Differentiation of the Ca2+-stimulated binding from the Cl- -dependent binding of [3H]glutamate in synaptic membranes from rat brain.
The effect of Ca2+ as well as Cl- ions on [3H]glutamate (Glu) binding was re-examined using rat brain synaptic membranes frozen at -80 degrees C in 0.32 M sucrose. The inclusion of 20 mM ammonium chloride or 20 mM ammonium chloride plus 2.5 mM calcium acetate disclosed the Cl- -dependent binding or Ca2+-stimulated binding even at 2 min after the initiation of incubation at 30 degrees C and each binding reached a plateau within 30 min. In contrast, the binding reached its maximal value within 10 min followed by a progressive decline up to 60 min in the presence of 100 mM sodium acetate. Scatchard analysis revealed that Cl- as well as Cl-/Ca2+ ions invariably caused a significant increment of the number of binding sites without altering their affinity, whereas Na+ ions induced a prominent increment of the density of binding sites with a concomitant lowering of their affinity. DL-2-Amino-4-phosphonobutyric acid selectively abolished the Cl- -dependent and Ca2+-stimulated bindings without significantly affecting the basal or Na+-dependent binding. Quisqualic acid induced a profound inhibition of both Cl- -dependent and Ca2+-stimulated bindings, to a significantly greater extent than that of the basal and Na+-dependent bindings. D-Aspartic acid exhibited a potent inhibition of the Na+-dependent binding with a significantly less potent displacement of the basal, Cl- -dependent and Ca2+-stimulated bindings. An inhibitor of anion transport, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), not only eliminated the Cl- -dependent binding, but also completely abolished the Ca2+-stimulated binding. Scatchard analysis revealed that DIDS (0.1 mM) prevented the Cl- - and Cl-/Ca2+-induced increment of the density of binding sites with no significant change of their affinity. Pretreatment of the membranes with hydrophilic SH-reactive agents such as N-ethylmaleimide and 5,5'-dithiobis-(2-nitrobenzoic acid) invariably resulted in a more sensitive inhibition of the Ca2+-stimulated binding than that of the Cl- -dependent binding, while hydrophobic reagent p-chloromercuribenzoic acid produced a similarly potent elimination of the Cl- -dependent and Ca2+-stimulated bindings. Calcium-stimulated binding was also found to be sensitively diminished by dithiothreitol and dithioerythritol as compared with the Cl- -dependent binding. In vitro addition of L-ascorbic acid (10(-6)-10(-3) M) attenuated the Ca2+-stimulated binding to a significantly greater extent than the inhibition of the Cl- -dependent binding.(ABSTRACT TRUNCATED AT 400 WORDS)
D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000187:drug effects / Q000378:metabolism*; D002118:Calcium / Q000502:physiology*; D002712:Chlorides / Q000502:physiology*; D005971:Glutamates / Q000378:metabolism*; D018698:Glutamic Acid; D066298:In Vitro Techniques; D007700:Kinetics; D008297:Male; D011869:Radioligand Assay; D051381:Rats; D011919:Rats, Inbred Strains; D017470:Receptors, Glutamate; D017981:Receptors, Neurotransmitter / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000494:pharmacology; D013570:Synaptic Membranes / Q000378:metabolism
D000596:Amino Acids; D002712:Chlorides; D005971:Glutamates; D017470:Receptors, Glutamate; D017981:Receptors, Neurotransmitter; D013438:Sulfhydryl Compounds; D018698:Glutamic Acid; D002118:Calcium