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A B S TRA CT Changes in ionized calcium were studied in axons isolated from living squid by measuring absorbance of the Ca binding dye Arsenazo III using multiwavelength differential absorption spectroscopy. Absorption changes measured in situ were calibrated in vitro with media of ionic composition similar to axoplasm containing CaEGTA buffers. Calcium loads of 50-2,500 /zmol/kg axoplasm were induced by microinjection, by stimulation in 112 mM Ca seawater, or by soaking in choline saline with 1-10 mM Ca. Over this range of calcium loading of intact axoplasm, the ionized calcium in the axoplasm rose about 0.6 nM/p.M load. Similar loading in axons pretreated with carbonyl cyanide 4-trifluoromethoxyphenylhydrazone (FCCP) to inhibit the mitochondrial proton gradient increased the ionized calcium by 5-7% of the imposed load, i.e. 93-95% of the calcium load was buffered by a process insensitive to FCCP. This FCCP-insensitive buffer system was not saturated by the largest calcium loads imposed, indicating a capacity of at least several millimolar. Treatment of previously loaded axons with FCCP or apyrase plus cyanide produced rises in ionized calcium which could be correlated with the extent of the load. Analysis of results indicated that, whereas only 6% of the endogenous calcium
The Journal of General Physiology – Rockefeller University Press
Published: Sep 1, 1977
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