Calcium permeability changes and neurotransmitter release in cultured rat brain neurons. I. Effects of stimulation on calcium fluxes

The permeability of neuronal membranes to Ca²⁺ is of great importance for neurotransmitter release. The temporal characteristics of Ca²⁺ fluxes in intact brain neurons have not been completely defined. In the present study ⁴⁵Ca²⁺ was used to examine the kinetics of Ca²⁺ influx and efflux from unstimulated and depolarized rat brain neurons in culture. Under steady-state conditions three cellular exchangeable Ca²⁺ pools were identified in unstimulated cells: 1) a rapidly exchanging pool (t( 1/2 ) = 7 s) which represented about 10% of the total cellular Ca²⁺ and was unaffected by the presence of Co²⁺, verapamil, or tetrodotoxin; 2) a slowly exchanging pool (t( 1/2 ) = 360 s) which represented 42% of the total cellular Ca²⁺ and was inhibited by Co²⁺, but not by verapamil or tetrodotoxin; 3) a very slowly exchanging pool (t( 1/2 ) = 96 min) which represented 48% of the total cell Ca²⁺ was observed only in the prolonged efflux experiments. The rate of exchange of ⁴⁵Ca²⁺ in the unstimulated cells was dependent on the extracellular Ca²⁺ concentration (half-saturation) at 70 μM). Depolarization of the neurons with elevated K⁺ causes a rapid and sustained ⁴⁵Ca²⁺ uptake. The cellular Ca²⁺ content increased from 56 nmol/mg protein in unstimulated cells to 81 nmol/mg protein during 5 min of depolarization. The kinetics of the net ⁴⁵Ca²⁺ by the stimulated neurons was consistent with movement of the ion with a first order rate constant of 0.0096 s^-1 (t( 1/2 ) = 72 s) into a single additional compartment. The other cellular Ca²⁺ pools were apparently unaffected by stimulation. The stimulated ⁴⁵Ca²⁺ uptake was inhibited by Co²⁺ and by the Ca²⁺ channel blocker verapamil but not by the Na⁺ channel blocker tetrodotoxin. Ca²⁺ uptake into this compartment was dependent on the extracellular Ca²⁺ concentration (half-saturation at 0.80 mM Ca²⁺). Predepolarization of the cells with high K⁺ for 10-60 s prior to the addition of the radioactive calcium did not alter the rate of ⁴⁵Ca²⁺ incorporation into the stimulated cells. It is concluded that the rapidly exchanging, the slowly exchanging, and the depolarization-induced Ca²⁺ pools observed in intact brain neurons are physically as well as kinetically distinct from each other. In addition, the depolarization-induced component observed in stimulated cells represents movement of the Ca²⁺ ions through a single class of voltage-sensitive Ca²⁺ channels. These Ca²⁺ channels are inhibited by Co²⁺ ions and by verapamil and are not inactivated during depolarization of the brain neurons.