Calcium buffering in axons and axoplasm of Loligo.

1. Ca-selective micro-electrodes were used to measure free Ca concentration in axons and extruded axoplasm. 2. Free Ca in axons immersed in artificial sea water containing 3 mM-Ca averaged 77 nM in freshly dissected axons and 4.9 microM in cyanide- or azide-poisoned axons. 3. Extruded axoplasm maintained a free Ca only a little higher than that of the axons from which it was obtained. 4. Axoplasmic buffering was investigated by titrating isolated axoplasm with CaCl2 or K-EGTA and monitoring the change in free Ca. Energy-dependent and energy-independent components of Ca binding could be recognized in fresh axoplasm. The energy-dependent fraction could be further subdivided into Ruthenium Red-sensitive and Ruthenium Red-insensitive components and the energy-independent fraction into a component of high affinity and rather low capacity and another component of low affinity and large capacity. 5. The Ruthenium Red-sensitive process could accumulate many millimoles Ca per kilogram axoplasm while still maintaining a free Ca close to 100 nM. After injection of Ruthenium Red into fresh axoplasm, binding is dramatically altered so that it closely resembles that in a metabolically poisoned preparation. 6. The Ruthenium Red-insensitive process has a small capacity and appears to be capable of lowering free Ca to about 200 nM. It can, however, lower free Ca to 50-150 nM if oxalate is also present. 7. Simultaneous measurement of pH and free Ca showed that axoplasmic pH only begins to fall appreciably in response to added Ca when mitochondrial Ca buffering becomes impaired. 8. Raising axoplasmic levels of Na or Li, but not K, tends to bring about a concomitant rise in free Ca.