Showing papers on "Polyamine binding published in 1999"

Antagonists selective for NMDA receptors containing the NR2B subunit.

Abstract: In the late 1980s, a new class of N-methyl-D-aspartate (NMDA) receptor antagonists, exemplified by the phenylethanolamine ifenprodil (1), was identified. Initially, the mechanism of action of ifenprodil was a mystery as it was not a competitive antagonist at the glutamate or glycine (co-agonist) binding sites, nor was it a blocker of the calcium ion channel associated with the NMDA receptor. Early studies with a novel polyamine binding site associated with the NMDA receptor and functional studies in various brain regions suggested a unique and selective activity profile for 1. However, it was not until the NMDA receptor subunits were identified and expressed that ifenprodil was shown to be a selective antagonist for a subset of NMDA receptors containing the NR2B subunit. The wide range of potential therapeutic targets for NMDA antagonists coupled with the hope that NR2B selective agents might possess an improved clinical safety profile compared to non-selective compounds has supported an aggressive effort to develop the structure-activity relationships (SAR) of NR2B selective antagonists. This SAR and the basic physiology of the NMDA receptor form the basis of this review.

Antagonists Selective for NMDA Receptors Containing the NR2B Subunit

Abstract: In the late 1980s, a new class of N-methyl-D-aspartate (NMDA) receptor antagonists, exemplified by the phenylethanolamine ifenprodil (1), was identified. Initially, the mechanism of action of ifenprodil was a mystery as it was not a competitive antagonist at the glutamate or glycine (co-agonist) binding sites, nor was it a blocker of the calcium ion channel associated with the NMDA receptor. Early studies with a novel polyamine binding site associated with the NMDA receptor and functional studies in various brain regions suggested a unique and selective activity profile for 1. However, it was not until the NMDA receptor subunits were identified and expressed that ifenprodil was shown to be a selective antagonist for a subset of NMDA receptors containing the NR2B subunit. The wide range of potential therapeutic targets for NMDA antagonists coupled with the hope that NR2B selective agents might possess an improved clinical safety profile compared to non-selective compounds has supported an aggressive effort to develop the structure-activity relationships (SAR) of NR2B selective antagonists. This SAR and the basic physiology of the NMDA receptor form the basis of this review.

Direct block of native and cloned (Kir2.1) inward rectifier K+ channels by chloroethylclonidine.

Abstract: 1. We have investigated the inhibition of inwardly rectifying potassium channels by the alpha-adrenergic agonist/antagonist chloroethylclonidine (CEC). We used two preparations; two-electrode voltage-clamp of rat isolated flexor digitorum brevis muscle and whole-cell patch-clamp of cell lines transfected with Kir2.1 (IRK1). 2. In skeletal muscle and at a membrane potential of -50 mV, chloroethylclonidine (CEC), an agonist at alpha2-adrenergic receptors and an antagonist at alpha1x-receptors, was found to inhibit the inward rectifier current with a Ki of 30 microM. 3. The inhibition of skeletal muscle inward rectifier current by CEC was not mimicked by clonidine, adrenaline or noradrenaline and was not sensitive to high concentrations of alpha1-(prazosin) or alpha2-(rauwolscine) antagonists. 4. The degree of current inhibition by CEC was found to vary with the membrane potential (approximately 70% block at -50 mV c.f. approximately 10% block at -190 mV). The kinetics of this voltage dependence were further investigated using recombinant inward rectifier K+ channels (Kir2.1) expressed in the MEL cell line. Using a two pulse protocol, we calculated the time constant for block to be approximately 8 s at 0 mV, and the rate of unblock was described by the relationship tau=exp((Vm+149)/22) s. 5. This block was effective when CEC was applied to either the inside or the outside of patch clamped cells, but ineffective when a polyamine binding site (aspartate 172) was mutated to asparagine. 6. The data suggest that the clonidine-like imidazoline compound, CEC, inhibits inward rectifier K+ channels independently of alpha-receptors by directly blocking the channel pore, possibly at an intracellular polyamine binding site.

Dissecting subdomains involved in multiple functions of the CK2β subunit

Abstract: We have characterized several subdomains of the β subunit of protein kinase CK2. The N-terminal half of the protein exhibits a pseudo-substrate segment in tandem with a polyamine binding domain responsible for the activation of the kinase by these polybasic compounds. Study of the chemical features of this polyamine binding site showed that polyamine analogs exhibiting the highest affinity for CK2 are the best CK2 activators. Mutational analysis disclosed that glutamic residues lying in the polyacidic region of the CK2β subunit are involved in the interaction with polyamine molecules and allowed the delineation of an autonomous binding domain. Furthermore, this regulatory domain was shown to mediate the association of CK2 with plasma membrane.

