Mitochondria and other calcium buffers of squid axon studied in situ.

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Abstract
Continuous nondestructive monitoring of intracellular ionized Ca2+ in isolated squid [Loligo pealei] axons by differential absorption spectroscopy (using arsenazo III and antipyrylazo III) was used to study Ca uptake by carbonyl cyanide, p-trifluoromethoxy-phenylhydrazone (FCCP)- and (or) cyanide (CN)-sensitive and insensitive constituents of axoplasm. Known Ca loads imposed on the axon by stimulation produced proportional increments of free axoplasmic Ca. Measurement of increments in Ca2+ as a function of load confirmed earlier reports of buffering in normal and FCCP- and (or) CN-poisoned axons. Measurement of rates of Ca uptake by presumed mitochondria showed little uptake at ambient Ca below 200-400 nM, with sigmoidal rise to about 20-30 .mu.mol/kg axoplasm per min (calculated to be about 200 mmol/kg mitochondrial protein per min) at 50 .mu.M, indicating a functional threshold for presumed mitochondrial uptake well above physiological ionized Ca concentration. Treatment of stimulated axons with cyanide, to release Ca from presumed mitochondria, showed that the sensitivity to cyanide decreased progressively with time after stimulation (t1/2 = 3-10 min) implying transfer of sequestered Ca into a less metabolically labile form.