Both neuronal NO synthase and nitric oxide are required for PC12 cell differentiation: a cGMP independent pathway

PC12 cells are used as a model system to study neuronal differentiation. Nerve growth factor (NGF) triggers a differentiation pathway in PC12 cells. Neurite outgrowth (a morphological marker of differentiation) in PC12 cells is significantly reduced in the presence of the NOS inhibitor l-NAME, but not d-NAME, implicating NOS in the differentiation process. Previously we have shown that the neuronal NO synthase (nNOS) isoform is induced in PC12 cells in the presence of NGF. Thus, we wished to further evaluate the role of nNOS and NO in PC12 cell differentiation. When a dominant negative mutant nNOS expression vector was transiently transfected into NGF-treated PC12 cells, it significantly reduced PC12 cell neurite outgrowth. Thus, we concluded that the NO required for PC12 cell differentiation, in response to NGF, is produced by nNOS. NO alone was insufficient to induce differentiation as cells treated with the NO donor, sodium nitroprusside did not produce neurites. Treatment of PC12 cells with oxyhemoglobin (an NO scavenger) was also found to significantly reduce the number of neurites produced by PC12 cells treated with NGF. Thus, NO appears to be necessary, but not sufficient, to induce differentiation, and its mode of action appears to be extracellular. A well documented action of NO is to activate soluble guanylate cyclase. Thus, we determined the role of soluble guanylate cyclase activation as a means by which NO induces PC12 cell differentiation. However, in the presence of NGF (to prime PC12 cells for differentiation) and l-NAME (to specifically remove the NO component), 8Br-cGMP (a cGMP analog) failed to induce PC12 cell differentiation. In addition, blockade of sGC activity with specific inhibitors failed to block NGF-induced PC12 cell differentiation. We conclude that the NO required for PC12 cell differentiation is produced by nNOS and that the NO exerts its effects on surrounding PC12 cells in a sGC/cGMP independent manner.