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The composition of dark-adapted and illuminated retinal rod outer and inner segments and mitochondria was determined with electron probe X-ray micro-analysis of cryosections. The concentration of Ca in the outer segment was 0.4 mmol/kg dry wt. (0.1 Ca/rhodopsin) and did not measurably change upon illumination with saturating light for 5 min. The non-mitochondrial regions of the inner segment contained the highest concentrations (up to 13 mmol/kg dry wt.) of Ca in rods; these regions probably represent the endoplasmic reticulum. The equilibrium potentials estimated from the measured elemental concentrations and the known water content of dark-adapted outer segments were (mV): ENa = +17, EK = -83, ECl = -27. The respective values in the inner segment were: ENa = +20, EK = -89, ECl = -26. The above values were obtained in frog rods bathed in 0.18 mM-Ca Ringer solution. In the outer segment of toad rods bathed in 1.8 mM-Ca Ringer, ENa = +33 mV. The Mg content of the rods was high. The (computed) concentration in the dark-adapted retinae was 11 mM in the outer segment and 24 mM in the inner segment. Illumination caused a reduction in Mg to 9 mM (outer segment) and 16 mM (inner segment). Illumination caused a highly significant reduction in Na and Cl concentrations, and an increase in K concentration in both outer and inner segments. Exposure to Na-free (choline Ringer) solution resulted in reduction in Na to just-detectable levels (3 +/- 1 mmol/kg dry wt.) in the outer segment and to 5 +/- 1 mM in the inner segment. This was associated with a significant loss of Cl and decrease in ECl to -50 mV. The low Na content of the outer segment in the Na-depleted rods is not compatible with an extracellular concentration (105 mM) of inexchangeable Na in the intradiskal space. Mitochondrial Na and Mg paralleled the changes in the cytoplasmic concentrations: both mitochondrial Na and Mg were significantly decreased in illuminated, compared to dark-adapted rods. There was no detectable Ca (0 +/- 0.2 mmol/kg dry wt.) in mitochondria of dark-adapted rods containing high concentrations of Na; mitochondrial Ca was slightly higher (0.5 +/- 0.2 mmol/kg dry wt.) in the mitochondria that contained low Na following illumination.

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