Bistability in the activity of glutamine synthetase in ammonium-limiting chemostat cultures of Escherichia coli ML 30]

The approximative estimation of the function micron([NH+4]) in cultures of E. coli ML 30 had shown that bistability of the ammonium concentration in ammonium limited continuous cultures could be possible (BERGTER et al. 1977). This phenomenon suggested a bistability in the regulation of ammonia assimilation. Therefore, the activity of one key enzyme of the two ammonia assimilation systems was measured. The distribution of the activity of glutamine synthetase in ammonia limited continuous cultures after different transition states confirmed this suggestion.     

On the dissociation constant of ammonium: effects of using an incorrect pK a in calculations of the ammonia concentration in animal cell cultures

The dissociation constant of NH 4 + , pK a NH 4 + , was used to calculate the ammonia concentration in cell cultures. pK a NH 4 + is 8.95 at 35°C. As an incorrect pK a (e.g. 9.27-9.3 at 37°C) is often used, this results in errors. The probable origin for this appears to be a combination of pK w at 24°C and pK b at 35°C. At 35°C and any pH, the ammonia concen-tration calculated from pK a = 8.95 is twice the ammonia concentration calculated from pK a = 9.27, but the differences in ammonium concentration are negligible.     

Transient behavior of the ammonium-limited chemostatic cultures of Escherichia coli ML 30]

In connection with the bistability of pyruvate formation in ammonium limited continuous cultures of E. coli ML 30 (Bergter u. Roth 1977) the transient behaviour of cell density and pyruvate concentration were studied. Immediately after a shift up in the dilution rate from D = 0.15 h-1 to D = 0.6 h-1 the bacteria excreted pyruvate into the medium, followed by a resumption of pyruvate. The specific pyruvate formation rate as well as the specific growth rate reached the new steady state with damped oscillations. Possibly the excretion of pyruvate after the shift is caused by the higher non limiting concentrations of ammonium during the first of the transition. This hypothesis is supported by the transient behaviour of an ammonium limited continuous culture after a pulse of ammonium to the culture. The relations between ammonium metabolism and pyruvate formation are discussed.     

Bistability of pyruvate production by E. coli ML30 in continuous culture]

The pyruvate production of E. coli ML30 in continuous cultures was investigated. In a glucose mineralsalt medium with ammonium as the limiting substrate two stable stationary states (bistability) of pyruvate concentration were obtained. The bistability was limited to dilution rates lower than 0.3 h-1 and connected with a decrease of the yield coefficient (Y Glc) from approximately 0.45 to approximately 0.1.     

NH3 is involved in the NH4+ transport induced by the functional expression of the human Rh C glycoprotein

Renal ammonium (NH3 + NH4+) transport is a key process for body acid-base balance. It is well known that several ionic transport systems allow NH4+ transmembrane translocation without high specificity NH4+, but it is still debated whether NH3, and more generally, gas, may be transported by transmembrane proteins. The human Rh glycoproteins have been proposed to mediate ammonium transport. Transport of NH4+ and/or NH3 by the epithelial Rh C glycoprotein (RhCG) may be of physiological importance in renal ammonium excretion because RhCG is mainly expressed in the distal nephron. However, RhCG function is not yet established. In the present study, we search for ammonium transport by RhCG. RhCG function was investigated by electrophysiological approaches in RhCG-expressing Xenopus laevis oocytes. In the submillimolar concentration range, NH4Cl exposure induced inward currents (IAM) in voltage-clamped RhCG-expressing cells, but not in control cells. At physiological extracellular pH (pHo) = 7.5, the amplitude of IAM increased with NH4Cl concentration and membrane hyperpolarization. The amplitude of IAM was independent of external Na+ or K+ concentrations but was enhanced by alkaline pHo and decreased by acid pHo. The apparent affinity of RhCG for NH4+ was affected by NH3 concentration and by changing pHo, whereas the apparent affinity for NH3 was unchanged by pHo, consistent with direct NH3 involvement in RhCG function. The enhancement of methylammonium-induced current by NH3 further supported this conclusion. Exposure to 500 microm NH4Cl induced a biphasic intracellular pH change in RhCG-expressing oocytes, consistent with both NH3 and NH4+ enhanced influx. Our results support the hypothesis of a specific role for RhCG in NH3 and NH4+ transport.     

