The nitric oxide signaling pathway inhibits intracellular calcium release to prevent neurodevelopmental alcohol toxicity

In the context of fetal alcohol spectrum disorders, we investigated how the nitric oxide (NO) signaling pathway influences intracellular calcium (Ca) to mediate alcohol resistance, using a primary cell culture model of cerebellar granule neurons (CGN). Alcohol during fetal brain development triggers abnormally high apoptotic cell death in vulnerable neuronal populations, culminating in serious behavioral and cognitive deficits that persist into adulthood. Prior studies demonstrated that the NO signaling pathway [neuronal nitric oxide synthase → NO → soluble guanylyl cyclase → cyclic guanosine monophosphate → protein kinase G (PKG)] mitigates alcohol toxicity, consequently diminishing neuronal loss both in vivo and in vitro. Endoplasmic reticulum (ER) Ca release, a key apoptotic mechanism, requires the inositol 1,4,5-trisphosphate receptor (IP3R), a known PKG substrate. Our studies focused on this crucial intersection point where the NO signaling cascade can influence Ca-mediated apoptotic mechanisms, and exposed a downstream mechanism where NO can moderate alcohol neurotoxicity. We hypothesized that as alcohol disturbs neuronal Ca homeostasis to trigger cell death, the NO signaling pathway counters it by limiting Ca release from the ER. We examined first the role of the phospholipase C (PLC) pathway [PLC → inositol 1,4,5trisphosphate → IP3R → Ca] in developmental neurotoxicity through our in vitro CGN model, extending previous in vivo studies. We found that alcohol terminates developing neurons by eliciting abnormal Ca release from the ER rather than from an extracellular source, via a PLC – IP3R-dependent signaling mechanism. Inhibiting either calcineurin or Ca / calmodulin-dependent protein kinase ii (CaMKii), which participate in parallel Ca-activated apoptotic cascades, shielded CGN cultures from alcohol. Blocking the mitochondrial Ca uniporter or the mitochondrial permeability transition pore also provided neuroprotection. That the activated pathways must interact to generate cell