MAGNESIUM AND ZINC POTENTIATE ETHANOL INHIBITION OF N-METHYL-D-ASPARTATE-STIMULATED NITRIC-OXIDE SYNTHASE IN CORTICAL-NEURONS

The coupling of calcium mobilizing receptors to nitric oxide (NO) form ation was examined in cerebral cortical cultures. Of the various agent s tested, only glutamate, depolarization with KCI and the calcium iono phore ionomycin stimulated nitric oxide synthase (NOS) activity. Chara cterization of the glutamate response revealed that the ionotropic glu tamate receptor agonists N-methyl-D-aspartate (NMDA), kainate and alph a-amino-3-hydroxy-5-methyl-4-isoxalone propionic all stimulated NOS ac tivity with a relative maximal efficacy of NMDA > kainate > alpha-amin o-3-hydroxy-5-methyl-4-isoxalone propionic. Ethanol, Mg++ and Zn++ pro duced a concentration-dependent inhibition of NMDA stimulation of NOS. The Mg++ inhibition was reversed by increasing concentrations of NMDA , whereas Zn++ inhibition was not. Ethanol (100 mM) produced an appare nt competitive type inhibition as seen by a parallel right-shift in th e NMDA concentration-response curve. However, ethanol inhibition was d ependent upon the presence of Mg++ and/or Zn++ in a concentration-rela ted manner. Whereas 100 mM ethanol did not significantly inhibit NMDA stimulation of NOS activity in the absence of Mg++ and Zn++, inclusion of a combination of these cations increased the sensitivity to ethano l such that the NMDA response was completely blocked by 100 mM ethanol IC50 similar to 30 mM). The potency for inhibition of NMDA stimulatio n of NOS by several short-chain alcohols followed their hydrophobicity profile and showed a similar dependency upon Mg++ for inhibition, alp ha-amino-3-hydroxy-5-methy-4-isoxalone propionic, but not kainate, sti mulation of NOS was also inhibited by ethanol (100 mM). These results indicate that activation of NOS in cultured cerebral cortical neurons occurs primarily through ionotropic glutamate receptors that show a di fferential sensitivity to ethanol inhibition. Ethanol appears to act i n an interactive manner with Mg++ and Zn++ ions to inhibit NMDA recept ors that couple to stimulation of NOS by potentiating the blocking act ions of these cations.