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 Ca(2+)-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 microM at [Ca2+]o = 0.1 mM to 1 microM 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 Ca(2+)-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.
Hochstrate
P
P
Institut für Tierphysiologie, Ruhr-Universität Bochum, Germany.
Juse
A
A
D000221:Adaptation, Ocular; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002138:Calibration; D002414:Cations, Monovalent; D002463:Cell Membrane Permeability; D004175:Diptera / Q000378:metabolism*; D004533:Egtazic Acid / Q000737:chemistry; D016257:Fura-2 / Q000737:chemistry; D008027:Light; D008297:Male; D010786:Photoreceptor Cells / Q000378:metabolism*; D012160:Retina / Q000378:metabolism; D012964:Sodium / Q000494:pharmacology; D013050:Spectrometry, Fluorescence
D002414:Cations, Monovalent; D004533:Egtazic Acid; D012964:Sodium; D002118:Calcium; D016257:Fura-2