scholarly journals Amino acid deprivation leads to the emergence of System A activity and the synthesis of a specific membrane glycoprotein in the bovine renal epithelial cell line NBL-1

1992 ◽  
Vol 284 (2) ◽  
pp. 577-582 ◽  
Author(s):  
A Felipe ◽  
C Soler ◽  
J D McGivan
Keyword(s):  
Amino Acid ◽  
Epithelial Cell ◽  
Epithelial Cell Line ◽  
Membrane Glycoprotein ◽  
Amino Acid Deprivation ◽  
Transport Activity ◽  
Renal Epithelial Cell ◽  
System A ◽  
Alanine Transport ◽  
Renal Epithelial Cell Line

1. Amino acid deprivation of confluent monolayers of the bovine renal epithelial cell line NBL-1 causes a stimulation of Na(+)-dependent alanine transport. 2. This stimulation is mediated by a protein-synthesis-dependent induction of 2-(methylamino)isobutyric acid (methyl-AIB)-sensitive alanine transport activity (System A), which was not previously present in these cells. 3. Induction was prevented by the addition of methyl-AIB, alanine or glutamine. 4. Tunicamycin prevented the induction of alanine transport activity. 5. Induction of System A activity was accompanied by incorporation of [3H]mannose into a single membrane protein band of molecular mass 113-140 kDa. 6. These results are consistent with the possibility that induced System A activity in confluent NBL-1 cells is mediated by the synthesis of a 113-140 kDa membrane glycoprotein.

scholarly journals Regulation of the glutamate transporter by amino acid deprivation and associated effects on the level of EAAC1 mRNA in the renal epithelial cell line NBL-I

1993 ◽  
Vol 295 (3) ◽  
pp. 749-755 ◽  
Author(s):  
S Plakidou-Dymock ◽  
J D McGivan
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Epithelial Cell ◽  
Cell Line ◽  
Glutamate Transport ◽  
Epithelial Cell Line ◽  
Amino Acid Deprivation ◽  
Renal Epithelial Cell ◽  
High Affinity ◽  
Renal Epithelial Cell Line

The glutamate transport system of the bovine renal epithelial cell line NBL-1 was studied. The Km for Na(+)-dependent glutamate transport was found to be 13.8 +/- 2.4 microM (Vmax. 365 +/- 19.2 pmol/3 min per mg) and for Na(+)-dependent aspartate transport 4.5 +/- 1.1 microM (Vmax. 108 +/- 6.3 pmol/3 min per mg). The Km values are in close agreement with those expected for high-affinity Na(+)-dependent glutamate transport by System XAG-. Upon deprivation of amino acids, the Vmax. for Na+/aspartate co-transport rose to 203 +/- 6.0 pmol/3 min per mg (Km 3.8 +/- 0.5 microns). A probe was constructed to the high-affinity excitatory amino acid carrier (EAAC1) [Kanai and Hediger (1992) Nature (London) 360, 467-471]. The probe hybridized to a 3.5 kb transcript. On deprivation of amino acids, the level of EAAC1 mRNA decreased sharply before the measurable increase in transport levels, but was subsequently restored to control levels. A motif, which we propose is linked to amino acid deprivation, was found in the EAAC1 primary sequence.

scholarly journals Regulation of System B0 amino-acid-transport activity in the renal epithelial cell line NBL-1 and concomitant changes in SAAT1 hybridizing transcripts

