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.

[Effect of NH4+ on synaptosomal membrane potential in the rat brain].

The effect of NH4+ on synaptosomal membrane potential was examined using rat brains. The membrane potential was measured by the rhodamine 6G fluorescence method. Both NH4+ diffusion potential (NH4+-potential) and K+ diffusion potential (K+-potential) were observed in the synaptosomes. Upon replacement of medium Cl- with SCN-, both K+- and NH4+-potentials depolarized. On the other hand, replacement of medium Cl- with gluconate resulted in the hyperpolarization of K+-potential, but not of NH4+-potential. Ethacrynic acid (0.3 mM), a Cl- -ATPase inhibitor, depolarized both K+(Cl-)- and NH4+ (Cl-)-potentials. In the presence of ethacrynic acid, both of the potentials were further depolarized by replacement of medium Cl- with SCN-, but not with gluconate-. Picrotoxin (5 mM), a Cl- channel inhibitor, did not significantly affect either K+- or NH4+-potential. In the presence of picrotoxin, replacement of medium Cl- with SCN- depolarized both K+- and NH4+-potentials with or without ethacrynic acid. Gluconate depolarized the K+-potential with ethacrynic acid and the NH4+-potential with or without ethacrynic acid. These findings suggest that NH4+ forms a diffusion potential in nerve endings, and inhibits the anion-mediated hyperpolarization through mechanisms other than anion channels.

Cyclic AMP and NH3/NH4+ both regulate cell-type-specific mRNA accumulation in the cellular slime mold, Dictyostelium discoideum.

Dictyostelium discoideum cells plated for development until aggregation stage, and then dissociated into media containing glucose, albumin, and cAMP will form into clumps and undergo prespore and prestalk differentiation. Differentiation in this in vitro system is dependent on three components: cAMP, multicellularity, and the acquisition of "differentiation competence" which the cells acquire in a period between interphase and aggregation stage when plated on Millipore filters. We have used this system to explore aspects of the multicellular environment which are involved in regulation the accumulation of the different prespore- and prestalk-specific messenger RNAs. Two classes of prespore messenger RNA, as well as a prestalk-specific messenger RNA, all require the acquisition of differentiation competence in order to be expressed in response to cAMP. Additionally, all of these messenger RNAs require agglomerate formation for maximal expression. The addition of 33 mM ammonium sulfate (NH4)2SO4, however, can entirely replace the requirement for agglomerate formation for expression of the prestalk-specific messenger RNA, and can partially substitute for agglomerate formation in inducing the expression of both classes of prespore-specific messenger RNAs. In this system, cAMP is essential for the initial induction of expression of all three classes of messenger RNAs. In this system, cAMP is essential for the initial induction of expression of all three classes of messenger RNAs while agglomerate formation or elevated NH3/NH+4 is essential only for the maintenance of the elevated levels of the messenger RNAs.

Rett syndrome revisited: a patient with biotin dependency.

A patient with Rett syndrome (cerebral atrophy associated with hyperammonemia) was studied. Primary defects of urea cycle enzymes were excluded as causes of the disorder. The analysis of urinary organic acids showed a moderate increase of lactate, methylcitrate, tiglyglycine and 3-hydroxisovalerate, indicating an abnormality of multiple carboxylases. Biotin supplementation reversed the urinary abnormalities. In fibroblasts grown with a low biotin medium propionylCoA and 3-methylcrotonylCoA carboxylase activities were reduced. Holocarboxylase synthetase activity was normal (Vmax and Km). Surprisingly the biotinidase in fibroblasts was not decreased. The data indicate that some patients with Rett syndrome might suffer from a biotin-dependent defect of unknown nature.

Pathophysiology of chronic tubulo-interstitial disease in rats. Interactions of dietary acid load, ammonia, and complement component C3.

