Calcium content and calcium exchange in dark-adapted toad rods.

We have used laser-activated micro mass analysis (l.a.m.m.a.) and energy-dispersive X-ray analysis (e.d.x.) to measure Ca content and Ca movements in 'red' rod photoreceptors in the dark-adapted retina of the toad, Bufo marinus. Measurements with both l.a.m.m.a. and e.d.x. show that intact rod outer segments contain 4-5 mmol total Ca/l wet tissue volume, or 1-2 Ca per rhodopsin. We could detect no significant variation in the total Ca as a function of distance across or up and down the outer segment. In the inner segment, Ca could be detected only within the mitochondria-rich ellipsoid body, where the total Ca concentration was of the order of 100-400 mumol/l wet tissue volume. To measure the exchange of Ca in outer segments from intact photoreceptors, we exposed the dark-adapted retina to Ringer containing the stable isotope 44Ca. Since l.a.m.m.a. can measure separately the concentrations of each of the isotopes of the elements, and since native rods contain almost exclusively 40Ca, the increase in 44Ca and decrease in 40Ca could be used as a measure of Ca influx and efflux. Ca exchange in intact rod outer segments in darkness is very slow. The rate of accumulation of 44Ca was only 10(5) Ca/rod.s, or about 10% of the total outer segment Ca/h. This slow rate of exchange is apparently not the result of restricted movement of Ca across the plasma membrane. Ca exchange was also measured in outer segments which were either partially or entirely detached from the rest of the photoreceptor. In broken-off outer segments, Ca exchange is faster than in the intact organelles, and in 1 h, half of the 44Ca exchanges for 40Ca. When the retina was incubated in Ringer for which all of the Na was substituted with Li or choline, there was an increase in the rate of 44Ca accumulation in intact outer segments, probably due to an inhibition of Na-Ca counter transport across the plasma membrane. Our measurements indicate that the great majority of the Ca in the rod appears to be inaccessible to exchange under physiological conditions, probably because it is sequestered within the disks which in intact rods appear to be nearly impermeable to Ca in darkness.