The regulation of sodium-dependent transport of anionic amino acids in cultured human fibroblasts (original) (raw)
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Phorbol esters stimulate the transport of anionic amino acids in cultured human fibroblasts
Biochemical and Biophysical Research Communications, 1990
The effect of phorbol esters on the transport of amino acids has been evaluated in cultured human fibroblasts. The activity of the Na+-dependent system XAG for anionic amino acids is selectively and markedly stimulated by phorbol esters. The effect is maximal whithin 15 min; it is attributable to an increase in transport maximum (Vmax) and not prevented by inhibitors of protein synthesis. The half-maximal stimulation is observed at concentrations of phorbol 12,13-dibutyrate lower than 100 nM. Prolonged incubations in the presence of 1 pM phorbol 12,13-dibutyrate lower the binding of the ligand to its receptor with a loss of the stimulatory effect on transport. The results presented indicate that the stimulation of amino acid transport through system XAG by phorbol esters requires the activation of protein kinase C.
The relationship between sodium-dependent transport of anionic amino acids and cell proliferation
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1993
The relationship between the transport of anionic amino acids and the proliferative status of the cell population has been studied in NIH-3T3 cells. Proliferative quiescence, verified by determinations of growth-rate quotient and incorporation of thymidine, is associated with a marked increase of the influx of L-aspartate. After 7-10 days of serum starvation, the initial influx of L-aspartate increases by 8-10-times with respect to the transport activity determined in growing cells. The operational properties of the influx of L-aspartate are similar in growing and quiescent cells; in particular, the influx of the anionic amino acid is mostly Na+-dependent and completely suppressed by an excess of L-glutamate and D-aspartate, but not of D-glutamate. These features suggest that, in both cases, aspartate uptake occurs through system XxG. The quiescence-related increase in aspartate transport is gradual, sensitive to the inhibition of protein synthesis and referable to the enhanced maximal capacity of transport system XxG. Restoration of serum concentration in the culture medium of serum-starved cells causes a decrease in aspartate transport that is maximal in correspondence to late G~/S phases. It is concluded that the XXG system for anionic amino-acid uptake is sensitive to the proliferative status of the cell population and that, in particular, its transport activity is stimulated by the establishment of proliferative quiescence.
Regulation of Na,K-ATPase Transport Activity by Protein Kinase C
Journal of Membrane Biology, 1997
Considerable evidence indicates that the renal Na + ,K +-ATPase is regulated through phosphorylation/ dephosphorylation reactions by kinases and phosphatases stimulated by hormones and second messengers. Recently, it has been reported that amino acids close to the NH 2-terminal end of the Na + ,K +-ATPase ␣-subunit are phosphorylated by protein kinase C (PKC) without apparent effect of this phosphorylation on Na + ,K +-ATPase activity. To determine whether the ␣-subunit NH 2-terminus is involved in the regulation of Na + ,K +-ATPase activity by PKC, we have expressed the wildtype rodent Na + ,K +-ATPase ␣-subunit and a mutant of this protein that lacks the first thirty-one amino acids at the NH 2-terminal end in opossum kidney (OK) cells. Transfected cells expressed the ouabain-resistant phenotype characteristic of rodent kidney cells. The presence of the ␣-subunit NH 2-terminal segment was not necessary to express the maximal Na + ,K +-ATPase activity in cell membranes, and the sensitivity to ouabain and level of ouabain-sensitive Rb +-transport in intact cells were the same in cells transfected with the wild-type rodent ␣1 and the NH 2-deletion mutant cDNAs. Activation of PKC by phorbol 12-myristate 13-acetate increased the Na + ,K +-ATPase mediated Rb +-uptake and reduced the intracellular Na + concentration of cells transfected with wild-type ␣1 cDNA. In contrast, these effects were not observed in cells expressing the NH 2-deletion mutant of the ␣-subunit. Treatment with phorbol ester appears to affect specifically the Na + ,K +-ATPase activity and no evidence was observed that other proteins involved in Na +-transport were affected. These results indicate that amino acid(s) located at the ␣-subunit NH 2-terminus participate in the regulation of the Na + ,K +-ATPase activity by PKC.
