Doctoral Dissertations

Date of Award

3-1985

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biomedical Sciences

Major Professor

John S. Cook

Committee Members

Stephen Kennel, Peter Mazur, Wen Yang

Abstract

NA⁺-dependent hexose transport, a secondary active transport system found in the small intestine and the proximal tubule of the kidney was used as a marker for epithelial differentiation in LLC-PK₁, a cell line isolated from juvenile pig kidney by R. N. Hull. LLC-PK₁ acquires Na+-dependent hexose transport at confluence, when approximately 90% of the cells are quiescent, and transport levels increase continuously over time in culture. The appearance of this transport system can be stimulated by phosphodiesterase inhibitors, such as theophylline and MIX, and inhibited by TPA, a powerful tumor promoter. When the cells differentiate, the population acquires an increased capacity for NA⁺-dependent hexose transport, without a change in the rate constant for uptake. Several proposed mechanisms which could account for the observed increase in hexose transport levels were examined. The first possibility was that the efflux pathway for α-meG, a nonmetabolizable glucose analog specific for NA⁺ cotransport, declined over time in culture. Efflux was first order, with a rate constant of 0.48 hr⁻¹, and was not altered by phlorizin, a competitive inhibitor of this transport system, or by cytochalasin B, an inhibitor of the facilitated diffusion transporter. Therefore, it was concluded that the primary pathway for efflux of α-meG in this cell line was via passive diffusion. The transporter was shown to be reversible. When ouabain was added to discharge the NA⁺ electrical gradient, the efflux rate constant increased to 0.65 h⁻¹. This component could be transstimulated by adding a high concentration of nonradioactive extracellular α-meG and was inhibited to control levels by phlorizin. The rate constant for efflux remained the same at all levels of hexose transport, indicating that this was not the mechanism responsible for the development of NA⁺ cotransport capacity by these cells. Several mechanisms involving an increase in the influx component of the accumulation process were examined. An increase in the NA⁺ electrochemical gradient was ruled out by data from Kurt Amsler and Carolyn Shaffer, who showed that the gradient actually declined at confluence. The development of gap junctions between transporting and nontransporting cells might also generate an increased capacity for hexose transport but these cells had no gap junctions when examined by fluorescein dye transfer, electrical coupling and freeze fracture analysis. A method of separating transporting from nontransporting cells by density centrifugation in Percoll was developed in order to study the remaining two mechanisms: an increase in the number of transporters per cell, and a progressive recruitment of nontransporting cells to a transporting population. Cells were incubated in Na-gluconate medium containing no K⁺, Cl⁻, or phosphate in order to inhibit volume regulation. In this medium, cells were unable to accumulate α-meG. Attempts made to restore the electrical gradient by adding NaCl or NaCl plus bicarbonate were unsuccessful. When 2 mM (NH₄)₂SO₄, which is transported by the Na,K-ATPase in place of K⁺, was added, NA⁺ cotransport levels were restored, indicating that the maintenance of the NA⁺ gradient was necessary for transport. In this medium, cells that took up α-meG were less dense and banded at a lighter density in Percoll than control cells. Phlorizin inhibited the movement of these cells on the gradient. A mixture of transporting and nontransporting cells could only be separated in the presence of α-meG. A timecourse of the development of NA⁺-dependent hexose transport conducted over a 20-day period showed a development of a transporting peak at a lighter density which increased in size over time while the nontransporting peak declined. Although it appeared that the cells were recruited gradually to a differentiating population, the radioactive α-meG curve was skewed to the left, which indicated that it took several days for the recruited cells to acquire all their transporters and reach their full capacity for α-meG transport. The question of whether this cell line undergoes terminal differentiation was also examined. Fractions of cells from two Percoll gradients--with and without α-meG--were replated under sterile conditions. Plating efficiencies and ability to incorporate thymidine were the same for transporting and nontransporting cells. Therefore, these cells do not undergo irreversible loss of growth potential as a prerequisite for differentiation. However, this experiment was only carried out for two cell cycles. Therefore, it does not rule out the possibility that these cells undergo terminal differentiation.

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