Nitric Oxide Mediated Signal Transduction in Networks of Human ...

spheres to study the role of NO signaling in neuronal
precursor migration.
Agents that modulate cell migration via NO signaling do
not alter cell viability and differentiation
We initially examined the potential cytotoxicity of bioactive
chemical compounds affecting cell migration via NO/cGMP
signal transduction (Fig. 3a). Thus, we performed Alamar
Blue cell viability and the live/dead assay under identical
Fig. 3 Schematic drawing of NO signal
transduction together with an array of
chemical compounds that block or activate
the pathway (a). The enzyme nitric oxide
synthase (nNOS) is stimulated by calcium–
calmodulin (Ca/CaM) and can be blocked
by bath application of the inhibitor, 7-nitroindazole
(7NI). NO binds to the heme moiety
of soluble guanylyl cyclase (sGC)
resulting in the stimulation of the enzyme.
The sGC activity is blocked by the inhibitor
ODQ and stimulated independently from
NO by the sGC activator YC-1. Synthesis of
cGMP activates protein kinase G (PKG)
and regulates cell migration. The PKG
inhibitor, RP-8-Br-cGMP, blocks cellular
responses of the cGMP/PKG pathway. The
NO donors (SNP and NOC-18) and the
membrane-permeable cGMP analog (8-BrcGMP)
can be applied to raise cGMP
levels. The Live/Dead and Alamar Blue
viability/cytotoxicity assay was performed in
the presence of chemical compounds that
modulate the NO/cGMP signaling. Application
of 7NI (500 lM), ODQ (50 lM), RP-8-
Br-cGMP (100 nM), NOC-18 (50 lM), and
8-Br-cGMP (1 mM) to NT2 spheres did not
affect cell viability as determined by Live/
Dead assay (b) and Alamar Blue assay (c).
Relatively higher concentrations (1 mM) of
7NI, NOC-18, and ODQ significantly affected
cell viability as determined by Alamar
Blue assay (d–f). Application of 7NI
(500 lM), ODQ (50 lM), RP-8-Br-cGMP
(100 nM), NOC-18 (50 lM), and 8-BrcGMP
(1 mM) to NT2 spheres did not significantly
alter the bIII-tubulin-stained cells
(g). NOC-18, 2, 2-hydroxynitrosohydrazinobis-ethanamine;
RP-8-Br-cGMP, RP isomer
of 8-Br-cGMP; Ctrl, control (***p < 0.001).
(a)
experimental conditions as the migration assay. Both assay
showed that none of the pharmacological agents had any
effect on cell viability at the concentration used to modulate
cell migration (Fig. 3b and c). This was also further
confirmed by morphological observation of the spheres
including the migrating cells in the presence of the
compounds. However, when the NOS inhibitor, 7NI; sGC
inhibitor; ODQ; and NO donor, NOC-18 were used at the
rather high concentration of 1 mM, they significantly
(b) (c)
(d) (e)
(f) (g)
Ó 2009 The Authors
Journal Compilation Ó 2009 International Society for Neurochemistry, J. Neurochem. (2009) 110, 1828–1841
NO signaling regulates neuronal precursor motility | 1833