Doctoral Dissertations
Date of Award
6-1982
Degree Type
Dissertation
Degree Name
Doctor of Philosophy
Major
Biomedical Sciences
Major Professor
John S. Cook
Abstract
LLC-PK 1 cells in culture develop the ability to concentrate hexose. The concentrative ability is dependent on the Na + —electrochemical gradient, and is inhibited by phlorizin with K = I,0.5 =0.2 &mirco;M. Development of the transport function can be accelerated by addition of the Friend cell inducer hexamethylene bisacetamide and by the phosphodiesterase inhibitors dibutyryl cAMP, theophylline, and l-methyl-3- isobutyl xanthine. In cultures treated with any of these chemicals development of hexose concentrating capacity is inhibited by the tumor promoter 12-0-tetradecanoylphorbol-13-acetate (TPA). In all cases the development of hexose accumulating capacity is correlated with growth arrest and increased intracellular cAMP levels. However the results suggest that these changes may be necessary but are not sufficient conditions for induction of the hexose transport function.
The level of concentrative hexose uptake begins to increase only after the level of Na + -dependent amino acid transport has decreased (step-down). The decrease is accelerated by those compounds that accelerate appearance of concentrative hexose transport and is retarded by TPA which retards appearance of the hexose transport function. Addition of TPA to stepped-down cultures produces a rapid increase in the rate of amino acid transport. This increase is dependent on protein and RNA synthesis.
The LLC-PKj 1 cell line was cloned and three clones were derived. Each of these clones is capable of developing Na' + -dependent hexose transport. One of these clones was used to analyze the development of hexose accumulating capacity in a cell population using a mathematical model developed by J. S. Cook. Modulation of only two parameters can yield the experimentally obtained results. One of these two possible mechanisms, regulation through a changing driving force for uptake, has been ruled out. It is concluded that development of concentrative hexose transport is probably due to an increase in the fraction of the cell population which is capable of concentrative hexose uptake. Expression of this transport function is an irreversible process. Further, the decision of a cell to express concentrative hexose uptake occurs just prior to or at the same time as expression of the transport function.
The number of Na +-dependent hexose transport sites can be determined by measuring the number of Na +-dependent phlorizin binding sites. During development of hexose accumulating capacity there is a corresponding increase in the number of Na + —dependent phlorizin binding sites with no consistent alteration in the affinity of binding. Thus development of hexose accumulating capacity in a cell population appears to be due to the progressive expression of active Na + -dependent hexose transporters. The photoaffinity label for the Na + -dependent hexose transporter, 4 —azido—phlorizin, was studied to determine its usefulness in this system. The compound reversibly inhibits hexose uptake with K I,0.5 =1&mirco;M when the uptake is carried out in the dark. Exposure of cells to 4-azido-phlorizin and blacklight results in an irreversible inhibition of hexose uptake. This inhibition is dependent on the presence of Na +in the incubation medium and can be blocked by simultaneous inclusion of phlorizin or D-glucose during exposure to light. Therefore a radioactive derivative of this compound, at a high enough specific activity, can be used to specifically and irreversibly label the Na + —dependent hexose transporter expressed by these cells.
Recommended Citation
Amsler, Kurt, "Development of Na⁺-Dependent hexose transport in a pig renal epithelial cell line, LLC-PK₁₊. " PhD diss., University of Tennessee, 1982.
https://trace.tennessee.edu/utk_graddiss/13176