Rate Of Proline Uptake Research Articles

Parathyroid hormone regulation of proline uptake by cultured neonatal mouse osteoblastlike cells

Regulation of proline uptake by the synthetic amino-terminal fragment of bovine parathyroid hormone [bPTH-(1-34)] has been studied in confluent primary cultures of osteoblastlike cells isolated from neonatal mouse calvaria. The initial velocity of proline transport was increased by 85% in cultures treated with 24 nM bPTH-(1-34) for 6 h. Cycloheximide, at a concentration that inhibited protein synthesis by 97%, did not prevent this effect. However, adding the inhibitor during the first 1-2 h of hormone treatment did significantly reduce its magnitude. Exposure of cells to the inhibitor alone caused a time-dependent decrease in the basal rate of proline uptake. In the absence of protein synthesis, the maximal velocity (Vmax) of transport was 60% greater in cultures treated with 24 nM bPTH-(1-34) than in controls. The concentration of proline at which half-maximal transport occurred (Km) was unchanged. In cultures treated with cycloheximide alone, proline transport decreased as a first-order exponential with a half-life of 250-280 min. Parathyroid hormone significantly reduced this decline, increasing the half-life of proline transport activity about fourfold. These effects were duplicated by 1 mM DBcAMP. It is concluded that bPTH-(1-34) increases proline transport in osteoblastlike cells by decreasing the degradation of amino acid transport system A proteins. The hormone may also affect the synthesis of these molecules. These effects appear to be mediated by cAMP.

Regulation of amino acid transport in L6 myoblasts. II. Different chemical properties of transport after amino acid deprivation.

The mechanism of stimulation of amino acid transport system A caused by amino acid deprivation in L6 cells was investigated. In cells loaded with alpha-aminoisobutyric acid (AIB), amino acid deprivation increased the rate of proline uptake only after the intracellular [AIB] dropped below 7 mM. Efflux of proline was not sensitive to the presence of proline in the outer medium (with or without external Na+), suggesting that efflux through system A (and possibly uptake) is not susceptible to transinhibition. Transport (stimulated uptake) into amino acid-deprived cells and that into amino acid-supplemented cells differed in several chemical properties: 1) In the former group, transport was higher at lower pH values than in the latter, and the optimum pH values were 7.5 and 7.8, respectively. 2) Unlike proline uptake in supplemented cells, uptake in deprived cells was inhibited by 50% with N-ethylmaleimide (1 mM) or by 50 microM p-chloromercuribenzoate (PCMBS). Inhibition by PCMBS was not due to collapse of the Na+ gradient. The mercurial inhibited only the deprivation-induced stimulation of transport, bringing the rate of proline uptake to the "basal" uptake level observed in amino acid-supplemented cells. Proline uptake was not stimulated by a second deprivation following treatment with PCMBS and a supplementation-deprivation cycle. However, in untreated cells, or by reversing mercaptide formation with dithiotreitol, the second deprivation stimulated transport. Deprivation at 4 degrees C did not elicit stimulation of proline uptake. Cycloheximide prevented the stimulation and decreased the rate of proline uptake in deprived cells more efficiently than in supplemented cells. Actinomycin D prevented stimulation when added at the onset of deprivation. The above data indicate that stimulation of transport by deprivation is protein synthesis-dependent and that the stimulated transport had chemical properties distinct from the "basal" transport in supplemented cells. The evidence presented is consistent with a model of activation of a finite pool of transporters upon deprivation, the chemical characteristics of which differ from those of the "basal" transport system.

Regulation of amino acid transport in L6 muscle cells: I. Stimulation of transport system A by amino acid deprivation.

AbstractThe regulation of amino acid transport in L6 muscle cells by amino acid deprivation was investigated. Proline uptake was Na+‐dependent, saturable and concentrative, and was predominantly through system A. Proline uptake was inhibited by alanine, α‐amino isobutyric acid (AIB), and by α‐methylamino isobutyric acid, but not by lysine or valine. At 25°C, Km of proline uptake was 0.5 mM. Amino acid‐deprivation resulted in a progressive increase in the rate of proline uptake, reaching up to 6‐fold stimulation after 6 hours. The basal and stimulated transport were equally Na+‐dependent, and both were inhibited by competition with the same amino acids. Kinetic analysis showed that Km decreased by a factor of 2.4 and Vmax increased 1.9‐fold in deprived cells. Amino acid‐deprivation did not stimulate amino acid uptake through systems other than system A. This suggests that the higher Km in proline‐supplemented cells is not due to release of intracellular amino acids into unstirred layers surrounding the cells. The presence of amino acids which are substrates of system A (including AIB) during proline‐deprivation, prevented stimulation of proline uptake, whereas those transported by systems Ly+ or L exclusively were ineffective. The stimulation of the transport‐rate in deprived cells could be reversed by subsequent exposure to proline or other substrates of system A. L6 cells, deprived of proline for 6 hours, retained the stimulation of transport after detachment from the monolayers with trypsin. Uptake rates were comparable in suspended and attached cells in monolayer culture. Thus, amino acid‐depreivation of L6 cells results in an adaptive increase in proline uptake, which is not due to unstirred layers but appears to be mediated by other mechanisms of selective transport regulation.

