Brief illumination of the isolated toad retina produces a transient increase in the extracellular free Ca concentration in the photoreceptor layer. This change in concentration arises from the release of Ca.+ by the rod outer segments.The release begins within 200 msec after the stimulus flash. The peak amplitude of the concentration change increases linearly with light intensity up to approximately 100 absorbed photons per rod and saturates at about 107 absorbed photons per rod. In the linear range, the amount of Ca released corresponds to about 2 X 104 Ca2+ per absorbed photon per rod. The high stoichiometry of this release, in relation to the low free intracellular
MATERIALS AND METHODSMaterials. The organophosphate used in the electrodes (11) was the Ca salt of bis(di-n-octyl)phenyl phosphate (HDOPP) (the kind gift of S. Firstenberg, Radiometer, Copenhagen, Denmark). Di-octylphenyl phosphonate (DOPP) was obtained from Specialty Organics (Irwindale, CA). Tetrahydrofuran and poly(vinyl) chloride (PVC) were purchased from Aldrich. Li-Al hydride was added to the tetrahydrofuran to prevent accumulation of peroxides. Na tetraphenylboron was obtained from Sigma. The water used throughout was distilled and deionized.Electrode Construction. The Ca-selective membrane was made by mixing HDOPP with DOPP, 1:10 (wt/wt), and allowing the solution to stand overnight at room temperature. The solution was then incorporated into nonporous PVC (12) as follows. PVC (0.39 g) was dissolved in 13 ml of tetrahydrofuran and 0.83 g of the HDOPP/DOPP solution and 5.2 mg of Na tetraphenylboran were added. The resulting solution was poured into a clean glass petri dish (diameter, 9.5 cm), covered with several layers of filter paper, and allowed to dry for at least 24 hr. The resulting PVC membrane was 100,gm thick.For measurements from the isolated retina, a special electrode was constructed. Two holes were drilled through the top of a cylindrical PVC cap (diameter, 1.3 cm) and a disc (diameter, 0.8 cm) of the Ca membrane was glued over the holes with 0.3% PVC in tetrahydrofuran (Fig. 1). One of the holes was made into a reference electrode by cutting its overlaying PVC membrane and inserting into it polyethylene tubing containing 2% agar in Ringer's solution. The assembled PVC cap was glued onto the end of a U-shaped plexiglass tube. The tube was then filled with a Ca standard solution which contacted the lower surface of the uncut (active) membrane. The Ca electrode potential was recorded between Ag/AgCl pellets placed in the reference tubing and in the Ca standard solution with a lownoise differential amplifier (model 113, PAR Instruments, Princeton, NJ).Electrode Properties. As originally reported by Ruzicka et al. (11), the Ca membrane electrodes exhibited nearly Nernstian behavior-i.e., 29 mV per decade change in Ca concentration, down to <1 gM Ca. The electrodes exhibited a high selectivity for Ca over other biologically important ions [-log KCa/Na = 5.2; -log KCa/K = 5.7; -log Kca/Mg = 3.6 (11)]. In addition, the elect...