dizocilpine-maleate and ferrous-chloride

Rat cortical neurons cultured for 3 days in vitro were loaded with the fluorescent indicator fluo-3 for assessment of intracellular free calcium ion (Ca2+) concentrations with the aid of a confocal laser-scanning microscope. In the absence of added MgCl2, the addition of NMDA induced a rapid but sustained increase in the number of fluorescent neurons in a concentration-dependent manner at a concentration range of 1-100 micro m with the increase by KCl being transient. The addition of FeCl2, but not FeCl3, markedly inhibited the increase by NMDA in a reversible manner at concentrations of 10-200 micro m, without affecting that by KCl. Extensive analyses revealed clear differentiation between inhibitions by ferrous iron and other channel blockers known to date. The inhibition by FeCl2 was completely prevented by the addition of two different iron chelators. Exposure to NMDA alone did not lead to cell death in immature cultured neurons, however, while further addition of FeCl2 invariably induced neuronal cell death 24 h after exposure. These results give support to our previous proposal that NMDA receptor complex may contain a novel site sensitive to blockade by ferrous iron in rat brain.

Topics: Animals; Calcium; Cell Death; Cell Survival; Cells, Cultured; Cerebral Cortex; Chlorides; Dizocilpine Maleate; Dose-Response Relationship, Drug; Excitatory Amino Acid Antagonists; Ferric Compounds; Ferrous Compounds; Fluorescent Dyes; Ion Transport; Iron Chelating Agents; Magnesium Chloride; Microscopy, Confocal; N-Methylaspartate; Neurons; Potassium Chloride; Rats; Receptors, N-Methyl-D-Aspartate; Zinc Compounds

In vitro addition or pretreatment with >/=1 microM ferrous chloride markedly inhibited in a concentration-dependent manner [3H]dizocilpine (MK-801) binding to an open ion channel associated with the N-methyl-D-aspartate (NMDA) receptor in rat brain synaptic membranes. The addition of NMDA agonists invariably attenuated the inhibition of [3H]MK-801 binding in hippocampal synaptic membranes previously treated with ferrous chloride, without significantly affecting that in cerebellar synaptic membranes. In the absence of spermidine, ferrous chloride was more potent in inhibiting binding in the cerebral cortex and hippocampus in adult rats than in those in rats at 3 days after birth, while in the striatum [3H]MK-801 binding was 10 times more sensitive to inhibition by added ferrous chloride in neonatal rats than in adult rats. Addition of spermidine significantly attenuated the potency of ferrous chloride to inhibit binding in the cerebral cortex of adult rats, with facilitation of the inhibition in newborn rats. Moreover, spermidine significantly reduced the inhibitory potency of ferrous chloride in neonatal rat striatum, without markedly affecting that in adult rat striatum. These results suggest that ferrous ions may interfere with opening processes of the native NMDA channel through molecular mechanisms peculiar to neuronal development in a manner associated with the polyamine recognition domain.

Topics: Animals; Animals, Newborn; Brain; Dizocilpine Maleate; Ferrous Compounds; Ion Channels; Male; Radioligand Assay; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate; Synaptic Membranes; Tritium

Quinolinic acid (QUIN) displaced binding of agonist and antagonist ligands for the N-methyl-D-aspartate (NMDA) receptor in rat brain synaptic membranes. Both QUIN and glutamic acid (GLU) potentiated binding of [3H]dizocilpine (MK-801) in the presence of glycine (GLY) alone, whereas the potentiation by QUIN was in a bell-shaped fashion in contrast to that by GLU. However, further addition of spermidine (SPD) induced bell-shaped potentiation by GLU as well as QUIN. The potentiation by QUIN was markedly deteriorated by the further addition of FeCl2 irrespective of the presence of GLY and SPD added. These results suggest that QUIN may potentiate [3H]MK-801 binding to the open NMDA channel in rat brain synaptic membranes through a mechanism different from that underlying the potentiation by GLU.