Interaction of Putrescine with Nuclear Oligopeptides in the Enterocyte-Like Caco-2 Cells

Abstract: Intestinal cells are able both to synthesize and take up putrescine, the main compound of the metabolic polyamine pathway. Polyamine binding to nuclear macromolecules is thougth to modulate DNA synthesis and transcription. Our aim was to study the fate of putrescine when taken up from the medium in the enterocyte-like Caco-2 cells and to analyze its binding to nuclear proteins. After having incubated the cells with 14C-putrescine (0.8 μM), during cell replication and differentiation, the nuclei were separated by sequential centrifugations in a sucrose gradient. About 20% of the putrescine taken up by Caco-2 cells resulted in the nuclei in both proliferating and differentiated cells. The binding of polyamines to nuclear proteins was studied on nuclear extracts, separated by both alkaline polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) and gel permeation chromatography (GPC). No radioactivity was found in nuclear protein extracts when using the SDS-PAGE method. Conversely, in replicating cells, GPC showed that the greatest amount of radioactivity was present in the nuclear peaks corresponding to oligopeptides with a molecular weight of 4,800–8,000 daltons. High-performance liquid chromatography analysis showed the presence of putrescine and spermidine in the 8,000-dalton protein peak, whereas in the 4,800-dalton peak spermine was found in addition to putrescine. The radioactive count in the HPLC separated polyamines showed that a small percentage of the radioactivity present in the 8,000 and 4,800-dalton GPC peaks was linked to spermine and spermidine, suggesting an interconversion of the supplemented putrescine. Conversely, in differentiated cells, the nuclear oligopeptides did not reveal any radioactivity or any polyamines, suggesting that the binding of polyamines to nuclear oligopeptides is exclusively concerned with replicating cells.

Human retina contains polyamine sensitive [3H]-ifenprodil binding sites: implications for neuroprotection?

Abstract: AIMS—This study characterised the pharmacology of [3H]-ifenprodil binding to the polyamine binding sites (PBS) on the N-methyl-D-aspartate (NMDA) receptor channel complex on human retinas These data were correlated with the known neuroprotective effects of ifenprodil and eliprodil METHODS—Specific binding of [3H]-ifenprodil (under sigma site blockade) was investigated using human retinal homogenates and radioligand binding techniques Scatchard and competition analyses were utilised to define the pharmacology of the [3H]-ifenprodil binding sites RESULTS—Specific binding of [3H]-ifenprodil comprised 73% (SEM 3%) of total and reflected interaction with two affinity sites (Kds = 039 and 43 µM) of different densities (Bmax = 144 and 105 pmol/ mg protein) (n = 5) The rank order of affinity of compounds competing for [3H]-ifenprodil binding to the high affinity PBS was: ifenprodil > eliprodil > arcaine > spermine > diaminodecane > spermidine > putrescine >> MK-801 (n = 3-7) However, [3H]-ifenprodil binding was minimally inhibited by glutamate, NMDA, and kainate CONCLUSION—These studies have shown, for the first time, the presence of specific [3H]-ifenprodil binding sites in the human retina with pharmacological characteristics of PBS associated with the NMDA receptor ionophore complex The neuroprotective effects of eliprodil and ifenprodil may, in part, be mediated via these [3H]-ifenprodil labelled sites Keywords: retina; neuroprotection; eliprodil; NMDA receptors

The modulatory effect of spermine on the glutamate-NMDA receptor is regionally variable in normal human adult cerebral cortex.

Abstract: The MK-801, glutamate and polyamine binding sites on the N-methyl-D-aspartate class of glutamate receptors labelled with [3H]MK-801 were characterized in four cortical areas (sensorimotor, superior temporal, mid-frontal and occipital) from seven human adult control cases. Age, post-mortem delay, tissue storage time and sex had no significant effects on any of the parameters measured. Dissociation constants (K(D) values) for MK-801 showed similar mean values in the four cortical areas, whereas receptor densities (B(max) values) showed significant differences between sensorimotor or occipital and superior temporal or mid-frontal cortex. There were marked regional differences in the profiles of the spermine- and glutamate-incremented enhancement of specific [3H]MK-801 binding. The EC(50) for the glutamate enhancement was significantly higher in the occipital than in the mid-frontal and sensorimotor cortex, whereas maximal glutamate-enhanced binding values did not differ. The maximal enhancement of [3H]MK-801 binding by spermine and glutamate varied between the cases, ranging from zero to 40.4+/-9.3 fmol x mg protein(-1) for spermine, and from 85+/-5 to 111+/-10 fmol x mg protein(-1) for glutamate. Maximal spermine enhancement of [3H]MK-801 binding was significantly more variable in superior temporal or mid-frontal than in sensorimotor or occipital cortex. The results suggest that N-methyl-D-aspartate receptor sites, especially the polyamine site, are heterogeneous in human cerebral cortex, and show a high degree of regional and individual variability.

Effect of NMDA receptor ligands on mast cell histamine release, a reappraisal.

