Involvement of reactive nitrogen species production in diazoxide‐induced preconditioning in cultured cortical neurons of rats (841.1)

Abstract

Diazoxide (DZ), a mitochondrial ATP‐sensitive potassium (mito K‐ATP) channel opener, has been shown to be protective in models of brain ischemia. We have previously shown that reactive oxygen species production and mitochondrial depolarization are major initiating steps in DZ preconditioning, but the signaling pathway is incomplete. We hypothesized that other signaling pathways, like those involved in RNS production, may participate in the protective effects of DZ. Confluent rat primary neuronal cultures were pre‐treated with DZ (500µM) alone or co‐treated with DZ+ Nω‐Nitro‐L‐arginine methyl ester hydrochloride (L‐NAME, 100µM or 150µM) for 3 days to block nitric oxide (NO) synthase. Additionally, NO was exogenously supplied by co‐applying SNP (5µM) with DZ and L‐NAME to potentially rescue protection by DZ. After the 3 day treatment period, neurons were subjected to 3 h oxygen glucose deprivation (OGD) followed by re‐oxygenation, an in vitro model of stroke. Neuronal viability was assessed using the MTS colorimetric assay 24 h after OGD. OGD reduced neuronal viability 24 h from 100% (time control) to 54.1±1.4% (P<0.05; n=32 wells). DZ protected cells and increased viability after OGD (67±2.4%, P<0.05; n=32 wells). Blocking RNS using L‐NAME 100µM and 150µM decreased viability to the untreated control level (53.2±3.9% and 50.6±3.9% respectively, NS compared to time control; n=32 wells). Replacing NO using SNP rescued the effect of L‐NAME treatment at both doses (63.5±6.3% and 60.8±6.5%, respectively) (NS compared to time control; n=32 wells). This study shows for the first time that RNS production is a crucial step to preconditioning using DZ in neurons.Grant Funding Source: HL‐077731, HL‐030260, HL‐065380, and HL‐093554