Modulation of electroneutral Na transport in sheep rumen epithelium by luminal ammonia

Ammonia is an abundant fermentation product in the forestomachs of ruminants and the intestine of other species. Uptake as NH3 or NH4+ should modulate cytosolic pH and sodium-proton exchange via Na+/H+ exchanger (NHE). Transport rates of Na+, NH4+, and NH3 across the isolated rumen epithelium were studied at various luminal ammonia concentrations and pH values using the Ussing chamber method. The patch-clamp technique was used to identify an uptake route for NH4+. The data show that luminal ammonia inhibits electroneutral Na transport at pH 7.4 and abolishes it at 30 mM (P < 0.05). In contrast, at pH 6.4, ammonia stimulates Na transport (P < 0.05). Flux data reveal that at pH 6.4, approximately 70% of ammonia is absorbed in the form of NH4+, whereas at pH 7.4, uptake of NH3 exceeds that of NH4+ by a factor of approximately four. The patch-clamp data show a quinidine-sensitive permeability for NH4+ and K+ but not Na+. Conductance was 135 +/- 12 pS in symmetrical NH(4)Cl solution (130 mM). Permeability was modulated by the concentration of permeant ions, with P(K) > P(NH4) at high and P(NH4) > P(K) at lower external concentrations. Joint application of both ions led to anomalous mole fraction effects. In conclusion, the luminal pH determines the predominant form of ammonia absorption from the rumen and the effect of ammonia on electroneutral Na transport. Protons that enter the cytosol through potassium channels in the form of NH4+ stimulate and nonionic diffusion of NH3 blocks NHE, thus contributing to sodium transport and regulation of pH.     

Effects of Ammonium and Non-Ammonium Salt Additions on Methane Oxidation by Methylosinus trichosporium OB3b and Maine Forest Soils

Additions of ammonium and non-ammonium salts inhibit atmospheric methane consumption by soil at salt concentrations that do not significantly affect the soil water potential. The response of soils to non-ammonium salts has previously raised questions about the mechanism of ammonium inhibition. Results presented here show that inhibition of methane consumption by non-ammonium salts can be explained in part by ion-exchange reactions: cations desorb ammonium, with the level of desorption varying as a function of both the cation and anion added; differential desorption results in differential inhibition levels. Differences in the extent of inhibition among ammonium salts can also be explained in part by the effects of anions on ammonium exchange. In contrast, only minimal effects of cations and anions are observed in liquid cultures of Methylosinus trichosporium OB3b. The comparable level of inhibition by equinormal concentrations of NH(4)Cl and (NH(4))(2)SO(4) and the insensitivity of salt inhibition to increasing methane concentrations (from 10 to 100 ppm) are of particular interest, since both of these patterns are in contrast to results for soils. The greater inhibition of methane consumption for NH(4)Cl than (NH(4))(2)SO(4) in soils can be attributed to increased ammonium adsorption by sulfate; increasing inhibition by non-ammonium salts with increasing methane concentrations can be attributed to desorbed ammonium and a physiological mechanism proposed previously for pure cultures.     

