IP₃ induces intragranular pH oscillations in isolated secretory vesicles

Intracellular fluctuations of [Ca²⁺] are critical for signal transduction in secretory cells. We have recently demonstrated that following ATP stimulation the [Ca²⁺]_G inside mucin granules of goblet cells oscillate. In isolated mucin granules, IP₃ stimulation results in similar [Ca²⁺]_G oscillation and oscillatory Ca²⁺ release to the medium. The oscillation of [Ca²⁺]_G results from a Ca²⁺/K⁺ ion exchange process taking place in the polyanionic mucin matrix of the granule. Outflow of Ca²⁺ to the medium takes place via an IP₃-receptor-Ca-channel, and inflow of K⁺ into the granule takes place via a SK_Ca -channel present in the granular membrane (Nguyen, Chin & Verdugo, Nature in press). Since pH can strongly modulate the yield of ion-exchange processes in ion-exchange resins and in mucin gels (Katchalsky et al J. Polymer Sci. 7:571,1951; Tam & Verdugo Nature 292:340, 1981), the present experiments were designed to test if pH also oscillates in mucin granules. Isolated mucin granules were loaded with the pH-sensitive probe Lysosensor Green DND-189 (Molecular Probes). Using thin digital optical sectioning techniques (200 nm), we observed that the fluorescence ratio (F/F_i) of Lysosensor Green DND-189 inside mucin granules oscillates. These preliminary results indicate that intragranular pH also undergoes oscillations similar to the Ca²⁺-oscillations reported by Ca²⁺-sensitive probes. Thus, pH could play an important role in the modulation of IP₃-induced Ca²⁺-oscillations and release in/from secretory granules.