Molecular study of polyamines and the NMDA receptor complex

Native NMDA receptor activity is modulated by numerous endogenous ligands. Studies have shown that polyamines exhibit multiple complex effects, both positive and negative, on the activation of the NMDA receptor. These observations raised the possibility that polyamines are endogenous modulators of the NMDA receptor and may be a source of novel therapeutic agents. We used rat brain membranes, cultured neurons, and a stably transfected cell line to investigate the structure-function relationships and the binding sites of polyamines on the NMDA receptor complex.; The polyamine antagonist arcaine was used as a basis for synthesizing more potent polyamine antagonists. A more potent arcaine-like compound would allow us to use lower concentrations of drug to antagonize polyamine effects without producing channel blockade. The compounds were screened to determine the inhibitory effects on {dollar}rmlbracksp3Hrbrack MK{dollar}-801 binding. To ascertain if the novel compounds selectively bound to the arcaine-sensitive site, binding assays were generated in the presence of the polyamine agonist spermidine.; We characterized the most potent polyamine antagonist synthesized by this approach, N,N{dollar}spprime{dollar}-Bis(propyl)guanidinium (BPG). We determined the polyamine site at which BPG exhibits it effects by competition analysis. The data showed that BPG was competitive with the polyamine agonist spermidine but not the "specific" polyamine agonist DEAP. We next investigated the effects BPG had on NMDA receptor function. We measured intracellular free {dollar}rm Casp{lcub}2+{rcub}(lbrack Casp{lcub}2+{rcub}rbracksb{lcub}i{rcub}){dollar} using the fluorescent Ca{dollar}sp{lcub}2+{rcub}{dollar}-sensitive dye fura-2. An IC{dollar}sb{lcub}50{rcub}{dollar} value of 246.3 {dollar}mu{dollar}M was determined for BPG against NMDA-stimulated changes in {dollar}rm Casp{lcub}2+{rcub}rbracksb{lcub}i{rcub}.{dollar} We assessed the role of BPG on ketamine and PCP affinity and NMDA receptor blockade. BPG significantly reduced the rate of inhibition of {dollar}rmlbrack Casp{lcub}2+{rcub}rbracksb{lcub}rm i{rcub}{dollar} produced by ketamine, but did not alter the effect of PCP.; We used the novel glycine site antagonist radioligand {dollar}rmlbracksp3Hrbrack MDL{dollar} 105,519 to directly assay the influence of polyamines on the affinity of agonists and antagonists on the glycine sites. Neither polyamine agonists nor antagonists had a significant effect on displacement of {dollar}rmlbracksp3Hrbrack MDL{dollar} 105,519 binding by glycine or glycine site antagonists. These data suggested that {dollar}rmlbracksp3Hrbrack MDL{dollar} 105,519 preferentially binds to a polyamine insensitive form of the NMDA receptor.; Finally, we examined specific recombinant NMDA receptors involved in the effects of polyamine agonists and antagonists, using {dollar}rmlbracksp{lcub}125{rcub}Irbrack MK{dollar}-801 binding. Our findings suggested that the stimulatory effects of polyamines on recombinant receptors are influenced by the NR2 subunit, and that NR1a:NR2A does not contain a positive modulatory site. However, the inhibitory effects of polyamine antagonists were similas in both subunit combinations (NR1a:NR2A and NR1a:NR2B). Furthermore, native NMDA receptor pharmacology cannot be modeled by simple NR1a:NR2A or NR1a:NR2B combinations.