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 (t1/2 = 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 (t1/2 = 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 (t1/2 = 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 microM). 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+ uptake by the stimulated neurons was consistent with movement of the ion with a first order rate constant of 0.0096 s-1 (t1/2 = 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.
Yarom
M
M
Zurgil
N
N
Zisapel
N
N
D000818:Animals; D001921:Brain / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002132:Calcium Radioisotopes; D002463:Cell Membrane Permeability / Q000187:drug effects; D002478:Cells, Cultured; D003035:Cobalt / Q000494:pharmacology; D004553:Electric Conductivity; D004622:Embryo, Mammalian; D007700:Kinetics; D009474:Neurons / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D018377:Neurotransmitter Agents / Q000378:metabolism; D051381:Rats; D013779:Tetrodotoxin / Q000494:pharmacology; D014700:Verapamil / Q000494:pharmacology
D002132:Calcium Radioisotopes; D018377:Neurotransmitter Agents; D003035:Cobalt; D013779:Tetrodotoxin; D014700:Verapamil; D002118:Calcium