Nitric oxide (NO) is required for neuronal differentiation: A cGMP independent pathway. † 1710

Abstract

PC12 cells are used as a model system to study neuronal differentiation. Nerve growth factor (NGF) treatment of PC12 cells induces neurite outgrowth (a measure of differentiation) and neuronal nitric oxide synthase (nNOS) protein expression. Neurite outgrowth in PC12 cells is significantly reduced in the presence of the NOS inhibitor, L-NAME, implicating NO in the differentiation process. To further evaluate the role of NO in neuronal differentiation, PC12 cells were treated with either the NO donor, sodium nitroprusside, or the cGMP analog, 8Br-cGMP. In neither case was differentiation induced. Thus, NO is necessary but not sufficient for PC12 cell differentiation. Since NO has both para- and autocrine properties, the effect of oxyhemoglobin (a scavenger of NO in the medium) on NGF-treated PC12 cells was also determined. This treatment significantly reduced the number of neurites produced by NGF treated PC12 cells. Thus, cell-cell communication appears to be necessary for NO to exert its effect during the differentiation process. Most of the effects of NO are thought to be mediated by soluble guanylyl cyclase (sGC) activation with the resulting product, cGMP, acting as a secondary messenger. Thus, sGC activation as the means by which NO is involved in PC12 cell differentiation was evaluated. In the presence of NGF (to prime PC12 cells for differentiation) and L-NAME (to inhibit NO synthesis) the addition of SNP resulted in PC12 cell differentiation. However, in the presence of NGF and L-NAME, 8Br-cGMP failed to produce significant PC12 cell differentiation. Similarly, blockade of sGC activity with specific inhibitors failed to block NGF-induced PC12 cell differentiation. These results demonstrate that the action of NO is critical in PC12 cell differentiation but appears to be independent of sGC activation and cGMP production.