[FDA-hydrolysis for the fluorometrical evidence of a cell membrane alteration in peritoneal macrophages (author's transl)]

Abstract

The enzymatic hydrolysis of fluoresceindiacetate (FDA) and the intracellular accumulation of fluorescein (fluorochromasia) by murine peritoneal macrophages were photometrically measured in cell suspensions. The intensity of fluorescence (excitation at lambda = 485 nm) was recorded by a right angel detector as a function of time, number of cells per ml, and FDA concentration, respectively. The meter response was found to be directly proportional up to the concentration of the fluorophor of 1.125 x 10(-6) M. In suspension the intensity caused by cells other than macrophages could be neglected. The rate of the fluorescein formation corresponds to the Michaelis-Menten constants (km = 8,34 x 10(-6) M.) Repeated in vivo administrations of some N-mustard benzimidazole derivatives were followed by competitive and non-competitive inhibition of the FDA-hydrolysis, respectively. Consequently, the fluorescence intensity varied, but without an concomitant adequate alteration of the non-specific (alpha-naphthyl acetate) esterase as was being measured by means of photometry of single macrophages in cell smears. Thus, this method seems to be suitable for quantitation of membrane (permeability) alteration induced experimentelly in macrophages.