Effects of amino acids on calcium uptake by glial and neuroblastoma cells.

The uptake of [45Ca] has been studied in clonal glial and neuronal cells. It was somewhat more efficient in the neuroblastoma clone M1 compared to glial clones. In all cases [45Ca] uptake was shown to depend on the phosphate concentration in the incubation medium. It was decreased by the ionophore A 23187 at 200 microM concentration in both neuronal and glial clones. The influence of amino acids some of which are putative neurotransmitters was investigated; the interactions between [45Ca] uptake and these amino acids were related to their concentration and the type of cells used (neuronal or glial). L-aspartate and taurine for example had two opposite effects on [45Ca] uptake by the glial clone NN at two different concentrations; they could therefore play a role in the control of calcium level in the synaptic cleft.     

Effects of amino acids on calcium uptake by glial and neuroblastoma cells

The uptake of [⁴⁵Ca] has been studied in clonal glial and neuronal cells. It was somewhat more efficient in the neuroblastoma clone M₁ compared to glial clones. In all cases [⁴⁵Ca] uptake was shown to depend on the phosphate concentration in the incubation medium. It was decreased by the ionophore A 23187 at 200 μM concentration in both neuronal and glial clones. The influence of amino acids some of which are putative neurotransmitters was investigated; the interactions between [⁴⁵Ca] uptake and these amino acids were related to their concentration and the type of cells used (neuronal or glial). L-aspartate and taurine for example had two opposite effects on [⁴⁵Ca] uptake by the glial clone NN at two different concentrations; they could therefore play a role in the control of calcium level in the synaptic cleft.     

Thyroid hormone inhibition of synaptosome amino acid uptake and protein synthesis.

Abstract— 3,3′,5-Triiodothyronine (T₃) inhibited L-[¹⁴C]leucine uptake into synaptosomes. Inhibition was competitive with a K_i of 3.1 × 10^?5m . Hofstee plot revealed an inverted hyperbolic curve suggestive of a two carrier or carrier plus diffusion mediated system for amino acid uptake. Both the carrier mediated and diffusional components were inhibited by thyroid analogues. l -Thyroxine and analogues inhibited the incorporation of l -[¹⁴C] leucine into cerebral synaptosome protein. At 50 μm , the triiodo-compounds were more inhibitory than tetraiodo->3,5-triiodo-l -thyronine >3,3′,5-triiodothyropro-pionic> l -thyroxine >3,5-diiodo-l -tyrosine. Thyroid analogue inhibition was not seen in liver or brain mitochondrial protein synthesis. 3,3′,5-Triiodothyronine had no effect on respiratory control or 2,4-DNP stimulated synaptosome respiration supported by malate plus pyruvate. Ouabain did not inhibit [¹⁴C]leucine uptake into adult synaptosomes. There was synergistic inhibition of synaptosome protein synthesis by thyroid analogues in the presence of 0.2 mm -ouabain. 3,3′,5-Triiodothyronine had no effect on synaptosome fraction ATPase or Na-K ATPase. Addition of T₃ induced further inhibition of synaptosome protein synthesis in the presence of either chloramphenicol (100μm ) or cycloheximide (50μg/ml). [¹⁴C]Glycine uptake and incorporation into synaptosome protein was inhibited by 3,3′,5-triiodothyronine. There was no inhibition of [¹⁴C]proline uptake or incorporation. The above evidence and kinetic data strongly favor a selective competitive block in amino acid transport at the synaptosome membrane leading to a decreased rate of protein synthesis.     

Characterization of experimental phenylketonuria. Augmentation of hyperphenylalaninemia with alpha-methylphenylalanine and p-chlorophenylalanine

Phenylalanine in conjunction with p-chlorophenylalanine or alpha-methylphenylalanine was administered to suckling rats to induce hyperphenylalaninemia reminiscent of untreated phenylketonuria, and developmental parameters were monitored. The experimental model utilizing p-chlorophenylalanine was found to be unsatisfactory, in that the drug had general deleterious effects on growth, numerous side effects including increased mortality, and affected brain levels of biogenic monoamine neurotransmitters. The model utilizing alpha-methylphenylalanine was relatively free from nonspecific effects and thus, changes observed in the animals were attributable to experimental phenylketonuria. The latter animals had slightly decreased body and brain weights, and exhibited grossly elevated serum phenylalanine and urinary excretion of phenylketone metabolites. Hyperphenylalaninemia produced greatly disrupted brain amino acids at 10 days of age (prior to the formalization of the blood-brain barrier and specific transport systems) which was limited by 30 days of age to changes in glycine, gamma-aminobutyric acid and the aliphatic and aromatic amino acids which compete for uptake in the brain by a common carrier. These animals also exhibited a myelin deficit and changes in proteins from isolated nerve cell preparations. Mature animals which had daily treatment up to 60 days of age exhibited a long-term learning impairment. These observations are consistent with many aspects of the clinical picture of untreated phenylketonuric patients, and suggest that this animal model will be beneficial in studying the disease.     

