Pharmacological characterisation and autoradiographic distribution of polyamine-sensitive [3H]ifenprodil binding sites in the rat brain

doi:10.1016/0304-3940(91)90127-F

Neuroscience Letters

Volume 125, Issue 1, 15 April 1991, Pages 45-48

https://doi.org/10.1016/0304-3940(91)90127-F Get rights and content

Abstract

Saturation studies with [³H]ifenprodil (in the presence of 3 μM (+)-3-PPP and 10 μM GBR 12909) demonstrated the presence of a high-affinity (K_d = 0.45 μM) population of binding sites in sagittal rat brain sections. This binding was inhibited by spermine (IC₅₀ = 69 μM) and spermidine (IC₅₀ = 623 μM) but not by putrescine (1 mM). Ifenprodil displaced this binding in a biphasic fashion with a high affinity component (IC₅₀ = 0.992 μM) accounting for approximately 50% of the spermine-displaceable [³H]ifenprodil binding. The polyamine-sensitive [³H]ifenprodil binding sites were heterogenously distributed in the rat brain, the highest binding densities being found in the hippocampus and in the nucleus accumbens. The anatomical distribution of [³H]ifenprodil binding sites closely matches that previously reported for the $N- methyl- d -aspartate$ (NMDA) receptor.

References (11)

C. Carter et al.
Ifenprodil and SL 82.0715 are antagonists at the polyamine site of the $N- methyl- d -aspartate$ (NMDA) receptor
Eur. J. Pharmacol.
(1989)
P.C. Contreras et al.
Autoradiographic distribution of non-dopaminergic binding sites labeled by [³H]haloperidol in rat brain
Neurosci. Lett.
(1987)
E.W. Karbon et al.
Ifenprodil potently interacts with $[^{3} H](+)-3- PPP-labeled$ σ binding sites in guinea pig brain membranes
Eur. J. Pharmacol.
(1990)
H. Schoemaker et al.
Binding of [³H]ifenprodil, a novel NMDA antagonist, to a polyamine-sensitive site in the rat cerebral cortex
Eur. J. Pharmacol.
(1990)
C. Carter et al.
Ifenprodil and SL 82.0715 as cerebral anti-ischemic agents. II. Evidence for $N- methyl- d -aspartate$ receptor antagonist properties
J. Pharmacol. Exp. Ther.
(1988)

There are more references available in the full text version of this article.

Cited by (43)

