cytochalasin-d and calpeptin

When rolling adherent neutrophils are stimulated, they rapidly immobilize through activation of integrin CD11b/CD18, and then modulate attachment through this integrin to allow migration. We investigated links between cytoskeletal rearrangement and changes in function of integrin CD11b/CD18 in neutrophils stimulated with formyl peptide (fMLP). Neutrophils treated with the actin-polymerizing agent jasplakinolide became rolling adherent on monolayers of activated platelets, but could not use CD11b/CD18 to become immobilised when fMLP was perfused over them. If treated with jasplakinolide after fMLP, the cells stopped migrating but could not detach when fMLP was removed. Jasplakinolide did not inhibit changes in intracellular Ca(2+) seen after fMLP treatment, or inhibit neutrophil immobilisation induced by externally added Mn(2+). Thus cytoskeletal rearrangement was directly implicated in upregulation and, later, downregulation of CD11b/CD18 binding. Inhibition of RhoA with C3-transferase caused a dose-dependent reduction of initial rolling adhesion of neutrophils, and reduced the rate of migration after stimulation; however, neither the conversion of rolling to stationary adhesion, nor the ability of neutrophils to detach on removal of the stimulus, were inhibited. Thus, Rho may regulate actin polymerisation and motility in neutrophils, but did not appear to control integrin-mediated adhesion itself. Integrin binding may be promoted by disruption of links to the cytoskeleton, effected through depolymerisation of actin or cleavage of linking protein talin by calpain. Disruption of actin filaments with cytochalasin D did not, however, cause integrin-mediated immobilisation of rolling neutrophils. Although the calpain inhibitor calpeptin did inhibit the adhesion response to fMLP, this was only at doses where actin polymerisation was also ablated. We suggest that the cytoskeleton actively regulates binding conformation of CD11b/CD18 as well as its mobility in the membrane.

Topics: ADP Ribose Transferases; Amides; Antineoplastic Agents; Botulinum Toxins; Cell Adhesion; Cell Movement; Cysteine Proteinase Inhibitors; Cytochalasin D; Cytoskeleton; Depsipeptides; Dipeptides; Down-Regulation; Enzyme Inhibitors; Humans; Integrins; Intracellular Signaling Peptides and Proteins; Macrophage-1 Antigen; Neutrophils; Nucleic Acid Synthesis Inhibitors; Peptides, Cyclic; Protein Serine-Threonine Kinases; Pyridines; rho-Associated Kinases; Signal Transduction

Modifications in cytoskeleton organization (monitored by scanning electron microscopy study of platelet shape) and cytoskeleton proteolysis were investigated for their role in phosphatidylserine exposure (measured with spin-labeled analogues of phospholipids) during A23187-induced activation of human platelets. Resting platelets treated with combinations of calpeptin and cytoskeleton-disrupting agents (nocodazole or cytochalasin D) remained discoid, and there was no dense granule release, cytoskeleton proteolysis or vesicle shedding. Spin-labeled phosphatidylserine was fully and rapidly redistributed (t1/2 approximately 5 min) from the outer to the inner leaflet of the plasma membrane through ATP-dependent aminophospholipid translocase activity. In contrast, spin-labeled phosphatidylcholine was only partially and slowly redistributed (less than 20% within 60 min) to the inner leaflet. Filopod formation, vesicle shedding, and calpain-mediated proteolysis were inhibited during activation of platelets treated with calpeptin and cytoskeleton-disrupting agents. Moreover, regardless of whether platelets were treated or not, spin-labeled phosphatidylserine was rapidly (t1/2 < 1 min) and massively (50%) exposed on the outer leaflet of the plasma membrane, while the slow and slight spin-labeled phosphatidylcholine influx did not counterbalance spin-labeled phosphatidylserine outflux. These results demonstrated that phosphatidylserine exposure was not connected to the following activation-related processes: cytoskeleton modifications (actin and tubulin polymerization, submembrane skeleton proteolysis), inhibition of aminophospholipid translocase, and filopod formation. Moreover, the redistribution kinetics of spin-labeled phospholipids during activation strongly suggested the involvement of an aminophospholipid exposure mechanism that differs from a scrambling phenomenon.

Topics: Blood Platelets; Calcimycin; Cell Membrane; Cytochalasin D; Cytoskeleton; Dipeptides; Glycerophosphates; Glycerylphosphorylcholine; Humans; Nocodazole; Phosphatidylserines; Platelet Activation; Serotonin