Amino acid uptake and protein synthesis in preimplanatation mouse embryos

Amino acid uptake and cycloheximide inhibitable incorporation into protein are demonstrable in follicular ova, unfertilized eggs, and in mouse embryos ranging from the 1-cell to the late blastocyst stages. The rates of uptake and incorporation are low and relatively constant until the early blastocyst (day 3) stage of development when they increase 3- to 9-fold. The rate of uptake continues to increase during the fourth day (late blastocyst stage) of development, but, despite embryonic growth, incorporation into protein remains constant. By exposing embryos to leucine and lysine at different concentrations, it is possible to dissociate the processes of uptake and incorporation into protein from one another and to use the latter as a measure of protein synthesis. Taking the number of embryonic cells into account, it is postulated that the period between 8- to 16-cell stage (day 2) and the early blastocyst stage is the only one in which the synthesis of major amounts of protein based on new messenger RNA synthesis is occurring.Leucine and lysine uptake take place by a facilitated process, although lysine transport is not readily saturated. Inhibitors of energy metabolism do not significantly affect amino acid uptake, but they do inhibit protein synthesis. However, since the rate of transport is highly temperature sensitive (Q₁₀ ? 3) and leucine is accumulated against a concentration gradient, active amino acid transport appears to be present.     

Effects of ascorbic acid on the uptake of serotonin in differentiated neuroblastoma cells

Ascorbic acid causes concentration-dependent and time-dependent effects on [³H]-serotonin (³H-5HT) uptake into differentiated neuroblastoma N-2a cells. Preincubation of cells with ascorbic acid inhibits both passive diffusion and active transport of ³H-5HT (0.1 μM). The kinetic characteristics of the active uptake process change with ascorbic acid treatment, resulting in an increase in the Km from 0.27 μM to 3.0 μM and in the Vmax from 453 to 2369 fmol/min/10⁶ cells. This inhibitory effect of ascorbic acid appears to be due to its reducing properties.     

Comparison of alpha-methylphenylalanine and p-chlorophenylalanine as inducers of chronic hyperphenylalaninaemia in developing rats.

alpha-Methylphenylalanine is a very weak competitive inhibitor of rat liver phenylalanine hydroxylase in vitro but a potent suppressor in vivo. The loss of the hepatic activity (the renal one is unaffected) becomes maximal (70-75% decrease; cf. control) 18h after the administration (per 10g body wt.) of 24 mumol of alpha-methylphenylalanine with or without 52 mumol of phenylalanine. Chronic suppression of hepatic phenylalanine hydroxylase was obtained by injections of alpha-methylphenylalanine plus phenylalanine to suckling rats, and by their addition to the diet after weaning. A series of comparisons of the effects of this treatment, and one with p-chlorophenylalanine, was then carried out. In both cases there was a rise (1.3-2-fold) in phenylalanine-pyruvate amino-transferase activity (but no change in four other enzyme activities) in the liver; in brain there was a rise in phosphoserine phosphatase activity, but the total activity and subcellular distribution of nine enzymes revealed no other abnormalities in cerebral development. Striking increases in the concentration of plasma phenylalanine during 26 of the 31 experimental days (with a transient fall at 18-22 days) were maintained by treatment with both analogues plus phenylalanine. However, p-chlorophenylalanine-treated animals had a 30-60% mortality rate and 27-52% decrease in body weight. Developing rats treated with alpha-methylphenylalanine, showing no growth deficit or signs of toxicity (e.g. cataracts), appear to be a more suitable model for the human disease of phenylketonuria. Their phenylalanine concentrations exhibited at least 20-40-fold increase during 50% of each of the first 18 days of life, and 30-fold after weaning.     

Effect of ouabain on amino acid uptake by mouse ascites-tumour cells in the presence of nigericin.

Mouse ascites-tumour cells oxidizing lactate, in a modified Ringer solution, concentrated 2-aminoisobutyrate, L-methionine or 2-(methylamino)isobutyrate about 20-fold from a 0.4 mM solution in the presence of 2-3 micrograms of nigericin/mg cellular dry wt. The ionophore increased cellular [Na+] to almost 100 mM when extracellular [Na+] was about 45 mM. Either valinomycin or the two mitochondrial inhibitors oligomycin and antimycin acting together each markedly lowered the extent to which the tumour cells concentrated amino acid, from the above factor of about 20 to roughly 2-fold. Ouabain (1 mM) had a similar effect, and further raised cellular [Na+]. The sodium pump appeared to be closely involved in amino acid uptake under these conditions.     

