dizocilpine-maleate and arcaine

Spermidine is an endogenous polyamine with a polycationic structure present in the central nervous system of mammals. Spermidine regulates biological processes, such as Ca(2+) influx by glutamatergic N-methyl-d-aspartate receptor (NMDA receptor), which has been associated with nitric oxide synthase (NOS) and cGMP/PKG pathway activation and a decrease of Na(+),K(+)-ATPase activity in rats' cerebral cortex synaptosomes. Na(+),K(+)-ATPase establishes Na(+) and K(+) gradients across membranes of excitable cells and by this means maintains membrane potential and controls intracellular pH and volume. However, it has not been defined whether spermidine modulates Na(+),K(+)-ATPase activity in the hippocampus. In this study we investigated whether spermidine alters Na(+),K(+)-ATPase activity in slices of hippocampus from rats, and possible underlying mechanisms. Hippocampal slices and homogenates were incubated with spermidine (0.05-10 μM) for 30 min. Spermidine (0.5 and 1 μM) decreased Na(+),K(+)-ATPase activity in slices, but not in homogenates. MK-801 (100 and 10 μM), a non-competitive antagonist of NMDA receptor, arcaine (0.5μM), an antagonist of the polyamine binding site at the NMDA receptor, and L-NAME (100μM), a NOS inhibitor, prevented the inhibitory effect of spermidine (0.5 μM). ODQ (10 μM), a guanylate cyclase inhibitor, and KT5823 (2 μM), a protein kinase G inhibitor, also prevented the inhibitory effect of spermidine on Na(+),K(+)-ATPase activity. Spermidine (0.5 and 1.0 μM) increased NO(2) plus NO(3) (NOx) levels in slices, and MK-801 (100 μM) and arcaine (0.5 μM) prevented the effect of spermidine (0.5 μM) on the NOx content. These results suggest that spermidine-induced decrease of Na(+),K(+)-ATPase activity involves NMDA receptor/NOS/cGMP/PKG pathway.

Topics: Animals; Biguanides; Carbazoles; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Dizocilpine Maleate; Enzyme Activation; Hippocampus; In Vitro Techniques; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Oxadiazoles; Quinoxalines; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate; Signal Transduction; Sodium-Potassium-Exchanging ATPase; Spermidine

The polyamines putrescine, spermidine, and spermine are a group of aliphatic amines that may act as physiological modulators of the N-methyl-D-aspartate (NMDA) receptor, a glutamate receptor implicated in memory formation and consolidation. Arcaine is a competitive antagonist of the polyamine binding site at the NMDA receptor, the post-training administration of which impairs memory of various tasks.. In this study, we investigated whether the administration of arcaine and MK-801 alters the memory of the step-down inhibitory avoidance task, and whether the effects of these NMDA antagonists involve state-dependency mechanisms, in adult male Wistar rats.. The administration of arcaine (30 mg/kg, i.p.) or MK-801 (0.03 mg/kg, i.p.) immediately after training impaired inhibitory avoidance performance at testing. Arcaine- and MK-801-induced performance impairment was reversed by the administration of arcaine (30 mg/kg, i.p.) and MK-801 (0.03 mg/kg, i.p.), respectively, 30 min before testing. Response transfer also occurred if arcaine substituted MK-801 at testing, and vice-versa.. These results suggest that arcaine and MK-801 induce state-dependent recall and that, probably due to their ability to decrease NMDA receptor function, one drug can substitute for the other at testing, demonstrating a cross-state dependency between arcaine and MK-801.

