Neuroprotection and intracellular Ca2+ modulation with fructose-1,6-bisphosphate during in vitro hypoxia-ischemia involves phospholipase C-dependent signaling

The neuroprotectant fructose-1,6-bisphosphate (FBP) preserves cellular [ATP] and prevents catastrophic increases in [Ca2+](i) during hypoxia. Because FBP does not enter neurons or glia, the mechanism of protection is not clear. In this study, we show that FBP's capacity to protect neurons and stabilize [Ca2+](i) during hypoxia derives from signaling by a phospholipase-C-intracellular Ca2+-protein kinases pathway, rather than Ca2+ chelation or glutamate receptor inhibition. FBP reduced [Ca2+](i) changes in hypoxic hippocampal neurons, regardless of [Ca2+](e), and preserved cellular integrity as measured by trypan blue or propidium iodide exclusion and [ATP]. FBP also prevented hypoxia-induced increases in [Ca2+](i) when glucose was absent and when [Ca2+](e) was increased to negate Ca2+ chelation by FBP. These protective effects were observed equally in postnatal day 2 (P2) and P16 neurons. Inhibiting glycolysis with iodoacetate eliminated the protective effects of FBP in P16 neurons. FBP did not alter Ca2+ influx stimulated by brief applications of NMDA or glutamate during normoxia or hypoxia, but did reduce the increase in [Ca2+](i) produced by 10 min of glutamate exposure during hypoxia. Because FBP increases basal [Ca2+](i) and stimulates membrane lipid hydrolysis, we tested whether FBP's protective action was dependent on phospholipase C signaling. The phospholipase C inhibitor U73122 prevented FBP-induced increases in [Ca2+](i) and eliminated FBP's ability to stabilize [Ca2+](i) and increase survival during anoxia. Similarly. FBP's protection was eliminated in the presence of the mitogen/extracellular signal protein kinase (MEK) inhibitor U0126. We conclude that FBP may produce neuroprotection via activation of neuroprotective signaling pathways that modulate Ca2+ homeostasis. (C) 2001 Elsevier Science BY. All rights reserved.