Vol. 296, Issue 1, 150-159, January 2001

Use of Trifluoroperazine Isolates a [³H]Ifenprodil Binding Site in Rat Brain Membranes with the Pharmacology of the Voltage-Independent Ifenprodil Site on N-Methyl-D-aspartate Receptors Containing NR2B Subunits

Linda L. Coughenour and Bridget M. Barr

Department of Neuroscience Therapeutics, Pfizer Global Research and Development, Ann Arbor Laboratories, Ann Arbor, Michigan

The use of trifluoroperazine in a well washed rat brain membrane preparation revealed [³H]ifenprodil binding to a single high affinity state with the pharmacology of N-methyl-D-aspartate (NMDA) receptors containing NR2B subunits. Inhibition of [³H]ifenprodil binding in the presence of trifluoroperazine by 10 NR1a/NR2B selective agents was highly correlated with their inhibition at rat NR1a/NR2B receptors expressed in Xenopus ooctyes and [³H]TCP binding to rat brain NR2B subunit containing NMDA receptors but not with their inhibition of [³H]DTG binding. Allosteric interactions with polyamines, Mg²⁺, Zn²⁺, glutamate, glycine, and their antagonists were consistent with NMDA receptors with NR2B subtype pharmacology. The rank order of polyamine inhibition was spermine > spermidine > 1,5-(diethylamino)piperidine > arcaine > agmatine > putrescine. Both spermidine and MgCl₂ shifted the inhibition curve of ifenprodil to the right in a parallel manner, but Mg²⁺ did not appear to be additive to spermidine. Glutamate increased and glycine decreased the binding. Conversely, CPP decreased the binding, and MDL 105,519 increased the binding in an agonist reversible manner. The increase with MDL 105,519 and glutamate appeared to be additive as did the decrease with glycine and CPP. Changes in the buffer pH between 6.5 and 8.0 did not affect the affinity of NR2B agents. Cirazoline but not clonidine inhibited the binding. MK-801 and agents from various other pharmacological classes did not significantly inhibit [³H]ifenprodil binding. [³H]Ifenprodil binding in the presence of trifluoroperazine appears to be selective for the voltage-independent ifenprodil site on NMDA receptors containing the NR2B subunit.