Fundamental role of nitric oxide in neuritogenesis of PC12h cells

We investigated the neuritogenic action of nitric oxide (NO)‐generating agents and their mechanisms of action in a subclone of rat pheochromocytoma, PC12h cells. NO donors such as sodium nitroprusside (SNP, 0.05–1  μ M ), NOR1 (5–100  μ M ), NOR2 (5–20  μ M ), NOR3 (5–20  μ M ), NOR4 (5–100  μ M ), or S ‐nitroso‐ N ‐acetyl‐ DL ‐penicillamine (SNAP, 10–100  μ M ) significantly induced neurite outgrowth. NOR4‐induced neurite outgrowth was accompanied by expression of neurofilament 200 kDa subunit (NF200) protein, an axonal marker, and was significantly inhibited by an NO scavenger, a soluble GC inhibitor, and a PKG inhibitor: 2‐(4‐carboxyphenyl)‐4,4,5,5‐tetramethylimidazole‐1‐oxyl‐3‐oxide (carboxy‐PTIO, 20–100  μ M ), 1 H ‐[1,2,4]oxadiazolo[4,3‐ a ] quinoxalin‐1‐one (ODQ, 100  μ M ) and KT5823 (0.2–1  μ M ), respectively. The intracellular cGMP concentration of cells was markedly increased by treatment with NOR4 (100  μ M ). A mitogen‐activated protein kinase (MAPK) kinase inhibitor, PD98059 (10–50  μ M ), abolished the NOR4‐induced neurite outgrowth. In agreement with this observation, NOR4 did phosphorylate extracellular signal‐regulated kinase (ERK) 1 and 2, substrates of MAPK kinase. A membrane‐permeable cGMP analog, 8‐Br‐cGMP (1 m M ) also induced significant neurite outgrowth. The 8‐Br‐cGMP‐induced neurite outgrowth was almost completely inhibited by both KT5823 (0.5  μ M ) and PD98059 (50  μ M ). Moreover, sustained ERK phosphorylation was observed in the 8‐Br‐cGMP‐treated PC12h cells. These results suggest that NO itself has the ability to induce neurite outgrowth and that NO‐induced ERK activation involves the NO‐cGMP‐PKG signaling pathway in PC12h cells.British Journal of Pharmacology (2005) 146 , 662–669. doi: 10.1038/sj.bjp.0706370