3H]ifenprodil binding to NMDA receptors in porcine hippocampal brain membranes

Abstract

(±)-2-(4-Benzylpiperidino)-1-(4-hydroxyphenyl)propan-1-ol ([ 3H]ifenprodil) binding to a subcellular fraction of porcine hippocampus, which was obtained by centrifugation on a discontinuous sucrose gradient, was investigated with the objective to label selectively the ifenprodil recognition site of native NMDA receptors. Saturation experiments revealed high-affinity sites for [ 3H]ifenprodil in this membrane fraction which could be characterised by a K d value of 23.0±1.8 nM using a one-site model. Calculation of saturation isotherms on the basis of a two-site model yielded a K d1 value of 10.4±2.4 nM and a K d2 value of 2200±1300 nM, respectively. Inhibition of [ 3H]ifenprodil binding by NR2B subunit-selective NMDA receptor antagonists, by polyamines, by σ receptor ligands, by a variety of ligands acting at different NMDA receptor recognition sites and by several cations was studied and compared with the effects of these compounds on (5 R,10 S)-(+)-5-methyl-10,11-dihydro-5 H-dibenzo[ a,d]cyclohepten-5,10-imine ([ 3H]MK-801) binding under non-equilibrium conditions. It turned out that σ receptor ligands such as 1,3-di(2-tolyl)-guanidine (DTG), (+)-3-(3-hydroxyphenyl)- N-propylpiperidine ( R)-3-PPP, ( S)-3-PPP and (1-{2-[bis(4-fluorophenyl)methoxy]ethyl})(-4-[3-phenylpropyl]piperazine) (GBR-12909) did not affect [ 3H]ifenprodil binding in the nanomolar range or only slightly. In contrast, ifenprodil, eliprodil, nylidrin and haloperidol inhibited [ 3H]ifenprodil binding in the nanomolar range and in the same rank order and with the same potency as observed for the inhibition of the high-affinity fraction of [ 3H]MK-801 binding. The polyamines, which activate NMDA receptors, inhibited [ 3H]ifenprodil binding in a biphasic manner. Their potency to inhibit the high-affinity fraction of [ 3H]ifenprodil binding was found to be in the same range as their potency to enhance [ 3H]MK-801 binding. In the presence of 10 μM spermine a significantly enhanced ( P=0.0097) rate of dissociation of [ 3H]ifenprodil binding was found, suggesting that inhibition of [ 3H]ifenprodil binding by spermine is not, or at least not exclusively mediated by a competitive interaction.