dizocilpine-maleate and diethylenetriamine

The binding of [3H]spermine to synaptosomal membranes from chick retina was examined. Saturable specific binding of [3H]spermine to synaptosomal membranes from plexiform layers of retina (P1 and P2) has been characterized, and found to concentrate in the inner plexiform layer compared to the outer plexiform layer (Bmax=9.3 and 37 pmol/mg protein for P1 and P2, respectively). Kinetics of specific [3H]spermine binding yield a sigmoidal saturation curve, indicating positive cooperativity (nH: 2.4 and 3.2 for P1 and P2, respectively) with high affinity: Kapp=61 and 67 nM for P1 and P2. The time required to attain equilibrium at room temperature was less than 5 min in both fractions. Dose-response curves for spermine, spermidine, and diethylene-triamine (DET) show different potencies for inhibiting [3HDET. Our results support a role for polyamines (PA) as neurotransmitters or neuromodulators in the vertebrate retina.

Topics: Animals; Chickens; Dizocilpine Maleate; Dose-Response Relationship, Drug; Excitatory Amino Acid Antagonists; Kinetics; Polyamines; Radioligand Assay; Receptors, N-Methyl-D-Aspartate; Retina; Spermidine; Spermine; Synaptosomes; Tritium

Polyamines are thought to modulate the activation of NMDA receptors through a unique allosteric regulatory site. The effects of polyamines on the binding of [3H]MK-801 were measured in cortical and hippocampal tissue surgically removed from patients with temporal lobe epilepsy (TLE). The polyamine agonist spermidine increased the binding of [3H]MK-801 in the cortex in a dose-dependent manner and this effect could be blocked by the weak partial agonist diethylenetriamine (DET). Spermidine decreased the Kd of [3H]MK-801 for the NMDA receptor but did not alter the density of receptors. Spermidine had essentially the same effect on Kd and Bmax measured in the dentate gyrus of TLE subjects and the cortex and dentate gyrus of postmortem controls. Moreover, there was no difference in the density of binding sites between postmortem and TLE subjects in either region. The binding of [3H]MK-801 in human cortex was decreased by 30% by incubation with DET or by prewashing the tissue sections. In contrast, DET did not alter the binding of [3H]MK-801 in rat cortex and prewashing sections produced an increase rather than a decrease in binding. These results suggest that there are different endogenous modulators for the polyamine site in rat and human tissue. The inverse agonist 1,10-diaminodecane decreased the binding of [3H]MK-801 in a dose-dependent manner. These results suggest that the fundamental modulatory properties of polyamines in rat and human tissues are essentially the same and that endogenous polyamines may regulate human NMDA receptors.

Topics: Adult; Animals; Brain; Cadaver; Cerebral Cortex; Diamines; Dizocilpine Maleate; Dose-Response Relationship, Drug; Epilepsy, Temporal Lobe; Female; Hippocampus; Humans; Male; Middle Aged; Polyamines; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Spermidine

Spermine and other polyamines both stimulate and inhibit N-methyl-D-aspartate receptor function, probably by interacting with two separate sites. To characterize these two actions, the effect of spermine on the binding kinetics of the channel blocker [3H]dizocilpine was studied in the presence of glutamate and glycine. Low concentrations (10 microM) of spermine increased the association and dissociation rates without modifying equilibrium binding, indicating that spermine increases the accessibility of [3H]dizocilpine to the channel by interacting with a high-affinity, stimulatory site. At higher concentrations (1 mM), spermine markedly decreased equilibrium [3H]dizocilpine binding by decreasing both affinity and Bmax, indicating that spermine allosterically inhibits binding by interacting with a second, low-affinity site. The presumed polyamine antagonists arcaine, diethylenetriamine, and 1,10-diaminodecane completely inhibited equilibrium [3H]dizocilpine binding, probably by interacting with the inhibitory polyamine site or other sites, but not with the stimulatory polyamine site. Low concentrations (10 microM) of ifenprodil completely reversed the increase in association rate produced by spermine, whereas higher concentrations (IC50 = 123 microM) inhibited equilibrium binding, indicating that ifenprodil is both a potent antagonist of the stimulatory site and a low-affinity ligand of the inhibitory site. The polyamine agonists spermine, spermidine, and neomycin interacted with the inhibitory site, but produced only partial inhibition of equilibrium [3H]dizocilpine binding.

