Detection of light‐induced changes of intracellular ionized calcium concentration in Limulus ventral photoreceptors using arsenazo III

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Abstract
The metallochromic indicator dye arsenazo III, was injected intracellularly into L. polyphemus ventral photoreceptor cells to concentrations > 1 mM. The absorption spectrum (450-750 nm) of the dye in single dark-adapted cells was measured by a scanning microspectrophotometer. When a cell was light-adapted, dye absorption changed; the difference spectrum had 2 maxima at .apprx. 610 and 660 nm, a broad minimum at .apprx. 540 nm and an isosbestic point at .apprx. 585 nm. When intracellular Ca concentration was raised in dark-adapted cells previously injected arsenazo III, the difference spectrum had 2 maxima at .apprx. 610 and 660 nm, a broad minimum at .apprx. 530 nm and an isosbestic point at .apprx. 585 nm. The injection of Mg2+ into dark-adapted cells previously injected with dye induced a difference spectrum that had a single maximum at .apprx. 620 nm. Decreasing the intracellular pH of previously injected cells induced a difference spectrum that had a minimum at .apprx. 620 nm. There seems to be a rise of intracellular Ca2+ when a Limulus ventral photoreceptor is light-adapted. The intracellular Ca2+ concentration, [Ca2+]i, in light-adapted photoreceptors reached at least 10-4 M on comparison of light-induced difference spectra measured in ventral photoreceptors with a standard curve determined in microcuvettes containing 2 mM arsenazo III in 400 mM-KCl, 1 mM-MgCl2 and 25 mM MOPS [3-(N-morpholino)propane sulfonic acid] at pH 7.0. In cells injected with < 3 mM arsenazo III, light induced a transient decrease in optical transmission at 660 nm (T660), indicating that illumination of a ventral photoreceptor normally causes a transient increase of [Ca2+]i. Arsenazo III was sensitive, selective and rapid enough to measure light-induced changes of intracellular Ca2+ in Limulus ventral photoreceptor cells.