INTRACELLULAR FREE CALCIUM-CONCENTRATION IN THE BLOWFLY RETINA STUDIED BY FURA-2

The retinal tissue of blowflies was loaded with the fluorescent Ca2+ indicator Fura-2 by incubating cut heads in saline solutions which contained the membrane permeable acetoxymethylester of Fura-2 (Fura-2/AM). The spectral analysis of the tissue fluorescence showed that Fura-2/AM was intracellularly hydrolysed to Fura-2. In order to monitor the intracellular free Ca2+ concentration ([Ca2+]i) the Fura-2 fluorescence was excited by short light flashes. The fluorescence was calibrated by incubating the tissue in Ca2+ buffers of high buffering capacity and subsequent disruption of the cell membranes by freeze/thawing, which gave a dissociation constant for the Ca2+-Fura-2 complex of 100 nM. When the extracellular Ca2+ concentration ([Ca2+]o) was altered [Ca2+]i reversibly changed. The changes were most pronounced when [Ca2+]o was varied in the millimolar range, e.g. [Ca2+]i increased from 0.07-mu-M at [Ca2+]o = 0.1 mM to 1-mu-M at [Ca2+]o = 10 mM. When extracellular Na+ was replaced by Li+ or other monovalent ions, [Ca2+]i rapidly increased which supports the view that electrogenic Na+/Ca2+ exchange contributes to the control of [Ca2+]i. However, [Ca2+]i decreased again when the tissue was superfused with Na+-free media for longer periods, which points to a Ca2+-transporting system different from Na+/Ca2+ exchange. Light adaptation had only a small effect on [Ca2+]i. Even after intense stimulation [Ca2+]i increased by a factor of 1.5 only, which is in line with results obtained in the photoreceptors of Balanus and Apis.