Measurement of luminal pH of acidic stores as a readout for NAADP action.

Abstract

In addition to mobilizing Ca²⁺, NAADP plays a role in modulating the luminal pH (pHL) of acidic stores of the endolysosomal system. The effects of NAADP on pHL have been most extensively studied in the sea urchin egg, both in the intact egg and in egg homogenates. Related observations have also been made in mammalian systems (e.g., guinea pig atrial myocytes and pancreatic acinar cells). Although the connection between Ca²⁺ mobilization and increase in pHL is not understood, pHL can be a useful parameter to measure when studying NAADP-mediated signaling. This protocol describes the fluorescent measurement of pHL of acidic stores. It relies on the use of acridine orange (AO), a standard dye for pHL. AO selectively accumulates to high concentrations in the lumen of organelles as a function of acidity; at these high concentrations it self-quenches. When pHL increases, some AO is lost from the vesicle. As a result, the lower luminal AO concentration relieves the quenching and fluorescence increases in the lumen.