[3H]ifenprodil binding to NMDA receptors in porcine hippocampal brain membranes.
(+/-)-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 sigma 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 (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5, 10-imine ([3H]MK-801) binding under non-equilibrium conditions. It turned out that sigma 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]piperazi ne) (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 microM 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.
Höfner
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Institut für Pharmazie, Zentrum für Pharmaforschung, Ludwig-Maximilians-Universität München, Butenandtstr. 5-13, D-81377, München, Germany. ghoef@cup.uni-muenchen.de
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D000818:Animals; D001386:Azides / Q000378:metabolism / Q000494:pharmacology; D001667:Binding, Competitive / Q000187:drug effects; D016291:Dizocilpine Maleate / Q000378:metabolism / Q000494:pharmacology; D018691:Excitatory Amino Acid Antagonists / Q000378:metabolism* / Q000494:pharmacology; D006146:Guanidines / Q000378:metabolism / Q000494:pharmacology; D006624:Hippocampus / Q000187:drug effects / Q000378:metabolism*; D066298:In Vitro Techniques; D007700:Kinetics; D008566:Membranes / Q000187:drug effects / Q000378:metabolism; D010880:Piperidines / Q000378:metabolism*; D011485:Protein Binding; D016194:Receptors, N-Methyl-D-Aspartate / Q000037:antagonists & inhibitors / Q000378:metabolism*; D017480:Receptors, sigma / Q000187:drug effects / Q000378:metabolism; D013347:Subcellular Fractions / Q000187:drug effects / Q000378:metabolism; D013552:Swine
D001386:Azides; D018691:Excitatory Amino Acid Antagonists; D006146:Guanidines; D010880:Piperidines; D016194:Receptors, N-Methyl-D-Aspartate; D017480:Receptors, sigma; C055550:1-(4-azido-2-methylphenyl)-3-(2-methylphenyl)guanidine; D016291:Dizocilpine Maleate; C010739:ifenprodil