Protection by imidazol(ine) drugs and agmatine of glutamate‐induced neurotoxicity in cultured cerebellar granule cells through blockade of NMDA receptor

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 μ M ) caused a concentration‐dependent neurotoxicity, as determined 24 h later by a decrease in the ability of the cells to metabolize 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazoliumbromide (MTT) into a reduced formazan product. L ‐glutamate‐induced neurotoxicity (EC 50 =5 μ M ) was blocked by the specific NMDA receptor antagonist MK‐801 (dizocilpine). Imidazol(ine) drugs and agmatine fully prevented neurotoxicity induced by 20 μ M (EC 100 ) L ‐glutamate with the rank order (EC 50 in μ M ): 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)>RX821002 (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 [ 3 H]‐(+)‐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 [ 3 H]‐(+)‐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 IC 50 of 10–12 μ M 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.British Journal of Pharmacology (1999) 127 , 1317–1326; doi: 10.1038/sj.bjp.0702679