Topics: Animals; Biguanides; Binding Sites; Dizocilpine Maleate; Glutamates; Glutamic Acid; Glycine; Kinetics; Male; Piperidines; Polyamines; Prosencephalon; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Spermine; Tritium

The NMDA receptor exhibits increased sensitivity to stimulation during early development compared with the adult. In this study, we examined modulation of the NMDA receptor by polyamines during development to see if it correlates with differences in the functional responsiveness of the NMDA receptor. [3H]MK-801 binding was measured in discrete brain regions in the presence and absence of polyamines in 3-, 7-, 15-, 25-, and 60-day-old Sprague-Dawley rats. [3H]MK-801 binding increased between postnatal days 3 and 15, with adult levels of binding being reached between days 15 and 25. Spermidine (75 microM) caused maximal stimulation of [3H]MK-801 binding during early development, ranging from 250% in the thalamus to 450% in the caudate putamen at postnatal day 3. This effect gradually declined to levels seen in the adult by postnatal days 15-25. During all developmental stages, the stimulation seen was greater in the caudate putamen compared with the hippocampus. Diethylenetriamine (1 mM) exhibited similar developmental and regional heterogeneity in its effects on [3H]MK-801 binding, producing substantial stimulation of binding in the neonate, but not in the adult. The EC50 and Emax values for the stimulatory effect of spermidine were significantly higher at day 7 compared with the adult. Unlike spermidine and diethylenetriamine, there was no regional variation in the effects of the putative "polyamine site" inverse agonist 1,10-diaminodecane at any age and only a slightly attenuated inhibition at postnatal day 3 compared with the adult.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Aging; Animals; Brain; Dizocilpine Maleate; Hippocampus; Polyamines; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Spermidine; Thalamus

We examined the block of N-methyl-D-aspartate (NMDA) receptors by n-alkyl (straight chain) diamines and related monoamines and triamines using whole-cell voltage clamp recording of NMDA receptor currents in cultured rat hippocampal neurons and [3H] dizocilpine binding to rat forebrain homogenates. At -60 mV, the diamines (carbon chain lengths 3-12) produced a concentration-dependent inhibition of NMDA receptor current (IC50 values, 6128-7.3 microM). For diamines of carbon chain lengths greater than 6, the inhibition was partially, but not completely, relieved by depolarization, indicating that the block occurs at distinct voltage-dependent and voltage-independent sites. The block produced by short-chain diamines (carbon chain lengths 3-6) was completely relieved by depolarization, indicating little or no interaction with the voltage-independent site. In comparison with the corresponding diamines, homologous monoamines exhibited very low potency, whereas homologous triamines were of equal or lower potency. For long-chain diamines, inhibitory potency at both the voltage-dependent and voltage-independent sites was correlated with carbon chain length (binding energy increasing 600-700 cal/mol-CH2), suggesting that binding to each of the sites is stabilized by a hydrophobic interaction. Affinities for the voltage-dependent blocking site (transformed to 0 mV) and for the voltage-independent blocking site were similar. These values were also similar to the inhibitory potencies of the diamines in the [3H]dizocilpine binding assay. Analysis of the voltage-dependence of block at the voltage-dependent site yielded z delta values for diamines of intermediate length (carbon chain lengths 7-9) that decreased with increasing length from 0.91 to 0.63 [approaching the z delta values of monovalent blockers (approximately 0.54) and one-half of the z delta values of shorter diamines (approximately 1.1)], suggesting that the intermediate length diamines block in a linear, extended chain conformation with one of the charges having incomplete access to a deep binding site. Longer chain diamines (carbon chain lengths 10 and 12) exhibited larger z delta values (0.78 and 0.98, respectively), presumably because enhanced conformational flexibility permitted a folded-over conformation. From the interchange distances of the intermediate length diamines in their lowest energy conformation, we estimated that the total voltage drop within the NMDA receptor channel occurs over a distance

