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Abstract: Nitric oxide (NO) generated by endothelial NO synthase (eNOS) is a key regulator of endothelial cell (EC) migration. Whereas the effects of acute NO generation are generally stimulatory, the role of chronic basal NO release has not been explored so far. Here, we addressed this question both in HeLa and in human endothelial cells. In stably transfected HeLa cells, inducibly expressing eNOS, expression of the enzyme per se blunted the phosphorylation of Akt/PKB in response to serum and strongly inhibited chemotaxis, an effect partially blocked by eNOS- and soluble guanylyl cyclase (sGC) inhibitors. Likewise, long-term pre-treatment of non-transfected HeLa cells with nanomolar concentrations of an NO donor inhibited subsequent migration, an effect blocked by sGC inhibition and mimicked by a cGMP analog. Finally, EC migration was stimulated by chronic pre-treatment with an eNOS inhibitor. Thus, in addition to its well-known stimulatory role, eNOS attenuates migration through basal long-term NO release. [Copyright &y& Elsevier]

Abstract: The enzyme endothelial Nitric Oxide Synthase (eNOS) is involved in key physiological and pathological processes, including cell motility and apoptosis. It is widely believed that at the cell surface eNOS is localized in caveolae, where caveolin-1 negatively regulates its activity, however, there are still uncertainties on its intracellular distribution. Here, we applied high resolution confocal microscopy to investigate the surface distribution of eNOS in transfected HeLa cells and in human umbilical vein endothelial cells (HUVEC) endogenously expressing the enzyme. In confluent and non-confluent HUVEC and HeLa cells, we failed to detect substantial colocalization between eNOS and caveolin-1 at the cell surface. Instead, in non-confluent cells, eNOS was concentrated in ruffles and at the leading edge of migrating cells, colocalizing with actin filaments and with the raft marker ganglioside GM1, and well segregated from caveolin-1, which was restricted to the posterior region of the cells. Treatments that disrupted microfilaments caused loss of eNOS from the cell surface and decreased Ca2+-stimulated activity, suggesting a role of the cytoskeleton in the localization and function of the enzyme. Our results provide a morphological correlate for the role of eNOS in cell migration and raise questions on the site of interaction between eNOS and caveolin-1. [Copyright &y& Elsevier]