Measuring calcium uptake and release by invertebrate photoreceptor cells by laser microprobe mass spectroscopy

Electroretinogramms (ERG) of isolated crayfish retinas in salines differing in their Ca2+ concentration were recorded to monitor changes in the ERG induced by changes in the extracellular Ca2+ concentration. Laser microprobe mass spectroscopy and electron microscopy of (a) shock-frozen and (b) chemically fixed retinas were used to analyse the distribution of Ca in the photoreceptor cells. For quantitative analysis a new standardisation procedure using vacuum deposition onto the specimen of thin films as an internal standard was developed. For the first time stable isotopes were used in microbeam analysis allowing direct measurements of Ca transport and metabolism on the cellular level. The major portion of Ca was found in the black distal shielding pigment granules (DP) within the retinular photoreceptor cells. Untreated retinas and retinas preincubated in physiological saline (with 10 mmol/1 Ca2+) contained up to 100 mmol/1 Ca in the DP, while in DP-free places within the same cell Ca was as low as < 40 mumol/1. If the Ca-concentration of the saline was increased (decreased), a rise (fall) of Ca in the DP was observed. Careful Ca-depletion of the DP under ERG control allowed removal of estimated 60--70% of the 49Ca originally present and refilling with 44Ca. The maximum amplitude of the ERG-response decreased under these conditions to 50% in low Ca saline, but could be reestablished to some 70% in physiological saline containing 44Ca. We conclude, that in the living cell the DP acts sa a Ca store possibly regulating the intracellular and/or extracellular Ca level.