Topics: Animals; Binding Sites; Cells, Cultured; Diamines; Dizocilpine Maleate; Electrochemistry; Polyamines; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Structure-Activity Relationship

N-Methyl-D-aspartic acid (NMDA, 500 microM) stimulated the net release of [3H]norepinephrine from rat hippocampal slices. The putative polyamine inverse agonist 1,10-diaminodecane (DA10) dose-dependently inhibited NMDA-stimulated release with a calculated IC50 value of 33 microM. The putative polyamine competitive antagonist diethylenetriamine (DET) partially inhibited (maximum effect 40%) NMDA-stimulated release at 1 mM which was the highest concentration tested. DET (1 mM) but not DA10 also partially inhibited potassium-stimulated release of norepinephrine from hippocampal slices. Neither DET or DA10 significantly altered the nonstimulated basal efflux of neurotransmitter. The inhibition of NMDA-stimulated release produced by 100 microM DA10 or 1 mM DET was not attenuated by addition of the polyamines spermine or spermidine up to 1 mM. In addition, the IC50 value for DA10-induced inhibition of NMDA-stimulated neurotransmitter release was not altered by the addition of DET (100-1000 microM). The combination of the glycine antagonist 7-chlorokynurenic acid (3 microM) and DA10 (100 microM) inhibited NMDA-stimulated release by approximately 70%. The addition of the glycine agonist D-serine (3-100 microM) partially attenuated the inhibition produced by these two compounds. No further enhancement was observed when D-serine was added in the presence of spermine or spermidine. Finally, the NMDA open channel blocker (+)-5-methyl-10,11- dihydro-5H-dibenzo[1,d]cyclo-hepten-5,10-imine maleate dose-dependently inhibited NMDA-stimulated release with an IC50 value of 25 nM. The inhibitory potency of (+)-5-methyl-10,11- dihydro-5H-dibenzo[1,d]cyclo-hepten-5,10-imine maleate was not significantly altered by the presence of either DET (100 microM) or DA10 (30 or 100 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Diamines; Dizocilpine Maleate; Hippocampus; In Vitro Techniques; Male; N-Methylaspartate; Norepinephrine; Polyamines; Rats; Rats, Sprague-Dawley

The ability of polyamines to alter NMDA-induced neurotoxicity in neonatal rats was examined to determine whether polyamines modulate NMDA receptor activity in vivo. Unilateral injections of NMDA and/or polyamines were made into the striatum of 7-day-old rats. After 5 days, the brains were removed and 20 microns thick coronal sections were cut and stained with Cresyl violet. A computer-based image analysis system was used to densitometrically measure the cross-sectional area of intact tissue in the control and injected hemispheres. Administration of NMDA (5-40 nmol) produced a dose-dependent tissue damage that ranged from 7 to 52% of the area of the uninjected hemisphere. The polyamine agonist spermine (10-500 nmol) dose-dependently exacerbated the toxicity of a 15 nmol dose of NMDA, increasing the size of the lesion by up to 50%. Administration of spermine alone produced dose-dependent tissue damage that ranged from 9 to 52%. The damage produced by both NMDA and spermine could be completely inhibited by co-administration of the NMDA antagonist MK-801. The polyamine inverse agonist 1,10-diaminodecane (DA-10, 50-400 nmol) inhibited the damage produced by NMDA in a dose-dependent manner, with a maximal inhibition of 50%. Administration of DA-10 alone produced limited damage at doses above 100 nmol. The weak partial agonist diethylenetriamine had no effect by itself or on NMDA-induced toxicity at the doses tested. These results indicate that polyamines can modulate the activity of NMDA receptors in vivo and suggest that polyamines or related compounds may have important therapeutic potential as neuroprotective agents.