Topics: 2-Amino-5-phosphonovalerate; Allosteric Regulation; Animals; Binding Sites; Brain; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Ferrous Compounds; Glutamic Acid; Glycine; Inhibitory Concentration 50; Kynurenic Acid; Male; Quinolinic Acid; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate; Spermidine; Synaptic Membranes

Prior treatment with ferrous chloride led to marked inhibition of [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imi ne (MK-801) binding to an open ion channel associated with the N-methyl-D-aspartate (NMDA) receptor in a concentration-dependent manner at concentrations of higher than 1 microM in rat brain synaptic membranes. Both phospholipases A2 and C significantly prevented the inhibition when treated before the treatment with ferrous chloride, while neither superoxide dismutase nor alpha-tocopherol affected the inhibition even when treated simultaneously with ferrous chloride. Of various saturated and unsaturated free fatty acids, moreover, both oleic and arachidonic acids exclusively decreased the potency of ferrous chloride to inhibit binding when membranes were first treated with fatty acids, followed by the second treatment with ferrous chloride. These results suggest that membrane phospholipids may be at least in part responsible for interference by ferrous ions with opening processes of the native NMDA channel through molecular mechanisms associated with the liberation of unsaturated free fatty acids in rat brain.

Topics: Animals; Brain; Dizocilpine Maleate; Fatty Acids; Ferrous Compounds; Free Radical Scavengers; Iron Chelating Agents; Male; Phospholipases; Radioligand Assay; Rats; Rats, Wistar; Synaptic Membranes; Tritium

Prior treatment with ferrous chloride led to the marked inhibition of [3H](+)-5-methyl-10, 11-dihydro-5H-dibenzo[a, d]cyclohepten-5, 10-imine (MK-801) binding to an open ion channel associated with the N-methyl-D-aspartate (NMDA) receptor in a concentration-dependent manner at concentrations of higher than 1 microM in rat brain synaptic membranes. Both phospholipases A2 and C significantly prevented the inhibition when treated before the treatment with ferrous chloride, while neither superoxide dismutase nor alpha-tocopherol affected the inhibition even when treated simultaneously with ferrous chloride. Of various saturated and unsaturated fatty acids, moreover, both oleic and arachidonic acids exclusively decreased the potency of ferrous chloride to inhibit binding when membranes were treated with fatty acids, followed by a second treatment with ferrous chloride. These results suggest that membrane phospholipids may be, at least in part, responsible for the interference by ferrous ions in the opening processes of the native NMDA channel through molecular mechanisms associated with the release of unsaturated fatty acids in rat brain.

Topics: Animals; Brain; Dizocilpine Maleate; Fatty Acids; Ferrous Compounds; In Vitro Techniques; Ion Channels; Iron; Male; Membrane Lipids; Phospholipases; Phospholipids; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate; Superoxide Dismutase; Synaptic Membranes; Vitamin E

The addition of sodium nitroprusside (SNP) significantly inhibited binding of (+)-5- [3H]methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine ([3H]MK-801) to an ion channel associated with the N-methyl-D-aspartate (NMDA) receptor in a concentration-dependent manner at concentrations of >1 microM in rat brain synaptic membranes not extensively washed. However, neither S-nitroso-N-acetylpenicillamine nor S-nitroso-L-glutathione inhibited binding even at 100 microM. Of the two compounds structurally related to SNP (II), similarly potent inhibition was induced by potassium ferrocyanide (II) but not by potassium ferricyanide (III). In addition, ferrous chloride (II) induced much more potent inhibition of binding than ferric chloride (III), at a similar concentration range. In contrast, iron chelators prevented the inhibition by ferrous chloride (II) without markedly affecting that by SNP (II) and potassium ferrocyanide (II). Pretreatment with ferrous chloride (II) also led to potent inhibition of [3H]MK-801 binding in a manner insensitive to subsequent addition of the iron chelators. Pretreatment with Triton X-100 resulted in significant potentiation of the ability of ferrous chloride (II) to inhibit [3H]MK-801 binding irrespective of the addition of agonists, moreover, although binding of other radioligands to the non-NMDA receptors was unaltered after pretreatment first with Triton X-100 and then with ferrous chloride (II). These results suggest that ferrous ions (II) may interfere selectively with opening processes of the NMDA channel through mechanisms entirely different from those underlying the inhibition by both SNP (II) and potassium ferrocyanide (II) in rat brain.

Topics: alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Brain; Chlorides; Dizocilpine Maleate; Ferric Compounds; Ferricyanides; Ferrocyanides; Ferrous Compounds; Ion Channels; Iron Chelating Agents; Kainic Acid; Male; Nitroprusside; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate; Synaptic Membranes