MTOR Pathway Mediates Diazoxide Preconditioning in Cultured Neurons

Abstract

Diazoxide (DZ), a mitochondrial ATP‐sensitive potassium channel opener, is protective in models of brain ischemia but the signaling pathway is not clear. The mammalian target of Rapamycin (mTOR) pathway is a major regulator of protein synthesis and has been shown to be involved in protection during cerebral ischemia. We tested the hypothesis that DZ protects neurons via mechanisms involving the mTOR pathway. Cultured cortical neurons were treated with DZ, and Rapamycin (R) or Wortmannin (W) and exposed to 3 hours of oxygen‐glucose deprivation (OGD) followed by reoxygenation. Viability, mitochondrial membrane potential (Ψm), of neurons and western blots of cell homogenates were performed. OGD reduced viability (100% to 46.7% ± 0.9, p<0.05) and DZ partially rescued the viability (64.9% ± 4.5, p<0.05). W, R, DZ + R, DZ + W, DZ + R + W, however, did not alter viability after OGD. Ψm decreased after OGD (to 49.9% ± 9.5, p<0.05 vs. Ctrl) compared to untreated controls (Ctrl). DZ reduced Ψm under normoxic conditions (65.3% ± 6.2, p<0.05 vs. Ctrl) and further decreased it after OGD (26.2% ± 2.9, p<0.05 vs. Ctrl). R or W did not affect Ψm at normoxia but decreased it with OGD (50.9% ± 8.4, p<0.05 and 53.9% ± 8.9, p<0.05; respectively; both vs. Ctrl). Immuno‐blot analysis showed decreased phosphorylated S6‐ Kinase (phospho‐S6K) expression (29.9% ± 6.9, p<0.05) following OGD that was rescued by DZ (206.5% ± 52.8, p<0.05 vs. OGD). DZ + R, DZ+W, DZ + R + W decreased phospho‐S6K when compared to Ctrl. Thus, DZ preconditioning is mediated by mTOR pathway by phosphorylation of S6K. Supported by HL‐030260, HL‐065380, HL077731, & HL093554.