Regulation of ammonia assimilation in ammonia-limited chemostat cultures of Escherichia coli ML 30: evidence of bistability

In a preceding paper evidence of two stationary stable states (bistability) in the specific activity of glutamine synthetase (GS) in ammonia-limited steady-state cultures of Escherichia coli ML 30 at dilution rates (D) about 0.15 h-1 was described (Müller et al. 1977). For better understanding of the regulation mechanisms leading to GS bistability chemostat experiments were performed over a wide range of dilution rates up to D = 0.8 h-1. For each steady state the specific activities of GS and glutamate dehydrogenase (GDH)--the other key enzyme of the two NH3 assimilation routes in E. coli--and in addition the remaining NH3 concentration in the culture liquid were determined. Parallel to GS bistability two states of GDH activity and NH3 concentration are found. The higher state of GS is connected with a lower GDH activity and NH3 concentration. With rising D the GS activities decrease whereas GDH activities and NH3 concentrations increase. Since no adenylation of the GS is detectable GS bistability seems to be regulated on the level of enzyme synthesis like GDH bistability. From the experimental findings a mathematical model is derived based on the bottle neck enzyme theory of growth. It describes the dependence between the specific growth rates on the one hand and the specific enzyme activities and NH3 concentration on the other. It is shown that the specific uptake rate of the limiting NH3 and the specific growth rates, respectively, depend on the simultaneous action of two bottle neck enzymes which are connected by a regulative link.     

耐铵型产琥珀酸放线杆菌的选育及铵离子对其生长代谢的影响

经硫酸二乙酯(DES)诱变,在含61～242mmol/LNH4+梯度平板中,筛选到一株耐铵型突变株YZ25,该菌株在含121mmol/LNH4+发酵培养基中,琥珀酸产量达32.68g/L,转化率为65.4%,比出发菌提高了180.5%。进一步考察了不同形态铵盐对YZ25生长的影响,结果表明添加少量铵盐能够提高突变菌的生长速率,但当超过一定量后菌株生长受到抑制,不同铵盐对菌株的抑制程度不同,硫酸铵、碳酸氢铵、氯化铵和硝酸铵对突变株YZ25的半抑制浓度分别为:215mmol/L、265mmol/L、235mmol/L、210mmol/L。为了考察铵离子对YZ25发酵产琥珀酸的影响过程,在3.0L发酵罐以氨水作为pH的调控剂发酵,结果表明在稳定期前菌株生长基本不受铵离子抑制,生物量能够达到正常水平,但是进入稳定期后铵离子抑制作用越来越明显,导致菌株生长提前结束,耗糖不完全,产酸受阻。最后结合产琥珀酸放线杆菌Actinobacillus succinogenes代谢途径分析了铵离子对菌株抑制作用的机理。     

Isotope-exchange enhancement studies of Escherichia coli glutamine synthetase

Isotope-exchange enhancement studies, a variation on positional isotope-exchange enhancement as described by Raushel and Garrard [Raushel, F. M., & Garrard, L. J. (1984) Biochemistry 23, 1791-1795], are used to establish the point in the biosynthetic reaction of Escherichia coli glutamine synthetase at which gamma-glutamyl phosphate is formed. In these experiments, the behavior of the reverse biosynthetic reaction, i.e., the reaction of ADP, L-glutamine, and phosphate to form NH4+, L-glutamate, and ATP, is examined as a function of the concentration of ammonium ion. By varying the concentration of NH4+, the ratio of the velocity of isotope exchange to the velocity of net reaction, as measured by the rate of 18O depletion from labeled phosphate and the rate of production of L-glutamate, respectively, can be modulated in a mechanism-dependent manner. Evidence is presented demonstrating the presence of a branch point in the mechanism. The enzyme-ATP-glutamate complex may partition in two ways, one involving binding of ammonium ion and the other involving the chemical transformation to form the enzyme-ADP-gamma-glutamyl phosphate complex. The alternate pathways then rejoin upon formation of the enzyme-ADP-NH4+-gamma-glutamyl phosphate complex. Because of the branch point, there is no absolute requirement that ammonium ion be absent or present in order for the formation of gamma-glutamyl phosphate to occur. At high concentrations of ammonia, one pathway through the branch can be eliminated, effectively making that portion of the pathway ordered, with ATP, L-glutamate, and NH4+ binding consistent with our previously reported steady-state kinetic mechanism [Meek, T. D., & Villafranca, J. J. (1980) Biochemistry 19, 5513-5519].     