Topics: Animals; Avoidance Learning; Behavior, Animal; Biguanides; Buffers; Dizocilpine Maleate; Dose-Response Relationship, Drug; Drug Administration Schedule; Excitatory Amino Acid Antagonists; Exploratory Behavior; Injections, Intraperitoneal; Male; Mental Recall; Neuroprotective Agents; Phosphates; Polyamines; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate; Time Factors; Transfer, Psychology

The polyamines putrescine, spermine, and spermidine are a group of aliphatic amines that physiologically modulate the N-methyl-D-aspartate (NMDA) receptor, a glutamate receptor implicated in memory formation.. Given the potential application of these drugs in the treatment of memory disorders, we investigated whether agonists and/or antagonists of the NMDA receptor polyamine binding site alters the memory of fear conditioning and determined the time window in which fear conditioning is modulated by polyaminergic agents given by the systemic route.. Post-training intraperitoneal administration of spermidine (10-100 mg/kg) immediately after training increased, whereas arcaine (10 mg/kg) and MK-801 (0.01-0.1 mg/kg) decreased contextual and auditory fear conditioning. Arcaine and MK-801, at doses that had no effect per se, reversed the facilitatory effect of spermidine. Memory of fear conditioning was impaired by polyaminergic blockade up to 180 min but not at 360 min after training.. These results provide evidence that systemic administration of polyamine binding site ligands modulate early consolidation of fear conditioning.

Topics: Animals; Behavior, Animal; Biguanides; Conditioning, Classical; Dizocilpine Maleate; Fear; Injections, Intraperitoneal; Male; Memory; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate; Spermidine; Time Factors

Intrathecal (i.t.) administration into mice of N-ethylmaleimide (NEM), a cysteine protease inhibitor, produced a characteristic behavioral response, the biting and/or licking of the hindpaw and the tail along with slight hindlimb scratching directed toward the flank. The behavior induced by NEM was inhibited by the intraperitoneal injection of morphine. We have recently reported that dynorphin A and, more potently big dynorphin, consisting of dynorphins A and B, produce the same type of nociceptive response whereas dynorphin B does not [Tan-No K, Esashi A, Nakagawasai O, Niijima F, Tadano T, Sakurada C, Sakurada T, Bakalkin G, Terenius L, Kisara K. Intrathecally administered big dynorphin, a prodynorphin-derived peptide, produces nociceptive behavior through an N-methyl-d-aspartate receptor mechanism. Brain Res 2002;952:7-14]. The NEM-induced nociceptive behavior was inhibited by pretreatment with dynorphin A- or dynorphin B-antiserum and each antiserum also reduced the nociceptive effects of i.t.-injected synthetic big dynorphin. The characteristic NEM-evoked response was not observed in prodynorphin knockout mice. Naloxone, an opioid receptor antagonist, had no effects on the NEM-induced behavior. Ifenprodil, arcaine and agmatine, antagonists at the polyamine recognition site on the N-methyl-D-aspartate (NMDA) receptor ion-channel complex, and MK-801, an NMDA ion-channel blocker inhibited the NEM-induced effects. Ro25-6981, an antagonist of the NMDA receptor subtype containing NR2B subunit was not active. NEM completely inhibited degradation of dynorphin A by soluble and particulate fractions of mouse spinal cord. Collectively, the results demonstrate that endogenous prodynorphin-derived peptides are pronociceptive in uninjured animals, and required for the NEM-induced behavior. The NEM effects may be mediated through inhibition of the degradation of endogenous dynorphins, presumably big dynorphin that in turn activates the NMDA receptor ion-channel complex by acting on the polyamine recognition site.

Topics: Agmatine; Analysis of Variance; Animals; Behavior, Animal; Biguanides; Dizocilpine Maleate; Dose-Response Relationship, Drug; Drug Interactions; Dynorphins; Enkephalins; Enzyme Inhibitors; Ethylmaleimide; Excitatory Amino Acid Antagonists; Immune Sera; Injections, Spinal; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Morphine; Narcotics; Nociceptin Receptor; Piperidines; Protein Precursors; Receptors, Opioid; Spinal Cord; Time Factors

Polyamines, among other functions, are considered to act as a free radical scavenger and antioxidant. The quinolinic acid (QA), sodium nitroprusside (SNP) and iron (Fe+2) stimulate production of free radicals and lipid peroxidation. In the present study, we investigated the free radical and/or aldehyde scavenger effects of polyamines spermine and spermidine on thiobarbituric acid reactive species (TBARS) production induced by QA, SNP, Fe+2/EDTA system and free Fe2+ in rat brain. Spermine and spermidine inhibited QA-induced TBARS production; however spermine was a better antioxidant than spermidine. Spermine also inhibited SNP-, Fe+2/EDTA- and free Fe2+-induced TBARS production, but had a modest effect. Spermidine, in turn, also discretely inhibited SNP-, Fe+2/EDTA- and free Fe2+-induced TBARS production. In the presence of MK-801, QA-induced TBARS production was considerably more inhibited by polyamines. In addition, arcaine does not affect the reducer effect of polyamines. The present findings suggest that the observed effects of polyamines are not related to the activation of NMDA receptor but with their antioxidant and free radical scavenger properties.

