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

(+/-)-2-(4-Benzylpiperidino)-1 -(4-hydroxyphenyl)propan-1-ol ([H-3]ifenprod il) binding to a subcellular fraction of porcine hippocampus, which was obt ained by centrifugation on a discontinuous sucrose gradient. was investigat ed with the objective to label selectively the ifenprodil recognition site of native NMDA receptors. Saturation experiments revealed high-affinity sit es for [H-3]ifenprodil in this membrane fraction which could be characteris ed 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. Inh ibition of [H-3]ifenprodil binding by NR2B subunit-selective NMDA receptor antagonists, by polyamines, by sigma receptor ligands, by a variety of liga nds acting at different NMDA receptor recognition sites and by several cati ons was studied and compared with the effects of these compounds on (5R,10S )-( +)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine ([H-3]M K-801) binding under non-equilibrium conditions. It turned out that sigma r eceptor ligands such as 1,3-di(2-tolyl)-guanidine (DTG), (+)-3-(3-hydroxyph enyl)-N-propylpiperidine (R)-3-PPP, (S)-3-PPP and (1-{2-[bis(4-fluorophenyl )methoxy]ethyl})(-4-[3-phenylproply]piperazine) (GBR-12909) did not affect [H-3]ifenprodil binding in the nanomolar range or only slightly. In contras t, ifenprodil, eliprodil, nylidrin and haloperidol inhibited [H-3]ifenprodi l binding in the nanomolar range and in the same rank order and with the sa me potency as observed for the inhibition of the high-affinity fraction of [3H]MK-801 binding. The polyamines, which activate NMDA receptors, inhibite d [H-3]ifenprodil binding in a biphasic manner. Their potency to inhibit th e high-affinity fraction of [H-3]ifenprodil binding was found to be in the same range as their potency to enhance [H-3]MK-801 binding. In the presence of 10 mu M spermine a significantly enhanced (P = 0.0097) rate of dissocia tion of [H-3]ifenprodil binding was found, suggesting that inhibition of [3 H]ifenprodil binding by spermine is not, or at least not exclusively mediat ed by a competitive interaction. (C) 2000 Elsevier Science B.V. All rights reserved.