Changes in the intracellular concentration of free calcium ions in a pace-maker neurone, measured with the metallochromic indicator dye arsenazo III

Abstract

1. The bursting pace-maker R-15 cell of Aplysia was injected with the Ca2+ sensitive dye arsenazo III. Changes in absorbance were measured with a differential spectrophotometer to monitor changes in free intracellular Ca2+, [Ca-a], during activity. 2. Dye absorbance increased during each pace-maker-induced burst of action potentials and decreased during the hyperpolarizing phase of the pace-maker cycle. 3. The increase in dye absorbance was, at least in part, dependent upon action potential discharge and was greater when action potential duration was prolonged by treatment with tetraethylammonium chloride. 4. Changes in dye absorbance occurred under voltage clamp conditions when the membrance was depolarized 5-15 mV from a holding potential near the resting potential and were larger with greater step depolarizations. 5. These changes were completely blocked by the addition of 3mM-La3+ to normal ASW. 6. The ratio of the absorbance change between two pairs of wave-lengths during the pace-maker cycle was compared with the ratio observed following injection of Ca2+, Mg2+ and H+ ions. The ratio for the pace-maker cycle was well matched by that for Ca2+ injection, but not by that for injection of Mg2+ or H+. 7. Intracellular Ca2+ injections which increased [Ca]1 to the same amount as occurred during the pace-maker cycle also produced an outward current of sufficient magnitude to account for the post-burst hyperpolarization. 8. Depolarization of the cell membrane by extrinsic current during the burst increased and prolonged the change in dye absorbance as well as the post-burst hyperpolarization. 9 It is suggested that Ca2+ enters during the pace-maker cycle, thereby increasing [Ca]i, and that this increase is sufficient to activate an outward current carried by K+ ions which causes or contributes to the post-burst hyperpolarization.