L-NAME-induced dispersion of melanosomes in melanophores activates PKC, MEK and ERK1.

Melanosome movement represents a good model of cytoskeleton-mediated transport of organelles in eukaryotic cells. We recently observed that inhibiting nitric oxide synthase (NOS) with Nomega-nitro-L-arginine methyl ester (L-NAME) induced dispersion in melanophores pre-aggregated with melatonin. Activation of cyclic adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase (PKA) or calcium-dependent protein kinase (PKC) is known to cause dispersion. Also, PKC and NO have been shown to regulate the mitogen/extracellular signal-regulated kinase (MEK)-ERK pathway. Accordingly, our objective was to further characterize the signaling pathway of L-NAME-induced dispersion. We found that the dispersion was decreased by staurosporine and PD98059, which respectively inhibit PKC and MEK, but not by the PKA inhibitor H89. Furthermore, Western blotting revealed that ERK1 kinase was phosphorylated in L-NAME-dispersed melanophores. L-NAME also caused dispersion in latrunculin-B-treated cells, suggesting that this effect is not due to inhibition of the melatonin signaling pathway. Summarizing, we observed that PKC and MEK inhibitors decreased the L-NAME-induced dispersion, which caused phosphorylation of ERK1. Our results also suggest that NO is a negative regulator of phosphorylations that leads to organelle transport.

Nitric oxide modulates intracellular translocation of pigment organelles in Xenopus laevis melanophores.

Pigment organelles in Xenopus laevis melanophores are used by the animal to change skin color, and they provide a good model for studying intracellular organelle transport. Movement of organelles and vesicles along the cytoskeleton is essential for many processes, such as axonal transport, endocytosis, and intercompartmental trafficking. Nitric oxide (NO) is a signaling molecule that plays a role in, among other things, relaxation of blood vessels, sperm motility, and polymerization of actin. Our study focused on the effect NO exerts on cytoskeleton-mediated transport, which has previously received little attention. We found that an inhibitor of NO synthesis, N-nitro-L-arginine methyl ester (L-NAME), reduced the melatonin-induced aggregation of the pigment organelles, melanosomes. Preaggregated melanosomes dispersed after treatment with L-NAME but not after exposure to the inactive stereoisomer (D-NAME) or the substrate for NO synthesis (L-arginine). Signal transduction by NO can be mediated through the activation of soluble guanylate cyclase (sGC), which leads to increased production of cGMP and activation of cGMP-dependent kinases (PKG). We found that both the sGC inhibitor 1H-(1,2,4) oxadiazolo(4,3-a)quinoxalin-1-one (ODQ) and the cGMP analogue 8-bromoguanosine 3':5'-cyclic monophosphate (8-Br-cGMP) reduced melanosome aggregation, whereas the PKG inhibitor KT582 did not. Our results demonstrate that melanosome aggregation depends on synthesis of NO, and NO deprivation causes dispersion. It seems, thus, as if NO and cGMP are essential and can regulate melanosome translocation.

Nitric oxide regulates the aggregation of stimulated human neutrophils.

Neutrophil aggregation is mediated by both CD18 integrin and L-selectin. Nitric oxide attenuates the integrin-mediated adhesion of neutrophils to collagen and to endothelium and may therefore affect aggregation as well. FMLP-stimulated neutrophils exposed to l-arginine showed increased and prolonged aggregation, whereas cells pretreated with L-NAME did not differ from FMLP-stimulated controls. Nitric oxide is known to induce ADP ribosylation of G-actin, which inhibits polymerization. We detected equivalent levels of total F-actin in cells pretreated with l-arginine or L-NAME and non-pretreated controls. However, neutrophils pretreated with l-arginine and stimulated by CD18 integrin cross-linking exhibited a more limited increase in total F-actin, compared to control and L-NAME-pretreated cells. Thus at least two signaling pathways may be involved FMLP-stimulated aggregation, mediated by CD18 integrins. More specifically, it is plausible that FMLP-receptor signaling upregulates CD18 integrins and endogenous NO subsequently modulates CD18-mediated signaling to prolong aggregation, possibly through ADP-ribosylation of actin.