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

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.