Considerable controversy exists in the literature with regard to the nature of the agent mediating the biological effects of nitroxyl (NO-) donors. Here it is demonstrated that Angeli's salt (AS), a generator of NO-, enhanced human neutrophil migration. Under aerobic conditions, AS was converted to peroxynitrite to a small extent. However, using methionine, a scavenger of peroxynitrite, it was shown that peroxynitrite was not involved in AS-induced migration. AS equally enhanced human neutrophil migration under aerobic and anaerobic conditions, which strongly suggests that extracellular conversion of NO- to .NO by oxygen was not required. Furthermore, metHb and L-cysteine, which react more readily with NO- than with .NO, inhibited AS-induced migration, whereas the response towards gaseous .NO remained unaffected. AS induced an increase in the intracellular level of cGMP, although the curves for migration and cGMP level appeared to be slightly different in their concentration dependence. An inhibitor of soluble guanylate cyclase and antagonists of cGMP-dependent protein kinase had a more pronounced inhibitory effect on .NO-induced migration than on AS-induced migration. This suggests that the cGMP signalling cascade is partially, but not solely, responsible for AS-induced migration. As it has been demonstrated that soluble guanylate cyclase can only be activated by .NO, and not by NO-, these data indicate that NO- is at least partly converted intracellularly to .NO.

The monomeric G-protein Rac1, a member of the family of Rho/Rac/ Cdc42 GTPases, is involved in light-induced photoreceptor degeneration, but its specific role remains elusive. In particular, reports on Rac1 interacting with the visual pigment rhodopsin are puzzling and need a more quantitative examination. We probed the presence of Rac1 in rod outer segments by immunohistochemical staining of bovine retinae and western blot analysis of isolated rod outer segments. Rac1 was present throughout the whole retina except in the outer and inner nuclear layers, but was strongly expressed in photoreceptor cells. Rac1 was distributed in three different fractions of rod outer segments: one fraction was soluble in detergents, a second fraction cosegregated with lipid rafts, and a third fraction was associated with lipid bilayer free axonemal/cytoskeletal structures. We also investigated the interaction between rhodopsin and Rac1 by using surface plasmon resonance spectroscopy under dark and light conditions. Biophysical interaction studies revealed that Rac1 could interact with rhodopsin, but in a light-independent manner, and kinetic analysis indicated that binding of Rac1 occurred with lower affinity and speed than the association of transducin and rhodopsin. Thus, in dark-adapted rod cells, Rac1 cannot compete with transducin for binding to rhodopsin, and signalling can proceed normally. Instead, the concentration of transducin has to drop significantly so that Rac1 can bind to rhodopsin; in the outer segment, this occurs only under intense illumination, when transducin is translocated to the inner segment. Structured digital abstractRac1 binds rhodopsin to by surface plasmon resonance (View interaction)