Intrasynaptosomal free Mg2+ concentration measured with the fluorescent indicator mag-fura-2: modulation by Na+ gradient and by extrasynaptosomal ATP.
Synaptosomes can be loaded with mag-fura-2 without significant perturbation of their ATP content by incubation for 10 min at 37 degrees C with 10 microM mag-fura-2 acetoxymethyl ester in Hanks'-HEPES buffer (pH 7.45). The intrasynaptosomal free Mg2+ concentration ([Mg2+]i) was found to be dependent on external Mg2+ concentration, increasing from 0.8 to 1.25 mM when the concentration of Mg2+ in the incubation medium increased from 1 to 8 mM. Dissipation of the Na+ gradient across the plasma membrane of synaptosomes by treatment with the Na+ ionophore monensin (0.2 mM) or with veratridine (0.2 mM) and ouabain (0.6 mM) produced a moderate increase of [Mg2+]i, from 1.0 to 1.2-1.3 mM in an incubation medium containing 5 mM Mg2+. Plasma membrane depolarization by incubation of synaptosomes in a medium containing 68 mM KCl and 68 mM NaCl had no effect on [Mg2+]i. Reversal of the Na+ gradient by incubation of synaptosomes in a medium in which external Na+ was replaced by choline increased [Mg2+]i up to 1.6 and 2.2 mM for extrasynaptosomal Mg2+ concentrations of 1 and 8 mM, respectively. We conclude that a Na+/Mg2+ exchange operates in the plasma membrane of synaptosomes. In the presence of Mg2+ in the incubation medium, extrasynaptosomal ATP, but not ADP or adenosine, increased [Mg2+]i from 1.1 +/- 0.1 up to 1.6 +/- 0.1 mM. The nonhydrolyzable ATP analogue adenosine 5'-(beta gamma-imido)triphosphate antagonized the effect of ATP, but had no effect by itself on [Mg2+]i. It is concluded that Mg2+ transport across the plasma membrane of synaptosomes is modulated by the activity of an ecto-ATPase or an ecto-protein kinase.
García-Martín
E
E
Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias, Universidad de Extremadura, Badajoz, Spain.
Martín-Romero
F J
FJ
Gutiérrez-Merino
C
C
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism; D005110:Extracellular Space / Q000378:metabolism; D005456:Fluorescent Dyes; D016257:Fura-2 / Q000031:analogs & derivatives* / Q000378:metabolism; D006868:Hydrolysis; D008274:Magnesium / Q000378:metabolism*; D009994:Osmolar Concentration; D051381:Rats; D017208:Rats, Wistar; D012964:Sodium / Q000378:metabolism*; D013574:Synaptosomes / Q000378:metabolism*
D005456:Fluorescent Dyes; C058776:2-(2-(5-carboxy)oxazole)-5-hydroxy-6-aminobenzofuran-N,N,O-triacetic acid; D000255:Adenosine Triphosphate; D012964:Sodium; D008274:Magnesium; D002118:Calcium; D016257:Fura-2