Effect of Chemicals on the Cell Membrane Transport of Nucleosides.
Final scientific rept. 1 Aug 82-31 Jul 83,
TENNESSEE UNIV MEMORIAL RESEARCH CENTER KNOXVILLE DEPT OF MEDICAL BIOLOGY
Pagination or Media Count:
An apparatus and methodology for a high speed kinetic assay of purine efflux has been developed. The procedure is based on a flow system with a membrane filter to remove preloaded L5178Y cells and a sensitive rapid detector of the fluorescence emission of a buffer that contains a transport substrate, 2-aminopurine AP. Two other purines interact rapidly with the AP carrier, hypoxanthine and uric acid. The rate of AP efflux from preloaded cells is increased by hypoxanthine in the external buffer and the efflux rate is decreased by uric acid in the buffer. Perfluorodecanoic acid PFDA, adenine, or xanthine in the external buffer have no direct effect on the rate of AP efflux, in comparison with the controls. L5178Y cells were given a prior incubation with 200 microgramml PFDA at 30 C for 24 hr. These cells were preloaded with 100 micromolar AP and the excess substrate was removed by rinsing the cells with cold buffer. The prior incubation of the cells with PFDA produces a total inhibition of the efflux of AP. On the other hand, a prior incubation of the cells with PFDA plus 50 microunitml bovine insulin produces approximately 40 inhibition in comparison with controls. These findings suggest that purine carrier exists in an active and an inactive form PFDA treatment inhibits formation of active carrier. Insulin appears to stabilize the active carrier and protect against the effects of PFDA.