Effect of hyperammonaemia on blood glucose and plasma insulin levels in sheep.

The effects of continuous intravenous infusions (6 h) of ammonium chloride (5.6; 11.2; and 16.8 mumol.kg-1.min) on plasma glucose and immunoreactive insulin (I.R.I.) levels were studied in three adult sheep. Infusions of 5.6 and 11.2 mumol.kg-1.min elevated ammonia levels in circulating blood from 100 to 150 and 300 microgram.100 ml-1, respectively, but showed no appreciable effect on plasma glucose and I.R.I. concentrations. Infusion of 16.8 mumol.kg-1.min-1 resulted in a blood ammonia concentration of about 400 microgram.100 ml-1 after six hours of infusion. Blood ammonia returned to normal 1 to 2 hours after the end of infusion. Plasma glucose concentration tended to increase slightly from 65 to 75 mg . 100 ml-1 when 16.8 mumol of NH4Cl were infused kg-1.min-1 and remained at the elevated level at least for two additional hours when ammonia infusions were stopped. Plasma I.R.I. tended to decrease from 48 to 38 microunits . ml-1 during the time of the NH4Cl infusion and increased continually to 82 microunits . ml-1 when NH4Cl infusions were stopped. It is concluded from the time courses of plasma glucose and plasma I.R.I. that the effect of ammonia infusion of these parameters cannot entirely be explained by a regulatory release of adrenaline.     

The effect of nitrate and ammonium concentrations on growth and alkaloid accumulation of Atropa belladonna hairy roots

The growth, the alkaloid production, as well as the scopolamine/hyoscyamine ratio of two clones of belladonna hairy roots were studied. The effects of nitrate and ammonium concentrations on these cultures were investigated. A rise in ammonium concentration caused the decline of the hairy roots, while nitrate had a marked effect on the alkaloid content. The alkaloid production obtained with 15.8 mM of NO3- and 20.5 mM of NH4+ was 1.2-1.4 times higher than that obtained when the roots were grown in the standard Murashige and Skoog medium (MS medium, 39.5 mM of NO3- and 20.5 mM of NH4+). The nitrate and ammonium concentrations in the culture medium also had a strong influence on the scopolamine/hyoscyamine ratio. When nitrate or ammonium concentrations were raised, that ratio also was increased 2-3-fold. The hairy root clones originating from transformations with two distinct strains of Agrobacterium had similar responses.     

Use of electrodialysis and reverse osmosis for the recovery and concentration of ammonia from swine manure

This project aimed at producing a concentrated nitrogen fertilizer from liquid swine manure using electrodialysis (ED) and reverse osmosis (RO), as a mean to help resolve the excess nutrient problem faced by many swine producers, and offer an alternative to chemical nitrogen fertilizer production. Different types of ED membranes were evaluated based on the NH4+ transfer rate, current efficiency and membrane stability. A combination of CMB/AMX membranes was retained due to its high NH4+ transfer rate and chemical stability. The maximum total ammonia concentration (NH3-N) achievable by ED was limited by water transport from the manure to the concentrate compartment, and ammonia volatilization (17%) from the open concentrate compartment. Results suggested that, under the conditions of this experiment, a maximum total NH3-N concentration of about 16g/L could be reached with the ED system. An ED concentrate (8.7g/L of total NH3-N) was also fed to TFC-HF reverse osmosis membranes. A mass balance analysis revealed that the RO permeate, which represented 49.6% of the initial volume, contained 8.6% of the ammonia. However, the RO concentrate contained only 66.6% of the initial total NH3-N, suggesting that 21.2% of the ammonia was volatilized during the concentration test with RO membranes. Ammonia concentration in the RO concentrate reached approximately 13g/L, which is similar to the maximum concentration that could be achieved by ED. These results suggest that the use of ED and RO membranes to recover and concentrate ammonia is potentially interesting but the process must include an approach to minimize ammonia volatilization or trap volatilized ammonia.     