Topics: Animals; Antioxidants; Biguanides; Biogenic Polyamines; Dizocilpine Maleate; Edetic Acid; Iron; Lipid Peroxidation; Nitroprusside; Oxidants; Quinolinic Acid; Rats; Rats, Wistar; Spermidine; Spermine; Thiobarbituric Acid Reactive Substances

The glucose deprivation-induced release of [3H]D-aspartate was studied in bovine and human retinas in a superfusion apparatus. [3H]D-aspartate release was significantly increased upon omitting glucose in the superfusion buffer. This effect was dependent on external Ca2+ because L- and N-type Ca2+-channel blockers, such as diltiazem (1 microM), nitrendipine (1 microM), and omega-conotoxin (100 nM), significantly reduced the effect of glucose-deprivation induced release of [3H]D-aspartate. Furthermore, while glutamate receptor agonists (L-glutamate, N-methyl-D-aspartate, but not kainate) potentiated the effects of glucose deprivation, antagonists (MK-801, MCPG, ifenprodil, and L-AP3) at these receptors blocked the glucose deprivation-induced release process. Taken together, these studies have demonstrated that under conditions of glucose deprivation, as may happen during ischemic events in vivo, the retinal glutamatergic nerve endings and/or glial cells promote the efflux of [3H]D-aspartate into the extracellular environment. This process appears to be receptor-mediated and dependent on extracellular Ca2+ and is similar to previous reports pertaining to brain tissues.

Topics: Alanine; Animals; Biguanides; Calcium Channel Blockers; Cattle; D-Aspartic Acid; Diltiazem; Dizocilpine Maleate; Drug Synergism; Glucose; Glutamic Acid; Glycine; Humans; Kainic Acid; N-Methylaspartate; Nitrendipine; omega-Conotoxins; Perfusion; Piperidines; Polyamines; Receptors, Glutamate; Retina; Tritium; Verapamil

Ifenprodil, arcaine and agmatine have all been reported to inhibit the NMDA receptor by actions at polyamine-sites, however the specific sites with which these compounds interact is unknown. Here we used radioligand binding of [3H]MK-801 to a membrane preparation from rat cerebral cortex to investigate the interactions of these compounds with the NMDA receptor complex. In the absence of exogenous polyamines, agmatine reduced [3H]MK-801 binding only at concentrations over 500 micro M, as opposed to the putative polyamine-site antagonists arcaine and ifenprodil which directly reduce ligand binding at much lower concentrations (5 micro M) in the absence of polyamines. In our studies, all three compounds significantly reduced spermidine-potentiated [3H]MK-801 binding, however agmatine was the only compound effective at concentrations below those that produced direct inhibition of [3H]MK-801 binding. Under these conditions, agmatine had a K(i)=14.8 micro M for spermidine-potentiated [3H]MK-801 binding and displayed characteristics of a competitive antagonist. Agmatine, as well as ifenprodil and arcaine, also displaced [3H]spermidine from rat cortical membranes at concentrations similar to those that were effective at reducing spermidine-potentiated [3H]MK-801 binding. In conclusion, these data suggest that agmatine reduces the potentiating effects of polyamines by competitive antagonism at a specific site on the NMDA receptor complex, and that these actions of agmatine differ from those of ifenprodil and arcaine.

