Fundamental role of nitric oxide in neuritogenesis of PC12h cells

1 We investigated the neuritogenic action of nitric oxide (NO)-generating agents and their mechanisms of action in a subclone of rat pheochromocytoma, PC12h cells. 2 NO donors such as sodium nitroprusside (SNP, 0.05-1 microM), NOR1 (5-100 microM), NOR2 (5-20 microM), NOR3 (5-20 microM), NOR4 (5-100 microM), or S-nitroso-N-acetyl-DL-penicillamine (SNAP, 10-100 microM) significantly induced neurite outgrowth. 3 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 microM), 1H-[1,2,4]oxadiazolo[4,3-a] quinoxalin-1-one (ODQ, 100 microM) and KT5823 (0.2-1 microM), respectively. 4 The intracellular cGMP concentration of cells was markedly increased by treatment with NOR4 (100 microM). 5 A mitogen-activated protein kinase (MAPK) kinase inhibitor, PD98059 (10-50 microM), 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. 6 A membrane-permeable cGMP analog, 8-Br-cGMP (1 mM) also induced significant neurite outgrowth. The 8-Br-cGMP-induced neurite outgrowth was almost completely inhibited by both KT5823 (0.5 microM) and PD98059 (50 microM). Moreover, sustained ERK phosphorylation was observed in the 8-Br-cGMP-treated PC12h cells. 7 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.