| Abstract: | When dihydrocytochalasin (dhCB) was added either prior to or after CHO-Met-Leu-Phe (fMLP), the rate and duration of superoxide production in human granulocytes stimulated by fMLP was augmented. This effect was maximal when dhCB was added before fMLP, increasing the rate 1.5-3-fold. The effect of dhCB was progressively diminished for later additions and was undetectable after 6-10 min. The effects of dhCB could be blocked by the additional presence of 10 microM t-Boc-Phe-Leu-Phe-Leu-Phe (where t-Boc is t-butoxycarbonyl) indicating a requirement for receptor occupancy. In the presence of dhCB, the reversible binding of fML[3H]P was elevated and the formation of slowly dissociating surface complexes of occupied receptor and cytoskeleton was inhibited. Myeloperoxidase and lactoferrin release from fMLP-stimulated cells was induced by dhCB but was only partially correlated with the potentiating effects of dhCB on superoxide production and receptor expression. To circumvent the complicating effects of degranulation on the analysis of the functional consequences of receptor-cytoskeletal associations, cells were also preincubated with 100 nM fMLP at 15 degrees C. Under these conditions, the majority of the surface receptors became irreversibly occupied and coisolated with the cytoskeletal fraction of the cell. Subsequent exposure of the cells to fMLP at 37 degrees C resulted in no superoxide production. This desensitization was blocked by dhCB which also inhibited coisolation of the ligand and cytoskeletons. Conversion of receptor to a slowly dissociating state may represent its trapping in an inactive form and would provide a role for receptor-cytoskeleton interactions in the termination of the granulocyte response to chemoattractants. The inhibition of such receptor "sequestration" and the induction of new receptor expression could, therefore, partially account for dhCB-induced potentiation of the fMLP response. |
