Membrane permeability transition promoted by phosphate enhances 1-anilino-8-naphthalene sulfonate fluorescence in calcium-loaded liver mitochondria

Phosphate and a number of other compounds induce membrane permeability transition (MBT) in Ca(2+)-loaded mitochondria. 1-Anilino-8-naphthalene sulfonate (ANS) was used as a fluorescent probe to investigate perturbations on the inner membrane during MBT. Induction of MBT caused ANS fluorescence enhancement with a biphasic rate that reached a plateau. The enhancement is analogous to that reported for de-energization of mitochondria. The fluorescence level was independent of whether ANS was added before or at different times after phosphate. In the absence of ANS, fluorescence was low and remained unchanged. The initial time course of MBT, as followed by large-amplitude swelling, was similar to that of fluorescence enhancement. Ruthenium red, EGTA, ADP, and cyclosporin A inhibited the enhancement. Only EGTA + ADP (or ATP) reversed the enhancement when added after phosphate. Efflux of matrix Ca2+ by sodium acetate or A23187 did not alter ANS fluorescence. The binding parameters (Kd and number of binding sites) were not significantly different, but the fluorescence maximum was more than doubled after MBT. Although the fluorescence of bound ANS showed a nonlinear relationship, it was always higher (73.0 +/- 19.0%) after reaching the plateau. Since ANS binding to membranes is nonspecific, the exact mechanism of the enhanced fluorescence is not apparent. The dependence of the initial rate of fluorescence enhancement on Ca2+ concentration was nonlinear, with 45 microM at half-maximal rate. The dependence on phosphate was hyperbolic with 0.7 mM at half-maximal rate, which is close to the Km value of phosphate carrier. The kinetics is compatible with Ca2+ binding to some membrane component(s) during MBT and cause ANS fluorescence enhancement.