Membrane perturbation: studies employing a calcium-sensitive dye, arsenazo III, in liposomes

Abstract

A metallochromic dye, arsenazo III [2,7-bis-(2-arsonophenylazo)-1,8-dihydroxynaphthalene-3,6-disulfonic acid], has been incorporated into the aquenous interspaces of multilamellar liposomes. multilamellar liposomes. Addition of Ca produced no shift in the absorbance spectrum of dye captured by liposomes, whereas disruption of liposomes by Triton X-100, followed by Ca, produced the spectrum chracteristic of the dye-Ca complex: evidence of latency. Addition of excess ethyleneglycol-bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) reversed the spectal shift. Differences between spectra obtained in this sequence yielded dye efflus. To measure Ca efflux, difference spectra (+/-EGTA) were obtained from cationic liposomes containing Ca after detergent lysis (sensitivity less than 10 mmol/ml). Since liposomes were impermeable either to dye or Ca until perturbed, it was possible to test a variety of membrane-active steroids (diethylstilbesterol, deoxycorticosterone, etiocholanolone) for their capacity to provoke dye efflux from liposomes; preincorporation of cortisol stablized liposomes against dye leak. Immunoglobulin-coated liposomes containing dye were taken up by phagocytes of Mustelus canis, and phagocytic vacuoles stained red-purple after ingestions. Liposomes containing the calcium-sensitive dye constitute a simple, accurate means for determining membrane perturbation and Ca fluxes; their uptake by cells or organelles remains to be exploited further.