The effects of rapid increases in osmotic pressure on Escherichia coli [proceedings

Actively growing Escherichia coli cells respond to increased osmotic pressure by increasing their internal pools of K+ (Epstein & Schultz, 1965) and of the amino acids glutamate, proline and a-aminobutyric acid (Britten & McClure, 1962; Measures, 1975). The change in amino acid pools may be brought about either by increased rates of their synthesis or by stimulation of the rate of their uptake. The present study was initiated to discover how the transport rates of amino acids were controlled by osmotic pressure. In particular the role of K+ in the control mechanism was investigated because of the role of this ion in stimulation of amino acid synthesis (Measures, 1975; Gould & Measures, 1977). The effect of osmotic pressure on transport of amino acids into endogenously respiring E. coli cells was investigated by using concentrated solutions of sorbitol (0.5 M) and lactose (0.4~). In control experiments with E. coli strain 7 (Hayashi et al., 1969), neither sorbitol nor lactose were transported; however, in subsequent experiments it became apparent that, at high concentrations of sorbitol, some metabolism occurs (see below). In experiments in which the osmotic pressure was rapidly increased 3-fold, only aspartate and proline transport systems were stimulated in both the presence and absence of K+. Glutamate transport only occurred in the presence of K+, but under these conditions was stimulated 4-fold by a 3-fold increase in osmotic pressure. The transport systems for alanine, lysine and isoleucine were all inhibited by increases in osmotic pressure. Amino acid uptake was studied over a range of osmotic pressure to determine the relationship between the magnitude of the increase in osmotic pressure and transport activity. The results of proline-transport assays in media of increasing osmotic strength are shown in Figs. l(a) and l(b) similar results were obtained with aspartate (not shown). It is apparent that lactose and sorbitol were not equivalent as agents to increase the osmotic pressure. Lactose-mediated pressure increases resulted in a gradual 4-fold increase in the rate of transport, and this effect showed no K+-dependence (Fig. la). In the absence of K+, sorbitol gave qualitatively similar results to lactose (Fig. lb). However, in the presence of K+, addition of only 10mM-sorbitol (compared with 0 . 4 ~ lactose) caused a 5-fold increase in the rate of uptake of proline (Fig. lb). The sorbitol effects could be simulated by lactose in the presence of 0.5 mM-glucose (Fig. la), suggesting that the effects of sorbitol were influenced by metabolism of this compound. Subsequently it was observed that sorbitol at lOmM caused an increase in the respiration rate of E. coli cells from 54.4 to 89nmol of 0, consumed/min per mg of cells. Osmotic pressure increases themselves were found not to have any effect on the rate of respiration. K+ appears to play a role in coupling sorbitol or glucose metabolism to proline uptake in E. coli. It was observed that there was no direct correlation between the respiration rate and the rate of proline uptake. Proline uptake is energized by the protonmotive force generated by respiration (Hamilton, 1975). Thus in the presence of glucose and independent of the presence of Kt, the respiration rate of the cells was increased over 5-fold. Proline transport, however, was only significantly stimulated by glucose in the presence of Kt (Fig. la). Therefore there appears to be a direct effect of external K+ on proline uptake, which is dependent upon the presence externally of a metabolizable carbon source. This effect requires further investigation. In conclusion, it is apparent that external K+ has no regulatory role in the response of amino acid transport to osmotic pressure, but does have other effects on energy coupling.

Variant Chinese hamster cells resistant to the proline analog L-azetidine 2-carboxylic acid.

Variants resistant to the toxic effects of the proline analog L-azetidine 2-carboxylic acid (AZCA) have been isolated from the Chinese hamster tissue culture line G3 by a three-step selection procedure using increasing concentrations of AZCA. Cells surviving each of the three selective steps have been examined for AZCA resistance and for proline uptake, biosynthesis, and degradation. The largest increment in AZCA resistance is acquired in the third step and is due to overproduction of proline as a result of increased activity of the enzyme system responsible for the conversion of glutamic acid to glutamic gamma-semialdehyde. It is not accompanied by an increase in the rate of formation of proline from ornithine or in the rate of proline uptake or degradation.

Water relations of Salmonella oranienburg: accumulation of potassium and amino acids during respiration.

SUMMARY Potassium and 14C-labelled proline, aspartic acid, glutamic acid, and alanine were accumulated by Salmonella oranienburg, during glucose oxidation, over a range of water activity (a w) values. In the absence of amino acids, potassium accumulation increased to a maximum as a w was decreased to about 0·975 but then dropped to a low value at 0·960 a w; with an amino acid, potassium accumulation was much increased and maximum uptake occurred at 0·960 a w. [14C]Proline uptake increased linearly with decrease in a w, the accumulated proline being metabolized in part to glutamic acid and to deaminated compounds; at 0·95 a w uptake of [14C]-proline reached 1·4mmol/g dry bacteria. Uptake of aspartate was comparable to that of proline at a w down to 0·98 but then decreased. Relatively little exogenously supplied glutamate or alanine was accumulated at any water activity. At a w levels below 0·98, oxygen uptake by non-growing bacterial suspensions increased with time and accompanied the linear uptake of proline. Proline accumulation ceased when a maximum rate of oxygen consumption was reached. At 0·97 a w the rate of proline uptake was unaffected by a mixture of 19 amino acids, but the total proline uptake decreased to one-half. In contrast, the proline homologue azetidine-2-carboxylic acid lowered the rate and extent of proline accumulation by one-third.

Intravenous proline tolerance in osteogenesis imperfecta.

The biological half-life of proline in blood has been determined in osteogenesis imperfecta (188±30 min., mean ±1 SD), in pediatric controls (409±130 min.), and in adult control subjects (511±163 minutes). Renal clearances (ml./min./1.73 m 2 ) of free and bound proline have been calculated for the 3 groups. The loss of free proline in urine does not appear to be sufficient to account for the short half-life in blood. Creatinine clearances (corrected to 1.73 m 2 ) were not significantly different in the 3 groups studied. The difference in proline half-life in blood in patients with osteogenesis imperfecta and in normal subjects can most probably be explained by an increased rate of proline uptake in cells or by more rapid incorporation of the amino acid into intermediary metabolic pathways in patients with the disorder. The higher level of bound proline in urine in patients with the disorder may be a reflection of increased turnover or catabolism of collagen.