NITRIC-OXIDE DONORS ENHANCE NEUROTROPHIN-INDUCED NEURITE OUTGROWTH THROUGH A CGMP-DEPENDENT MECHANISM

Nitric oxide (NO), a diffusible and unstable gas, has been implicated in inter- and intra-cellular communication in the nervous system. NO a lso plays a role in neural development, plasticity and alterations of synaptic function such as long-term potentiation and longterm depressi on (Gally et al.: Proc NY Acad Sci, 87: 354-355, 1990; Zhuo et al.: Sc ience 260:1946-1950, 1993; Schuman and Madison.: Science 254:1503-1506 , 1991; Bruhwyler et al.: Neurosci Biobehav Rev 17:373-384, 1993) some of which likely involve growth and remodelling of neurites. Some acti ons of NO are mediated directly by protein modification (e.g., nitrosy lation) and others by activation of soluble guanylyl cyclase (soluble GC), which increases intracellular levels of guanosine 3',5'-cyclic mo nophosphate (cGMP). NO is synthesized by the enzyme nitric oxide synth ase (NOS), which is induced by treatment of CNS neurons (Holtzman et a l.: Neurobiol Disease 1:51-60, 1994) or pheochromocytoma PC12 cells (H irsch et al.: Curr Biol 3:749-754, 1993) with NGF. NO has been propose d to mediate some of the effects of NGF on PC12 cells by inhibiting ce ll division (Peunova and Enikolopov: Nature 374:68-73, 1995). In addit ion, NO can substitute for NGF by delaying the death of trophic factor -deprived PC12 cells through a mechanism that does not involve a cytos tatic action (Farinelli et al.: J Neurosci 16:2325-2334, 1996). We inv estigated whether NO stimulated neurite outgrowth from hippocampal neu rons and PC12 cells. Primary cultures of E17 mouse hippocampal neurons co-cultured with neopallial astrocytes were exposed to the NO donors sodium nitrite (100 mu M) or sodium nitroprusside (100 nM). After 48 h r, NO donor-treated cultures contained a greater proportion of cells b earing neurites and neurites that were much longer than those found in control cultures. In cultures of PC12 cells, NO donors also enhanced the neuritogenic effects of NGF. The proportion of PC12 cells with neu rites 48 hr after exposure to NO donors sodium nitrite (100 mu M-10 mM ) or sodium nitroprusside (100 nM-1 mu M) plus 2.5S nerve growth facto r (NGF) was approximately twice the proportion of cells with neurites in sister cultures grown in NGF alone. Neither of the NO donors elicit ed neurites from the PC12 cells in the absence of NGF. The effects of the NO donors were likely mediated by release of NO since their effect s were antagonized by addition of hemoglobin, which avidly binds NO, t o the culture medium. The enhancement by NO of NGF-mediated neurite ou tgrowth in PC12 cells appeared to occur through a cGMP-dependent mecha nism. The NO donors stimulated a prompt increase in intracellular cGMP in PC12 cells. Moreover their action was mimicked by addition of the membrane-permeant cGMP analogs 8-Bromo-cGMP (8-Br-cGMP) and para (chlo rophenylthio)-cGMP (pCPT-cGMP) to the culture medium and by atrial nat riuretic factor which stimulates particulate guanylyl cyclase. The neu ritogenic activity of the NO donors was inhibited by LY83583 and methy lene blue, inhibitors of guanylyl cyclase. These data imply that NO ma y act alone or with other growth factors to regulate synapse formation and maintenance by stimulating neurite outgrowth. (C) 1997 Wiley-Liss , Inc.