Topics: Agmatine; Animals; Biguanides; Binding Sites; Binding, Competitive; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Male; Piperidines; Radioligand Assay; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Spermidine; Tritium

Human immunodeficiency virus type-I (HIV-1) infection is often associated with neuronal loss in cortical and subcortical regions that may manifest as motor dysfunction and dementia. The function of the HIV-1 transcription protein Tat and subsequent activation of N-methyl-D-aspartate receptors (NMDAr) have been implicated in this form of neurodegeneration. However, it is unclear if Tat interacts directly with the NMDAr and the role of specific NMDAr subunit composition in mediating effects of Tat is also unclear. The present studies examined the ability of HIV-1 Tat1-72 protein (10 pM-1.0 microM) to displace [3H]MK-801 binding and to attenuate spermidine-induced potentiation of this binding in rat brain homogenate comprised of cerebellum, hippocampus, and cerebral cortex. The role of NMDAr polyamine-site function in the neurotoxic effects of Tat was determined using organotypic hippocampal slice cultures. Binding of [3H]MK-801 in adult rat brain homogenate was not reduced by Tat at concentrations below 1 microM. Tat potently inhibited the potentiation of [3H]MK-801 binding produced by co-exposure of membranes to the NMDAr co-agonist spermidine (IC(50)=3.74 nM). In hippocampal explants, Tat produced neurotoxicity in the CA3 and CA1 pyramidal cell layers, as well as in the dentate gyrus, that was significantly reduced by co-exposure to MK-801 (20 microM) and the NMDAr polyamine-site antagonist arcaine (10 microM). Exposure to the HIV-1 Tat deletion mutant (Tatdelta31-61) did not produce neurotoxicity in hippocampal explants. These data suggest that the neurotoxic effects of HIV-1 Tat are mediated, in part, by direct interactions with a polyamine-sensitive site on the NMDAr that positively modulates the function of this receptor.

Topics: Animals; ATPases Associated with Diverse Cellular Activities; Biguanides; Brain; Cerebellum; Cerebral Cortex; Culture Techniques; Dizocilpine Maleate; DNA-Binding Proteins; Excitatory Amino Acid Antagonists; Female; Hippocampus; HIV-1; Male; Neurons; Neurotoxins; Proteasome Endopeptidase Complex; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Spermidine

The pharmacology of N-methyl-D-aspartate (NMDA) receptors shows regional differences in affinity for various agonists and antagonists. We have investigated the modulatory mechanisms acting via the polyamine, redox and proton sites in the cerebral cortex and the spinal cord of adult, male rats using [3H]MK-801 binding. The affinity for glycine-independent spermine stimulation was one magnitude higher in cerebrocortical than in spinal cord membranes while the affinity for the spermine antagonist arcaine was similar. Spermine abolished the inhibiting effect of low pH in both regions. Thus, the difference in the polyamine site between the two regions seems to be restricted to agonist binding. The proportion of high affinity/total ifenprodil binding was approximately 35% both in the spinal cord and the cerebral cortex, suggesting similar relative amounts of the NMDA receptor subunit 2B. The affinity of ifenprodil to the high affinity site was however significantly higher in the cerebral cortex. Redox modulatory agents had similar effects in the two regions but spermine fully counteracted the inhibiting effect of 0.2 mM 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) in the cerebral cortex while there was only a partial effect in the spinal cord. These data show that the regional pharmacological heterogeneity involves several of the mechanisms regulating the function of the NMDA receptor. The data also indicate that the NMDA receptor subunit 2B is much more common in spinal cord than previously suggested.

Topics: Animals; Biguanides; Cerebral Cortex; Dizocilpine Maleate; Hydrogen-Ion Concentration; Male; Neuroprotective Agents; Oxidation-Reduction; Piperidines; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Spermine; Spinal Cord; Tritium

Aromatic analogs of arcaine were shown to have inhibitory effects on the binding of the channel blocking drug [3H]MK-801 to the NMDA receptor complex. The most potent compound of the series was an N,N'-bis(propyl)guanidinium which inhibited [3H]MK-801 binding with an IC50 of 0.58 microM and an IC50 of 12.17 microM upon addition of 100 microM spermidine. The increase in IC50 upon addition of spermidine suggests competitive antagonism between the inhibitor and spermidine at the arcaine-sensitive polyamine site of the NMDA receptor complex.