The human end-stage kidney and its experimental analogue, the remnant kidney in the rat, exhibit widespread tubulo-interstitial disease. We investigated whether the pathogenesis of such tubulo-interstitial injury is dependent upon adaptive changes in tubular function and, in particular, in ammonia production when renal mass is reduced. Dietary acid load was reduced in 1 3/4-nephrectomized rats by dietary supplementation with sodium bicarbonate (NaHCO3), while control rats, paired for serum creatinine after 1 3/4 nephrectomy, were supplemented with equimolar sodium chloride. After 4-6 wk, NaHCO3-supplemented rats demonstrated less impairment of tubular function as measured by urinary excretory rates for total protein and low molecular weight protein and higher transport maximum for para-aminohippurate per unit glomerular filtration rate, less histologic evidence of tubulo-interstitial damage, less deposition of complement components C3 and C5b-9, and a lower renal vein total ammonia concentration. Such differences in tubular function could not be accounted for simply on the basis of systemic alkalinization, and differences in tubular injury could not be ascribed to differences in glomerular function. Because nitrogen nucleophiles such as ammonia react with C3 to form a convertase for the alternative complement pathway, and because increased tissue levels of ammonia are associated with increased tubulo-interstitial injury, we propose that augmented intrarenal levels of ammonia are injurious because of activation of the alternative complement pathway. Chemotactic and cytolytic complement components are thereby generated, leading to tubulo-interstitial inflammation. Thus, alkali supplementation reduces chronic tubulo-interstitial disease in the remnant kidney of the rat, and we propose that this results, at least in part, from reduction in cortical ammonia and its interaction with the alternative complement pathway.

Influence of ammonium ions on hepatic de novo pyrimidine biosynthesis.

Carbamyl phosphate (CP) is synthesized in the liver by two separate enzymes, CPS I and CPS II. CPS I, an intramitochondrial enzyme involved in ureogenesis, has a relative activity of 500- to 1000-fold greater than CPS II, a cytoplasmic enzyme which initiates the sequence of reactions for pyrimidine biosynthesis. The contributions of NH4Cl (substrate for CPS I) ang glutamine (substrate for CPS II) as precursors for pyrimidine biosynthesis in isolated hepatocytes were compared by measuring their effect on uracil nucleotide pool size, the incorporation of NaH14CO3 into these pools, and the accumulation of orotic acid. Physiological concentrations of NH4Cl caused a marked stimulation of incorporation of radioactivity into uracil nucleotides (6-fold increase at 0.5 mM NH4Cl), and radioactive orotate appeared in both the cells and the medium. In contrast, glutamine (at concentrations up to 10 mM) had no effect on the incorporation of radioactivity into uracil nucleotides, and no orotic acid was detected. Uracil nucleotide pools were expanded up to 50% by low levels of NH4Cl, but there was no expansion of this pool in the presence of added glutamine. NH4Cl-driven pyrimidine de novo biosynthesis was insensitive to feedback inhibition by an expanded uracil nucleotide pool, to galactosamine treatment, and to acivicin treatment, indicating that NH+4 stimulated pyrimidine biosynthesis as a result of CP synthesis by mitochondrial CPS I. The consequence of intramitochondrially produced CP being available for pyrimidine biosynthesis is that the controlling step of this pathway (CPS II) is bypassed. The appearance of orotic acid following NH4Cl stimulation indicated that the rate-controlling step of hepatic de novo pyrimidine synthesis under these conditions was orotate phosphoribosyl transferase. These data indicate that, at physiological concentrations of NH+4, the majority of uracil nucleotides synthesized in isolated rat hepatocytes was derived from intramitochondrially generated CP. The effect of NH4Cl on the output of uridine by the isolated perfused rat liver was examined. In the presence of a single addition of 20 mM NH4Cl, the excretion of uridine was increased from 100-200 to 375 nmol h-1 g-1 liver and orotic acid was released into the circulating perfusate reaching a maximum of 2 microM (in 220 ml of perfusate) after 2 h. With 40 mM NH4Cl, uridine export was increased to 450 nmol h-1 g-1 and a maximum of 5 microM orotic acid was released into the perfusate after 2 h.(ABSTRACT TRUNCATED AT 400 WORDS)

Correlation of increased intraacrosomal pH with the hamster sperm acrosome reaction.

