Abstract

The assembly of heterogeneous populations of nativeN-methyl-d-aspartate receptors results in receptors with multiple pharmacological properties dependent on subunit combinations. Using stably transfected ML(tk−) mouse fibroblasts expressing N-methyl-d-aspartate R1a and either R2A or R2B, we evaluated polyamine effects on [¹²⁵I]dizocilpine (MK-801) binding to determine subunit-specific pharmacological characteristics. The polyamine agonists spermine and spermidine produced biphasic concentration response curves in rat brain membrane: low concentrations (<100 μM) enhanced [¹²⁵I]MK-801 binding and higher concentrations (>100 μM) inhibited binding. Polyamine agonists did not affect [¹²⁵I]MK-801 binding in NR1a/NR2A, whereas spermine and spermidine did produce enhancement, and, at higher concentrations, inhibition of binding in NR1a/NR2B. The polyamine 1,5-(diethylamino)piperidine is thought to be selective for the agonist polyamine site and only enhanced [¹²⁵I]MK-801 binding in brain membranes (EC₅₀ = 9.6 μM). However, 1,5-(diethylamino)piperidine inhibited [¹²⁵I]MK-801 binding (IC₅₀ = 8.0 μM) in NR1:NR2A receptors and produced a small increase followed by a modest decrease in binding to NR1a/NR2B receptors. In brain membranes, the polyamine antagonist arcaine inhibited [¹²⁵I]MK-801 binding (IC₅₀= 4.6 μM). Similar effects were demonstrated in both NR1:NR2A and NR1:NR2B receptors (IC₅₀ = 8.4 and 14.1 μM, respectively) and agonists decreased the affinity of arcaine in both receptor preparations. These results suggest that the stimulatory effects of polyamines on recombinant receptors are influenced by the NR2 subunit, and that NR1:NR2A does not contain a positive modulatory site. However, the inhibitory effects of polyamine antagonists are similar in both subunit combinations. Furthermore, native NMDA receptors pharmacology cannot be modeled by simple NR1:NR2A or NR1:NR2B combinations.