sq-23377 and monodansylcadaverine

We have recently reported that disruption of the actin cytoskeleton enhanced N-formylmethionyl-leucyl-phenylalanine (fMLP)-stimulated granule exocytosis in human neutrophils but decreased plasma membrane expression of complement receptor 1 (CR1), a marker of secretory vesicles. The present study was initiated to determine if reduced CR1 expression was due to fMLP-stimulated endocytosis, to determine the mechanism of this endocytosis, and to examine its impact on neutrophil functional responses. Stimulation of neutrophils with fMLP or ionomycin in the presence of latrunculin A resulted in the uptake of Alexa fluor 488-labeled albumin and transferrin and reduced plasma membrane expression of CR1. These effects were prevented by preincubation of the cells with sucrose, chlorpromazine, or monodansylcadaverine (MDC), inhibitors of clathrin-mediated endocytosis. Sucrose, chlorpromazine, and MDC also significantly inhibited fMLP- and ionomycin-stimulated specific and azurophil granule exocytosis. Disruption of microtubules with nocodazole inhibited endocytosis and azurophil granule exocytosis stimulated by fMLP in the presence of latrunculin A. Pharmacological inhibition of phosphatidylinositol 3-kinase, ERK1/2, and PKC significantly reduced fMLP-stimulated transferrin uptake in the presence of latrunculin A. Blockade of clathrin-mediated endocytosis had no significant effect on fMLP-stimulated phosphorylation of ERK1/2 in neutrophils pretreated with latrunculin A. From these data, we conclude that the actin cytoskeleton functions to limit microtubule-dependent, clathrin-mediated endocytosis in stimulated human neutrophils. The limitation of clathrin-mediated endocytosis by actin regulates the extent of both specific and azurophilic granule exocytosis.

Topics: Actins; Bridged Bicyclo Compounds, Heterocyclic; Cadaverine; Chlorpromazine; Clathrin; Endocytosis; Exocytosis; Humans; Ionomycin; Microtubules; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; N-Formylmethionine Leucyl-Phenylalanine; Neutrophil Activation; Neutrophils; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase Inhibitors; Receptors, Complement 3b; Secretory Vesicles; Serum Albumin, Bovine; Sucrose; Thiazolidines; Transferrin; Tubulin Modulators

Kinetic analysis of transferrin receptor properties in 6-8 day rat reticulocytes showed the existence of a single class of high-affinity receptors (Kd 3-10 nM), of which 20-25% were located at the cell surface and the remainder within an intracellular pool. Total transferrin receptor cycling time was 3.9 min. These studies examined the effects of various inhibitors on receptor-mediated transferrin iron delivery in order to define critical steps and events necessary to maintain the functional integrity of the pathway. Dansylcadaverine inhibited iron uptake by blocking exocytic release of transferrin and return of receptors to the cell surface, but did not affect transferrin endocytosis; this action served to deplete the surface pool of transferrin receptors, leading to shutdown of iron uptake. Calmidazolium and other putative calmodulin antagonists exerted an identical action on iron uptake and receptor recycling. The inhibitory effects of these agents on receptor recycling were overcome by the timely addition of Ca2+/ionomycin. From correlative analyses of the effects of these and other inhibitors, it was concluded that: (1) dansylcadaverine and calmodulin antagonists inhibit iron uptake by suppression of receptor recycling and exocytic transferrin release, (2) protein kinase C, transglutaminase, protein synthesis and release of transferrin-bound iron are not necessary for the functional integrity of the iron delivery pathway, (3) exocytic transferrin release and concomitant receptor recycling in rat reticulocytes is dependent upon Ca2+/calmodulin, (4) dansylcadaverine, dimethyldansylcadaverine and calmidazolium act on iron uptake by interfering with calmodulin function, and (5) the endocytotic and exocytotic arms of the iron delivery pathway are under separate regulatory control.

Topics: Animals; Biological Transport; Cadaverine; Calcium; Calmodulin; Cell Membrane; Endocytosis; Exocytosis; Imidazoles; Ionomycin; Iron; Kinetics; Microscopy, Electron; Rats; Rats, Inbred Strains; Receptors, Transferrin; Reticulocytes