Die Rolle von Serin-Threonin-Kinasen für epitheliale Transportvorgänge
2008
The phosphoinositide dependent kinase PDK1 activates the SGK isoforms SGK1, SGK2 and SGK3 and protein kinase B isoforms which in turn are known to stimulate a variety of sodium coupled transporters, such as the renal and intestinal Na+-dependent glucose transporter SGLT1. SGK1 is known to be up-regulated by mineralocorticoids and to enhance ENaC activity in several expression systems. Moreover, the amiloride-sensitive transepithelial potential difference in collecting duct is lower in gene-targeted mice lacking SGK1 (sgk1-/-) than in their wild type littermates (sgk1+/+). Accordingly, the ability of sgk1-/- mice to decrease urinary sodium output during salt depletion is impaired. ENaC activity and thus transepithelial potential difference in the colon are similarly influenced by mineralocorticoids. The first aim of the present study was to explore the role of PDK1 in in electrogenic glucose and amino-acid transport in small intestine and proximal renal tubules As mice completely lac...
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1986
The distribution ratio of the lipophilic cation tetraphenylphosphonium (TPP +) has been used to estimate the electrical potential difference across the plasma membrane in cultured human fibroblasts. These cells exhibit a membrane potential markedly influenced by the diffusion potential of K +. High extracellular potassium concentrations depolarize human fibroblasts and depress the activity of transport systems A, ASC (both serving for zwitterionic amino acids), XAG (for anionic amino acids), and y + (for cationic amino acids). High doses (I00 ~tM) of the K÷-ionophore valinomycin hyperpolarize the cells. This condition enhances the activity of systems A, ASC and y +. Transport systems L (for neutral amino acids) and x c (for anionic amino acids) are insensitive to changes in extracellular K + or to valinomycin. System XAC is inhibited by the addition of I00/tM valinomycin, but the effect of the ionophore appears to be potential-independent. These results indicate that: (a) the activity of systems L and x c is potential-independent and (b) the activity of systems A, ASC, XAc and y + is sensitive to alterations of external IK +I associated to changes in membrane potential.
Adaptive Increase of Amino Acid Transport System A Requires ERK1/2 Activation
Journal of Biological Chemistry, 1999
Amino acid starvation markedly stimulates the activity of system A, a widely distributed transport route for neutral amino acids. The involvement of MAPK (mitogen-activated protein kinase) pathways in this adaptive increase of transport activity was studied in cultured human fibroblasts. In these cells, a 3-fold stimulation of system A transport activity required a 6-h amino acidfree incubation. However, a rapid tyrosine phosphorylation of ERK (extracellular regulated kinase) 1 and 2, and JNK (Jun N-terminal kinase) 1, but not of p38, was observed after the substitution of complete medium with amino acid-free saline solution. ERK1/2 activity was 4-fold enhanced after a 15-min amino acid-free incubation and maintained at stimulated values thereafter. A transient, less evident stimulation of JNK1 activity was also detected, while the activity of p38 was not affected by amino acid deprivation. PD98059, an inhibitor of ERK1/2 activation, completely suppressed the adaptive increase of system A transport activity that, conversely, was unaffected by inhibitors of other transduction pathways, such as rapamycin and wortmannin, as well as by chronic treatment with phorbol esters. In the presence of either L-proline or 2-(methylaminoisobutyric) acid, two substrates of system A, the transport increase was prevented and no sustained stimulation of ERK1/2 was observed. To identify the stimulus that maintains MAPK activation, cell volume was monitored
Activation of classical protein kinase C decreases transport via systems y+ and y+L
AJP: Cell Physiology, 2007
