Flow cytometric analysis of membrane permeability properties influencing intracellular accumulation and efflux of fluorescein

A flow cytometric investigation has been made on the membrane permeability properties that mediate intracellular turnover of fluorogenic substrates. The accumulation and efflux of fluorescein, consequent to the enzymatic turnover of fluorescein diacetate, were assessed in the presence of metabolic inhibitors and after treatment with membrane-active compounds. The metabolic poisons KCN and rotenone greatly inhibited only the fluorescein efflux, reducing the rate constant to as little as one-tenth in relation to control cells; in the presence of glucose such inhibition was partially removed. Glucose availability also affected fluorescein efflux: an increase of the rate constant was observed in cells treated with 20 mM glucose, and a decrease was measured in cells incubated for 1 hr in glucose-free buffer. Membrane-active compounds Triton X-100 and hydrocortisone reduced fluorescein accumulation. Hydrocortisone strongly blocked also the efflux; the addition of glucose did not restore the rate significantly. The major evidence of these results is that fluorescein efflux is dependent on membrane integrity and on availability of metabolic energy. Fluorescein accumulation is only partially related to permeability properties regulating FDA uptake, due to the influence that treatments exhibit at the same time on FDA hydrolysis and/or fluorescein release.