DIFFERENTIATION BETWEEN ALTERATIONS IN PLASMA AND MITOCHONDRIAL-MEMBRANE POTENTIALS IN SYNAPTOSOMES USING A CARBOCYANINE DYE

The cationic potentiometric fluorescent probe 3,3'-diethylthiadicarbocyanine iodide [DiS-C2(5)] was used in synaptosomes to assess the relative contributions of plasma and mitochondrial membrane potentials (PSI(p) and PSI(m), respectively) to overall fluorescence. Addition of synaptosomes to media containing 0.5-mu-M dye caused a decrease in fluorescence intensity due to dye accumulation, which equilibrated usually within 5 min. Depolarization of mitochondria by combined treatment with cyanide and oligomycin increased fluorescence by 42%, indicating significant prior accumulation of dye into intrasynaptosomal mitochondria. PSI(p) was calculated to be -54 mV and was not altered significantly by prior depolarization of PSI(m) with cyanide and oligomycin (hereafter referred to as "poisoned" synaptosomes). Similarly, the linear relationship between dye fluorescence and PSI(p) was not altered by depolarization of PSI(m). Valinomycin, a K+ ionophore, caused a PSI(p)-dependent increase in fluorescence in control (nonpoisoned) synaptosomes, but did not alter fluorescence of poisoned synaptosomes except when the extracellular concentration of K+ ([K+]e) was 2 mM, in which case valinomycin hyperpolarized PSI(p) by about 5 mV. The poreforming antibiotic gramicidin depolarized both PSI(p) and PSI(m) maximally. Under these conditions, Triton X-100 further increased fluorescence by 40%, indicating significant dye binding to synaptosomal components. In poisoned synaptosomes depolarized by 75 mM K+, gramicidin caused a decrease in fluorescence intensity (hyperpolarization of PSI(p)). The organic solvent dimethyl sulfoxide, used as a vehicle for the hydrophobic ionophores, had voltage-dependent effects on PSI(p) and PSI(m). The magnitude of the fluorescence response of synaptosomes to depolarization with sufficient K+ to increase [K+]e by 40 mM was dependent on the initial PSI(p) and was not altered by prior depolarization of PSI(m). Thus, K+-induced depolarization may be useful for estimating PSI(p) in the face of fluorescence change associated with depolarization of both PSI(p) and PSI(m). Thus, carbocyanine dyes may be used to differentiate effects of experimental treatments on synaptosomal PSI(p) as compared with intrasynaptosomal PSI(m).