Prevention of glutamate neurotoxicity in cultured neurons by 3,4-dihydro-6-hydroxy-7-methoxy-2,2-dimethyl-1(2H)-benzopyran (CR-6), a scavenger of nitric oxide.

Abstract Glutamate neurotoxicity in cerebellar neurons in culture is mediated by excessive production of nitric oxide (NO). We anticipated that 3,4-dihydro-6-hydroxy-7-methoxy-2,2-dimethyl-1(2 H )-benzopyran (CR-6) could act as a scavenger of NO since it contains a position (C-5) highly activated towards nitration reaction. The aim of this work was to assess whether CR-6 acts as an NO scavenger and prevents glutamate neurotoxicity in cultures of cerebellar neurons. It was shown that CR-6 reduced, in a dose-dependent manner, glutamate-induced formation of cGMP ( ec 50 ≈ 15 μM) and prevented glutamate neurotoxicity. The protection was ≈50% at 3–10 μM and nearly complete at 100 μM. CR-6 did not prevent glutamate-induced activation of NO synthase, but interfered with the glutamate-NO-cGMP pathway at a later step. CR-6 reduced the formation of cGMP induced by S -nitroso- N -acetylpenicillamine (SNAP), an NO-generating agent, indicating that CR-6 acts as a scavenger of NO in cultured neurons. This was further supported by experiments showing that in neurons treated with CR-6 and glutamate, the 5-nitro derivative of CR-6 was formed, as determined by GC-MS analyses. Moreover, in vitro incubation of CR-6 with SNAP also produced the 5-nitroderivative, thus confirming that CR-6 directly reacts with NO. The results reported indicate that CR-6 acts as an NO scavenger in neurons and prevents glutamate neurotoxicity.