The effect of serum deprivation on the initiation of protein synthesis in mouse neuroblastoma cells

Growth of mouse neuroblastoma cells becomes stationary when cultured in serum-free medium. Within 60 h, the protein-synthesizing capacity of the cells declines to 25% as compared to that of exponentially growing cells. The transitional activity of the crude ribosomal salt washes from serum-deprived and control cells was compared in in vitro protein-synthesizing pH 5 systems. It appears that the ribosomal salt wash from serum-deprived cells has significantly (70%) lost its ability to support the translation of neuroblastoma poly(A)+ RNA. This activity of the ribosomal wash from serum-deprived cells can be restored to control level with rabbit reticulocyte initiation factor eIF-4B only. The ability of the ribosomal wash from serum-deprived cells to support the translation of encephalomyocarditis virus (EMC) and Semliki Forest virus (SFV) 42 S mRNA was tested. We found that EMC-mRNA is efficiently translated with the ribosomal salt wash from serum-deprived cells, whereas on the other hand the translation of SFV 42 S mRNA is severely impaired. Therefore, we conclude that in serum-deprived neuroblastoma cells protein synthesis is regulated in both a quantitative and a qualitative way. Modulation of the activity of initiation factor of protein synthesis eIF-4B is at least partly responsible for the observed (selective) blockade of protein synthesis in serum-deprived cells.     

A single hamster PrP amino acid blocks conversion to protease-resistant PrP in scrapie-infected mouse neuroblastoma cells.

Neurodegeneration caused by the transmissible spongiform encephalopathies is associated with the conversion of a normal host protein, PrP-sen, into an abnormal aggregated protease-resistant form, PrP-res. In scrapie-infected mouse neuroblastoma cells, mouse PrP-sen is converted into PrP-res but recombinant hamster PrP-sen expressed in these cells is not. In the present studies, recombinant hamster/mouse PrP-sen molecules were expressed in these scrapie-infected cells to define specific PrP amino acid residues critical for the conversion to PrP-res. The results showed that homology to the region of mouse PrP-sen from amino acid residues 112 to 138 was required for conversion of recombinant PrP-sen to PrP-res in scrapie-infected mouse cells. Furthermore, a single hamster-specific PrP amino acid at residue 138 could inhibit the conversion of the recombinant PrP-sen into PrP-res. The data are consistent with studies in humans which show that specific amino acid residue changes within PrP can influence disease pathogenesis and transmission of transmissible spongiform encephalopathies across species barriers.     

Dibutyryl cAMP-induced protein changes in differentiating mouse neuroblastoma cells.

Proteins from mouse neuroblastoma cells treated with dibutyryl adenosine-3',5'-monophosphate (B2cAMP) were analyzed by high resolution, two-dimensional gel electrophoresis. Quantitative changes in proteins and charge modifications of proteins apparently induced B2cAMP were detected by isoelectric focusing. Some proteins appeared to be modified and one protein was increased 7- to 8-fold in cells treated with B2cAMP. Since neuroblastoma cells differentiate when treated with B2cAMP, understanding the protein changes induced by B2cAMP may help to understand cellular differentiation in neural tissue.     

Mechanisms of amino acid uptake in cumulus-enclosed mouse oocytes

The nutritional role of mouse granulosa cells on antral dictyate mouse oocytes has been studied by measuring the transfer of different amino acids through gap junctional channels between somatic and germ cells. When present in the incubation medium at concentrations resembling in vivo conditions, glycine, alanine, proline, serine, tyrosine, glutamic acid and lysine entered cumulus-enclosed oocytes cooperatively, while valine, leucine and phenylalanine did not. However, cooperative uptake of leucine and phenylalanine was observed at higher external precursor concentrations. We conclude that in vivo antral mouse oocytes depend on surrounding granulosa cells for amino acid uptake, with the exception of amino acids carried by the leucine exchange transport system, and propose that amino acid transfer between granulosa cells and oocytes is dependent on precursor concentrations in the coupled cells.     

Effects of adenosine 3':5'-monophosphate and related agents on ribonucleic acid synthesis and morphological differentiation in mouse neuroblastoma cells in culture.

