title: Protection by imidazol(ine) drugs and agmatine of glutamate-induced neurotoxicity in cultured cerebellar granule cells through blockade of NMDA receptor.
abstract: This study was designed to assess the potential neuroprotective effect of several imidazol(ine) drugs and agmatine on glutamate-induced necrosis and on apoptosis induced by low extracellular K+ in cultured cerebellar granule cells. Exposure (30 min) of energy deprived cells to L-glutamate (1-100 microM) caused a concentration-dependent neurotoxicity, as determined 24 h later by a decrease in the ability of the cells to metabolize 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) into a reduced formazan product. L-glutamate-induced neurotoxicity (EC50=5 microM) was blocked by the specific NMDA receptor antagonist MK-801 (dizocilpine). Imidazol(ine) drugs and agmatine fully prevented neurotoxicity induced by 20 microM (EC100) L-glutamate with the rank order (EC50 in microM): antazoline (13)>cirazoline (44)>LSL 61122 [2-styryl-2-imidazoline] (54)>LSL 60101 [2-(2-benzofuranyl) imidazole] (75)>idazoxan (90)>LSL 60129 [2-(1,4-benzodioxan-6-yl)-4,5-dihydroimidazole](101)>RX82 1002 (2-methoxy idazoxan) (106)>agmatine (196). No neuroprotective effect of these drugs was observed in a model of apoptotic neuronal cell death (reduction of extracellular K+) which does not involve stimulation of NMDA receptors. Imidazol(ine) drugs and agmatine fully inhibited [3H]-(+)-MK-801 binding to the phencyclidine site of NMDA receptors in rat brain. The profile of drug potency protecting against L-glutamate neurotoxicity correlated well (r=0.90) with the potency of the same compounds competing against [3H]-(+)-MK-801 binding. In HEK-293 cells transfected to express the NR1-1a and NR2C subunits of the NMDA receptor, antazoline and agmatine produced a voltage- and concentration-dependent block of glutamate-induced currents. Analysis of the voltage dependence of the block was consistent with the presence of a binding site for antazoline located within the NMDA channel pore with an IC50 of 10-12 microM at 0 mV. It is concluded that imidazol(ine) drugs and agmatine are neuroprotective against glutamate-induced necrotic neuronal cell death in vitro and that this effect is mediated through NMDA receptor blockade by interacting with a site located within the NMDA channel pore.
authors.affiliation: Associate Unit of the Institute Cajal/Consejo Superior de Investigaciones Científicas, Department of Biology, University of the Balearic Islands, Palma de Mallorca, Spain.
authors.forename: G
authors.initials: G
authors.lastname: Olmos
authors.forename: N
authors.initials: N
authors.lastname: DeGregorio-Rocasolano
authors.forename: M
authors.initials: M
authors.lastname: Paz Regalado
authors.forename: T
authors.initials: T
authors.lastname: Gasull
authors.forename: M
authors.initials: M
authors.lastname: Assumpció Boronat
authors.forename: R
authors.initials: R
authors.lastname: Trullas
authors.forename: A
authors.initials: A
authors.lastname: Villarroel
authors.forename: J
authors.initials: J
authors.lastname: Lerma
authors.forename: J A
authors.initials: JA
authors.lastname: García-Sevilla
mesh_terms: D000376:Agmatine / Q000494:pharmacology*; D000818:Animals; D000865:Antazoline / Q000494:pharmacology; D001572:Benzofurans / Q000494:pharmacology; D002460:Cell Line; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology / Q000187:drug effects* / Q000473:pathology; D004146:Dioxanes / Q000494:pharmacology*; D016291:Dizocilpine Maleate / Q000378:metabolism / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004594:Electrophysiology; D018698:Glutamic Acid / Q000494:pharmacology*; D006801:Humans; D019329:Idazoxan / Q000031:analogs & derivatives / Q000494:pharmacology; D007093:Imidazoles / Q000494:pharmacology*; D018696:Neuroprotective Agents / Q000494:pharmacology*; D018408:Patch-Clamp Techniques; D011188:Potassium / Q000494:pharmacology; D011869:Radioligand Assay; D051381:Rats; D017208:Rats, Wistar; D016194:Receptors, N-Methyl-D-Aspartate / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000502:physiology; D013343:Styrenes / Q000494:pharmacology*
chemical_list: C121753:2-(1,4-benzodioxan-6-yl)-4,5-dihydroimidazole; C121754:2-styryl-2-imidazoline; D001572:Benzofurans; D004146:Dioxanes; D007093:Imidazoles; C095677:LSL 60101; D018696:Neuroprotective Agents; D016194:Receptors, N-Methyl-D-Aspartate; D013343:Styrenes; D018698:Glutamic Acid; D016291:Dizocilpine Maleate; D000376:Agmatine; C029899:imidazole; D000865:Antazoline; C058525:2-methoxyidazoxan; C014282:cirazoline; D011188:Potassium; D019329:Idazoxan