Pharmacological characterization of rat brain excitatory amino acid receptors expressed in Xenopus oocytes

The Xenopus oocyte mRNA translation system was used to study the pharmacology of rat brain excitatory amino acid (EAA) receptors. mRNA was isolated from rat brain or primary cultures of rat brain tissue and injected into Xenopus oocytes. The presence of EAA receptors was assayed electrophysiologically by measuring agonist-induced currents under voltage clamp. The application of EAA agonists to mRNA-injected cells, but not to control oocytes, produced several different inward currents, two of which are characteristic of neuronal EAA receptors. Currents with properties expected from activation of N-methyl-D-aspartate (NMDA) receptors were evoked by L-glutamate (EC{dollar}sb{lcub}50{rcub}{dollar} = 2.3 {dollar}mu{dollar}M), D-aspartate (10 {dollar}mu{dollar}M), L-aspartate (13 {dollar}mu{dollar}M), NMDA (31 {dollar}mu{dollar}M) and ibotenate (35 {dollar}mu{dollar}M). Inward currents activated by these agonists were blocked by Mg{dollar}sp{lcub}++{rcub}{dollar} in a voltage-dependent manner, and antagonized by D-2-amino-5-phosphonovaleric acid (D-APV). The antagonism of NMDA and L-aspartate responses by D-APV was competitive as judged by linear Schild regressions with slopes of unity. The D-APV pA{dollar}sb2{dollar}s for both agonists (5.87 against NMDA and 5.86 against L-aspartate) were identical. The non-NMDA receptor antagonist, 6-nitro-7-cyano-quinoxaline-2,3-dione (CNQX), significantly reduced the maximum NMDA response indicating its mode of action on NMDA receptors was not competitive. A second type of inward current was produced by kainate, domoate, (RS)-{dollar}alpha{dollar}-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and L-glutamate. This smooth current was insensitive to Mg{dollar}sp{lcub}++{rcub}{dollar}. L-glutamate and domoate were equipotent for activating this current (EC{dollar}sb{lcub}50{rcub}{dollar} = 14{dollar}mu{dollar}M) whereas kainate was less potent (98 {dollar}mu{dollar}M). D-APV weakly blocked the current evoked by kainate (pA{dollar}sb2{dollar} = 3.39), but the slope of the Schild regression was significantly less than one (0.903) suggesting the antagonism was not entirely competitive. CNQX was found to be a potent and competitive antagonist of kainate-evoked currents with a pA{dollar}sb2{dollar} of 6.53. A third type of inward current was produced by quisqualate. This current, consisting of oscillating and smooth components, was carried by Cl{dollar}sp-{dollar} and not evoked by AMPA indicating that activation of the conventional quisqualate receptor/ion channel complex was unlikely. These data illustrate a clear pharmacological distinction between receptors mediating the NMDA and non-NMDA smooth currents in mRNA-injected Xenopus oocytes, and provide important quantitative information concerning the effects of agonists and antagonists on this family of neurotransmitter receptors.