GUANINE-NUCLEOTIDES ARE COMPETITIVE INHIBITORS OF N-METHYL-D-ASPARTATE AT ITS RECEPTOR-SITE BOTH INVITRO AND INVIVO

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  • 1 July 1989
    • journal article
    • research article
    • Vol. 250  (1) , 162-169
Abstract
Guanine nucleotides were shown to alter N-methyl-d-aspartate (NMDA) receptor-effector coupling by competitive antagonism at the glutamate binding site, rather than via interaction with an intracellularly located GTP-binding protein. Thus, in contrast to known G-protein linked receptors, micromolar concentrations of guanine nucleotides and their analogs decreased both agonist ([3H]glutamate) and antagonist ([3H]-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid binding to the NMDA receptor complex. The most potent compound, the GDP analog guanosine-5''-O-(2-thiodiphosphate) (GDP.beta.S), was studied in detail. GDP.beta.S exhibited almost 200-fold selectivity for the glutamate recognition site vs. the strychnine-insensitive glycine binding site. IC50 values were 27.7 UU 1.4 and 484 .+-. 97 .mu.M, respectively. GDP.beta.S also inhibited N-[1-(2-thienyl)cyclohexyl-3H]piperidine binding (IC50 was 28.0 .+-. 3.7 .mu.M) in an NMDA-reversible fashion. [3H]-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid saturation binding studies revealed an increase in Kd from 263 .+-. 49 (control to 552 .+-. 134 nM (8 .mu.M GDP.beta.S) without any change in maximum binding (4.94 .+-. 0.34 and 5.19 .+-. 0.58 pmol/mg of protein, respectively) GDP.beta.S was also a competitive inhibitor of the following NMDA-stimulated responses: elevation of cyclic GMP in neonatal rat cerebellar slices, release of preloaded [3H]norepinephrine from superfused rat hippocampal slices and elevation of cytosolic calcium concentration in fura-2-loaded cultured rat forebrain neurons. IC50 values were 78.4, 53.4 and 1.6 .mu.M, respectively. Finally, GDP.beta.S resembed known NMDA receptor antagonists in its ability to block NMDA receptor-induced seizures after i.c.v. administration. IC50 values for 2-amino-5-phosphovaleric acid, 2-amino-7-phosphoheptanoic acid and GDP.beta.S were 4.77, 1.49 and 44.53 nmol, respectively.