The number of Na/K-ATPase sites in the plasma membrane of Hela cells have previously been demonstrated to be regulated by changes in intracellular electrolyte levels. Preliminary evidence suggests that a similar regulatory mechanism is demonstrable in a superfused suspension of outer medullary kidney tubule segments, as described in this proposal. The superfused kidney tubular preparation provides an ideal system in which to study this mechanism. Intracellular electrolyte levels can be manipulated through precisely regulated changes in extracellular composition and measured directly using techniques which have been established in this particular preparation. Intracellular Na+ levels will be measured using NMR techniques and intracellular Ca2+ levels will be measured using the fluorescent indicator 'Quin 2'. In addition, it is proposed to establish the NMR technique of intracellular K+ level measurement. Intracellular K+ levels will also be determined in the characterization of the response described. In vivo, the changes required would be very complex and difficult to interpret. Finally, the yield of cells, characterized by high Na/K-ATPase concentrations, will provide the material necessary for the investigation of the cellular mechanisms involved. The first goal of the proposed project is to characterize the changes in Na/K-ATPase activity observed after chronic changes in intracellular electrolyte levels. This characterization will contribute to the definition of steroidal cellular actions, which are believed to implicate changes in intracellular electrolyte levels. The second goal is to define the ion species responsible for the changes in Na/K-ATPase activity observed. Finally, the third goal is to investigate the possible mechanisms involved in the regulation of Na/K-ATPase activity by chronic changes in intracellular electrolyte levels. (i) The possible association between changes in Na/K-ATPase activity and changes in plasma membrane area will be investigated to determine whether the response observed is a reflection of an increased concentration of transporting sites. (ii) To test whether changes in intracellular electrolyte levels unmask latent Na/K-ATPase sites. (iii) To determine whether changes in either enzyme synthetic or degradative rates are involved, and if so (iv) to examine possible mechanisms implicated in such changes.
| Rayson, B M (1989) Rates of synthesis and degradation of Na+-K+-ATPase during chronic ouabain treatment. Am J Physiol 256:C75-80 |
| Laury-Kleintop, L D; Alhadeff, J A; Damjanov, I (1985) Isoelectric forms of alpha-L-fucosidase in mouse teratocarcinoma-derived cell lines. Dev Biol 111:520-4 |