A variety of compounds were assessed for their ability to induce morphological differentiation and to affect the synthesis of RNA in uncloned mouse neuroblastoma cells in culture. The stimulation of morphological differentiation in uncloned cells after exposure for 48 hours to concentrations of 3 times 10-7 to 3 times 10-4 M papavarine or 10-9 to 10-3 M dibutyryl adenosine 3':5'-monophosphate (dibutyryl-cAMP) was associated, in part, with a concentration-dependent decrease in incorporation of [5-3H]uridine into ribosomal RNA (rRNA) and heterogeneous RNA (HnRNA). The latter effect on cellular RNA produced by papavarine occurred within 1 hour after its addition to the medium and was associated with impaired uptake of radioactive precursor into uridine nucleotides and reduction in the intracellular concentration of uridine 5'-triphosphate (UTP). Dibutytyl-cAMP produced a decreased in the specific radioactivity of UTP without affecting the concentration of UTP in the tumor cells. The effects of papavarine and dibutyryl-cAMP could be distinguished further by the 50% reduction of acetylcholinesterase activity produced by papavarine, but not by dibutyryl-cAMP. Papavarine did not, however, reduce the cellular level of the soluble enzyme, adenine phosphoribosyltransferase. Sodium butyrate, while producing morphological effects similar to those of papavarine and dibutyryl-cAMP at equimolar concentrations, caused no significant changes in the incorporation of [5-3H]uridine into rRNA and HnRNA; however, acetylcholinesterase activity was stimulated 6- to 7-fold above control levels. In contrast to the other differentiating agents examined, addition of 10-9 to 3 times 10-4 M concentrations of cAMP to the tissue culture medium enhanced morphological differentiation of nueroblastoma cells, and caused a 10- to 20-fold stimulation of the incorporation of [5-3H]uridine into rRNA and HnRNA at concentrations of 10-4 M and higher. This effect observed only at high concentrations of cyclic nucleotide was accompanied by an elevation in the specific acitivty of UTP, These studies suggest that the morphological response of neuroblastoma cells is not necessarily associated with concomitant alterations in the synthesis of RNA with agents other than cAMP. Observed changes in incorporation of [5-3H]uridine into RNA appear in most instances to be due to alterations in the uptake of uridine, and in the pool size and specific radioactivity of UTP.     

Comparative studies on the heat-induced thermotolerance of protein synthesis and cell division in synchronized mouse neuroblastoma cells

Mouse neuroblastoma (N2A) cells react to a heat treatment by inhibition of DNA and protein synthesis and induction of cell cycle progression delay. Mitotic delay of heat-treated G1 cells correlates with reduction of protein synthesis and is due to an extensive delay of entrance into S phase, while the G2 phase of these cells is shortened. Mitotic delay of heat-treated G2 cells is more than in G1 cells and no correlation with protein synthesis reduction is found. In heat-treated G1 phase cells, both protein synthesis and cell cycle progression become thermotolerant to a second incubation at increased temperature. Moreover, the process of DNA synthesis becomes thermotolerant. In contrast, when heat-treated G1 phase cells have progressed into G2 phase and are then incubated at increased temperature, this G2 phase delay is not diminished. Apparently, additional targets for hyperthermia are present in late S and G2 phase cells.     

Characterization of the polyamine transport system in mouse neuroblastoma cells. Effects of sodium and system A amino acids

The biochemical properties of polyamine transport system have been studied in detail in NB-15 mouse neuroblastoma cells in culture by measuring the uptake of [14C]putrescine under various experimentally imposed pharmacological conditions. Putrescine uptake in the NB-15 mouse neuroblastoma cells appeared to be a sodium-dependent process. Iso-osmotic displacement of Na+ in the assay medium with either choline or Li+ resulted in a linear decrease of putrescine uptake. Gramicidin, a channel-former ionophore, inhibited putrescine uptake by more than 90% at 20 nM. N-Ethylmaleimide at 5 mM or p-chloromercuribenzene sulfonate at 50 microM completely abolished putrescine uptake. Conversely, oxidized glutathione at 10 mM or 5,5'-dithiobis-(2-nitrobenzoic acid) at 5 microM gave a 1.3-1.4-fold stimulation after a 1-h incubation. This polyamine transport system appeared to be subjected to adaptive regulation. Polyamine antimetabolites such as alpha-difluoromethyl ornithine stimulated putrescine uptake whereas preloading of cells with polyamines inhibited putrescine uptake. Preloading cells with neutral amino acids that belong to sodium-dependent transport System A stimulated putrescine uptake by more than 8-10-fold. These results suggested that the polyamine transport system in NB-15 mouse neuroblastoma cells was sodium dependent and shared some characteristics common to other known sodium-dependent transport systems. These characteristics included (a) sensitivity to ionophores, (b) sensitivity to sulfhydryl reagents, and (c) sensitivity to intracellular contents of substrate molecules. Our data also indicated that polyamine transport may be regulated by transport System A amino acids.