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 microM 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 pore-forming 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Hare
M F
MF
Department of Pharmacology and Toxicology, Michigan State University, East Lansing 48824-1317.
Atchison
W D
WD
D000818:Animals; D002412:Cations / Q000378:metabolism; D002462:Cell Membrane / Q000502:physiology; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004225:Dithiazanine; D008564:Membrane Potentials; D008928:Mitochondria / Q000502:physiology*; D009994:Osmolar Concentration; D011188:Potassium / Q000494:pharmacology; D013574:Synaptosomes / Q000378:metabolism / Q000502:physiology*; D014634:Valinomycin / Q000494:pharmacology
D002412:Cations; D014634:Valinomycin; D004225:Dithiazanine; D011188:Potassium; D004121:Dimethyl Sulfoxide