1994 ◽  
Vol 301 (2) ◽  
pp. 399-405 ◽  
Author(s):  
S Plakidou-Dymock ◽  
M J Tanner ◽  
J D McGivan
Keyword(s):  
Amino Acid ◽  
Cell Line ◽  
Physiological Stress ◽  
Sequence Similarity ◽  
Isobutyric Acid ◽  
Amino Acid Starvation ◽  
Epithelial Cell Line ◽  
Transport Activity ◽  
Renal Epithelial Cell ◽  
Renal Epithelial Cell Line

alpha-(Methylamino)isobutyric acid (MeAIB) insensitive Na(+)-dependent alanine transport activity in the bovine kidney cell line NBL-1 was increased upon amino acid starvation (> or = 20% over control levels). When L-phenylalanine (3 mM) was included in the starvation medium the increase was further enhanced (> or = 85% over control levels). In cells grown in control medium the Vmax, for MeAIB-insensitive Na+/alanine co-transport was found to be 6.0 +/- 0.7 nmol/3 min per mg (Km 41 +/- 12 microM) and for L-phenylalanine-treated amino-acid-starved cells the Vmax. was 21 +/- 5 nmol/3 min per mg (Km 92 +/- 40 microM). The increase in Vmax. was prevented by cycloheximide. Substrate specificity analysis identified the L-phenylalanine-induced transport system as System B0. [35S]Methionine labelling of cells during the amino acid starvation/phenylalanine treatments resulted in the differential labelling of a protein of 78 kDa. Northern-blot analysis using a SAAT1-specific probe revealed the presence of a new transcript (3.2 kb) in RNA extracted from cells incubated in amino acid starvation medium with L-phenylalanine included. The present findings suggest a novel means of control for System B0 by the use of physiological stress. It is also proposed that SAAT1 and System-B0 transcripts have considerable sequence similarity.

scholarly journals Identification and partial characterization of a novel membrane glycoprotein induced by amino acid deprivation in renal epithelial cells

1997 ◽  
Vol 322 (2) ◽  
pp. 551-555 ◽  
Author(s):  
Judy BURSTON ◽  
John McGIVAN
Keyword(s):  
Amino Acid ◽  
Rat Liver ◽  
Plasma Membranes ◽  
Partial Purification ◽  
Epithelial Cell Line ◽  
Amino Acid Deprivation ◽  
Renal Epithelial Cell ◽  
Western Blots ◽  
Renal Epithelial Cell Line ◽  
Relationship Of

We have identified a protein of 110 kDa in the renal epithelial cell line NBL-1, which is induced on incubation of the cells in an amino-acid-free medium. The protein was purified on conA–Sepharose and subjected to N-terminal sequencing. The sequence obtained, VDRINFKT, does not correspond to any protein in the databases. Antipeptide antibodies made to this sequence recognised a single protein of 110 kDa in whole cell membranes and in a conconavalin A protein extract. Using the antibody on Western blots, the protein was induced 2.5–3 fold in 10–15 h and the induction was inhibited by cycloheximide and tunicamycin. The protein was found also in rat liver plasma membranes. A procedure for the partial purification of this protein from rat liver is described, and some internal sequence is reported. The possible relationship of the induction of this novel protein to the induction of amino acid transport in these cells by amino acid deprivation is discussed.

scholarly journals Hyperosmolarity leads to an increase in derepressed system A activity in the renal epithelial cell line NBL-1

1993 ◽  
Vol 289 (3) ◽  
pp. 653-658 ◽  
Author(s):  
C Soler ◽  
A Felipe ◽  
F J Casado ◽  
J D McGivan ◽  
M Pastor-Anglada
Keyword(s):  
Epithelial Cell ◽  
Cell Line ◽  
Culture Medium ◽  
Epithelial Cell Line ◽  
Control Medium ◽  
Renal Epithelial Cell ◽  
Adaptive Regulation ◽  
Cell Monolayers ◽  
System A ◽  
Renal Epithelial Cell Line

Hyperosmolarity induced an increase in Na(+)-dependent L-alanine uptake in confluent monolayers of the established renal epithelial cell line NBL-1. This induction was attributable to system A and was only seen when the cells had been previously deprived of amino acids in the culture medium to derepress system A activity. It was additive to the adaptive regulation induction, and both were inhibited by cycloheximide. However, the hyperosmolarity effect was inhibited by colcemid (an inhibitor of microtubular function), but adaptive regulation was not. Otherwise, when cell monolayers were incubated in a control medium, basal Na(+)-dependent L-alanine uptake mediated by system B0 decreased. The results of this study show that: (i) system A activity was not induced by cell shrinkage and subsequent swelling due to extracellular hyperosmolarity when cells were incubated in control medium; (ii) previous expression of system A activity induced by amino acid starvation seems to be a prerequisite for further induction due to hyperosmolarity; and (iii) the effects of adaptive regulation and hyperosmotic stress are mediated by different mechanisms.

