dizocilpine-maleate and ferric-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

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

Excitatory amino acids and oxygen free radicals have been reported to cooperate in the genesis of brain injury in vivo and in vitro. In this study, we tested the capacity of a noncompetitive N-methyl-D-aspartate receptor antagonist, MK-801, and a 21-aminosteroid, U-74006F, tirilazad mesylate, to block the opening of the blood-brain barrier after subarachnoid injection of FeCl2, which is believed to cause a primarily "pure" free radical insult. Subarachnoid injection of FeCl2 resulted in a significant 10-fold increase in Evans blue extravasation while sham injection or NaCl injection had no effect. Pretreatment with either MK-801 or U-74006F significantly reduced the FeCl2-induced increase in capillary permeability by 43 and 63%, respectively (p < 0.05). Combined treatment with MK-801 and U-74006F resulted in a 65% reduction in vascular leakage that was not significantly greater than pretreatment with either drug alone. These results show that both excitatory amino acids and free radicals can damage the cerebral microvasculature and that an excitatory amino acid antagonist can partially protect the blood-brain barrier after free radical-induced injury.

Topics: Amino Acids; Animals; Blood Pressure; Blood-Brain Barrier; Chlorides; Dizocilpine Maleate; Evans Blue; Ferric Compounds; Free Radical Scavengers; Free Radicals; Injections; Intracranial Pressure; Male; Pregnatrienes; Rats; Rats, Sprague-Dawley; Subarachnoid Space