Pharmacological characterization of interactions of RO 25-6981 with the NR2B (ε2) subunit
2001, European Journal of Pharmacology
We used ligand binding to ascertain whether the pharmacological actions of RO 25-6981 [(R:(*), S:(*))-α-(4-hydroxyphenyl)-β-methyl-4-(phenylmethyl)-1-piperidinepropanol] match those of other NR2B (ε2) subunit specific agents. RO 25-6981 inhibited binding of ¹²⁵I-MK801 [iodo-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohept-5,10-imine maleate] to receptors made from NR1a/ε2 but not NR1a/ε1. Increasing the concentration of spermidine did not change the efficacy of RO 25-6981 and minimally changed the IC₅₀ value. Chimeric ε1/ε2 receptors demonstrated that the structural determinants for high affinity actions of RO 25-6981 were contained completely within the first 464 amino acids, but no receptor retained wildtype features when the size of the ε2 component was decreased further. ε1Q336R receptors were more inhibited by ifenprodil and RO 25-9681 than wildtype ε1 receptors in ligand binding assays but not in functional assays. Selected mutations of ε2E200 and ε2E201 also decreased the sensitivity of receptors to ifenprodil and RO 25-6981. These results suggest that RO 25-6981 shares structural determinants with ifenprodil and other modulators in the NR2B subunit.
[<sup>3</sup>H]ifenprodil binding to NMDA receptors in porcine hippocampal brain membranes
2000, European Journal of Pharmacology
(±)-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 (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 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.
Effects of the NMDA antagonist CP-98,113 on regional cerebral edema and cardiovascular, cognitive, and neurobehavioral function following experimental brain injury in the rat
1998, Brain Research
The present study examined the effects of CP-98,113, an N-methyl-d-aspartate (NMDA) receptor blocker, on cardiovascular variables, neurobehavioral motor function, spatial memory deficits, and cerebral edema formation following lateral (parasagittal) fluid-percussion (FP) brain injury in the rat. In Study 1, we compared the cardiovascular effects of i.p. administration of CP-98,113 at 15 min postinjury at doses of 1 mg/kg, 2 mg/kg, 5 mg/kg, or 20 mg/kg (n=8/dose). Animals receiving 1 mg/kg to 5 mg/kg CP-98,113 showed slight but nonsignificant decreases in blood pressure, while those receiving the highest dose (20 mg/kg) showed significant hypotension. Based upon those observations, the 5 mg/kg dose was chosen as the optimal dose for subsequent behavioral studies. In Study 2, 15 min following lateral FP brain injury of moderate severity (2.5 atm), animals randomly received either CP-98,113 (5 mg/kg, i.p., n=23) followed by a 24-h subcutaneous infusion (1.5 mg kg⁻¹ h⁻¹) by means of a miniature osmotic pump, or identical volume of vehicle (n=24), and were evaluated for neurologic motor function (n=11/drug vs. 11/vehicle), memory function, and cerebral edema (n=12/drug vs. 13/vehicle). CP-98,113 (5 mg/kg) significantly attenuated neurologic motor dysfunction at 24 h (p<0.01) and 2 weeks (p<0.05) postinjury, reduced posttraumatic impairment in spatial memory observed at 48 h postinjury (p<0.001), and significantly reduced focal brain edema in the cortex adjacent to the site of maximal injury at 48 h postinjury (injury penumbra) (p<0.001). These results suggest that blockade of the NMDA receptor may attenuate the deleterious sequelae of traumatic brain injury.
Molecular determinants of NMDA receptor pharmacological diversity
1998, Progress in Brain Research
This chapter describes the molecular basis of the known pharmacologically distinct N-methyl-D-aspartate (NMDA) receptor subtypes. The potent excitotoxic effect of NMDA receptor overactivation has made this receptor a prime suspect in various neurodegenerative diseases such as Alzheimer's, Parkinson's, and AIDS dementia. Two general approaches have been taken to develop agents with a higher therapeutic index—using compounds that act at different regulatory domains on the NMDA receptor or identifying compounds that act at subtypes of NMDA receptors. NMDA receptors display a rich diversity of sites at which pharmacological agents can modify activity. In addition to the glutamate and glycine agonist binding sites, there are sites for channel blockers, polyamines, redox reagents, ifenprodil, protons, steroids, Zn²⁺, Mg²⁺, and histamine. Two families of NMDA receptor subunits have been cloned, together these consist of 12 different subunits. In specific nuclei of the midline thalamus, for example, the paraventricular and intermediodorsal thalamic nuclei and other diencephalic structures, NMDA receptors display a distinct pharmacological profile at both the glutamate recognition site and the channel blocker-binding site.
Neurotoxicity of Polyamines and Pharmacological Neuroprotection in Cultures of Rat Cerebellar Granule Cells
1997, Experimental Neurology
We have studied in a well-characterizedin vitroneuronal system, cultures of cerebellar granule cells, the toxicity of polyamines endogenously present in the brain: spermine, spermidine, and putrescine. Twenty-four-hour exposure of mature (8 daysin vitro) cultures to 1–500 μMspermine resulted in a dose-dependent death of granule cells, with the half-maximal effect being reached below 50 μMconcentration. Putrescine was moderately toxic but only at 500 μMconcentration. Spermidine was tested at 50 and 100 μMconcentration and its toxicity was evaluated to be about 50% that of spermine. Neuronal death caused by spermine occurred, at least in part, by apoptosis. Spermine toxicity was completely prevented by competitive (CGP 39551) and noncompetitive (MK-801) antagonists of the NMDA receptor, but was unaffected by a non-NMDA antagonist (NBQX) or by antagonists of the polyamine site present on the NMDA receptor complex, such as ifenprodil. A partial protection from spermine toxicity was obtained through the simultaneous presence of free radical scavengers or through inhibition of the free radical-generating enzyme nitric oxide synthase, known to be partially effective against direct glutamate toxicity. The link between spermine toxicity and glutamate was further strengthened by the fact that, under culture conditions in which glutamate toxicity was ineffective or much reduced, spermine toxicity was absent or very much decreased. Exposure to spermine was accompanied by a progressive accumulation of glutamate in the medium of granule cell cultures. This was attributed to glutamate leaking out from dying or dead cells and was substantially prevented by the simultaneous presence of MK-801 or CGP 39551. The present results demonstrate that polyamines are toxic to granule cells in culture and that this toxicity is mediated through the NMDA receptor by interaction of exogenously added polyamines with endogenous glutamate released by neurons in the medium. The involvement of brain polyamines, in particular spermine and spermidine, in excitotoxic neuronal death is strongly supported by our present results.
CP-101,606, a potent neuroprotectant selective for forebrain neurons
1997, European Journal of Pharmacology
The neuroprotective activity of (1S,2S)-1-(4-hydroxyphenyl)-2-(4-hydroxy-4-phenylpiperidino)-1-propanol (CP-101,606), an N-methyl-d-aspartate (NMDA) receptor antagonist structurally similar to ((±)-(R^*,S^*)-α-(4-hydroxyphenyl)-β-methyl-4-(phenylmethyl)-1-piperidineethanol (ifenprodil), was investigated in neurons in primary culture. CP-101,606 potently and efficaciously protected hippocampal neurons from glutamate toxicity but was >900-fold less effective for cerebellar granule neurons. The neuroprotective activity in the hippocampal neurons is mediated through a high affinity binding site distinct from the agonist and thienylcyclohexylpiperidine (TCP) binding sites of the NMDA receptor. Autoradiography indicates the CP-101,606 binding site is localized in forebrain, most notably in hippocampus and the outer layers of cortex. The functional selectivity for hippocampal neurons, forebrain localization of binding sites, and structural relation to ifenprodil suggest that CP-101,606 is an NMDA antagonist highly selective for NR2B subunit containing receptors.

View all citing articles on Scopus

View full text

Pharmacological characterisation and autoradiographic distribution of polyamine-sensitive [3H]ifenprodil binding sites in the rat brain

Abstract

Eur. J. Pharmacol.

Neurosci. Lett.

Eur. J. Pharmacol.

Eur. J. Pharmacol.

Ifenprodil and SL 82.0715 as cerebral anti-ischemic agents. II. Evidence for N-methyl-d-aspartate receptor antagonist properties

J. Pharmacol. Exp. Ther.

Pharmacological characterisation and autoradiographic distribution of polyamine-sensitive [³H]ifenprodil binding sites in the rat brain

Ifenprodil and SL 82.0715 as cerebral anti-ischemic agents. II. Evidence for $N- methyl- d -aspartate$ receptor antagonist properties