Measurements of intracellular ionized calcium in squid giant axons using calcium-selective electrodes

Ca²⁺-selective electrodes have been used to measure free intracellular Ca²⁺ concentrations in squid giant axons. Electrodes made of glass cannulas of about 20 μm in diameter, plugged with a poly(vinyl chloride) gelled sensor were used to impale the axons axially. They showed a Nernstian response to Ca²⁺ down to about 3 μM in solutions containing 0.3 M K⁺ and 0.025 M Na⁺. Sub-Nernstian but useful responses were obtained up to pCa 8. The electrodes showed adequate selectivity to Ca²⁺ over Mg²⁺, H⁺, K⁺ and Na⁺. To calibrate them properly, a set of standard solutions were prepared using different Ca²⁺ buffers (EGTA, HEEDTA, nitrilotriacetic acid) after carefully characterizing their apparent Ca²⁺ association constants under conditions resembling the axoplasmic environment. In fresh axons incubated in artificial seawater containing 4 mM Ca²⁺, the mean resting intracellular ionized calcium concentration was 0.106 μM ($\text{n = 15}$). The Ca²⁺-electrodes were used to investigate effects of different experimental procedures on the [Ca²⁺]_i. The main conclusions are: (i) intact axons can extrude calcium ions at low [Ca²⁺]_i levels by a process independent of external Na⁺; (ii) poisoned axons can extrude calcium ions at high levels of [Ca²⁺]_i by an external Na⁺-dependent process. The level of free intracellular Ca attained at these latter conditions is about an order to magnitude greater than the resting physiological value.     

Neutral carrier ion-selective microelectrodes for measurement of intracellular free calcium

This paper describes the making and testing of calcium-selective microelectrodes for measurement of intracellular [free Ca²⁺] levels. Pipettes of tip outer diameter down to 0.4 μm were siliconized by a novel and easy method of vapor treatment. The tips were filled with a sensor mixture using the neutral ligand and solvent of Oehme et al. (Oehme, M., Kessler, M. and Simon, W. (1976) Chimia (Aarau) 30, 204–206) but with very hydrophobic cations replacing Na⁺ in the salt component. This change improved electrode stability and greatly reduced hysteresis in the responses to changing [Ca²⁺] levels. Lowering the Ca²⁺ concentration in the internal electrolyte also increased electrode lifetime.Electrodes showed a Nernstian response to [Ca²⁺] down to 1 μM free concentration in 0.1 M KCl, and usually a useful response to below 100 nM Ca²⁺. Selectivity for Ca²⁺ over Mg²⁺ and H⁺ was sufficiently high that typical free intracellular levels of Mg²⁺ and H⁺ caused negligible interference. Selectivity for Ca²⁺ over Na⁺ was adequate for cells with 10^?2 M free Na⁺, but higher levels could raise significantly the detection limit for Ca²⁺. Preliminary measurements of [free Ca²⁺] have been made in frog skeletal muscle, ferret ventricular myocardium, and early embryos of Xenopus laevis. Relative merits of Ca²⁺ microelectrodes and optical indicators are discussed.