Amino acid transport in placental slices. Mechanism of increased accumulation by prolonged incubation.

The accumulation of alpha-aminoisobutyric acid by placental slices is increased dramatically upon prior incubation of the slices in amino acid-free, buffered saline. This increase is inhibited by inhibitors of protein synthesis and is accompanied by an increased V for the transport process. While alternative explanations are discussed, these data suggest that the incubation effect may be mediated through an increase in the number of available transport sites which are synthesized during the incubation period. Incubation with an amino acid mixture diminishes the increase as well as general protein synthesis, suggesting that a reduced availability of amino acids may initiate compensatory changes in the synthesis of cellular transport proteins.     

The effects of different concentrations of glycerol and dimethylsulfoxide on the metabolic activities of kidney slices

Kidney slices either were exposed to the cryoprotectants for 1 hr at room temperature and subsequently washed and incubated in fresh KR buffer containing only the radioactive metabolic tracers, or were immediately incubated for 2 hr at 37 °C in KR buffer containing the cryoprotectant and the tracers. Exposure to glycerol by incubation of kidney slices in Krebs-Ringer bicarbonate buffer containing varying concentrations of glycerol from 0 to 70% ($\text{v}\text{v}$) resulted in a pronounced inhibitory effect on the protein synthesizing activity, while thymidine incorporation into DNA and the α-aminoisobutyric acid uptake through the cell membranes were less affected. Exposure of the tissue to buffer containing dimethylsulfoxide (Me₂SO) in concentrations of 10 to 20% ($\text{v}\text{v}$) resulted in a stimulatory effect on metabolism. At higher concentrations, Me₂SO was toxic resulting in damaging effects on protein and DNA synthesis as well as on membrane integrity. The stimulatory effects of exposure to low concentrations of Me₂SO on protein and DNA synthesis in kidney slices were concluded to be the result of an increased transport of precursors through the cell membranes.     

Inhibition of protein kinase activity and amino acid and α-methyl-d-glucoside transport by diamide

Dizene dicarboxylic acid $\text{bis}\text{-(N,N-}\text{dimethylamide}\text{)}$, commonly called diamide, is known to oxidize stoichiometrically intracellular pools of reduced glutathione and inhibit the accumulation of sugars and amino acids by rat kidney slices. Incubation of rat renal cortical slices in diamide also leads to a significant decrease in the level of endogenous protein kinase activity. The inhibition of sugar and amino acid transport and protein kinase activity by diamide is partially reversible by the addition of exogenous glutathione or other thiols. A comparison of protein kinase activity with amino acid and sugar transport at various concentrations of diamide indicates that there is a high degree of correlation between these two processes.     

Regulatory characteristics of amino acid transport in newborn rat renal cortex cells

The uptake of α-aminoisobutyric acid by slices of kidney cortex from newborn rats is enhanced by a preliminary incubation of the tissue in buffer at 37 °C. This effect is abolished by anaerobiosis, the presence of dinitrophenol or the removal of Na⁺ during the preliminary incubation. Cycloheximide (50 μM) and purimycin (1 mM) as well as α-aminoisobutyric acid, glycine and proline (5 mM) in the pre- incubation buffer also abolish the effect, while actinomycin D (0.8 μM) partially inhibits the enhancement due to preliminary incubation. A kinetic examination of the phenomenon indicates that the enhanced uptake is due to an increased entry rate into the cells without a change in efflux. There is no alteration in the apparent transport $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ but an increase in the $\text{V}$ for entry. The effect is dependent on tissue age being observed between birth and 22 days, after which there is a decrease in response to preliminary incubation with no effect seen in adult tissues.     

Testosterone induces a rapid stimulation of endocytosis,amino acid and hexose transport in mouse kidney cortex

Testosterone induced a rapid (<1 min) stimulation of endocytosis, amino acid and hexose transport, measured by the temperature-sensitive uptake of HRP, ¹⁴C-AIB and ³H-DG, in mouse kidney cortex slices. The hormonal increment in uptake persisted for at least 60–120 min, showed time-, energy-, and Na⁺-dependence, and varied with substrate and testosterone concentration. Testosterone was maximally effective at 10^?8 to 10^?7 M. Peroxidase histochemistry indicated that the hormonal increase in HRP uptake is restricted to proximal tubules. Testosterone was more effective than DHT, whereas cyproterone acetate, androsterone and dexamethasone had little or no stimulating effect on this uptake. Kidney slices from androgen-insensitive $\text{tfm}\text{Y}$ mice did not respond to testosterone. The rapid increase in endocytosis, amino acid and hexose transport may represent a direct, receptor-mediated response of the surface membrane of target cells to testosterone.     

