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pubmed:abstractText |
Changes in fluorescence were measured with an argon ion laser from the outer segments of isolated salamander rods containing the Ca(2+)-sensitive fluorescent dye fluo-5F. When the outer segments were exposed to a 0Ca(2+)/0Na(+) solution designed to minimise surface membrane Ca(2+) fluxes, exposure to intense light from the laser evoked a slow increase in fluorescence, reflecting a light-induced rise in outer segment [Ca(2+)](i). The time course of this slow fluorescence rise could be fitted with the sum of two asymptotic exponential functions of approximately equal amplitude, having time constants of approximately 200 ms and 5.7 s. When rods were exposed to saturating background light to reduce outer segment [Ca(2+)](i) before laser illumination, the relative amplitude of the two exponentials was altered so as to reduce the contribution from the one with the shorter time constant. Examination of the initial time course of fluorescence when recording at high temporal resolution revealed a further rapid rise with a time constant of 1-2 ms, which could be observed even from rods in Ringer solution. This initial rapid rise could be abolished by pre-exposing the rod to bleaching illumination, whether the bleach was given in Ringer solution or in 0Ca(2+)/0Na(+) solution. It would therefore appear that the rapid rise in fluorescence is generated in some way by the bleaching of the photopigment. Unlike the slower components of fluorescence increase, the rapid initial rise was virtually unaffected in waveform or amplitude when rods were pre-exposed in Ringer solution to light which was bright enough to suppress completely the circulating current but which bleached a negligible fraction of the photopigment. Furthermore, pre-incubation with the AM ester of the Ca(2+) chelator BAPTA, although completely abolishing the slower components of fluorescence increase, had virtually no effect on the rapid rise. These results indicate that the rapid component, though triggered by rhodopsin bleaching, does not reflect an increase in outer segment [Ca(2+)](i). Neither the rapid nor the slower components of fluorescence increase were affected by exposure of the outer segment to 10 microM of the membrane-permeant compound N,N,N',N'-tetrakis(2-pyridyl-methyl)ethylenediamine (TPEN), which chelates heavy metals such as Zn(2+), or 100 microM 2-aminoethoxydiphenylborate (2-APB), a membrane-permeant blocker of IP(3) receptors. These results appear to exclude a role for changes in heavy metal concentration or Ca(2+) release via IP(3) receptors in the light-induced increases in dye fluorescence. Estimates of absolute Ca(2+) concentration and of rod buffering capacity suggest that the slower components of fluorescence increase represent the release of around 10-50 micromoles Ca(2+) per litre cytoplasmic volume from bound or sequestered stores after bleaching.
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