[4] Intracellular ion activities and membrane transport in parietal cells measured with fluorescent dyes

This chapter discusses the methods used for measuring intracellular pH, [Cai2+] (Cai2+, with Fura-2) and [Na +] in single, identified cells of gastric glands isolated from rabbits. General methods for loading the dyes into cells, attaching the glands to coverslips and perfusing them with different solutions using flow-through chambers will be described. Techniques for assessing intracellular dye behavior and calibrating the dye fluorescence in cells will be discussed. In addition, approaches for identifying specific membrane transport systems and for measuring rates of transport by these mechanisms will be presented. One advantage of using fluorescent probes is that the same rationing system can be used for measuring any of the dyes discussed in this chapter. This is particularly advantageous because there are many potential interactions among the various ions and ion transporters. With such considerations as guidelines, fluorescent ion indicators should continue to provide unique insights into cellular physiology for many years.