Activation of classical protein kinase C decreases transport via systems y ϩ and y ϩ L. tion of protein kinase C (PKC) downregulates the human cationic amino acid transporters hCAT-1 (SLC7A1) and hCAT-3 (SLC7A3) (Rotmann A, Strand D, Martiné U, Closs EI. J Biol Chem 279: 54185-54192, 2004; Rotmann A, Vekony N, Gassner D, Niegisch G, Strand D, Martine U, Closs EI. Biochem J 395: 117-123, 2006). However, others found that PKC increased arginine transport in various mammalian cell types, suggesting that the expression of different arginine transporters might be responsible for the opposite PKC effects. We thus investigated the consequence of PKC activation by phorbol-12-myristate-13-acetate (PMA) in various human cell lines expressing leucine-insensitive system y ϩ [hCAT-1, hCAT-2B (SLC7A2), or hCAT-3] as well as leucinesensitive system y ϩ L [y ϩ LAT1 (SLC7A7) or y ϩ LAT2 (SLC7A6)] arginine transporters. PMA reduced system y ϩ activity in all cell lines tested, independent of the hCAT isoform expressed, while mRNAs encoding the individual hCAT isoforms were either unchanged or increased. System y ϩ L activity was also inhibited by PMA. The extent and onset of inhibition varied between cell lines; however, a PMA-induced increase in arginine transport was never observed. In addition, when expressed in Xenopus laevis oocytes, y ϩ LAT1 and y ϩ LAT2 activity was reduced by PMA, and this inhibition could be prevented by the PKC inhibitor bisindolylmaleimide I. In ECV304 cells, PMA-induced inhibition of systems y ϩ and y ϩ L could be prevented by Gö 6976, a specific inhibitor of conventional PKCs. Thymelea toxin, which activates preferentially classical PKC, had a similar inhibitory effect as PMA. In contrast, phosphatidylinositol-3,4,5-triphosphate-dipalmitoyl, an activator of atypical PKC, had no effect. These data demonstrate that systems y ϩ and y ϩ L are both downregulated by classical PKC.
Kidney International, 1994
Complex subcellular distribution of sodium-dependent amino acid transport systems hi kidney cortex and LLC-PK1/Cl,4 cells. To characterize the amino acid transport system in basalateral membranes and to test for possible intracellular loci of amino acid transport activity, we surveyed the distribution of L-alanine transport activity in rabbit proximal tubular cells and LLC-PK1/C14 cells. A three-dimensional separation procedure based on differential sedimentation, density gradient centrifugation, and counter-current distribution resolved 21 physically and biochemically distinct membrane populations from rabbit cortex. Inhibition of L-alanine transport by phenylalanine and N-(methylamino)isobutyric acid was used to delineate parallel amino acid transport pathways. Population n was identified as brush border membranes by virtue of its 16-fold maltase enrichment; 94% of its Nadependent alanine transport activity was mediated by systems previously shown to be characteristic of brush border membranes. Two populations, c' and c", which accounted for 25% of the total Na,K-ATPase activity, were identified as basalateral membranes on the basis of Na,K-ATPase cumulative enrichment factors of 15 and 21; 82% of the total alanine transport in these populations was mediated by a Na4-independent system similar to the classical system L. Na,K-ATPase, Na-independent and Na-dependent alanine transport activities were associated with intracellular membrane populations as well as with the plasma membranes. The major intracellular locus of Na,K-ATPase activity, population i accounted for roughly 31% of the Na,K-ATPase, maximally enriched ninefold; it contained 29% of the total system L transport activity. Population I, which was identified as endoplasmic reticulum because it was the major locus of membranebound NADPH cytochrome c reductase activity, contained 44% of the total system A transport. Three distinct Golgi-derived populations, m', m", and o, accounted for 39% of the total system A transport. A survey of the amino acid transport systems in LLC-PK1/Cl4 cells showed that the majority of system A-mediated amino acid transport was present in membranes of intracellular and possibly apical origin. The presence of large intracellular pools of amino acid transport activities might reflect newly synthesized transport proteins, ongoing membrane recycling or, perhaps, intracellular reserves available for rapid recruitment to the plasma membrane.