Studies on the role of protein synthesis and of sodium on the regulation of ornithine decarboxylase activity

The minimum requirements for eliciting or enhancing ornithine decarboxylase activity (EC. 4.1.1.17); L-ornithine carboxylase) in neuroblastoma cells incubated in salts-glucose solutions have been investigated. These incubation conditions permit the study of changes in ornithine decarboxylase activity independently of the growth-associated reactions that occur in cell culture media (Chen, K.Y. and Canellakis, E.S. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 3791–3795). Ornithine decarboxylase activity can be elicited by a variety of asparagine and other amino acid analogs, including α-aminoisobutyric acid, that cannot participate in protein synthesis. Of the eleven asparagine analogs tested. $\text{α-N-}\text{CH}{}_3\text{-}\text{DL}\text{-asparagine}$ is the most potent in eliciting ornithine decarboxylase activity and is equivalent to asparagine in this regard. Inclusion of polar groups into the asparagine molecule results in the loss of its ability to elicit ornithine decarboxylase activity. With the use of these analogs and of analogs of other amino acids it is shown that the rapid fall in ornithine decarboxylase activity that is noted following cycloheximide treatment may not be a consequence of the inhibition of protein synthesis. The rapid fall in ornithine decarboxylase activity is primarily due to the removal of the agent that elicits and stabilizes its activity. These results, the finding that α-amminoisobutyric acid stimulates ornithine decarboxylase activity and that sodium is required for the stimulation of ornithine decarboxylase activity are discussed in relation to the ‘A’ amino acid transport system.     

On the mechanism of the glucagon-induced inhibition of hepatic protein synthesis.

The intraperitoneal administration of glucagon (200 μg) to rats produced a transient increase of the hepatic polypeptide chain completion time, the increase being maximum at 5 min returning to control values at 20 min. This inhibitory effect was sustained when glucagon was constantly supplied by continuous infusion. Postmitochondrial supernatants from livers of the control group or rats treated with glucagon for 5 min showed no difference in their protein synthetic activity. After 20 min of intraperitoneal administration of the hormone, that is, when the effect on protein synthesis had vanished, the levels of cAMP were still 40% above those of the control group, and the ribosomal proteins were 110% more phosphorylated. These results suggest that the observed effect of glucagon is not due to its direct action on the protein synthesis machinery. On the other hand, the variations in the hepatic amino acid content brought about by glucagon do not appear to be quantitatively significant to account for the observed inhibition of protein synthesis. The effect of glucagon was always paralleled by a decrease in the $\text{[ATP]}\text{[ADP]}$ ratio which may be responsible for the observed decrease in the rates of elongation and/or termination steps of protein synthesis. Glucagon also produced a rise in the $\text{[NADH]}\text{[NAD}{}^{\mathrm{+}}\text{]}$ ratio in both cellular compartments, cytosol and mitochondria, as reflected by the rise in the lactate to pyruvate and the β-hydroxybutyrate to acetoacetate ratios. This shift of the NAD⁺ couple to a more reduced state seems to be the result of an increased mobilization and oxidation of fatty acids brought about by the hormone. It is postulated then that the primary effect of glucagon leading to a decrease in protein synthesis is probably to increase the state of reduction of the hepatic nicotinamide nucleotide system. This point of view is supported by the fact that the nicotinamide and adenine nucleotide systems in rat liver are in equilibrium through cytosolic equilibrium reactions, so that a decrease in the $\text{[ATP]}\text{[ADP]}$ ratio brought about by glucagon may be secondary to the increase in the $\text{[NADH]}\text{[NAD}{}^{\mathrm{+}}\text{]}$ ratio. This hypothesis is supported by the fact that glucagon was not effective in inhibiting hepatic protein synthesis in rats pretreated with a drug, 2-benzene-sulfonamido-5-(β-methoxy-ethoxy)pyrimidine, that prevents fatty acid mobilization and the subsequent changes in the $\text{[NADH]}\text{[NAD}{}^{\mathrm{+}}\text{]}$ and $\text{[ATP]}\text{[ADP]}$ ratios. Furthermore, the administration of exogenous fatty acid brings about an inhibition of the rate of hepatic protein synthesis accompanied by a decrease in the ATP levels and an increase in the state of reduction of the NAD⁺ system.     