Topics: Animals; Brain; Corpus Striatum; Diamines; Dizocilpine Maleate; Dose-Response Relationship, Drug; Female; Male; N-Methylaspartate; Neurotoxins; Polyamines; Rats; Rats, Sprague-Dawley; Spermine

The distribution and properties of N-methyl-D-aspartate (NMDA) receptors, labeled with [3H](+)-5-methyl-10, 11-dihydro-5H-dibenzo[a,d]cyclohepten-5 5,10-imine (MK-801), were examined in rat brain. In sections of brain mash, the kinetics of association and dissociation of [3H]MK-801 were monophasic in the presence of 100 microM glutamate and glycine, and Scatchard transformations of saturation isotherms resulted in linear plots. Inhibition of the binding of [3H]MK-801 by other noncompetitive antagonists produced competition curves with Hill coefficients close to 1.0, consistent with a simple bimolecular interaction between the radioligand and the receptor. Scatchard plots based upon densitometric measurements of [3H]MK-801 binding in serial sections of rat brain were also linear, with dissociation constant values ranging from 5.0 to 8.4 nM in different regions at the level of the hippocampus. The distribution of [3H]MK-801 binding sites paralleled the distribution of NMDA displaceable L-[3H]glutamate binding sites. One exception was the cerebellar granule cell layer, where the density of binding sites for [3H]MK-801 was extremely low. The relative density of [3H]MK-801 to NMDA displaceable L-[3H]glutamate binding sites was approximately 1 to 2, consistent with the existence of two transmitter recognition sites per NMDA receptor. The modulatory effects of polyamines on [3H]MK-801 binding were studied in washed brain sections. The polyamine agonists spermine and spermidine enhanced [3H]MK-801 binding in all regions studied, with increases ranging from 18% in the thalamus to 106% in the ventromedial striatum. The effects of spermine and spermidine in these regions were highly correlated. Diethylenetriamine, which blocks the effects of spermidine, by itself produced decreases in the binding of [3H]MK-801 in most regions ranging from 5 to 21% but increased binding in parts of the striatum by 3 to 22%. The decrease in binding produced by diethylenetriamine in different brain regions was negatively correlated with the increase in binding produced by the agonists, suggesting that variability in the residual concentration of endogenous polyamines contributes to the regional variability of agonist effects.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Autoradiography; Binding Sites; Brain Chemistry; Dizocilpine Maleate; Glutamates; Glutamic Acid; Male; Polyamines; Rats; Rats, Inbred Strains; Receptors, N-Methyl-D-Aspartate

The endogenous polyamines spermine and spermidine increase the binding of [3H]MK-801 to NMDA receptors. This effect is antagonized by diethylenetriamine (DET). We report here that spermine increases the rates of both association and dissociation of binding of [3H]MK-801, suggesting that it increases the accessibility of the binding site for MK-801 within the ion channel of the receptor complex. 1,10-Diaminodecane (DA10) inhibited the binding of [3H]MK-801. This effect was due to a decrease in the rate of association with no change in the rate of dissociation of [3H]MK-801. The effect of DA10 was not mediated by an action of DA10 at the binding sites for glutamate, glycine, Mg2+, or Zn2+, and was attenuated by DET. This suggests that DA10 acts at the polyamine recognition site. In hippocampal neurons the NMDA-elicited current was decreased by DA10, an effect opposite to that of spermine. The effects of spermine and DA10 were selectively blocked by DET. It is concluded that DA10 acts as a negative allosteric modulator or inverse agonist at the polyamine recognition site of the NMDA receptor.

Topics: Animals; Binding Sites; Cells, Cultured; Diamines; Dibenzocycloheptenes; Dizocilpine Maleate; Electric Conductivity; Glutamates; Glycine; Hippocampus; Kinetics; Magnesium; Polyamines; Rats; Receptors, N-Methyl-D-Aspartate; Receptors, Neurotransmitter; Spermidine; Spermine; Zinc