J. Membrane Biol. 91, 157-164 (1986) The Journal of MembraneBiology 9 Sprioger-Vetlag New York Inc. 1986 Continuous Measurement of Calcium Influx in Mammalian Nonmyelinated Nerve Fibers: Effects of Nao, Cao, and Electrical Activity P. Jirounek, W.F. Pralong, J. Vitus, and R.W. Straub D6partement de Pharmacologie, Centre M6dical Universitaire, CH 1211 Gen6ve 4, Switzerland Summary. A new technique for continuous monitoring of the cellular calcium was developed and used for studying the effects of external and internal Na (Nao and Nai), external Ca (Cao), Ca ionophore A23187, and electrical activity on membrane-bound and intracellular Ca in mammalian nonmyelinated nerve fibers. Increasing Cao increased both the membrane-bound and the in- tracellular Ca. Lowering Nao increased the membrane-bound fraction of Ca indicating that lack of Na, enhanced the capacity of the plasma membrane to bind Ca, and produced an increase of the internal Ca pool. Increasing Nai by treatment with ouabain enhanced the Ca inflow in both, the presence and absence of Nao, presumably by stimulating the Cao/Nai exchange. The Ca ionophore A23187 produced a large and irreversible increase in the intracellular Ca without affecting the membrane-bound frac- tion. On the other hand, electrical activity, which is known to produce a large increase of the total Ca in squid axon, had no measurable effect on the total calcium content in our prepara- tion. It is concluded that in mammalian nerve fibers a Ca load by exposition to Na-free solution or to A23187 produces an accumu- lation of Ca into the intracellular Ca stores, whereas during elec- trical activity the membrane-associated extrusion mechanisms are able to maintain the intracellular Ca 2+ below the threshold for intracellular sequestration. Furthermore, the results indicate that the intracellular sequestration mechanisms are dependent on the internal concentration of Na. Key Words mammalian nerve 9 intracellular calcium 9 mem- brane-bound calcium . calcium buffering 9 Na/Ca exchange 9 electrical activity 9 A23187 Introduction The concentration of free calcium in the cytoplasm of virtually all living cells is maintained within the range of 10 .8 to l0 -7 M, compared to about 10 -3 M in the extracellular fluid. It is thus not surprising that cells are endowed with systems, both intracellular and membrane-associated, that maintain and regu- late the intracellular calcium ions at this extremely low concentration. Most of our knowledge on cal- cium regulation in excitable cells originates from studies in intact, injected, internally perfused or dia- lyzed giant axons or giant muscle fibers, and little is known about calcium homeostasis in mammalian nerve fibers. We have therefore directed our attention to cal- cium fluxes across the cell plasma membrane in rab- bit nerve fibers, in order to examine whether the mechanisms which have been described in giant ax- ons are also found in mammalian nonmyelinated nerves. The present study is based on results obtained by a method which allows continuous monitoring of the radioactivity of a preparation during superfu- sion with solutions containing 45Ca (Jirounek et al., 1983). The results are compared to measurements of efflux of 45Ca in the same tissue (Jirounek, Vitus, Pralong & Straub, in preparation) and of intracellu- lar free calcium with the quin2 method (Pral0ng & Straub, 1985). Materials and Methods PREPARATION OF NERVES; APPARATUS FOR RECORDING THE RADIOACTIVITY Rabbits weighing between 2 and 3 kg were shot and their cervical vagus nerves rapidly removed and desheathed with scissors. The preparation was then inserted into a 5-cm length of plastic scintil- lator tubing (Nuclear Enterprises NE102A, internal diameter 0.7, external 0.9 ram), connected to a small chamber where the nerve fibers could be stimulated and the action potential recorded (see Fig. 1). The scintillating materials served to transform the weak beta radiation of 45Ca into light flashes which were detected by a pair of photomultipfiers placed on either side of the scintillator tube. The photomultipliers were connected via a coincidence circuit to a rate meter, and further to an integrator and pen recorder. After mounting, the preparation was first superfused during about 30 rain with a nonradioactive solution of the same calcium concentration that was later used in the influx experi- ment. The flow of the solution was maintained at a rate of 1 ml/ rain by a peristaltic pump. After the period of equilibration in the