The influence of mitochondria on ribosomes. Cycloheximide resistance of ribosomes from petite mutants of saccharomyces cerevisiae

The influence of cycloheximide on amino acid incorporation into protein of standard strain ? + and cytoplasmic mutant ? ? of $\text{S.}\text{cerevisiae}$ was determined $\text{in}\text{vivo}$ and $\text{in}\text{vitro}$. $\text{In}\text{vivo}$ cycloheximide at the concentration which inhibits protein synthesis in ? + strain by over 60% has litle or no effect in mutant ? ? strain. $\text{In}\text{vitro}$ cycloheximide in the range of 0.05 to 0.3 ug/ml of incubation medium inhibits polyphenylalanine synthesis in ? + strain by 50% and in ? ? strain by less than 10%. Similar resistance to this antibiotic are shown in standard strain grown in anaerobic conditions. It has been found that the resistance to cycloheximide is associated with changes in cytoplasmic ribosomes and may depend on the integrity of mitochondrial system.     

Carnitine-induced uptake of l-carnitine into cells from an established cell line from human heart (CCL 27)

l-Carnitine is actively transported into Girardi human heart cells, an established cell line from human heart. The present study was undertaken to investigate the effect of different concentrations of l-carnitine in the growth medium on the rate of uptake of l-[³H]carnitine.Increasing the concentration of l-carnitine from 2 to 100 μmol/1 in the growth medium of the cells, increased the rate of uptake of l-[³H]carnitine by about 50%. The maximal effect was reached after approx. 72 h incubation. The increase in rate seemed to be caused by synthesis of increased number of carriers, as judged by the increase in $\text{V}$ with unchanged apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for the transport process. This effect of l-carnitine could be inhibited by cycloheximide, indicating the dependence on intact protein synthesis. The morphology of the cells was studied by electron microscopy. No myofilaments were found, thus the cells are dedifferentiated and no longer typical muscular cells.     

Increase of cyclic AMP-dependent protein kinase type II as an early event in hormone-dependent mammary tumor regression.

Primary, 7,12-dimethylbenz(α)anthracene (DMBA)-induced mammary carcinoma in the rat contains cyclic adenosine 3′,5′-monophosphate (cAMP)-dependent and -independent forms of protein kinase. When growth of DMBA-induced tumors was arrested by either ovariectomy or N⁶,O²′-dibutyryl cAMP treatment of the host, the activity of cAMP-dependent protein kinase type II markedly increased in the tumor cytosol, as shown by DEAE-cellulose chromatography and autophosphorylation. The increase in activity of cAMP-dependent protein kinase was also demonstrable in the tumor cytosol and nuclei following $\text{in}$$\text{vitro}$ incubation of tumor slices with cAMP. These results suggest that protein kinase type II is involved in the regression of hormone-dependent mammary tumors.     

Effect of alkali ions on the active transport of neutral amino acids into Streptomyces hydrogenans

The active transport of neutral amino acids into Streptomyces hydrogenans is inhibited by external Na⁺. There is no indication that in these cells amino acid accumulation is driven by an inward gradient of Na⁺. The extent of transport inhibition by Na⁺ depends on the nature of the amino acid. It decreases with increasing chain length of the amino acid molecules i.e. with increasing non-polar properties of the side chain. Kinetic studies show that Na⁺ competes with the amino acid for a binding site at the amino acid carrier. There is a close relation between the $\text{K}{}_{\mathrm{<mtext>i</mtext>}}$ values for Na⁺ and the number of C atoms of the amino acids. Other cations also inhibit neutral amino acid uptake competitively; the effectiveness decreases in the order $\text{Li}{}^{\mathrm{+}}\text{> Na}{}^{\mathrm{+}}\text{> K}{}^{\mathrm{+}}\text{> Rb}{}^{\mathrm{+}}\text{> Cs}{}^{\mathrm{+}}$. Anions do not have a significant effect on the uptake of neutral amino acids. After prolonged incubation of the cells with 150 mM Na⁺, in addition to the competitive inhibition of transport Na⁺ induces an increase in membrane permeability for amino acids.     

Bimodal effects of cellular amino acids on Na+-dependent amino acid transport in ehrlich cells

Cells depleted of amino acids show lower rates of glycine or aminoisobutyric acid uptake than do freshly isolated cells. In the amino acid-depleted cells, addition of valinomycin stimulates amino acid influx at least to the level observed in freshly isolated cells. In cells containing high levels of cellular amino acids, valinomycin has little effect on influx of amino acids. It is concluded that the transport of amino acids in freshly isolated cells is elevated compared to depleted cells because the cells are hyperpolarized by the continuous loss of cellular amino acids during the transport assay. During this hyperpolarization by amino acid loss, transport of amino acids is not further stimulated by valinomycin at low external [K⁺] ($\text{10 mM ± 5 mM}$).With the exception of preloading with glycine, cells preloaded with a single amino acid to a concentration greater than 20 mM show reduced rates of glycine and aminoisobutyric acid influx at early times (less than 15 min) compared to amino acid-depleted cells. The reduction of infiux is transient and by 30 min, influx is greater in preloaded than in amino acid-depleted cells.Knowing that increases and decreases in the membrane potential are achieved by using varying external [K⁺] in the presence of valinomycin and propranolol, and using amino acid-depleted cells, it can be shown that an increased membrane potential increases the $\text{V}$ for glycine and aminoisobutyric acid influx. A decrease in the potential difference results in a decreased $\text{V}$. Changes in $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ also occur when the membrane potential is varied.     

