Carrier-mediated sodium-dependent and calcium-dependent calcium efflux from pinched-off presynaptic nerve terminals (synaptosomes) in vitro

Abstract

The influence of external cations on 45Ca2+ efflux from Ca2+ loaded synaptosomes has been examined. The synaptosomes were pre-loaded with 45Ca2+ by incubating the suspensions in potassium-rich media for 2 min. The suspensions were then diluted into "efflux" media containing a "normal" (5mM) K+ concentration; the content of Na+ and Ca2+ was varied, as noted below. Efflux of 45Ca2+ was measured for a 2-min period (except for "zero-time" samples), and was terminated by filtering the suspensions on 0.3 mum cellulose acetate filters. 45Ca2+ retained on the filters was determined by liquid scintillation spectroscopy. The difference between the 45Ca2+ in the "zero-time" samples (="Ca2+ load") and in the samples incubated for 2 min was taken as the 45Ca2+ efflux. 45Ca2+ loss into Ca2+ -free efflux media containing ethyleneglycol-bis-(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA) was markedly influenced by the Na+ concentration: nearly 80% of the 45Ca2+ was lost from the synaptosomes if the media contained 132 mM Na+, but only about 7% was lost in 2 min if 97% of the Na+ was replaced mol-for-mol by choline. In media containing 1.2 mM Ca2+ and 132 mM Na+, the 45Ca2+ uptake by synaptosomes previously loaded with 40Ca2+ was significantly less than 45Ca2+ loss from synaptosomes loaded with 45Ca2+. Thus there was a net efflux of Ca2+ from the Ca2+ -loaded synaptosomes; this efflux was, presumably, Na+ dependent. In media containing 1.2 mM 40Ca2+ and only 4 mM Na+, the 45Ca2+ efflux from 45Ca2+ -loaded synaptosomes was significantly greater if most of the external Na+ (128 mM) was replaced isomotically by Li+ rather than by choline, guanidine or glucose. This observation may be evidence for a Ca2+ -Ca2+ exchange which is promoted by Li+. Both the Na+ -dependent and the Ca2+ -dependent Ca2+ effluxes were inhibited by Mn2+. The data are consistent with a Ca2+ carrier mechanism which can extrude Ca2+ in exchange for Na+ or for Ca2+, the latter being activated by Li+. These properties bear a striking resemblance to those of a Ca2+ efflux mechanism which has been characterized in squid axons. This mechanism may there fore have evolved fairly early on in the history of the animal kingdom.