Topics: Biguanides; Dizocilpine Maleate; Protein Binding; Receptors, N-Methyl-D-Aspartate; Structure-Activity Relationship

The influence of noncompetitive (MK-801), competitive (AP-7) and the antagonist of polyamines site of NMDA receptor (arcaine) on the central activity of angiotensin II (A II) was studied. The open field test, conditioning of active avoidance responses (CARs) and passive avoidance situation was used to investigate learning and memory in rats. All used antagonists decreased beneficial action of A II on these processes.

Topics: Angiotensin II; Animals; Avoidance Learning; Biguanides; Brain; Conditioning, Psychological; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Male; Memory; Motor Activity; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate

Arginine-vasopressin (AVP) is a neuropeptide which facilitates learning and memory processes. We examinated the participation of NMDA receptors in beneficial effects of peptide. The results of our study show that noncompetitive antagonist of NMDA receptor-MK-801 impairs the effect of AVP on the consolidation of conditioned avoidance responses and antagonist of polyamines site-arcaine reduced advantageous effect of AVP on the retrieval of memory in passive avoidance situation.

Topics: 2-Amino-5-phosphonovalerate; Animals; Arginine Vasopressin; Biguanides; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Learning; Male; Memory; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate

We have investigated the effects of polyamine agonists and antagonists on the modulation of the N-methyl-D- aspartate receptor by glycine using a [3H]dizocilpine ([3H]MK801) binding assay. We monitored the non-equilibrium binding of [3H]dizocilipine in the presence of 5,7-dichlorokynurenate to preclude occupation of the glycine site by endogenous glycine. Using this assay, spermine and spermidine increased both the affinity and the maximum effect of glycine. Similar effects are produced by other polyamine agonists including 1,5-diethylaminopiperidine, neomycin and Ca2+. These actions are reversed by the polyamine glycine produced by polyamine agonists appears to be due to an increase in the equilibrium affinity of [3H]dizocilpine, and cannot therefore be attributed solely to modulation of glycine binding. Interestingly, 1,5-diethylaminopiperidine increases the maximum effect of glycine to a greater extent than it alters glycine affinity, suggesting that the two effects may be mediated by different sites or mechanisms. These studies will help to define the role of the glycine site in the modulation of the N-methyl-D-aspartate receptor by polyamines.

Topics: Animals; Biguanides; Binding, Competitive; Dizocilpine Maleate; Dose-Response Relationship, Drug; Glycine; Polyamines; Rats; Rats, Inbred Strains; Receptors, N-Methyl-D-Aspartate

Topics: Animals; Biguanides; Dizocilpine Maleate; Histamine Release; In Vitro Techniques; Kinetics; Mast Cells; p-Methoxy-N-methylphenethylamine; Piperidines; Rats; Receptors, N-Methyl-D-Aspartate; Spermine

In the present study the influence of arcaine (0.01-1 microgram/rat), an in vitro putative non-competitive antagonist of the NMDA receptors, on cardiovascular changes induced by intracerebral administration of N-methyl-D-aspartate (NMDA) (0.1 microgram/rat) has been evaluated. Both NMDA and arcaine were microinjected into the periaqueductal gray (PAG) area of anesthetized rats. Arcaine did not decrease NMDA-induced arterial hypertension and tachycardia, but, in a dose-related manner, increased the NMDA-induced cardiovascular effects. Moreover, treatment with arcaine was not able per se to modify arterial blood pressure and heart rate basal values. Although updated in vitro reports indicate arcaine as a blocker of the NMDA receptors by an open channel mechanism, our in vivo results, at the level of the PAG area, show this not to be true. Indeed the drug may facilitate NMDA receptor activation.

