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Journal of Neuroscience, Vol 8, 3240-3246, Copyright © 1988 by Society for Neuroscience
The concentration of cytosolic free calcium in vertebrate rod outer segments measured with fura-2
GM Ratto, R Payne, WG Owen and RY Tsien
Department of Biophysics and Medical Physics, University of California, Berkeley 94720.
The use of fluorescent indicators such as fura-2 (Grynkiewicz et al., 1985)
to measure the cytosolic free calcium activity in retinal rods is
complicated by the rods' sensitivity both to the fluorescence and to the
light that excites it. By stimulating fluorescence from large numbers of
rods in whole, loaded retinas and averaging repeated measurements, however,
we have been able to monitor changes in free [Ca2+]i during exposure to
nonsaturating lights under physiological conditions. Retinas, isolated from
the bullfrog Rana catesbeiana, were loaded with fura-2 by incubation and
mounted, receptor-side up, in a perfusion chamber placed on the stage of a
specially designed apparatus. A step of light delivered from above, whose
wavelength alternated between 340 and 380 nm every 110 msec, excited
fluorescence from 24 mm2 of retina and evoked a light response (the
aspartate- isolated pIII component of the electroretinogram--ERG). By
comparing the fluorescence intensities excited by the 2 wavelengths
(corrected for background and dark-noise), the free [Ca2+]i of the rod
outer segment was determined. In darkness, the [Ca2+]i of the outer segment
was found to be approximately 220 nM. A bright light caused it to fall
exponentially to approximately 140 nM, with a time constant of
approximately 1.6 sec. The value of [Ca2+]i at the onset of illumination
was independent of stimulus intensity over a 2 log-unit range, and in all
cases the fall was monotonic. After terminating the illumination, [Ca2+]i
rose again to its time-zero value.(ABSTRACT TRUNCATED AT 250 WORDS)
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