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

Abstract

SUMMARY1. We have used laser-activated micro mass analysis (l.a.m.m.a.) and energydispersive 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 marines.2. 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.3. 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 ,tmol/l wet tissue volume.4. 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.5. Ca exchange in intact rod outer segments in darkness is very slow. The rate of accumulation of 44Ca was only 105 Ca/rod . s, or about 100 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.6. 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.7. 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.8. Our measurements indicate that the great majority of the Ca in the rod appearsAuthors' names are in alphabetical order. * To whom reprint requests should be sent. t Experiments carried out in Julich. 21P HY 368

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

“…However, in addition to cyclic GMP, rod outer segments also contain large quantities of another putative messenger substance, Ca (Hagins & Yoshikami, 1975; Schnetkamp, 1979Schnetkamp, , 1980Schnetkamp, , 1986 but see Somlyo & Walz, 1985). We have shown that rods in the intact retina contain 4-5 mmol Ca/l tissue volume, or 1-2 Ca for each rhodopsin molecule in the outer segment (Schr6der & Fain, 1984;Fain & Schr6der, 1985). This result has been recently confirmed using a different method, for intact rods isolated in normal Ringer solution from frog retina (Nicol, Kaupp & Bownds, 1986).…”

“…5 was in fact no significant difference in the 4"Ca measurements between these two sets of experiments. The decline of 4"Ca content in both these experiments was larger than previously observed for rods incubated in darkness in normal Ringer solution (Fain & Schr6der, 1985). It was also larger than that seen in elevated Ca concentrations in Fig.…”

“…Both control and experimental pieces were dehydrated and prepared for analysis as previously described (see Methods and Fain & Schr6der, 1985). Fain & Schr6der (1985).…”

“…The Wechsung et al 1979;Vogt et al 1981). Peak at m/e = 40 corresponds to approximately 4-5 mmol total Ca/l tissue volume (see Fain & Schr6der, 1985 protocol for these experiments was as follows. A retina from a dark-adapted animal was cut into eight pieces, and one or two pieces were immediately shock-frozen as a control (see Methods).…”

“…In all of the experiments in this paper, the retina was dehydrated by cryo-substitution in acetone-0s04 and embedded in an Epon-Araldite mixture, as described in detail in Fain & Schr6der (1985).…”