Topics: 2-Amino-5-phosphonovalerate; Anesthesia; Animals; Biguanides; Blood Pressure; Dizocilpine Maleate; Dose-Response Relationship, Drug; Heart Rate; Hemodynamics; Male; N-Methylaspartate; Periaqueductal Gray; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate

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

This study used [3H]dizocilpine ([3H]MK-801) binding to the N-methyl-D-aspartate (NMDA) receptor to examine redox, polyamine, and glycine modulatory sites in membranes derived from the superior frontal and the superior temporal cortex of patients with Alzheimer's disease. In control subjects the competitive polyamine site antagonist arcaine inhibited [3H]dizocilpine binding in a dose-dependent fashion and this curve was shifted to the right by the addition of 50 microM spermidine. Arcaine inhibition of binding was more potent in the temporal cortex than in the frontal cortex, in both the absence and presence of 50 microM spermidine. In Alzheimer's disease, arcaine inhibition of [3H]dizocilpine binding (in both the absence and the presence of spermidine) was not different from control in either of the two brain areas examined. The sulfhydryl redox site of the NMDA receptor was assessed using the oxidizing agent 5,5'-dithio-bis(2-nitrobenzoic acid), which inhibited binding in a dose-dependent fashion. This inhibition was similar in patients with Alzheimer's disease and control subjects. Glycine-stimulated [3H]dizocilpine binding was also unaffected in patients with Alzheimer's disease. However, in the temporal cortex there was a significant age-associated decline in [3H]dizocilpine binding in the presence of 100 microM glutamate (Rs = -0.71) and 100 microM glutamate plus 30 microM glycine (Rs = -0.90). There was also an age-related increase in arcaine IC50 (which reflects an age-related decrease in arcaine affinity) in the frontal cortex, determined both in the absence (Rs = 0.83) and the presence (Rs = 0.79) of spermidine. These data indicate that the NMDA receptor and its modulatory redox, polyamine, and glycine subsites are intact in patients with Alzheimer's disease and that the modulatory activity of polyamine and glycine sites decline with aging.

Topics: Aged; Aged, 80 and over; Alzheimer Disease; Analysis of Variance; Biguanides; Binding Sites; Brain; Cell Membrane; Cerebral Cortex; Dithionitrobenzoic Acid; Dizocilpine Maleate; Female; Frontal Lobe; Glycine; Humans; Male; Middle Aged; Organ Specificity; Oxidation-Reduction; Radioligand Assay; Receptors, N-Methyl-D-Aspartate; Spermidine; Temporal Lobe

Micromolar concentrations of beta-amyloid (25-35) or substance P stimulated [3H]MK-801 binding in the presence of low concentrations of glutamate (1 microM) and glycine (0.02 microM). Unlike polyamines spermine and spermidine, neither beta-amyloid (25-35) nor substance P increased [3H]MK-801 binding in the presence of maximally stimulating concentrations of glutamate and glycine. 5,7-Dichloro-kynurenic acid, CGS-19755, and arcaine completely inhibited the stimulated [3H]MK-801 binding. There was an apparent decreased potency of the [3H]MK-801 binding inhibition curve for 5,7-dichlorokynurenic acid, but not CGS-19755 or arcaine, in the presence of either beta-amyloid (25-35) or substance P. The compounds do not appear to act through the strychnine-insensitive glycine binding site because neither beta-amyloid (25-35) nor substance P displaced [3H]glycine binding. Full-length beta-amyloid (1-40), up to 10 microM, did not stimulate [3H]MK-801 binding. Concentrations > 10 microM could not be tested because they formed large aggregate precipitates in the assay. The data indicate that beta-amyloid (25-35) or substance P does not stimulate [3H]MK-801 binding at either the N-methyl-D-aspartate, glycine, or polyamine binding sites. Furthermore, the nonpeptide substance P receptor (NK1) antagonist, CP-96,345, did not block beta-amyloid (25-35)- or substance P-stimulated [3H]MK-801 binding. Therefore, the effect is not due to an interaction between the substance P receptors and the N-methyl-D-aspartate receptor-operated ionophore. Finally, if these observations can be verified using single-channel recording techniques, they may have implications in the pattern of selective neuronal loss observed in patients with neurodegenerative processes such as Alzheimer's, Parkinson's, and Huntington's diseases.