Electrophysiological Measurements of a Toad Renal Epithelial Cell Line (A6) as an Assay for Predicting Ocular Eye Irritancy

1992 ◽  
Vol 20 (2) ◽  
pp. 218-221
Author(s):  
Henning F. Bjerregaard
Keyword(s):  
Epithelial Cell ◽  
Cell Line ◽  
South African ◽  
Mdck Cells ◽  
Epithelial Cell Line ◽  
Renal Epithelial Cell ◽  
Electrophysiological Measurements ◽  
Renal Epithelial Cell Line ◽  
Darby Canine Kidney

An established epithelial cell line (A6) from a South African clawed toad (Xenopus laevis) kidney was used as a model for the corneal epithelium of the eye in order to determine ocular irritancy. When grown on Millipore filter inserts, A6 cells form a monolayer epithelium of high electrical resistance and generate a trans-epithelial potential difference. These two easily-measured electrophysiological endpoints showed a dose-related decrease after exposure for 24 hours to seven selected chemicals of different ocular irritancy potential. It was demonstrated that both trans-epithelial resistance and potential ranked closely with in vivo eye irritancy data and correlated well (r = 0.96) with loss of trans-epithelial impermeability of Madin-Darby canine kidney (MDCK) cells, detected by use of a fluorescein leakage assay.

scholarly journals Expression of phosphoenolpyruvate carboxykinase (PEPCK) chimeras in renal epithelial cells. Retention of appropriate physiological responsiveness using enhancerless retroviral vectors

1992 ◽  
Vol 284 (3) ◽  
pp. 725-732 ◽  
Author(s):  
A S Pollock ◽  
D H Lovett
Keyword(s):  
Epithelial Cell ◽  
Cyclic Amp ◽  
Cell Line ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Large Fraction ◽  
Expression System ◽  
Retroviral Vectors ◽  
Epithelial Cell Line ◽  
Renal Epithelial Cell ◽  
Renal Epithelial Cell Line

We used an enhancerless U3 mutant retroviral vector to deliver chimeras of the phosphoenolpyruvate carboxykinase (PEPCK) promoter region to a renal epithelial cell line capable of expressing PEPCK mRNA. Chimeras consisting of the PEPCK promoter and chloramphenicol acetyltransferase, neomycin phosphotransferase or human growth hormone genes were expressed after viral infection of the NRK52E renal epithelial cell line. Virus-delivered sequences in which the direction of PEPCK promoter transcription was antegrade to the normal direction of the long terminal repeat (LTR)-initiated transcription correctly upon stimulation with dexamethasone or 8-bromo cyclic AMP and upon lowering of the extracellular pH. Fluorescent primer extension in situ using primers specific for virus-delivered sequences of antegrade constructs indicated that a large fraction of NRK52E cells could be infected by co-cultivation with virus-producing psi-2 cells without G418 selection. Virus-delivered constructs whose orientation was opposite to that of the LTRs were expressed at very low levels, with transcripts detectable by PCR only in RNA from cyclic AMP-treated cells. Using reverse transcription/PCR, we demonstrated that the chimeric transcripts were from the internal PEPCK promoter rather than a functional or reconstituted Moloney LTR. PEPCK-reporter chimeras delivered by retroviral vectors demonstrated a level of expression more consistent with the level of expression of the native PEPCK gene than did transfected chimeras. This expression system should prove useful for studies of the physiological modulation of gene expression in renal tissues.

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