Simultaneous nitrification and formaldehyde biodegradation in an activated sludge unit

The simultaneous removal of formaldehyde and ammonium in a lab-scale activated sludge unit was investigated. The unit was operated at a hydraulic retention time of 2.4 days with an ammonium concentration in the influent of 350 mg NH4+-N/L, maintaining the ammonium loading rate at 0.15 g NH4+-N/Ld during the operation time. However, the applied organic loading rate was increased stepwise by increasing the formaldehyde concentration from 26 up to 3168 mg/L, corresponding to 0.01-1.40 g COD/Ld. High formaldehyde removal efficiencies, around 99.5% (+/-0.38), were maintained at all the formaldehyde concentrations. Ammonium removal was also very high during the operation period, around 99.9% (+/-0.01). The ammonium concentration in the effluent was lower than 0.1 mg NH4+-N/L at all applied organic loading rates, indicating that there was no inhibition of nitrification by formaldehyde.     

Effects of ammonia on the de novo synthesis of polypeptides in cells of Nitrosomonas europaea denied ammonia as an energy source.

The effects of ammonium on the de novo synthesis of polypeptides in the soil-nitrifying bacterium Nitrosomonas europaea have been investigated. Cells were incubated in the presence of both acetylene and NH4+. Under these conditions, the cells were unable to utilize NH4+ as an energy source. Energy to support protein synthesis was supplied by the oxidation of hydroxylamine or other alternative substrates for hydroxylamine oxidoreductase. De novo protein synthesis was detected by 14C incorporation from 14CO2 into polypeptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. In the presence of NH4+, acetylene-treated cells synthesized the 27-kDa polypeptide of ammonia monoxygenase (AMO) and two other major polypeptides (with sizes of 55 and 65 kDa). The synthesis of these polypeptides was completely inhibited by chloramphenicol and attenuated by rifampin. The optimal concentration of hydroxylamine for the in vivo 14C-labeling reaction was found to be 2 mM. The effect of NH4+ concentration was also examined. It was shown to cause a saturable response with a Ks of approximately 2.0 mM NH4+. Labeling studies conducted at different pH values suggest cells respond to NH3 rather than NH4+. No other compounds tested were able to influence the synthesis of the 27-kDa component of AMO, although we have also demonstrated that this polypeptide can be synthesized under anaerobic conditions in cells utilizing pyruvate- or hydrazine-dependent nitrite reduction as an energy source. We conclude that ammonia has a regulatory effect on the synthesis of a subunit of AMO in addition to providing nitrogen for protein synthesis.     

Responses to reversed NH3 and NH4+ gradients in a teleost (Ictalurus punctatus), an elasmobranch (Raja erinacea), and a crustacean (Callinectes sapidus):evidence for NH4+/H+ exchange in the teleost and the elasmobranch

Ammonia excretion rates of channel catfish, Ictalurus punctatus, little skate (Raja erinacea), and blue crab (Callinectes sapidus) were measured in experimental regimes which permitted simultaneous assessment of the partial pressure gradients for nonionized NH3 and the chemical concentration gradients of NH4+. Under conditions of low external ammonia, the average ammonia excretion was +295 microM kg-1 h-1 for catfish, +149 microM kg-1 h-1 for blue crabs, and +59 microM kg-1 h1 for skates with partial pressure gradients of +72.5 mu Torr, +413 mu Torr, and +24.4 mu Torr, respectively; and [NH4+] gradients of +189 microM l-1, +643 microM l-1, and +107 microM l-1 (positive indicating greater from inside to medium). When the external ammonia was increased to 1.15 mM l-1, both gradients were reversed, and the net ammonia movement was initially from the external water into all three species. In the catfish the inward movement ceased, however, and ammonia excretion eventually resumed in the face of reversed gradients of both NH3 partial pressure and [NH4+]. Unidirectional Na+ influx, indicative of a Na+/NH4+ exchange, did not increase. The ammonia data, changes in titratable acidity, and net apparent H+ efflux were all consistent with a linked extrusion of internal NH4+ for external H+. Incorporation of such an exchange into a computer simulation model of the ammonia equilibrium and exchange system duplicated the experimental data. Other hypotheses failed to match experimental data, or failed to predict internal ammonia levels lower than outside. In the crab, internal ammonia levels rose rapidly to concentrations and partial pressures above the external medium until excretion was reestablished, with no evidence of maintenance of a reversed gradient. In the skate, internal concentrations rose appreciably in the first hour and continued to rise for 6-8 h, with no resumption of ammonia excretion. The interspecies differences appear to be due at least partly to differences in ammonia permeability of the gills.     