Topics: Amino Acid Sequence; Amyloid beta-Peptides; Animals; Biguanides; Cell Membrane; Cerebral Cortex; Dizocilpine Maleate; Glutamates; Glutamic Acid; Glycine; Kinetics; Molecular Sequence Data; N-Methylaspartate; Peptide Fragments; Pipecolic Acids; Rats; Receptors, N-Methyl-D-Aspartate; Sequence Homology, Amino Acid; Substance P

Arcaine, a putative competitive antagonist at the polyamine site on the N-methyl-D-aspartate (NMDA) receptor complex, not only inhibits polyamine enhancement of NMDA-induced [3H]dizocilpine (MK-801) binding but also depresses binding in the absence of polyamines. In the present experiments, we investigated the mechanism of this latter effect in whole-cell and single-channel recordings from cultured rat hippocampal neurons. Arcaine produced a concentration-dependent block of NMDA-evoked inward currents (KD, 61 microM at -60 mV) but not those induced by kainate, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, or gamma-aminobutyric acid. The arcaine block was strongly voltage dependent and was almost completely relieved at positive holding potentials. Analysis of the voltage dependence indicated that the arcaine acceptor site appeared to sense 67% of the transmembrane electric field. In support of an open channel blocking mechanism, arcaine, like Mg2+, prevented dizocilpine from blocking the NMDA receptor channel. Moreover, increasing the dizocilpine concentration partially overcame the arcaine effect, indicating a competitive interaction between arcaine and dizocilpine. Spermine, which in our preparation usually produced only an arcaine-like voltage-dependent block of NMDA currents at high concentrations (greater than 100 microM), had no effect on the block by arcaine at lower concentrations. In single-channel recordings, arcaine caused a concentration- and voltage-dependent decrease in apparent channel amplitude. Assuming a simple model of open channel block, we estimate the arcaine binding and unbinding rates as 4.4 x 10(8) M-1 sec-1 and 1.8 x 10(4) sec-1, respectively, which are comparable to the rates for open channel block by Zn2+ and substantially faster than those of Mg2+. These results indicate that arcaine inhibits NMDA-induced [3H]dizocilpine binding by blocking the open NMDA receptor channel, an action that is independent of the polyamine site.

Topics: alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Biguanides; Cations, Divalent; Cells, Cultured; Dizocilpine Maleate; gamma-Aminobutyric Acid; Glycine; Hippocampus; Ibotenic Acid; Ion Channels; Kainic Acid; Magnesium; Membrane Potentials; N-Methylaspartate; Neurons; Polyamines; Rats; Rats, Inbred Strains; Receptors, N-Methyl-D-Aspartate

This study investigated the mechanism of action of two novel, noncompetitive antagonists of the N-methyl-D-aspartate (NMDA) receptor using [3H]dizocilpine binding to rat brain membranes. Pentamidine and 1,10(bisguanidino)decane (BG10) inhibited [3H]dizocilpine binding in the concentration range of 1 to 100 microM, as did Zn++ and the competitive polyamine antagonist arcaine. The action of each of these agents was sensitive to the addition of spermidine to the assay. However, only arcaine interacted with spermidine in a competitive fashion. Spermidine decreased the apparent affinity of Zn++ and also increased the Hill slope of the Zn++ inhibition curves. BG10 and pentamidine inhibition of [3H]dizocilpine binding was less sensitive to spermidine. Calcium had similar effects to spermidine on the inhibition of [3H]dizocilpine binding by Zn++, arcaine, pentamidine and GB10, but was less potent than spermidine. Treating membranes with diethylpyrocarbonate, a histidine-modifying reagent, decreased the apparent affinity of Zn++ by more than 4-fold while having very modest effects on the actions of BG10, pentamidine and arcaine. In addition, Zn++ failed to slow the dissociation of [3H]dizocilpine in diethylpyrocarbonate-treated tissue, whereas the action of BG10 and pentamidine was qualitatively unaffected. These data show that the effects of BG10 and pentamidine on the NMDA receptor are complex and may involve more than one binding site for each drug. In addition, this study shows that the action of Zn++ on the NMDA receptor is modulated by polyamines. Finally, the mechanism of action of pentamidine and BG10 cannot be attributed to an action at the Zn++ recognition site.

