Mechanisms of glutamate-stimulated Mg2+ influx and subsequent Mg2+ efflux in rat forebrain neurones in culture.
1. Mag-fura-2 fluorescence microscopy and whole-cell patch-clamp recordings were used to measure glutamate-induced changes in the intracellular free Mg2+ concentration ([Mg2+]i) and Mg2+ currents, respectively, in cultured forebrain neurones from fetal rats in the absence of extracellular Na+ (Nao+) and Ca2+ (Cao2+). 2. Increasing the extracellular Mg2+ concentration ([Mg2+]o) from 9 to 70 mM significantly enhanced the maximum [Mg2+]i induced by a 5 min 100 microM glutamate plus 1 microM glycine stimulation ([Mg2+]i,5 min) from 2.04 +/- 0.07 to 2.98 +/- 0.20 mM. Increasing [Mg2+]o from 9 to 70 mM also significantly enhanced the initial rate of rise in [Mg2+]i upon glutamate stimulation from 0.41 +/- 0.02 to 0.81 +/- 0.08 mM min-1. 3. The glutamate-stimulated increase in [Mg2+]i was not altered by prior depletion of intracellular free Na+ (Nai+). For paired stimulations in single neurones, the mean [Mg2+]i,5 min was 1.95 +/- 0.17 mM under Na(+)-depleted conditions and 1.94 +/- 0.16 mM under control conditions. 4. The glutamate-stimulated increase in [Mg2+]i was significantly reduced when NMDA channel-permeant Cs+ or K+ ions were used as the Na+ substitute instead of the presumably NMDA channel-impermeant ions N-methyl-D-glucamine (NMDG), Tris or sucrose. The mean [Mg2+]i,5 min was 0.56 +/- 0.06 and 0.74 +/- 0.08 mM in the presence of Cs+ or K+, respectively, compared with 2.13 +/- 0.10, 1.93 +/- 0.11 and 2.07 +/- 0.22 mM in the presence of NMDG, Tris or sucrose, respectively. 5. In whole-cell recordings performed with Cs+ as the primary intracellular cation, application of 100 microM NMDA plus 10 microM glycine induced inward currents that reversed around -55 mV in an extracellular solution containing 70 mM Mg2+ and 31 mM NMDG as the only cations. The currents were reversibly inhibited by DL-2-amino-5-phosphonovaleric acid (APV). In an extracellular solution containing 2 mM Mg2+ and 140 mM NMDG, NMDA plus glycine activated outward currents at potentials more depolarized than -90 mV. 6. In whole-cell recordings made with NMDG as the principal cation in the patch pipette, application of NMDA plus glycine in the 70 mM Mg2+ extracellular solution induced inward currents at voltages more negative than +15 mV. The ratio of the current measured under these conditions to the current measured in an extracellular solution containing Na+ as the principal cation (0.073:1) was nearly constant from cell to cell. 7. Following a 5 min glutamate stimulation in the presence of 9 mM Mg2+, [Mg2+]i returned to basal levels at a mean rate of 58.1 +/- 2.1 microM min-1. Complete removal of Nao+ significantly inhibited the rate of recovery to 31% of control. Raising [Mg2+]o to 30 mM in combination with removal of Nao+ did not inhibit recovery significantly more than either manipulation alone (28% of control). 8. These results suggest that glutamate-stimulated increases in [Mg2+]i that occur in the absence of Nao+ and Cao2+ result from Mg2+ entry through NMDA-activated ion channels. Furthermore, recovery from a glutamate-induced Mg2+ load appears to be primarily due to Mg2+ efflux via a mechanism whose characteristics are consistent with Na(+)-Mg2+ exchange.
Stout
A K
AK
Department of Pharmacology, University of Pittsburgh, PA 15261, USA.
Li-Smerin
Y
Y
Johnson
J W
JW
Reynolds
I J
IJ
D000818:Animals; D017920:Antiporters / Q000378:metabolism; D001572:Benzofurans / Q000378:metabolism; D001921:Brain / Q000187:drug effects* / Q000378:metabolism; D002478:Cells, Cultured; D004594:Electrophysiology; D004988:Ethers, Cyclic / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D016257:Fura-2 / Q000031:analogs & derivatives / Q000378:metabolism; D018698:Glutamic Acid / Q000494:pharmacology*; D006096:Gramicidin / Q000494:pharmacology; D008274:Magnesium / Q000378:metabolism*; D008564:Membrane Potentials; D016202:N-Methylaspartate / Q000494:pharmacology; D009474:Neurons / Q000187:drug effects*; D011188:Potassium / Q000378:metabolism / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D016194:Receptors, N-Methyl-D-Aspartate / Q000378:metabolism; D012964:Sodium / Q000378:metabolism
D017920:Antiporters; D001572:Benzofurans; D004988:Ethers, Cyclic; D005456:Fluorescent Dyes; D016194:Receptors, N-Methyl-D-Aspartate; C046250:sodium-magnesium antiporter; C058776:2-(2-(5-carboxy)oxazole)-5-hydroxy-6-aminobenzofuran-N,N,O-triacetic acid; C061665:sodium-binding benzofuran isophthalate; D006096:Gramicidin; D018698:Glutamic Acid; D016202:N-Methylaspartate; D012964:Sodium; D008274:Magnesium; D011188:Potassium; D016257:Fura-2