced lactate aerobically and anaerobically at high rates. Incubation of the cells for 2-4 h with 0.1-1 mM L-glutamate or D-aspartate did not alter the rate of production of lactate. ATP content in the cells at the end of the incubation period was unchanged by addition of L-glutamate or D-aspartate to the incubation media. Na(+)-dependent L-glutamate uptake was observed in the Müller cells, but the rate of uptake was very low relative to the rate of lactic acid production. Ouabain (1 mM) decreased the rate of lactic acid production by 30-35% in Müller cells, indicating that energy demand is enhanced by the activity of the Na(+)-K(+) pump or depressed by its inhibition. Incubation of Müller cells with 0.01 mM monensin, a Na(+) ionophore, caused a twofold increase in aerobic lactic acid production, but monensin did not alter the rate of anaerobic lactic acid production. Aerobic ATP content in cells incubated with monensin was not different from that found in control cells, but anaerobic ATP content decreased by 40%. These results show that Na(+)-dependent L-glutamate/D-aspartate uptake by cultured retinal Müller cells causes negligible changes in lactic acid production, apparently because the rates of uptake are low relative to the basal rates of lactic acid production. In contrast, the marked stimulation of aerobic lactic acid production caused by monensin opening Na(+) channels shows that glycolysis is an effective source of ATP production for the Na(+)-K(+) ATPase. A previous report suggests that coupled Na(+)-L-glutamate transport stimulates glycolysis in freshly dissociated salamander Müller cells by activation of glutamine synthetase. The Müller cell line used in this study does not express glutamine synthetase; consequently these cells could only be used to examine the linkage between Na(+) entry and the Na(+) pump. As normal and RCS retinas express glutamine synthetase, the role of this enzyme was examined by coapplication of L-glutamate and NH(4) (+) in the presence and absence of methionine sulfoximine, an inhibitor of glutamine synthetase. In normal retinas, neither the addition of L-glutamate alone or together with NH(4) (+) caused a significant change in the glycolytic rate, an effect linked to the low rate of uptake of this amino acid relative to the basal rate of retinal glycolysis. However, incubation of the RCS retinas in media containing L-glutamate and NH(4)(+) did produce a small (15%) increase in the rate of glycolysis above the rate found with L-glutamate alone and controls. It is unlikely that this increase was the result of conversion of L-glutamate to L-glutamine, as it was not suppressed by inhibition of glutamine synthetase with 5 mm methionine sulfoximine. It appears that the magnitude of Müller cell glycolysis required to sustain the coupled transport of Na(+) and L-glutamate and synthesis of L-glutamine is small relative to the basal glycolytic activity in a rat retina.     

Simultaneous synthesis and degradation of glutamine in isolated rat liver cells. Effect of vasopressin

When hepatocytes suspensions obtained from whole livers of 48-h-fasted rats were incubated in Krebs-Henseleit buffer with a near-physiological concentration (1 mM) of L-[1-14C]glutamine as substrate, the apparent removal of glutamine was low, but the release of 14CO2 was much larger than the enzymatically measured removal of glutamine. This indicates that glutamine was metabolized at rates much higher than those accounted for by the apparent removal of glutamine. This also suggests that glutamine utilization was, at least in part, masked by concomitant synthesis of glutamine from endogenous substrates via glutamine synthetase. Evidence that such synthesis occurred was obtained by: (i) addition of methionine sulfoximine, an inhibitor of glutamine synthetase, which caused a large increase in the apparent removal of glutamine; and (ii) measurement of the specific radioactivity of L-[1-14C]glutamine which was shown to decrease during incubation. Addition of vasopressin (10(-7) M) led to a marked increase in glutamine removal by a dual mechanism: it accelerated flux through glutaminase, the enzyme which initiates the hepatic degradation of glutamine, and inhibited flux through glutamine synthetase.