Topics: Animals; Biguanides; Binding Sites; Brain Chemistry; Diethyl Pyrocarbonate; Dizocilpine Maleate; Dose-Response Relationship, Drug; Drug Interactions; Guanidines; Pentamidine; Rats; Receptors, N-Methyl-D-Aspartate; Spermidine; Zinc

1. Agonists may act at any one of three sites on the N-methyl-D-aspartate (NMDA) receptor-effector complex to promote opening of the associated ion channel. The three sites are activated by i) NMDA, L-glutamate, aspartate, and other dicarboxylic amino acids; ii) glycine, D-serine, D-cycloserine, and others; iii) the polyamines spermine or spermidine, but not cadaverine or putrescine. 2. This opening by exogenous agonists is reflected by an enhanced binding of the phencyclidine-like dissociative anesthetic [3H]MK-801 to rat cortical membranes (well washed to remove endogenous agonists, e.g., L-glutamate, glycine). 3. The effects of adding combinations of agonists yielded stimulation approximately equal to the sum of each agonist's effect, suggesting that in the first approximation the three classes act at independent sites. 4. When the glutamate (E) site was antagonized with D-2-amino-5-phosphonopentanoate (D-AP5), no stimulation in binding could be elicited by agonists at the two other sites. Activation of the E site is therefore necessary but not sufficient for channel opening. 5. When the glycine (G) site was antagonized with 7-chlorokynurenate, no stimulation in binding could be elicited by agonists at the other two sites. Activation of the G site is therefore necessary but not sufficient for channel opening. 6. Of the two putative antagonists for the polyamine (PA) site, ifenprodil fails to completely inhibit the binding of [3H]MK-801, whereas arcaine inhibited [3H]MK-801 binding completely. We present data which question the selectivity of arcaine for the polyamine site, and propose that the polyamine site is merely modulatory, but neither necessary nor sufficient, for channel opening.

Topics: 2-Amino-5-phosphonovalerate; Adrenergic alpha-Antagonists; Animals; Biguanides; Binding, Competitive; Biogenic Polyamines; Biotransformation; Brain; Dizocilpine Maleate; Glutamates; Glutamic Acid; Glycine; In Vitro Techniques; Kynurenic Acid; Piperidines; Rats; Receptors, N-Methyl-D-Aspartate; Spermidine; Spermine

This study investigated the interaction between the polyamines spermine and spermidine and the N-methyl-D-aspartate (NMDA) receptor by using (+)-[3H]-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-im ine maleate ([3H]MK801) binding to well washed rat brain membranes. The actions of arcaine, agmatine, diethylenetriamine and 1,8-octanediamine as polyamine antagonists were compared to use as tools in this study. Arcaine was found to be the antagonist of choice due to its greater potency. Several divalent cations, including Ba++, Ca++ and Sr++, but not Zn++, decreased the apparent potency of arcaine. These cations enhance [3H]MK801 binding in a similar fashion to spermidine and spermine suggesting that they may share a common site and mechanism of action. Moreover, arcaine competitively reduced the enhancement of [3H]MK801 binding produced by Sr++ did not alter the inhibition produced by higher concentrations of this cation, a phenomenon that also occurs with spermidine. The distinct arcaine sensitivity of the two separate phases of the concentration-response curves of both spermidine and Sr++ suggests two separate mechanisms underlying the action of spermidine-like drugs on the NMDA receptor. Further investigation of the increase in [3H]MK801 binding produced by spermidine revealed that spermidine increased the equilibrium affinity of this ligand by 2-fold without significantly altering the density of binding sites. In contrast, polyamine induced increases in the dissociation of [3H]MK801 required higher polyamine concentrations than necessary to increase ligand binding and were relatively insensitive to arcaine. These findings suggest that polyamines do not activate or promote the activation of the NMDA receptor, but instead enhance [3H]MK801 binding by allosterically increasing ligand affinity.

Topics: Animals; Biguanides; Binding, Competitive; Cations, Divalent; Dizocilpine Maleate; Drug Interactions; Kinetics; Polyamines; Rats; Receptors, N-Methyl-D-Aspartate; Tritium

Topics: Biguanides; Binding, Competitive; Biogenic Polyamines; Dibenzocycloheptenes; Dizocilpine Maleate; Receptors, N-Methyl-D-Aspartate; Receptors, Neurotransmitter; Spermidine