Ammonia uptake and its effects on ionoregulation in the freshwater crayfish Pacifastacus leniusculus (Dana)

Exposure of adult crayfish Pacifastacus leniusculus to Artificial Freshwater (AFW) media containing 1.5 m and 0.15 mmol x l(-1) total ammonia [Tamm; 0.1 x acute lethal concentration (24 h LC50) and 0.01 x 24 h LC50] and adjusted to pH 6.5, pH 8.2 and pH 10.5 resulted in significant increases in haemolymph ammonia over a 24-h period. Ammonia accumulated most rapidly at pH 10.5. These media were chosen to expose animals to a range of different un-ionised ammonia (UIA) [NH3] and ionised ammonia [NH4+] concentrations. From comparisons of measured transepithelial potential differences (PDte) with calculated Nernst potentials (PDNH4+) for the known haemolymph-to-medium gradients of [NH4+], it was deduced that, in pH 8.2 and pH 6.5 AFW, NH4+ was not in thermodynamic equilibrium across the integument (presumably gill epithelium). In pH 10.5 AFW with 1.5 mmol x l(-1) Tamm (predominantly NH3), the accumulation of ammonia in the haemolymph was in the NH4+ form due to haemolymph pH regulation by the crayfish in this alkaline external medium. Measured net fluxes of ammonia (Jamm(net)) were inwardly directed and maximal when [NH3] was the main component externally, but were also significant at pH 8.2 with high [NH4+] ([NH4+]:[NH3] approximately 20:1). Haemolymph Na+ depletion was significant and, over the 24-h exposure period, most rapid in high [NH3] medium but [Cl-] was unaffected. However, paradoxically, sodium uptake (measured JNa(in) on immediate transfer to high Tamm medium) was not significantly inhibited when [NH3] was the predominant ammonia species. In 1.5 mmol x l(-1) Tamm (mainly [NH4+), VNa(in) (the active component of JNa(in)) was significantly inhibited, particularly at low external [Na+]. This inhibition could not be demonstrated as one of competition at an Na+/NH4+ apical gill exchange site. The resultant net efflux of sodium from the animal showed that the ability of the animals to balance sodium losses at low external [Na+] was severely affected. Longer exposure to pH 10.5 AFW with high [NH3] (12 h) resulted in significantly increased JNa(out), while not significantly affecting JNa(in). Analysis of urinary Na+ losses showed that, while urinary flow rate and water reabsorption was most likely unaffected by ammonia exposure, final urine [Na+] was significantly elevated. The resulting urinary Na+ loss accounted for 63% of the increased JNa(out) in high [NH3] medium.     

Incorporation of ammonium into intracellular UDP-activated N-acetylhexosamines and into carbohydrate structures in glycoproteins.