Analysis of two chloride requirements for sodium-dependent amino acid and glucose transport by intestinal brush-border membrane vesicles of fish

Intestinal brush border vesicles of a Mediterranean sea fish (Dicentrarchus labrax) were prepared using the Ca²⁺-sedimentation method. The transport of glucose, glycine and 2-aminoisobutyric acid is energized by an Na⁺ gradient ($\text{out > in}$). In addition, amino acid uptake requires Cl^? in the extravesicular medium (2-aminoisobutyric acid more than glycine). This Na⁺- and Cl^?-dependent uptake is electrogenic, since it can be stimulated by negative charges inside the vesicles. The specific Cl^? requirement of glycine and 2-aminoisobutyric acid transport is markedly influenced by pH, a change from 6.5 to 8.4 reducing the role played by Cl^?. In the presence of Cl^?, the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ of 2-aminoisobutyric acid uptake is reduced and its $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ is enhanced. Cl^? affects also a non-saturable Na⁺-dependent component of this amino acid uptake. Amino acid transport is also increased by intravesicular Cl^? (2-aminoisobutyric acid less than glycine). This effect is more concerned with glucose uptake, which can be then multiplied by 2.3. A concentration gradient ($\text{in > out}$) as well as the presence of Na⁺ in the incubation medium seems to enter into this requirement. This intravesicular Cl^? effect is not influenced by pH between 6.5 and 8.4.     

Inefficiency of the stringent response in the fungus Mucor

Removal of a required amino acid from the growth medium or addition of cycloheximide caused an immediate stoppage of growth and protein synthesis in the fungus $\text{Mucor}\text{racemosus}$. However, RNA synthesis persisted for several hours at rates that only gradually decreased under the same circumstances. An analysis of the major classes of RNA synthesized during the first hour of treatment showed that cycloheximide preferentially inhibited rRNA synthesis, whereas amino acid starvation slowed synthesis of all RNA species uniformly. Neither treatment affected the percentage of mRNA synthesized. The partial and delayed effects of amino acid starvation and cycloheximide treatment on RNA synthesis reported here suggest the absence of or the gross inefficiency of a classical stringent response in $\text{M.}\text{racemosus}$.     

Roles of sodium and potassium ions on p-aminohippurate transport in rabbit kidney slices

This investigation was principally undertaken to test the ionic gradient hypothesis as applied to active $\text{p-}\text{aminohippurate}$ uptake in the rabbit kidney cortical slice preparation. Efflux of $\text{p-}\text{aminohippurate}$ from the slice was shown to be independent of external Na⁺ concentration. Transferring slices from a low sodium preincubation to a high sodium incubation medium containing $\text{p-}\text{aminohippurate}$ increased intracellular concentrations of both Na⁺ and K⁺, and $\text{p-}\text{aminohippurate}$ accumulation occurred. Transferring slices from a low sodium preincubation to a high sodium incubation medium containing ouabain and $\text{p-}\text{aminohippurate}$ resulted in a net increase in intracellular Na⁺ concentration but no $\text{p-}\text{aminohippurate}$ accumulation occurred. Different combinations of preincubation and incubation media gave a high to low array of intracellular Na⁺ concentrations and these directly reflected their respective $\text{p-}\text{aminohippurate}$ uptake. These results suggest that the Na⁺-gradient hypothesis does not adequately explain the transport of organic acids in rabbit kidney. These results also suggest that Na⁺ possibly has an intracellular role through its stimulation of $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ channeled to energizing the $\text{p-}\text{aminohippurate}$ accumulative mechanism.     

