N-formylpeptide-receptor dynamics, cytoskeletal activation, and intracellular calcium response in human neutrophil cytoplasts.
Cytoplasts (enucleated neutrophils which are depleted of dense granules) were prepared from human neutrophils with a modified procedure which employed dihydrocytochalasin B instead of cytochalasin B. These cytoplasts retained an activatable cytoskeletal network similar to cells in that filamentous actin polymerization in response to an N-formylpeptide (fluoresceinated N-formyl-nle-leu-phe-nle-tyr-lys, FLPEP) occurred with similar dose-response characteristics and was inhibitable by cytochalasin B and dihydrocytochalasin B. Cytoplasts had the same number of receptors per surface area as cells and binding constants and dissociation kinetics were the same for cells and cytoplasts. The conversion of receptors from a rapidly dissociating form to a slowly dissociating form was comparable in cells and cytoplasts. This conversion was not inhibited by cytochalasins and thus did not require actin polymerization. Cytoplasts were capable of internalizing 30% of bound FLPEP after 3 min of binding. Cytochalasins did not block this internalization which thus did not appear to require actin polymerization. After 5 min of binding, [3H]-N-formyl-met-leu-phe cosedimented with the Golgi marker enzymes when cytoplasts were fractionated on sucrose density gradients after N2 cavitation. These results indicate that the internalization mechanism is functional in cytoplasts. The Indo-1-detectable calcium response in cytoplasts had a ED50 similar to cells, though the maximum increase in Ca2+ concentration was about one-half that of cells. The response recovered with time after stimulation and the calcium detected was primarily from intracellular stores. The decay of responses after addition of formylpeptide antagonists was parallel for cells and cytoplasts, and leukotriene B4-induced responses in both cells and cytoplasts. Thus the regulation of the responses in cells and cytoplasts was analogous.
Omann
G M
GM
Department of Immunology, Scripps Clinic and Research Foundation, La Jolla, CA 92037.
Swann
W N
WN
Oades
Z G
ZG
Parkos
C A
CA
Jesaitis
A J
AJ
Sklar
L A
LA
D000199:Actins / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D003572:Cytochalasins / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism*; D003599:Cytoskeleton / Q000187:drug effects / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D007424:Intracellular Fluid / Q000378:metabolism; D007975:Leukotriene B4 / Q000494:pharmacology; D009504:Neutrophils / Q000378:metabolism* / Q000648:ultrastructure; D009842:Oligopeptides / Q000378:metabolism* / Q000494:pharmacology; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic / Q000378:metabolism*
D000199:Actins; D003572:Cytochalasins; D009842:Oligopeptides; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic; D007975:Leukotriene B4; C021642:F-chemotactic peptide; D002118:Calcium