Intracellular calcium buffering capacity in isolated squid axons.

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Abstract
Changes in Ca2+ were studied in axons isolated from living squid [Loligo pealei] 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 [calcium ethylene glycol bis(.beta.-aminoethyl ether) tetraacetate] buffers. Ca2+ loads of 50-2500 .mu.mol/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 Ca2+ loading of intact axoplasm, the Ca2+ in the axoplasm rose .apprx. 0.6 nM/.mu.M load. Similar loading in axons pretreated with carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) to inhibit the mitochondrial proton gradient increased the ionized Ca2+ by 5-7% of the imposed load, i.e., 93-95% of the Ca2+ load was buffered a process insensitive to FCCP. This FCCP-insensitive buffer system was not saturated by the largest Ca2+ loads imposed, indicating a capacity of at least several millimolar. Treatment of previously loaded axons with FCCP or apyrase plus cyanide produced rises in Ca2+ which could be correlated with the extent of the load. Whereas only 6% of the endogenous Ca2+ in fresh axons was stored in the FCCP-sensitive (presumably mitochondrial) buffer system, .apprx. 30% of an imposed exogenous load in the range of 50-2500 .mu.M was taken up by this system.