A light-dependent increase in free Ca2+ concentration in the salamander rod outer segment
H. R. Matthews: Physiological Laboratory, Downing Street, Cambridge CB2 3EG, UK.
- 1The Ca2+ indicator dye fluo-5F was excited by an argon ion laser to measure changes in free Ca2+ concentration ([Ca2+]i) in the outer segments of isolated salamander rods rapidly exposed to a 0 Ca2+, 0 Na+ solution designed to minimise surface membrane Ca2+ fluxes. Over 30-60 s of laser illumination, the fluorescence first increased rapidly and then declined at a rate that was much slower than in Ringer solution and consistent with previous physiological evidence that 0 Ca2+, 0 Na+ solution greatly retards light-induced changes in [Ca2+]i.
- 2The initial increase in fluorescence was investigated with a sequence of 100 ms laser flashes presented at 5 s intervals. The fluorescence evoked by the second laser flash was on average 30 % larger than the first, and subsequent responses exhibited a slow decline like that measured with continuous laser exposures. The initial increase in fluorescence did not depend upon the timing of exposure to 0 Ca2+, 0 Na+ solution but appeared to be evoked by exposure to the laser light.
- 3Both the increase and subsequent decline in fluorescence measured with brief laser flashes could be reduced by incorporation of the Ca2+ chelator BAPTA. This and other results indicate that the fluorescence increase was unlikely to have been caused by a change in the affinity of fluo-5F for Ca2+ or an increase in the quantity of incorporated dye available to bind Ca2+ but reflects an actual release of intracellular Ca2+ within the outer segment.
- 4The pool of Ca2+ available to be released could be decreased if, before the first laser flash, the rod was exposed to light bright enough to bleach a substantial fraction of the photopigment. The releasable pool could also be depleted by exposure to saturating light of much lower intensity if delivered in Ringer solution but not if delivered in 0 Ca2+, 0 Na+ solution. We conclude that Ca2+ can be released within the outer segment both by the bleaching of rhodopsin and by the reduction in [Ca2+]i which normally accompanies illumination in Ringer solution.
- 5The activation of rhodopsin appears somehow to induce the release of Ca2+ from a binding site or store within the outer segment. Substantial release, however, required stimulating light of an intensity sufficient to bleach a considerable fraction of the visual pigment. It therefore seems unlikely that such release contributes to the normal Ca2+-mediated modulation of transduction during light adaptation. The mechanism and physiological function of light-induced Ca2+ release are unknown.