Effect of temperature and ionophores on the permeability of synaptosomes.

Synaptosomes swell rapidly in isosmotic solutions of glycerol or urea, but the swelling in solutions of larger non-electrolytes. such as erythritol, glucose or sucrose is slower. The permeability of synaptosomes to non-electrolytes is temperature dependent, and the low activation energies for the permeation of urea (13 kcdl/mol) and erythritol (9.5 kcal/mol) indicate that the penetration of nonelectrolytes into the synaptosomes does not imply complete dehydration of the molecules. The relative permeability of synaptosomes to cations, as measured by the rate of swelling in isosmotic solutions of acetate salts is in the order: NH: > Na' > Lit > K' > Ca". The ionophores, X-537A and nigericin. or valinomycin + FCCP, which promote exchange of cations for H + , cause swelling of synaptosomes in solutions of potassium salts of acetate or propionate. but not in KCI, whereas H t release is higher in KCI medium. This suggests that the organic anions cross the membrane after combining with H + to form the respective weak acids. The relative permeability to anions is in the order: acetate 2 propionate > C1> SO$= maleate 2 succinate. The energies of activation for the permeability of synaptosomes to potassium acetate in the presence of X-537A or gramicidin D are 13 kcal/mol and 7.5 kcal/mol, respectively, which reflects different mechanisms of action for the two ionophores in the membranes. SYNAPTOSOMES have been used for a wide range of neurochemical studies, especially in experimental approaches designed to study transport and release of neurotransmitters at nerve endings (RAITERI & LEVI, 1978: KUHAR, 1973; COTMAN et ul., 1976; OLSEN et ul., 1977; CAHILL & MEDZIHRADSKY, 1976; HOLZ, 1975 ; BRADFORD, 1970, 1975). More recently, the role of ions in these phenomena has been recognized, and synaptosomes have again been the preferred biological system to study the mechanism of action of Ca2 ', Na+, Li+ and many other cations (DE BELLEROCHE & BRADFORD, 1972; OSBORNE et al., 1973; OSBORNE & BRADFORD, 1975; BLAUSTEIN, 1974. 1975; BLAUSTEIN & OBORN, 1975; BLAUSTEIN & ECTOR, 1976; STEFANINI et al., 1976). Furthermore, it now appears that synaptosomes can also be used to study the intracellular cationic regulation at nerve terminals. Thus, recent studies have been reported on the role of Na' and other cations on the regulation of intracellular Ca2' in nerve using synaptosomes as the experimental biological system (BLAUSTEIN & ECTOR, 1976; STEFANINI et al., 1976; SILBERGELD, 1977; ICHIDA et a!., 1976). and Ca'+-ATPase and Ca2' transport systems found in .synaptic membranes have also been implicated in Ca2 ' regulation by nerve cells BLAUSTEIN et al., 1978; GODDARD & ROBINSON, 1976; VICKERS & DOWDALL, 1976). (DUNCAN, 1976; RAHAMIMOFF & A3RAMOVIT2, 1978; Abbreviations used. FCCP, p-trifluorometoxycarbonylcyanide phenylhydrazone; HEPES, N-2-hydroxyethylpiperazone-N'-2-ethanesulfonic acid. In spite of this wide utilization of synaptosomes to study nerve membrane phenomena, it is surprising that little information exists about the passive permeability properties of synaptosomal membranes (KEEN & WHITE, 1970, 1971; MARCHBANKS, 1967). Thus, the osmotic response of synaptosomes to various solutions, which can be utilized as an index of simple permeability to ions and non-electrolytes, has not been studied to the same extent as for other isolated vesicular fractions of secretory systems, such as chromaffin granules (PERLMAN, 1976; JOHNSON & SCARPA, 1976; PHILLIPS, 1977). The osmotic respome of these vesicular fractions can be followed by a simple light scattering technique which has been successfully applied in various membrane vesicle systems such as liposomes (BLOK et al., 1976; DE GIER rt al., 1968; ANTUNES-MADEIRA & MADEIRA, 1979; McELHANEY et al., 1973; DE GIER et al., 1971), mitochondria (MITCHELL & MOYLE, 1969; BRIERLEY et ul., 1977), chromaffin granules (JOHNSON & SCARPA, 1976; PHILLIPS, 1977; PHILLIPS & ALLISON, 1978; CASEY et al., 1976), sarcoplasmic reticulum (KOMETANI & KASAI, 1978) and, to some extent, synaptosomes We present here the results of experiments in which we studied the effect of temperature on the passive permeability of synaptosome fractions isolated from sheep brain cortex to various electrolytes and nonelectrolytes. Furthermore, by utilizing selective ionophores and measuring the H + fluxes coupled to the fluxes of other cations, we were able to measure the net proton production during ionic fluxes. (KEEN & WHITE, 1970, 1971; MARCHBANKS, 1967).