Energization of sulfate transport in yeast

Sulfate uptake by Saccharomyces cerevisiae is stimulated about 12-fold by preincubation of cells with 1% d-glucose or 1% ethanol. The $\text{K}{}_{\mathrm{<mtext>T</mtext>}}$ remains unchanged (0.34–0.38 mM), the $\text{J}{}_{\mathrm{<mtext>mar</mtext>}}$ increase from 18–20 to 195–230 and 170–185 nmol/min per g dry wt., respectively, after glucose and ethanol preincubation. The stimulation involves protein synthesis (it is suppressed by cycloheximide), has a half-time of 18 min and requires mitochondrial respiration (no or low effect in respiration-deficient mutants and those lacking ADP-ATP transport in mitochondria, as well as after anaerobic preincubation of the wild-type strain, and in low-phosphate cells). The presence of NH₄⁺ and some amino acids (e.g., leucine, aspartate, cysteine and methionine) depressed the stimulation while that of cationic amino acids (typically arginine and lysine) and of K⁺ increased it by 50–80%. The stimulated (i.e., newly synthesized) transport system was degraded with a half-life of about 10 min.     

Mechanism of glycogenolytic action of cycloheximide in rat liver

Cycloheximide (0.1 to 0.2 m$\text{M}$) increases cAMP concentration 3 to 4-fold in isolated rat liver slices $\text{in vitro}$. This increase in cAMP concentration parallels an increase in phosphorylase activity. When cycloheximide, at a concentration used to inhibit protein synthesis (1 to 2 μg/g body weight), is administered to whole animals, phosphorylase is activated up to 13-fold by 6 hours. This leads to almost complete depletion of liver glycogen (from about 40 mg/g liver to 0.4 mg/g liver).     

The effect of diamide and glutathione on the uptake of α-methyl-d-glucoside by slices of rat kidney cortex

The uptake of α-methyl-d-glucoside was stimulated in slices of rat kidney cortex by pretreatment with reduced glutathione. Diamide, an oxidizing agent with high specificity for GSH, caused an inhibition of α-methyl-d-glucoside uptake. These effects appeared to be related specifically to GSH, since dithiothreitol and mercaptoethanol did not increase α-methyl-d-glucoside uptake, and were not as effective as GSH in reversing the effects of diamide. GSH and diamide had no effect on the uptake of another sugar analog, $\text{3-O-}\text{methylglucose}$, which is not actively transported. Kinetic studies indicated that GSH increased the apparent $\text{V}$ without affecting $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$. The results are discussed in terms of the possible role of GSH in the process of sugar transport.     

Parathyroid mRNA directs the synthesis of pre-proparathyroid hormone and proparathyroid hormone in the Krebs ascites cell-free system.

Translation in a cell-free extract of Krebs II ascites cells of a mRNA fraction prepared from bovine parathyroid glands results in the synthesis of two radioactive products that appear identical to pre-proparathyroid hormone (Pre-ProPTH) (M.W. ～ 14,000), the suspected earliest biosynthetic precursor of parathyroid hormone (PTH) (M.W. 9,500), and to proparathyroid hormone (ProPTH) (M.W. 10,200), the immediate biosynthetic precursor of PTH. The two products of synthesis in the ascites extract co-electrophoresed on both urea-acetate and urea-SDS acrylamide gels with Pre-ProPTH obtained from cell-free translation of parathyroid RNA in extracts of wheat-germ and with ProPTH isolated from parathyroid slices. Both products were precipitated with an antiserum to PTH. Partial analysis of the amino acid sequence of [³⁵S]methionine-labeled Pre-ProPTH synthesized by the ascites extract indicates that a substantial fraction of the product is lacking the two N-terminal methionines present in the Pre-ProPTH synthesized by the wheat-germ system. The results indicate that, ($\text{i}$), unlike the wheat-germ, ascites extracts contain enzymes that remove the initiator methionine from Pre-ProPTH and convert Pre-ProPTH into ProPTH (no ProPTH was observed in the wheat-germ system) and ($\text{ii}$) the cleavage processes appear to occur in association with synthesis, inasmuch as neither removal of NH₂-terminal methionine nor formation of ProPTH was observed upon incubation of Pre-ProPTH isolated from either the wheat-germ system or from the ascites system when put back into the ascites system.     

Phosphorylation of tyrosine aminotransferase in vitro by cyclic nucleotide-dependent protein kinases

An intraperitoneal injection of either leucine (1.57 mg/g body wt) or valine (2 mg/g body wt) into newborn mice led to a rapid accumulation of inactive monoribosomes in their brains. $\text{In}\text{vitro}$ measurements of protein synthesis by the remaining active ribosomes in leucine-treated mice revealed that polypeptide chain elongation was also inhibited. When a mixture of the seven amino acids from the leucine transport system was injected (0.15 mg each amino acid/g body wt) following the valine or leucine treatment, brain monoribosomes did not accumulate and elongation rates in the leucine-treated mice were only slightly altered.