Abstract: Natural polyamines have been proposed to induce histamine release from mast cells through a direct interaction with G proteins. Alternatively, the polyamine binding site of ionotropic N-methyl-d-aspartate (NMDA) receptors has been suggested as a target for spermine on mast cells. We reexamined both hypotheses. Incubation of rat peritoneal mast cells with spermine resulted in a concentration-dependent histamine release (EC50 270 μM). Incubation with NMDA receptor agonists, glutamate or NMDA, associated to the co-agonist glycine, did not induce secretion. Western blot experiments did not reveal NMDA R1, R2a, R2b or R2c subunit expression in rat peritoneal mast cell membranes. The NMDA receptor antagonist at the glycine site, L-689,560, did not modify, at relevant concentrations, the spermine-induced secretion. The NMDA receptor antagonists, ifenprodil and LY 235959, and the NMDA channel blocker, MK801, slightly inhibited, at high concentrations, the secretory effect of spermine. The polyamine arcaine, an antagonist of the NMDA receptor polyamine binding site, induced histamine secretion (EC50 350 μM). Both spermine- and arcaine-induced effects were independent upon extracellular calcium and were largely inhibited by treatment of mast cells with pertussis toxin or benzalkonium chloride. The response to spermine and arcaine was prevented by the hydrolysis of sialic acid residues of the cell surface by neuraminidase, and was restored by permeabilization of the plasma membrane with streptolysine-O, indicating that polyamines act intracellularly. These results confirm the involvement of pertussis toxin-sensitive G proteins in the secretory effect of polyamines and demonstrate the absence of NMDA receptors on rat peritoneal mast cells. Nonselective effects of some NMDA receptor ligands on mast cells cannot be excluded.

Pharmacological Characterization of [3H]-Ifenprodil Binding to Polyamine Binding Sites on Rabbit and Rat Retinal Homogenates: Role in Neuroprotection?

Abstract: Polyamine binding sites (PBS) represent one of the modulatory sites on the N-methyl-D-aspartate (NMDA) receptor-channel complex. We have characterized [3H]-ifenprodil binding to the PBS on washed homogenates of rabbit and rat retinas. Specific binding of [3H]-ifenprodil (2 nM) (in the presence of 3 microM 1,3-Di [2-tolyl] guanidine HCl and 10 microM GBR12909 to block sigma sites) comprised 47-56% of the total binding. Scatchard analyses indicated interaction with apparent high- and low-affinity sites: dissociation constants (K(d)s) = 0.5-0.6 microM and apparent density of sites (Bmax) = 1.5-4.3 pmol/mg protein and K(d)s = 2.0-2.9 microM, and Bmax values = 15.8-17.8 pmol/mg protein (n = 3). Ifenprodil (Ki = 0.4-0.8 microM), eliprodil (Ki = 0.7-0.8 microM), spermine (Ki = 72-79 microM), spermidine (Ki = 283-330 microM), putrescine (Ki > 650 microM) and MK-801 (Ki > 1 mM) (n = 3-5) differentially competed for [3H]-ifenprodil binding. The biphasic competition curves for ifenprodil were resolved into two binding components: rat retinas, IC50high = 0.19 +/- 0.13 microM and IC50low = 8.7 +/- 1.3 microM; rabbit retinas, IC50high = 0.1 +/- 0.01 microM and IC50low = 16.0 +/- 7.8 microM. These studies have shown the presence of specific PBS labeled by [3H]-ifenprodil in the rabbit and rat retinas which may, in part, be responsible for mediating the neuroprotective effects of eliprodil and ifenprodil.

Recombinant anti-polyamine antibodies: identification of a conserved binding site motif

Abstract: Polyamines are small linear polycations found ubiquitously in eukaryotic cells. They are involved in nucleic acid and protein synthesis and rises in cellular polyamine levels have been correlated with cell proliferation. Antibodies to these molecules have potential as prognostic indicators of disease conditions and indicators of treatment efficacy. Antipolyamine monoclonal antibodies of differing but defined specificities have been generated in our laboratory using polyamine ovalbumin conjugates as immunogens. These antibodies show small but significant cross reactivities with other polyamine species; IAG-1 cross reacts with spermidine (8%), JAC-1 with spermine (6%) and JSJ-1 with both putrescine (11%) and spermine (6%). We have rescued and sequenced the heavy and light chain variable regions of all three of these antibodies. While the light chains of two antibodies, IAG-1 and JSJ-1, were 93% homologous at the amino acid level, none of the heavy chains displayed any significant sequence homology. However, computer-generated models of all three antibody binding sites revealed a three-dimensionally conserved polyamine binding site motif. The polyamine appears to bind into a negatively charged cleft lined with acidic and polar residues. The cleft is partially or completely closed at one end and the specificity of the interaction is determined by placement of acidic residues in the cleft. Aromatic residues contribute to polyamine binding interacting with the carbon backbone. The polyamine-binding motif we have identified is very similar to that observed in the crystal structure of PotD, the primary receptor of the polyamine transport system in Escherichia coli.