The negative effects of ammonia on animal cells, especially in vitro cultures, are well known, but the mechanism of how ammonia inhibits cell growth and influences the glycosylation of proteins is not completely understood. We investigated the ammonium action on the synthesis of the intracellular UDP-N-acetylhexos- amines (UDPGNAc), which are precursors of glycosylation as well as on N-linked oligosaccharides of a recombinant human IL-2 mutant variant model glycoprotein expressed in BHK-21 cells under defined and controlled culture conditions in a continuously perfused bioreactor. The examinations were based on our previous observations that increased ammonia concentrations in the medium lead to the intracellular formation and accumulation of UDPGNAc (Ryll et al., 1994). The kinetics of formation of the UDPGNAc pool after adding ammonia and its reconstitution to normal conditions are shown. To study the pathway leading to the intracellular increase of UDPGNAc, the uptake and incorporation of 15NH4+ was confirmed by the detection of 15N in UDP-N-acetylglucosamine (UDP-GlcNAc). UDP-GlcNAc was purified using high pH anion-exchange chromatography with pulsed amperometric detection and analyzed by GC/MS. The proportion of UDP-GlcNAc containing 15N was approximately 60% and corresponds quantitatively to the increased intracellular concentration of UDP-GlcNAc. In order to confirm the direct influence of ammonia on protein glycosylation, the human IL-2 mutant glycoprotein variant IL-Mu6, bearing a novel N-glycosylation site, has been produced under defined protein-free medium conditions in the presence of 15NH4Cl. IL-Mu6 glycoprotein was purified and N-glycans released were analyzed by matrix-assisted laser desorption ionization time of flight mass spectroscopy. Maximally 60-80% of N-acetylated sugars in N-glycan structures contained 15N indicating that ammonium is used as a building block during synthesis of the carbohydrate structures expressed from in vitro cultivated mammalian cells.     

The effect of various ambient ammonia concentrations on the nitrogen metabolism of carp fry (Cyprinus carpio L.)

Authors studied how 125, 375 and 625 micrograms/l amounts of NH4Cl added to the water influenced the ammonia excretion of carp fry with an average weight of 2.4 g, compared to the control. During the course of the experiments the NH4N concentration, the pH and the temperature were measured for three days, twice daily. On the basis of our results the threshold concentration exerting harmful effects on the ammonia household of carp fry (disturbing the normal rate of metabolism in the fishes and decreasing their growth rate) is between 125 and 375 micrograms/l. NH4H (50-100 micrograms/l NH3). The ammonia concentrations exceeding 375 micrograms/l NH4+ (and 100 micrograms/l NH3, respectively) can be regarded as undesirable and harmful in frybreeding fish ponds. With regard to the ambient ammonia, a daily cycle developed in the excretion of ammonia: contrary to the control, a minimum in ammonia excretion was measured in the morning, while a maximum was measured in the afternoon.     

Ammonia production and amino acid metabolism by rat renal papillary epithelial cells in culture

A significant percentage of excreted ammonium is added to tubular fluid along the medullary collecting duct. However, it is not clear whether this ammonia is produced in the cortex and delivered into the medulla or is produced directly by medullary cells. To address this issue, rat epithelial cells derived from the renal papilla were grown in continuous culture and their ability to generate ammonia was examined. When grown in Dulbecco's modified Eagle's medium with 4 mM glutamine, these cells produced ammonia at a rate of approximately 27 nmol/10(6) cells/h. When these cells were grown in minimum essential medium without glutamine, ammonia production fell to 7 nmol/10(6) cells/ h. Increasing the glutamine concentrations of minimum essential medium to 4 mM increased ammonia production to slightly greater than 30 nmol/10(6) cells/ h. Increasing the media concentration of glutamate, glycine, or asparagine resulted in no significant increase in ammoniagenesis. Analysis of media amino acid concentration revealed that glutamine was the main amino acid consumed while alanine was the predominant amino acid produced. The glutaminase activity of these cells appears to be primarily phosphate-dependent, similar to that observed in vitro in papillary tubules. Alterations of K+ or H+ ion concentration did not alter ammoniagenesis, but addition of 2.5 mM ammonium chloride significantly reduced net ammonia production. It is concluded that rat papillary epithelial cells have the intrinsic ability to utilize glutamine to generate ammonia and alanine. In vivo ammonia produced locally in the medulla may contribute to final urinary ammonium excretion.