Effects of Na+ and Ca2+ gradients on intracellular free Ca2+ in voltage-clamped Aplysia neurons.

Selected neurons of the abdominal ganglion of Aplysia californica were voltage-clamped and intracellular free Ca [( Ca2+]i) and Na [( Na+]i) concentrations were monitored with ion selective microelectrodes. Reducing [Na+]o from 500 mM (normal seawater, NSW) to 5 mM resulted in a decrease of the potential measured by the Ca electrode (VCa). Increasing [Ca2+]o from 10 to 50 mM increased [Ca2+]i two-fold, keeping [Ca2+]o at 50 mM and decreasing [Na+]o to 5 mM still led to a decrease in VCa. With 100 mM [Ca2+]o, which also increased [Ca2+]i, decreasing [Na+]o increased VCa in two of the eight cells tested. This indicates that in normal or moderately high resting [Ca2+]i, Ca2+ extrusion by Na/Ca exchange (forward mode) is not essential for [Ca2+]i buffering. [Na+]i was 12.9 +/- 3.6 mM (S.E.M., n = 7) in NSW; reducing [Na+]o to 5 mM decreased [Na+]i to 2.0 +/- 1.1 mM (S.E.M.). Keeping [Na+]o at 5 mM and increasing [Ca2+]o from 10 to 20 mM further decreased [Na+]i to about 1.0 mM, evidence of Na/Ca exchange operating in the reverse mode. Attempts to increase [Ca2+]i by bath application of the Ca ionophores A23187, X537A, ionomycin or ETH 1001 resulted in no measurable change of the resting [Ca2+]i. Application of Ouabain caused an apparent increase in [Ca2+]i in two of the six cells tested. In cells injected with the metallochromic indicator arsenazo III (AIII), the rate of the falling phase of the AIII absorbance increase, following a voltage-clamp pulse, was significantly slower in 5 mM [Na+]o. This indicates that in its forward mode Na-Ca exchange is active in clearing large submembrane increases in [Ca2+]i.