This study describes investigations of the importance of intraacrosomal pH in the hamster sperm acrosome reaction (AR). Washed cauda epididymal sperm were capacitated in vitro in a medium containing 2 mM Ca2+, 144 mM Na+, and 3 mM K+. Such sperm underwent a significant increase in the number of AR within 10 min after the addition of the Mg2+-ATPase (adenosine triphosphatase) inhibitors DCCD (20 microM) or NBD-Cl (10 microM) or the proton ionophore FCCP (6 micrograms/ml) at 3.5 hr of incubation or after addition of HN4Cl (3 mM) at 4 hr of incubation. Addition of the mitochondrial electron transport inhibitor rotenone (2.5 microM) at 3.5 hr or of NaCl (3 mM) or KCl (3 mM) at 4 hr did not stimulate AR over control levels, suggesting that the stimulation of AR by the other compounds was not directly due to depletion of acrosomal adenosine triphosphate (ATP) or alteration of the acrosomal transmembrane potential. The AR also was not stimulated by either DCCD or FCCP added prior to 3 hr of incubation of sperm, whereas both compounds were increasingly effective at stimulating AR with increasing length of preincubation of sperm before the addition of the test compounds. The intraacrosomal pH of sperm incubated in low [K+] (0.6-0.9 mM) for 3.5 hr rose by at least one pH unit (as measured with the fluorescent dye 9-aminoacridine) within 15-30 min after raising extracellular [K+] to 4.2-4.5 mM. The pH rise occurred even in the presence of the Ca2+-chelator EGTA (2 mM). Either FCCP (8 micrograms/ml) or DCCD (20 microM), but not rotenone (2.5 microM), plus K+ (3.6 mM), raised the intraacrosomal pH of sperm incubated for 3 hr in low [K+] within 10 min after addition. No pH rise occurred in the absence of additional K+. These results demonstrate that the intraacrosomal pH of the hamster sperm becomes more alkaline in a process not requiring high concentrations of external Ca2+, but requiring K+. The results of this and previous studies lead us to suggest here that the intraacrosomal pH rise may be mediated via a change in K+ and H+ permeability of sperm head membranes, which allows K+ influx and H+ efflux, and via inhibition of an acrosomal Mg2+-ATPase proton pump. We propose that the permeability changes and the consequent alkalinization of the acrosomal interior are important steps in late capacitation and/or the mammalian AR.

Intracellular pH and the control of cell differentiation in Dictyostelium discoideum.

During development in the cellular slime mould Dictyostelium discoideum starved amoebae aggregate to form multicellular structures that display a simple antero-posterior pattern: prestalk cells occupy the front 20% of the aggregate, and prespore cells occupy the remainder. We have attempted to elucidate the nature of the mechanism regulating the proportions of the two cell types by examining the factors that influence the pathway of differentiation of amoebae in vitro. Amoebae of D. discoideum strain V12 M2 form stalk cells efficiently in appropriate conditions and 'sporogenous' derivatives produce spores as well as stalk cells. Mature spores are formed in a medium containing only cyclic AMP and salts, whereas formation of stalk cells requires, in addition, a low molecular weight hydrophobic factor (DIF). Recent observations have led us to propose that DIF is a morphogen responsible for activating stalk cell differentiation. Here we present evidence that ammonia is a second morphogen, that acts antagonistically to DIF, and that the choice of differentiation pathway is mediated by intracellular pH.

Ammonia regulation of amino acid permeases in Saccharomyces cerevisiae.

The activities of the proline-specific permease (PUT4) and the general amino acid permease (GAP1) of Saccharomyces cerevisiae vary 70- to 140-fold in response to the nitrogen source of the growth medium. The PUT4 and GAP1 permease activities are regulated by control of synthesis and control of activity. These permeases are irreversibly inactivated by addition of ammonia or glutamine, lowering the activity to that found during steady-state growth on these nitrogen sources. Mutants altered in the regulation of the PUT4 permease (Per-) have been isolated. The mutations in these strains are pleiotropic and affect many other permeases, but have no direct effect on various cytoplasmic enzymes involved in nitrogen assimilation. In strains having one class of mutations (per1), ammonia inactivation of the PUT4 and GAP1 permeases did not occur, whereas glutamate and glutamine inactivation did. Thus, there appear to be two independent inactivation systems, one responding to ammonia and one responding to glutamate (or a metabolite of glutamate). The mutations were found to be nuclear and recessive. The inactivation systems are constitutive and do not require transport of the effector molecules per se, apparently operating on the inside of the cytoplasmic membrane. The ammonia inactivation was found not to require a functional glutamate dehydrogenase (NADP). These mutants were used to show that ammonia exerts control of arginase synthesis largely by inducer exclusion. This may be the primary mode of nitrogen regulation for most nitrogen-regulated enzymes of S. cerevisiae.

Continuous culture of Mycobacterium avium limited for ammonia.

Previous investigations demonstrated the utility of a cell-cycle technique for physiologic studies of lipid metabolism in Mycobacterium avium. Such a culture technique was not suitable for enzymic analyses. Therefore, continuous culture of M. avium was attempted. A modified culture medium, limited for ammonia nitrogen, was used to culture the organism at dilution rates of 0.08 to 0.175 per h. Measurements of colony-forming units, isotope incorporation, and cell protein during extended incubation times demonstrated that the organism was in steady state. Cells harvested from continuous culture had levels of glutamate synthase that increased proportionately to the dilution rate.