Calcium in dark‐adapted toad rods: evidence for pooling and cyclic‐guanosine‐3'‐5'‐monophosphate‐dependent release.

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Abstract
1. We have used laser micromass analysis (l.a.m.m.a.) to investigate Ca uptake and release in intact ''red'' rod photoreceptors in the dark-adapted retina of the toad, Bufo marinus. 2. With l.a.m.m.a. it it possible to measure separately the concentrations of each of the Ca isotopes. Rods normally containing almost exclusively 40Ca can be incubated in Ringer solution containing the stable isotopes 42Ca or 44Ca. In this way, the movements of Ca into and out of the rod can be separately determined. 3. When rods are incubated in darkness in high 44Ca (up to 20 mM), large amounts of 44Ca accumulate in the outer segment at a rate which increases with increasing external 44Ca concentration. However, this 44Ca appears not to exchange with the 40Ca originally present within the rod. This result suggests that the 40Ca may be sequestered within a pool which normally exchanges slowly with external Ca. 4. We explored Ca exchange in high-Ca solutions in more detail with double-isotope labelling. In these experiments, rods were first pre-loaded with Ca of one isotope (42Ca) and then incubated in Ringer solution containing another (44Ca). We could then measure separately the rate of exchange of the pre-loaded 42Ca with the 44Ca in the Ringer solution and with the 40Ca originally present within the rod in the sequestered pool. 5. These experients show that the pre-loaded-Ca exchanges rapidly with Ca in the Ringer solution, at least in part by Ca .sbd. Ca exchange, but much more slowly with the Ca originally present within the rod. Thus Ca in the outer segments can exist in (at least) two pools: one which exchanges rapidly across the plasma membrane and is probably Ca free or loosely bound within the cytosol, and another which exchanges slowly and is probably Ca within the disks. 6. Although Ca sequestered within the outer segment normally exchanges quite slowly, it can be rapidly released if the extracellular free Ca is buffered to low levels with EGTA. The rate-limiting step for Ca release under these conditions appears not to be Na .sbd. Ca exchange, since the rate of Ca efflux is unchanged if the Na in the Ringer solution is substituted with choline. 7. Ca can also be released from the sequestered pool if rods are incubated in Ringer solution containing 100 or 500 .mu.M-IBMX (3-isobutyl-1-methylxanthine). We speculate that the mechanism of release in both low-free-Ca solutions and IBMX solution involves the opening of cyclic-GMP-dependent channels in the disk membrane. 8. Rods incubated in IBMX show a dramatic increase in the rate of accumulation of Ca from the external medium. This is probably a direct result of the increase in the light-dependent conductance of the outer-segment plasma membrane in IBMX. 9. Under the assumption that the sequestered pool of Ca is within the disks, the rate of exchange of Ca across the disk membrane in darkness in physiological Ringer solution can be calculated to be no greater than 108 Ca cm-2 s-1 or about 100 Ca disk-1 s-1. This is more than three orders of magnitude smaller than the rate of Ca movement in darkness per square centimetre across the plasma membrane.